PMC:1942070 / 0-8942 JSON TXT

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2_test

Id	Subject	Object	Predicate	Lexical cue
17349631-10859332-30580518	1348-1349	10859332	denotes	3
17349631-12506120-30580518	1348-1349	12506120	denotes	3
17349631-12506120-30580519	1662-1663	12506120	denotes	6
17349631-9765302-30580519	1662-1663	9765302	denotes	6
17349631-11062248-30580519	1662-1663	11062248	denotes	6
17349631-11410586-30580519	1662-1663	11410586	denotes	6
17349631-15590638-30580520	1807-1808	15590638	denotes	7
17349631-10471840-30580521	1884-1886	10471840	denotes	12
17349631-10856238-30580521	1884-1886	10856238	denotes	12
17349631-11410587-30580521	1884-1886	11410587	denotes	12
17349631-12646243-30580521	1884-1886	12646243	denotes	12
17349631-12676944-30580521	1884-1886	12676944	denotes	12
17349631-11912133-30580522	1994-1996	11912133	denotes	16
17349631-11514571-30580522	1994-1996	11514571	denotes	16
17349631-16899224-30580522	1994-1996	16899224	denotes	16
17349631-12505989-30580523	2026-2028	12505989	denotes	18
17349631-14654790-30580523	2026-2028	14654790	denotes	18
17349631-14563314-30580524	2057-2059	14563314	denotes	19
17349631-16678913-30580525	2218-2220	16678913	denotes	20
17349631-12505989-30580526	2382-2384	12505989	denotes	23
17349631-15604256-30580526	2382-2384	15604256	denotes	23
17349631-15226414-30580526	2382-2384	15226414	denotes	23
17349631-15728188-30580527	2438-2440	15728188	denotes	24
17349631-16875491-30580528	2527-2529	16875491	denotes	25
17349631-16449666-30580529	2708-2710	16449666	denotes	28
17349631-15738054-30580529	2708-2710	15738054	denotes	28
17349631-15623513-30580529	2708-2710	15623513	denotes	28
17349631-15367659-30580529	2708-2710	15367659	denotes	28
17349631-16449666-30580530	2855-2856	16449666	denotes	1
17349631-16449666-30580531	3155-3156	16449666	denotes	1
17349631-16449666-30580532	3922-3923	16449666	denotes	1
17349631-16449666-30580533	4035-4036	16449666	denotes	1
17349631-7730364-30580534	4113-4115	7730364	denotes	29
17349631-16449666-30580535	4822-4823	16449666	denotes	1
17349631-16449666-30580536	5518-5519	16449666	denotes	1
17349631-15728188-30580537	5605-5607	15728188	denotes	24
17349631-16678913-30580538	7023-7025	16678913	denotes	20
17349631-12505989-30580539	8270-8272	12505989	denotes	32
17349631-16166634-30580539	8270-8272	16166634	denotes	32
17349631-16421204-30580539	8270-8272	16421204	denotes	32
17349631-15145937-30580539	8270-8272	15145937	denotes	32

MyTest

Id	Subject	Object	Predicate	Lexical cue
17349631-10859332-30580518	1348-1349	10859332	denotes	3
17349631-12506120-30580518	1348-1349	12506120	denotes	3
17349631-12506120-30580519	1662-1663	12506120	denotes	6
17349631-9765302-30580519	1662-1663	9765302	denotes	6
17349631-11062248-30580519	1662-1663	11062248	denotes	6
17349631-11410586-30580519	1662-1663	11410586	denotes	6
17349631-15590638-30580520	1807-1808	15590638	denotes	7
17349631-10471840-30580521	1884-1886	10471840	denotes	12
17349631-10856238-30580521	1884-1886	10856238	denotes	12
17349631-11410587-30580521	1884-1886	11410587	denotes	12
17349631-12646243-30580521	1884-1886	12646243	denotes	12
17349631-12676944-30580521	1884-1886	12676944	denotes	12
17349631-11912133-30580522	1994-1996	11912133	denotes	16
17349631-11514571-30580522	1994-1996	11514571	denotes	16
17349631-16899224-30580522	1994-1996	16899224	denotes	16
17349631-12505989-30580523	2026-2028	12505989	denotes	18
17349631-14654790-30580523	2026-2028	14654790	denotes	18
17349631-14563314-30580524	2057-2059	14563314	denotes	19
17349631-16678913-30580525	2218-2220	16678913	denotes	20
17349631-12505989-30580526	2382-2384	12505989	denotes	23
17349631-15604256-30580526	2382-2384	15604256	denotes	23
17349631-15226414-30580526	2382-2384	15226414	denotes	23
17349631-15728188-30580527	2438-2440	15728188	denotes	24
17349631-16875491-30580528	2527-2529	16875491	denotes	25
17349631-16449666-30580529	2708-2710	16449666	denotes	28
17349631-15738054-30580529	2708-2710	15738054	denotes	28
17349631-15623513-30580529	2708-2710	15623513	denotes	28
17349631-15367659-30580529	2708-2710	15367659	denotes	28
17349631-16449666-30580530	2855-2856	16449666	denotes	1
17349631-16449666-30580531	3155-3156	16449666	denotes	1
17349631-16449666-30580532	3922-3923	16449666	denotes	1
17349631-16449666-30580533	4035-4036	16449666	denotes	1
17349631-7730364-30580534	4113-4115	7730364	denotes	29
17349631-16449666-30580535	4822-4823	16449666	denotes	1
17349631-16449666-30580536	5518-5519	16449666	denotes	1
17349631-15728188-30580537	5605-5607	15728188	denotes	24
17349631-16678913-30580538	7023-7025	16678913	denotes	20
17349631-12505989-30580539	8270-8272	12505989	denotes	32
17349631-16166634-30580539	8270-8272	16166634	denotes	32
17349631-16421204-30580539	8270-8272	16421204	denotes	32
17349631-15145937-30580539	8270-8272	15145937	denotes	32

pmc-enju-pas

Id	Subject	Object	Predicate	Lexical cue
T49	180-187	NN	denotes	protein
T50	188-194	NN	denotes	kinase
T51	195-196	NN	denotes	D
T52	197-198	-LRB-	denotes	(
T53	198-201	NN	denotes	PKD
T54	201-202	-RRB-	denotes	)
T55	203-210	NN	denotes	enzymes
T56	211-213	PRP	denotes	we
T57	214-223	VB	denotes	generated
T58	224-225	DT	denotes	a
T59	226-234	JJ	denotes	PKD-null
T60	235-239	NN	denotes	DT40
T61	240-252	NN	denotes	B-lymphocyte
T62	253-257	NN	denotes	cell
T63	258-262	NN	denotes	line
T64	264-274	RB	denotes	Previously
T65	275-277	PRP	denotes	we
T66	278-282	VB	denotes	have
T67	283-288	VB	denotes	shown
T68	289-293	IN	denotes	that
T69	294-298	NN	denotes	PKDs
T70	299-303	VB	denotes	have
T71	304-306	DT	denotes	an
T72	307-316	JJ	denotes	essential
T73	317-321	NN	denotes	role
T74	322-324	IN	denotes	in
T75	325-335	VB	denotes	regulating
T76	336-341	NN	denotes	class
T77	342-344	CD	denotes	II
T78	345-352	NN	denotes	histone
T79	353-365	NN	denotes	deacetylases
T80	366-368	IN	denotes	in
T81	369-373	NN	denotes	DT40
T82	374-381	NN	denotes	B-cells
T83	382-383	-LRB-	denotes	[
T84	383-391	NNP	denotes	Matthews
T85	391-392	-COMMA-	denotes	,
T86	393-397	NNP	denotes	S.A.
T87	397-398	-COMMA-	denotes	,
T88	399-402	NNP	denotes	Liu
T89	402-403	-COMMA-	denotes	,
T90	404-406	NNP	denotes	P.
T91	406-407	-COMMA-	denotes	,
T92	408-416	NNP	denotes	Spitaler
T93	416-417	-COMMA-	denotes	,
T94	418-420	NNP	denotes	M.
T95	420-421	-COMMA-	denotes	,
T96	422-427	NNP	denotes	Olson
T97	427-428	-COMMA-	denotes	,
T98	429-433	NNP	denotes	E.N.
T99	433-434	-COMMA-	denotes	,
T100	435-443	NNP	denotes	McKinsey
T101	443-444	-COMMA-	denotes	,
T102	445-449	NNP	denotes	T.A.
T103	449-450	-COMMA-	denotes	,
T104	451-459	NNP	denotes	Cantrell
T105	459-460	-COMMA-	denotes	,
T106	461-465	NNP	denotes	D.A.
T107	466-469	CC	denotes	and
T108	470-481	NNP	denotes	Scharenberg
T109	481-482	-COMMA-	denotes	,
T110	483-487	NNP	denotes	A.M.
T111	488-489	-LRB-	denotes	(
T112	489-493	CD	denotes	2006
T113	493-494	-RRB-	denotes	)
T114	495-504	JJ	denotes	Essential
T115	505-509	NN	denotes	role
T116	510-513	IN	denotes	for
T117	514-521	NN	denotes	protein
T118	522-528	NN	denotes	kinase
T119	529-530	NN	denotes	D
T120	531-537	NN	denotes	family
T121	538-545	NN	denotes	kinases
T122	546-548	IN	denotes	in
T123	549-552	DT	denotes	the
T124	553-563	NN	denotes	regulation
T125	564-566	IN	denotes	of
T126	567-572	NN	denotes	class
T127	573-575	CD	denotes	II
T128	576-583	NN	denotes	histone
T129	584-596	NN	denotes	deacetylases
T130	597-599	IN	denotes	in
T131	600-601	NN	denotes	B
T132	602-613	NN	denotes	lymphocytes
T133	615-619	NNP	denotes	Mol.
T134	620-624	NNP	denotes	Cell
T135	625-630	NNP	denotes	Biol.
T136	631-633	CD	denotes	26
T137	633-634	-COMMA-	denotes	,
T138	635-644	CD	denotes	1569–1577
T139	644-645	-RRB-	denotes	]
T140	647-649	PRP	denotes	We
T141	650-653	RB	denotes	now
T142	654-658	VB	denotes	show
T143	659-663	IN	denotes	that
T144	664-668	NN	denotes	PKDs
T145	669-672	VB	denotes	are
T146	673-677	RB	denotes	also
T147	678-686	VB	denotes	required
T148	687-689	TO	denotes	to
T149	690-698	VB	denotes	regulate
T150	699-704	NN	denotes	HSP27
T151	705-720	NN	denotes	phosphorylation
T152	721-723	IN	denotes	in
T153	724-728	NN	denotes	DT40
T154	729-736	NN	denotes	B-cells
T155	738-745	RB	denotes	However
T156	745-746	-COMMA-	denotes	,
T157	747-749	IN	denotes	in
T158	750-758	NN	denotes	contrast
T159	759-761	TO	denotes	to
T160	762-770	JJ	denotes	previous
T161	771-783	NN	denotes	observations
T162	784-786	IN	denotes	in
T163	787-792	JJ	denotes	other
T164	793-797	NN	denotes	cell
T165	798-803	NN	denotes	types
T166	803-804	-COMMA-	denotes	,
T167	805-808	NN	denotes	PKD
T168	809-816	NN	denotes	enzymes
T169	817-819	VB	denotes	do
T170	820-823	RB	denotes	not
T171	824-832	VB	denotes	regulate
T172	833-838	JJ	denotes	basic
T173	839-847	JJ	denotes	cellular
T174	848-857	NN	denotes	processes
T175	858-862	JJ	denotes	such
T176	863-865	IN	denotes	as
T177	866-879	NN	denotes	proliferation
T178	880-882	CC	denotes	or
T179	883-891	NN	denotes	survival
T180	892-901	NN	denotes	responses
T181	901-902	-COMMA-	denotes	,
T182	903-906	CC	denotes	nor
T183	907-911	NN	denotes	NFκB
T184	912-927	JJ	denotes	transcriptional
T185	928-936	NN	denotes	activity
T186	937-947	RB	denotes	downstream
T187	948-950	IN	denotes	of
T188	951-954	DT	denotes	the
T189	955-956	NN	denotes	B
T190	957-961	NN	denotes	cell
T191	962-969	NN	denotes	antigen
T192	970-978	NN	denotes	receptor
T193	980-984	RB	denotes	Thus
T194	984-985	-COMMA-	denotes	,
T195	986-990	NN	denotes	PKDs
T196	991-995	VB	denotes	have
T197	996-997	DT	denotes	a
T198	998-1007	JJ	denotes	selective
T199	1008-1012	NN	denotes	role
T200	1013-1015	IN	denotes	in
T201	1016-1020	NN	denotes	DT40
T202	1021-1027	NN	denotes	B-cell
T203	1028-1035	NN	denotes	biology
T532	635-1057	NN	denotes	1569–1577]. We now show that PKDs are also required to regulate HSP27 phosphorylation in DT40 B-cells. However, in contrast to previous observations in other cell types, PKD enzymes do not regulate basic cellular processes such as proliferation or survival responses, nor NFκB transcriptional activity downstream of the B cell antigen receptor. Thus, PKDs have a selective role in DT40 B-cell biology. 1 Introduction The
T533	1058-1065	NN	denotes	protein
T534	1066-1072	NN	denotes	kinase
T535	1073-1074	NN	denotes	D
T536	1075-1076	-LRB-	denotes	(
T537	1076-1079	NN	denotes	PKD
T538	1079-1080	-RRB-	denotes	)
T539	1081-1097	NN	denotes	serine/threonine
T540	1098-1104	NN	denotes	kinase
T541	1105-1111	NN	denotes	family
T542	1112-1115	VB	denotes	has
T543	1116-1121	CD	denotes	three
T544	1122-1129	NN	denotes	members
T545	1129-1130	-COLON-	denotes	:
T546	1131-1135	NN	denotes	PKD1
T547	1135-1136	-COMMA-	denotes	,
T548	1137-1141	NN	denotes	PKD2
T549	1142-1145	CC	denotes	and
T550	1146-1150	NN	denotes	PKD3
T551	1152-1156	JJ	denotes	Most
T552	1157-1161	NN	denotes	cell
T553	1162-1167	NN	denotes	types
T554	1168-1175	VB	denotes	express
T555	1176-1178	IN	denotes	at
T556	1179-1184	JJ	denotes	least
T557	1185-1188	CD	denotes	two
T558	1189-1192	NN	denotes	PKD
T559	1193-1201	NN	denotes	isoforms
T560	1202-1205	CC	denotes	but
T561	1206-1209	NN	denotes	PKD
T562	1210-1217	NN	denotes	enzymes
T563	1218-1221	VB	denotes	are
T564	1222-1232	RB	denotes	especially
T565	1233-1239	RB	denotes	highly
T566	1240-1249	VB	denotes	expressed
T567	1250-1252	IN	denotes	in
T568	1253-1267	JJ	denotes	haematopoietic
T569	1268-1273	NN	denotes	cells
T570	1273-1274	-COMMA-	denotes	,
T571	1275-1280	WRB	denotes	where
T572	1281-1285	PRP	denotes	they
T573	1286-1289	VB	denotes	are
T574	1290-1299	VB	denotes	activated
T575	1300-1302	IN	denotes	in
T576	1303-1311	NN	denotes	response
T577	1312-1314	TO	denotes	to
T578	1315-1322	NN	denotes	antigen
T579	1323-1332	NN	denotes	receptors
T580	1333-1344	NN	denotes	stimulation
T581	1345-1346	-LRB-	denotes	[
T582	1346-1349	CD	denotes	2,3
T583	1349-1350	-RRB-	denotes	]
T584	1352-1353	DT	denotes	A
T585	1354-1363	VB	denotes	conserved
T586	1364-1374	NN	denotes	signalling
T587	1375-1382	NN	denotes	pathway
T588	1383-1390	VB	denotes	linking
T589	1391-1398	NN	denotes	antigen
T590	1399-1408	NN	denotes	receptors
T591	1409-1411	TO	denotes	to
T592	1412-1416	NN	denotes	PKDs
T593	1417-1425	VB	denotes	involves
T594	1426-1429	DT	denotes	the
T595	1430-1440	NN	denotes	activation
T596	1441-1443	IN	denotes	of
T597	1444-1448	NN	denotes	PLCγ
T598	1449-1452	CC	denotes	and
T599	1453-1456	DT	denotes	the
T600	1457-1467	JJ	denotes	subsequent
T601	1468-1478	NN	denotes	production
T602	1479-1481	IN	denotes	of
T603	1482-1496	NN	denotes	diacylglycerol
T604	1497-1498	-LRB-	denotes	(
T605	1498-1501	NN	denotes	DAG
T606	1501-1502	-RRB-	denotes	)
T607	1503-1508	WDT	denotes	which
T608	1509-1519	VB	denotes	stimulates
T609	1520-1529	JJ	denotes	classical
T610	1530-1536	CC	denotes	and/or
T611	1537-1542	JJ	denotes	novel
T612	1543-1550	NN	denotes	protein
T613	1551-1557	NN	denotes	kinase
T614	1558-1560	NN	denotes	Cs
T615	1561-1562	-LRB-	denotes	(
T616	1562-1565	NN	denotes	PKC
T617	1565-1566	-RRB-	denotes	)
T618	1567-1571	WDT	denotes	that
T619	1572-1585	VB	denotes	phosphorylate
T620	1586-1589	CD	denotes	two
T621	1590-1593	JJ	denotes	key
T622	1594-1604	JJ	denotes	regulatory
T623	1605-1611	NN	denotes	serine
T624	1612-1620	NN	denotes	residues
T625	1621-1623	IN	denotes	in
T626	1624-1627	DT	denotes	the
T627	1628-1638	NN	denotes	activation
T628	1639-1643	NN	denotes	loop
T629	1644-1646	IN	denotes	of
T630	1647-1650	NN	denotes	PKD
T631	1651-1658	NN	denotes	kinases
T632	1659-1660	-LRB-	denotes	[
T633	1660-1663	CD	denotes	3–6
T634	1663-1664	-RRB-	denotes	]
T635	1666-1669	DT	denotes	The
T636	1670-1680	JJ	denotes	N-terminal
T637	1681-1691	JJ	denotes	regulatory
T638	1692-1698	NN	denotes	region
T639	1699-1701	IN	denotes	of
T640	1702-1705	NN	denotes	PKD
T641	1706-1713	NN	denotes	enzymes
T642	1714-1722	VB	denotes	contains
T643	1723-1724	DT	denotes	a
T644	1725-1728	NN	denotes	DAG
T645	1729-1736	NN	denotes	binding
T646	1737-1743	NN	denotes	domain
T647	1744-1747	CC	denotes	and
T648	1748-1754	JJ	denotes	direct
T649	1755-1762	NN	denotes	binding
T650	1763-1765	IN	denotes	of
T651	1766-1769	NN	denotes	DAG
T652	1770-1774	RB	denotes	also
T653	1775-1786	VB	denotes	contributes
T654	1787-1789	TO	denotes	to
T655	1790-1794	NN	denotes	PKD1
T656	1795-1805	NN	denotes	activation
T657	1806-1807	-LRB-	denotes	[
T658	1807-1808	CD	denotes	7
T659	1808-1809	-RRB-	denotes	]
T660	1810-1812	RB	denotes	as
T661	1813-1817	RB	denotes	well
T662	1818-1820	IN	denotes	as
T663	1821-1831	VB	denotes	regulating
T664	1832-1835	DT	denotes	the
T665	1836-1843	JJ	denotes	spatial
T666	1844-1852	NN	denotes	location
T667	1853-1855	IN	denotes	of
T668	1856-1859	NN	denotes	PKD
T669	1860-1867	NN	denotes	enzymes
T670	1868-1874	IN	denotes	within
T671	1875-1880	NN	denotes	cells
T672	1881-1882	-LRB-	denotes	[
T673	1882-1886	CD	denotes	8–12
T674	1886-1887	-RRB-	denotes	]
T675	1889-1892	NN	denotes	PKD
T676	1893-1900	NN	denotes	enzymes
T677	1901-1905	VB	denotes	have
T678	1906-1910	VB	denotes	been
T679	1911-1919	VB	denotes	proposed
T680	1920-1922	TO	denotes	to
T681	1923-1931	VB	denotes	regulate
T682	1932-1940	JJ	denotes	numerous
T683	1941-1949	JJ	denotes	cellular
T684	1950-1959	NN	denotes	functions
T685	1959-1960	-COMMA-	denotes	,
T686	1961-1970	VB	denotes	including
T687	1971-1975	NN	denotes	cell
T688	1976-1989	NN	denotes	proliferation
T689	1990-1991	-LRB-	denotes	[
T690	1991-1996	CD	denotes	13–16
T691	1996-1997	-RRB-	denotes	]
T692	1997-1998	-COMMA-	denotes	,
T693	1999-2013	JJ	denotes	anti-apoptotic
T694	2014-2021	NN	denotes	signals
T695	2022-2023	-LRB-	denotes	[
T696	2023-2028	CD	denotes	17,18
T697	2028-2029	-RRB-	denotes	]
T698	2030-2033	CC	denotes	and
T699	2034-2043	NN	denotes	thymocyte
T700	2044-2055	NN	denotes	development
T701	2056-2057	-LRB-	denotes	[
T702	2057-2059	CD	denotes	19
T703	2059-2060	-RRB-	denotes	]
T704	2062-2072	NN	denotes	Expression
T705	2073-2075	IN	denotes	of
T706	2076-2082	JJ	denotes	mutant
T707	2083-2096	RB	denotes	catalytically
T708	2097-2105	JJ	denotes	inactive
T709	2106-2109	CC	denotes	and
T710	2110-2124	RB	denotes	constitutively
T711	2125-2134	VB	denotes	activated
T712	2135-2139	NN	denotes	PKDs
T713	2140-2143	MD	denotes	can
T714	2144-2148	RB	denotes	also
T715	2149-2155	VB	denotes	modify
T716	2156-2161	NNP	denotes	Golgi
T717	2162-2170	NN	denotes	function
T718	2170-2171	-COMMA-	denotes	,
T719	2172-2176	NN	denotes	cell
T720	2177-2185	NN	denotes	adhesion
T721	2186-2189	CC	denotes	and
T722	2190-2194	NN	denotes	cell
T723	2195-2203	NN	denotes	motility
T724	2204-2205	-LRB-	denotes	(
T725	2205-2213	VB	denotes	reviewed
T726	2214-2216	IN	denotes	in
T727	2217-2218	-LRB-	denotes	[
T728	2218-2220	CD	denotes	20
T729	2220-2221	-RRB-	denotes	]
T730	2221-2222	-RRB-	denotes	)
T731	2224-2226	IN	denotes	In
T732	2227-2237	JJ	denotes	particular
T733	2237-2238	-COMMA-	denotes	,
T734	2239-2243	NN	denotes	PKDs
T735	2244-2248	VB	denotes	have
T736	2249-2253	VB	denotes	been
T737	2254-2260	RB	denotes	widely
T738	2261-2267	VB	denotes	linked
T739	2268-2270	TO	denotes	to
T740	2271-2274	DT	denotes	the
T741	2275-2285	NN	denotes	activation
T742	2286-2288	IN	denotes	of
T743	2289-2292	DT	denotes	the
T744	2293-2297	NN	denotes	NFκB
T745	2298-2311	NN	denotes	transcription
T746	2312-2318	NN	denotes	factor
T747	2319-2322	CC	denotes	and
T748	2323-2325	IN	denotes	in
T749	2326-2336	VB	denotes	regulating
T750	2337-2341	NN	denotes	cell
T751	2342-2350	NN	denotes	survival
T752	2351-2357	IN	denotes	during
T753	2358-2367	JJ	denotes	oxidative
T754	2368-2374	NN	denotes	stress
T755	2375-2376	-LRB-	denotes	[
T756	2376-2384	CD	denotes	17,21–23
T757	2384-2385	-RRB-	denotes	]
T758	2387-2394	DT	denotes	Another
T759	2395-2403	RB	denotes	recently
T760	2404-2412	VB	denotes	proposed
T761	2413-2417	NN	denotes	PKD1
T762	2418-2427	NN	denotes	substrate
T763	2428-2430	VB	denotes	is
T764	2431-2436	NN	denotes	HSP27
T765	2437-2438	-LRB-	denotes	[
T766	2438-2440	CD	denotes	24
T767	2440-2441	-RRB-	denotes	]
T768	2441-2442	-COMMA-	denotes	,
T769	2443-2444	DT	denotes	a
T770	2445-2450	JJ	denotes	small
T771	2451-2455	NN	denotes	heat
T772	2456-2461	NN	denotes	shock
T773	2462-2469	NN	denotes	protein
T774	2470-2478	VB	denotes	involved
T775	2479-2481	IN	denotes	in
T776	2482-2492	VB	denotes	regulating
T777	2493-2497	NN	denotes	cell
T778	2498-2507	NN	denotes	migration
T779	2508-2511	CC	denotes	and
T780	2512-2516	NN	denotes	cell
T781	2517-2525	NN	denotes	survival
T782	2526-2527	-LRB-	denotes	[
T783	2527-2529	CD	denotes	25
T784	2529-2530	-RRB-	denotes	]
T785	2532-2534	DT	denotes	An
T786	2535-2544	JJ	denotes	essential
T787	2545-2549	NN	denotes	role
T788	2550-2553	IN	denotes	for
T789	2554-2557	NN	denotes	PKD
T790	2558-2565	NN	denotes	enzymes
T791	2566-2568	IN	denotes	in
T792	2569-2579	VB	denotes	regulating
T793	2580-2585	NN	denotes	class
T794	2586-2588	CD	denotes	II
T795	2589-2596	NN	denotes	histone
T796	2597-2609	NN	denotes	deacetylases
T797	2610-2611	-LRB-	denotes	(
T798	2611-2616	NN	denotes	HDACs
T799	2616-2617	-RRB-	denotes	)
T800	2617-2618	-COMMA-	denotes	,
T801	2619-2626	NN	denotes	enzymes
T802	2627-2631	WDT	denotes	that
T803	2632-2639	VB	denotes	repress
T804	2640-2654	JJ	denotes	MEF2-dependent
T805	2655-2659	NN	denotes	gene
T806	2660-2673	NN	denotes	transcription
T807	2673-2674	-COMMA-	denotes	,
T808	2675-2678	VB	denotes	has
T809	2679-2683	RB	denotes	also
T810	2684-2688	VB	denotes	been
T811	2689-2701	VB	denotes	demonstrated
T812	2702-2703	-LRB-	denotes	[
T813	2703-2710	CD	denotes	1,26–28
T814	2710-2711	-RRB-	denotes	]
T815	2713-2715	TO	denotes	To
T816	2716-2727	VB	denotes	investigate
T817	2728-2731	DT	denotes	the
T818	2732-2742	JJ	denotes	biological
T819	2743-2747	NN	denotes	role
T820	2748-2750	IN	denotes	of
T821	2751-2755	NN	denotes	PKDs
T822	2756-2758	PRP	denotes	we
T823	2759-2763	VB	denotes	have
T824	2764-2773	VB	denotes	generated
T825	2774-2778	NN	denotes	DT40
T826	2779-2780	NN	denotes	B
T827	2781-2785	NN	denotes	cell
T828	2786-2791	NN	denotes	lines
T829	2792-2796	WDT	denotes	that
T830	2797-2801	VB	denotes	lack
T831	2802-2812	NN	denotes	expression
T832	2813-2815	IN	denotes	of
T833	2816-2819	CD	denotes	one
T834	2820-2822	CC	denotes	or
T835	2823-2827	JJ	denotes	more
T836	2828-2835	NN	denotes	members
T837	2836-2838	IN	denotes	of
T838	2839-2842	DT	denotes	the
T839	2843-2846	NN	denotes	PKD
T840	2847-2853	NN	denotes	family
T841	2854-2855	-LRB-	denotes	[
T842	2855-2856	CD	denotes	1
T843	2856-2857	-RRB-	denotes	]
T844	2857-2858	-COMMA-	denotes	,
T845	2859-2867	VB	denotes	allowing
T846	2868-2870	PRP	denotes	us
T847	2871-2873	TO	denotes	to
T848	2874-2885	VB	denotes	investigate
T849	2886-2889	DT	denotes	the
T850	2890-2898	NN	denotes	function
T851	2898-2899	-LRB-	denotes	(
T852	2899-2900	NN	denotes	s
T853	2900-2901	-RRB-	denotes	)
T854	2902-2904	IN	denotes	of
T855	2905-2908	NN	denotes	PKD
T856	2909-2917	NN	denotes	isoforms
T857	2918-2927	VB	denotes	following
T858	2928-2929	NN	denotes	B
T859	2930-2934	NN	denotes	cell
T860	2935-2942	NN	denotes	antigen
T861	2943-2951	NN	denotes	receptor
T862	2952-2953	-LRB-	denotes	(
T863	2953-2956	NN	denotes	BCR
T864	2956-2957	-RRB-	denotes	)
T865	2958-2969	NN	denotes	stimulation
T866	2969-2970	-COMMA-	denotes	,
T867	2971-2973	RB	denotes	as
T868	2974-2978	RB	denotes	well
T869	2979-2989	VB	denotes	addressing
T870	2990-2993	DT	denotes	the
T871	2994-2999	NN	denotes	issue
T872	3000-3002	IN	denotes	of
T873	3003-3013	JJ	denotes	functional
T874	3014-3024	NN	denotes	redundancy
T875	3025-3032	IN	denotes	between
T876	3033-3036	DT	denotes	the
T877	3037-3046	JJ	denotes	different
T878	3047-3050	NN	denotes	PKD
T879	3051-3057	NN	denotes	family
T880	3058-3065	NN	denotes	members
T881	3067-3075	JJ	denotes	Previous
T882	3076-3083	NN	denotes	studies
T883	3084-3088	VB	denotes	have
T884	3089-3094	VB	denotes	shown
T885	3095-3099	IN	denotes	that
T886	3100-3104	NN	denotes	PKDs
T887	3105-3108	VB	denotes	are
T888	3109-3122	JJ	denotes	indispensable
T889	3123-3126	IN	denotes	for
T890	3127-3131	NN	denotes	HDAC
T891	3132-3142	NN	denotes	regulation
T892	3143-3145	IN	denotes	in
T893	3146-3147	NN	denotes	B
T894	3148-3153	NN	denotes	cells
T895	3154-3155	-LRB-	denotes	[
T896	3155-3156	CD	denotes	1
T897	3156-3157	-RRB-	denotes	]
T898	3159-3165	RB	denotes	Herein
T899	3166-3168	PRP	denotes	we
T900	3169-3173	VB	denotes	show
T901	3174-3178	IN	denotes	that
T902	3179-3183	NN	denotes	PKDs
T903	3184-3187	VB	denotes	are
T904	3188-3192	RB	denotes	also
T905	3193-3206	JJ	denotes	indispensable
T906	3207-3210	IN	denotes	for
T907	3211-3216	NN	denotes	HSP27
T908	3217-3232	NN	denotes	phosphorylation
T909	3233-3235	IN	denotes	in
T910	3236-3237	NN	denotes	B
T911	3238-3243	NN	denotes	cells
T912	3245-3252	RB	denotes	However
T913	3252-3253	-COMMA-	denotes	,
T914	3254-3262	JJ	denotes	PKD-null
T915	3263-3267	NN	denotes	DT40
T916	3268-3269	NN	denotes	B
T917	3270-3275	NN	denotes	cells
T918	3276-3279	VB	denotes	are
T919	3280-3286	JJ	denotes	viable
T920	3287-3290	CC	denotes	and
T921	3291-3302	VB	denotes	proliferate
T922	3303-3311	RB	denotes	normally
T923	3313-3321	RB	denotes	Moreover
T924	3321-3322	-COMMA-	denotes	,
T925	3323-3327	NN	denotes	loss
T926	3328-3330	IN	denotes	of
T927	3331-3334	DT	denotes	the
T928	3335-3341	JJ	denotes	entire
T929	3342-3350	JJ	denotes	cellular
T930	3351-3355	NN	denotes	pool
T931	3356-3358	IN	denotes	of
T932	3359-3362	NN	denotes	PKD
T933	3363-3367	VB	denotes	does
T934	3368-3371	RB	denotes	not
T935	3372-3382	RB	denotes	critically
T936	3383-3389	VB	denotes	affect
T937	3390-3399	JJ	denotes	oxidative
T938	3400-3406	NN	denotes	stress
T939	3407-3416	NN	denotes	responses
T940	3417-3419	IN	denotes	in
T941	3420-3421	NN	denotes	B
T942	3422-3427	NN	denotes	cells
T943	3428-3431	CC	denotes	nor
T944	3432-3434	VB	denotes	do
T945	3435-3438	NN	denotes	PKD
T946	3439-3446	NN	denotes	kinases
T947	3447-3451	VB	denotes	play
T948	3452-3454	DT	denotes	an
T949	3455-3464	JJ	denotes	essential
T950	3465-3469	NN	denotes	role
T951	3470-3472	IN	denotes	in
T952	3473-3483	VB	denotes	regulating
T953	3484-3488	NN	denotes	NFκB
T954	3489-3504	JJ	denotes	transcriptional
T955	3505-3513	NN	denotes	activity
T956	3515-3523	RB	denotes	Together
T957	3523-3524	-COMMA-	denotes	,
T958	3525-3530	DT	denotes	these
T959	3531-3539	NN	denotes	findings
T960	3540-3546	VB	denotes	reveal
T961	3547-3551	IN	denotes	that
T962	3552-3554	IN	denotes	in
T963	3555-3556	NN	denotes	B
T964	3557-3568	NN	denotes	lymphocytes
T965	3568-3569	-COMMA-	denotes	,
T966	3570-3573	NN	denotes	PKD
T967	3574-3581	NN	denotes	kinases
T968	3582-3585	VB	denotes	are
T969	3586-3589	RB	denotes	not
T970	3590-3598	JJ	denotes	critical
T971	3599-3609	NN	denotes	regulators
T972	3610-3612	IN	denotes	of
T973	3613-3617	JJ	denotes	many
T974	3618-3620	IN	denotes	of
T975	3621-3624	DT	denotes	the
T976	3625-3633	JJ	denotes	cellular
T977	3634-3643	NN	denotes	processes
T978	3644-3654	RB	denotes	previously
T979	3655-3663	VB	denotes	ascribed
T980	3664-3666	TO	denotes	to
T981	3667-3671	PRP	denotes	them
T982	3672-3674	IN	denotes	in
T983	3675-3680	JJ	denotes	other
T984	3681-3689	JJ	denotes	cellular
T985	3690-3697	NN	denotes	systems
T1674	0-3735	NN	denotes	Protein kinase D enzymes are dispensable for proliferation, survival and antigen receptor-regulated NFκB activity in vertebrate B-cells Abstract To investigate the importance of protein kinase D (PKD) enzymes we generated a PKD-null DT40 B-lymphocyte cell line. Previously we have shown that PKDs have an essential role in regulating class II histone deacetylases in DT40 B-cells [Matthews, S.A., Liu, P., Spitaler, M., Olson, E.N., McKinsey, T.A., Cantrell, D.A. and Scharenberg, A.M. (2006) Essential role for protein kinase D family kinases in the regulation of class II histone deacetylases in B lymphocytes. Mol. Cell Biol. 26, 1569–1577]. We now show that PKDs are also required to regulate HSP27 phosphorylation in DT40 B-cells. However, in contrast to previous observations in other cell types, PKD enzymes do not regulate basic cellular processes such as proliferation or survival responses, nor NFκB transcriptional activity downstream of the B cell antigen receptor. Thus, PKDs have a selective role in DT40 B-cell biology. 1 Introduction The protein kinase D (PKD) serine/threonine kinase family has three members: PKD1, PKD2 and PKD3. Most cell types express at least two PKD isoforms but PKD enzymes are especially highly expressed in haematopoietic cells, where they are activated in response to antigen receptors stimulation [2,3]. A conserved signalling pathway linking antigen receptors to PKDs involves the activation of PLCγ and the subsequent production of diacylglycerol (DAG) which stimulates classical and/or novel protein kinase Cs (PKC) that phosphorylate two key regulatory serine residues in the activation loop of PKD kinases [3–6]. The N-terminal regulatory region of PKD enzymes contains a DAG binding domain and direct binding of DAG also contributes to PKD1 activation [7] as well as regulating the spatial location of PKD enzymes within cells [8–12]. PKD enzymes have been proposed to regulate numerous cellular functions, including cell proliferation [13–16], anti-apoptotic signals [17,18] and thymocyte development [19]. Expression of mutant catalytically inactive and constitutively activated PKDs can also modify Golgi function, cell adhesion and cell motility (reviewed in [20]). In particular, PKDs have been widely linked to the activation of the NFκB transcription factor and in regulating cell survival during oxidative stress [17,21–23]. Another recently proposed PKD1 substrate is HSP27 [24], a small heat shock protein involved in regulating cell migration and cell survival [25]. An essential role for PKD enzymes in regulating class II histone deacetylases (HDACs), enzymes that repress MEF2-dependent gene transcription, has also been demonstrated [1,26–28]. To investigate the biological role of PKDs we have generated DT40 B cell lines that lack expression of one or more members of the PKD family [1], allowing us to investigate the function(s) of PKD isoforms following B cell antigen receptor (BCR) stimulation, as well addressing the issue of functional redundancy between the different PKD family members. Previous studies have shown that PKDs are indispensable for HDAC regulation in B cells [1]. Herein we show that PKDs are also indispensable for HSP27 phosphorylation in B cells. However, PKD-null DT40 B cells are viable and proliferate normally. Moreover, loss of the entire cellular pool of PKD does not critically affect oxidative stress responses in B cells nor do PKD kinases play an essential role in regulating NFκB transcriptional activity. Together, these findings reveal that in B lymphocytes, PKD kinases are not critical regulators of many of the cellular processes previously ascribed to them in other cellular systems. 2 Materials and methods 2.1 Cell
T1675	3736-3743	NN	denotes	culture
T1676	3743-3744	-COMMA-	denotes	,
T1677	3745-3754	JJ	denotes	transient
T1678	3755-3768	NN	denotes	transfections
T1679	3769-3772	CC	denotes	and
T1680	3773-3777	NN	denotes	cell
T1681	3778-3789	NN	denotes	stimulation
T1682	3790-3793	DT	denotes	The
T1683	3794-3804	NN	denotes	generation
T1684	3804-3805	-COMMA-	denotes	,
T1685	3806-3813	NN	denotes	culture
T1686	3814-3817	CC	denotes	and
T1687	3818-3828	NN	denotes	activation
T1688	3829-3831	IN	denotes	of
T1689	3832-3839	NN	denotes	PKD1−/−
T1690	3839-3840	-COMMA-	denotes	,
T1691	3841-3848	NN	denotes	PKD3−/−
T1692	3849-3852	CC	denotes	and
T1693	3853-3862	NN	denotes	PKD1/3−/−
T1694	3863-3871	NN	denotes	knockout
T1695	3872-3876	NN	denotes	DT40
T1696	3877-3878	NN	denotes	B
T1697	3879-3883	NN	denotes	cell
T1698	3884-3889	NN	denotes	lines
T1699	3890-3894	VB	denotes	have
T1700	3895-3899	VB	denotes	been
T1701	3900-3909	VB	denotes	described
T1702	3910-3920	RB	denotes	previously
T1703	3921-3922	-LRB-	denotes	[
T1704	3922-3923	CD	denotes	1
T1705	3923-3924	-RRB-	denotes	]
T1706	3926-3931	NN	denotes	Cells
T1707	3932-3936	VB	denotes	were
T1708	3937-3942	VB	denotes	lysed
T1709	3943-3946	CC	denotes	and
T1710	3947-3954	NN	denotes	protein
T1711	3955-3963	NN	denotes	extracts
T1712	3964-3968	VB	denotes	were
T1713	3969-3977	VB	denotes	analysed
T1714	3978-3980	IN	denotes	in
T1715	3981-3988	JJ	denotes	Western
T1716	3989-3997	NN	denotes	blotting
T1717	3998-4009	NN	denotes	experiments
T1718	4010-4012	IN	denotes	as
T1719	4013-4023	RB	denotes	previously
T1720	4024-4033	VB	denotes	described
T1721	4034-4035	-LRB-	denotes	[
T1873	4151-4152	-LRB-	denotes	(
T1874	4152-4159	CD	denotes	2 × 106
T1875	4160-4165	NN	denotes	cells
T1876	4166-4169	IN	denotes	per
T1877	4170-4175	NN	denotes	point
T1878	4175-4176	-RRB-	denotes	)
T1879	4177-4181	VB	denotes	were
T1880	4182-4193	VB	denotes	resuspended
T1881	4194-4196	IN	denotes	in
T1882	4197-4203	NN	denotes	200 μl
T1883	4204-4210	NN	denotes	buffer
T1884	4211-4212	-LRB-	denotes	(
T1885	4212-4216	NN	denotes	RPMI
T1886	4217-4221	CD	denotes	1640
T1887	4222-4227	NN	denotes	media
T1888	4227-4228	-COMMA-	denotes	,
T1889	4229-4230	CD	denotes	1
T1890	4230-4231	NN	denotes	%
T1891	4232-4238	JJ	denotes	foetal
T1892	4239-4243	NN	denotes	calf
T1893	4244-4249	NN	denotes	serum
T1894	4249-4250	-RRB-	denotes	)
T1895	4251-4261	VB	denotes	containing
T1896	4262-4274	JJ	denotes	anti-chicken
T1897	4275-4277	NN	denotes	M1
T1898	4278-4288	JJ	denotes	monoclonal
T1899	4289-4297	NN	denotes	antibody
T1900	4298-4308	VB	denotes	conjugated
T1901	4309-4311	TO	denotes	to
T1902	4312-4316	NN	denotes	FITC
T1903	4317-4320	IN	denotes	for
T1904	4321-4327	NN	denotes	20 min
T1905	4328-4330	IN	denotes	on
T1906	4331-4334	NN	denotes	ice
T1907	4336-4339	DT	denotes	The
T1908	4340-4345	NN	denotes	cells
T1909	4346-4350	VB	denotes	were
T1910	4351-4357	VB	denotes	washed
T1911	4358-4363	RB	denotes	twice
T1912	4364-4367	CC	denotes	and
T1913	4368-4379	JJ	denotes	fluorescent
T1914	4380-4389	NN	denotes	intensity
T1915	4390-4393	VB	denotes	was
T1916	4394-4402	VB	denotes	analysed
T1917	4403-4405	IN	denotes	by
T1918	4406-4410	NN	denotes	flow
T1919	4411-4420	NN	denotes	cytometry
T1920	4422-4425	DT	denotes	All
T1921	4426-4433	NN	denotes	results
T1922	4434-4439	VB	denotes	shown
T1923	4440-4443	VB	denotes	are
T1924	4444-4458	JJ	denotes	representative
T1925	4459-4461	IN	denotes	of
T1926	4462-4464	IN	denotes	at
T1927	4465-4468	CD	denotes	two
T1928	4469-4471	TO	denotes	to
T1929	4472-4476	CD	denotes	four
T1930	4477-4488	JJ	denotes	independent
T1931	4489-4500	NN	denotes	experiments
T1932	4501-4507	IN	denotes	unless
T1933	4508-4517	RB	denotes	otherwise
T1934	4518-4527	VB	denotes	indicated
T2288	0-4551	NN	denotes	Protein kinase D enzymes are dispensable for proliferation, survival and antigen receptor-regulated NFκB activity in vertebrate B-cells Abstract To investigate the importance of protein kinase D (PKD) enzymes we generated a PKD-null DT40 B-lymphocyte cell line. Previously we have shown that PKDs have an essential role in regulating class II histone deacetylases in DT40 B-cells [Matthews, S.A., Liu, P., Spitaler, M., Olson, E.N., McKinsey, T.A., Cantrell, D.A. and Scharenberg, A.M. (2006) Essential role for protein kinase D family kinases in the regulation of class II histone deacetylases in B lymphocytes. Mol. Cell Biol. 26, 1569–1577]. We now show that PKDs are also required to regulate HSP27 phosphorylation in DT40 B-cells. However, in contrast to previous observations in other cell types, PKD enzymes do not regulate basic cellular processes such as proliferation or survival responses, nor NFκB transcriptional activity downstream of the B cell antigen receptor. Thus, PKDs have a selective role in DT40 B-cell biology. 1 Introduction The protein kinase D (PKD) serine/threonine kinase family has three members: PKD1, PKD2 and PKD3. Most cell types express at least two PKD isoforms but PKD enzymes are especially highly expressed in haematopoietic cells, where they are activated in response to antigen receptors stimulation [2,3]. A conserved signalling pathway linking antigen receptors to PKDs involves the activation of PLCγ and the subsequent production of diacylglycerol (DAG) which stimulates classical and/or novel protein kinase Cs (PKC) that phosphorylate two key regulatory serine residues in the activation loop of PKD kinases [3–6]. The N-terminal regulatory region of PKD enzymes contains a DAG binding domain and direct binding of DAG also contributes to PKD1 activation [7] as well as regulating the spatial location of PKD enzymes within cells [8–12]. PKD enzymes have been proposed to regulate numerous cellular functions, including cell proliferation [13–16], anti-apoptotic signals [17,18] and thymocyte development [19]. Expression of mutant catalytically inactive and constitutively activated PKDs can also modify Golgi function, cell adhesion and cell motility (reviewed in [20]). In particular, PKDs have been widely linked to the activation of the NFκB transcription factor and in regulating cell survival during oxidative stress [17,21–23]. Another recently proposed PKD1 substrate is HSP27 [24], a small heat shock protein involved in regulating cell migration and cell survival [25]. An essential role for PKD enzymes in regulating class II histone deacetylases (HDACs), enzymes that repress MEF2-dependent gene transcription, has also been demonstrated [1,26–28]. To investigate the biological role of PKDs we have generated DT40 B cell lines that lack expression of one or more members of the PKD family [1], allowing us to investigate the function(s) of PKD isoforms following B cell antigen receptor (BCR) stimulation, as well addressing the issue of functional redundancy between the different PKD family members. Previous studies have shown that PKDs are indispensable for HDAC regulation in B cells [1]. Herein we show that PKDs are also indispensable for HSP27 phosphorylation in B cells. However, PKD-null DT40 B cells are viable and proliferate normally. Moreover, loss of the entire cellular pool of PKD does not critically affect oxidative stress responses in B cells nor do PKD kinases play an essential role in regulating NFκB transcriptional activity. Together, these findings reveal that in B lymphocytes, PKD kinases are not critical regulators of many of the cellular processes previously ascribed to them in other cellular systems. 2 Materials and methods 2.1 Cell culture, transient transfections and cell stimulation The generation, culture and activation of PKD1−/−, PKD3−/− and PKD1/3−/− knockout DT40 B cell lines have been described previously [1]. Cells were lysed and protein extracts were analysed in Western blotting experiments as previously described [1]. Chloramphenicol acetyl transferase assays have been described previously [29]. 2.2 sIgM staining DT40 B cells (2 × 106 cells per point) were resuspended in 200 μl buffer (RPMI 1640 media, 1% foetal calf serum) containing anti-chicken M1 monoclonal antibody conjugated to FITC for 20 min on ice. The cells were washed twice and fluorescent intensity was analysed by flow cytometry. All results shown are representative of at two to four independent experiments unless otherwise indicated. 3 Results 3.1 Loss
T2289	4552-4554	IN	denotes	of
T2290	4555-4560	NN	denotes	HSP27
T2291	4561-4576	NN	denotes	phosphorylation
T2292	4577-4579	IN	denotes	in
T2293	4580-4584	NN	denotes	DT40
T2294	4585-4586	NN	denotes	B
T2295	4587-4592	NN	denotes	cells
T2296	4593-4600	VB	denotes	lacking
T2297	4601-4611	NN	denotes	expression
T2298	4612-4614	IN	denotes	of
T2299	4615-4618	NN	denotes	PKD
T2300	4619-4625	NN	denotes	family
T2301	4626-4633	NN	denotes	kinases
T2302	4634-4638	NN	denotes	DT40
T2303	4639-4640	NN	denotes	B
T2304	4641-4646	NN	denotes	cells
T2305	4647-4654	VB	denotes	express
T2306	4655-4658	CD	denotes	two
T2307	4659-4662	NN	denotes	PKD
T2308	4663-4671	NN	denotes	isoforms
T2309	4671-4672	-COMMA-	denotes	,
T2310	4673-4677	NN	denotes	PKD1
T2311	4678-4681	CC	denotes	and
T2312	4682-4686	NN	denotes	PKD3
T2313	4686-4687	-COMMA-	denotes	,
T2314	4688-4691	CC	denotes	and
T2315	4692-4694	IN	denotes	as
T2316	4695-4705	RB	denotes	previously
T2317	4706-4715	VB	denotes	described
T2318	4716-4718	PRP	denotes	we
T2319	4719-4723	VB	denotes	have
T2320	4724-4732	RB	denotes	recently
T2321	4733-4742	VB	denotes	generated
T2322	4743-4747	NN	denotes	DT40
T2323	4748-4749	NN	denotes	B
T2324	4750-4754	NN	denotes	cell
T2325	4755-4760	NN	denotes	lines
T2326	4761-4765	WDT	denotes	that
T2327	4766-4770	VB	denotes	lack
T2328	4771-4781	NN	denotes	expression
T2329	4782-4784	IN	denotes	of
T2330	4785-4791	CC	denotes	either
T2331	4792-4796	NN	denotes	PKD1
T2332	4797-4799	CC	denotes	or
T2333	4800-4804	NN	denotes	PKD3
T2334	4805-4807	CC	denotes	or
T2335	4808-4812	DT	denotes	both
T2336	4813-4820	NN	denotes	enzymes
T2337	4821-4822	-LRB-	denotes	[
T2338	4822-4823	CD	denotes	1
T2339	4823-4824	-RRB-	denotes	]
T2340	4826-4828	IN	denotes	In
T2341	4829-4839	VB	denotes	generating
T2342	4840-4843	DT	denotes	the
T2343	4844-4850	JJ	denotes	double
T2344	4851-4859	NN	denotes	knockout
T2345	4860-4864	NN	denotes	cell
T2346	4865-4870	NN	denotes	lines
T2347	4871-4873	PRP	denotes	we
T2348	4874-4882	VB	denotes	targeted
T2349	4883-4886	DT	denotes	the
T2350	4887-4891	NN	denotes	PKD1
T2351	4892-4896	NN	denotes	loci
T2352	4897-4899	IN	denotes	in
T2353	4900-4901	DT	denotes	a
T2354	4902-4909	NN	denotes	PKD3−/−
T2355	4910-4914	NN	denotes	cell
T2356	4915-4919	NN	denotes	line
T2357	4920-4924	WDT	denotes	that
T2358	4925-4934	VB	denotes	expressed
T2359	4935-4936	DT	denotes	a
T2360	4937-4946	NN	denotes	Flag-PKD3
T2361	4947-4956	NN	denotes	transgene
T2362	4957-4962	IN	denotes	under
T2363	4963-4966	DT	denotes	the
T2364	4967-4974	NN	denotes	control
T2365	4975-4977	IN	denotes	of
T2366	4978-4979	DT	denotes	a
T2367	4980-5001	JJ	denotes	doxycycline-inducible
T2368	5002-5010	NN	denotes	promoter
T2369	5012-5017	RB	denotes	Hence
T2370	5017-5018	-COMMA-	denotes	,
T2371	5019-5021	IN	denotes	in
T2372	5022-5025	DT	denotes	the
T2373	5026-5034	NN	denotes	presence
T2374	5035-5037	IN	denotes	of
T2375	5038-5049	NN	denotes	doxycycline
T2376	5049-5050	-COMMA-	denotes	,
T2377	5051-5060	NN	denotes	Flag-PKD3
T2378	5061-5071	NN	denotes	expression
T2379	5072-5074	IN	denotes	in
T2380	5075-5081	NN	denotes	PKD1/3
T2381	5082-5088	JJ	denotes	double
T2382	5089-5097	JJ	denotes	knockout
T2383	5098-5103	NN	denotes	cells
T2384	5104-5106	VB	denotes	is
T2385	5107-5117	JJ	denotes	comparable
T2386	5118-5120	TO	denotes	to
T2387	5121-5131	JJ	denotes	endogenous
T2388	5132-5136	NN	denotes	PKD3
T2389	5137-5144	JJ	denotes	present
T2390	5145-5147	IN	denotes	in
T2391	5148-5157	JJ	denotes	wild-type
T2392	5158-5162	NN	denotes	DT40
T2393	5163-5168	NN	denotes	cells
T2394	5169-5172	CC	denotes	and
T2395	5173-5180	NN	denotes	removal
T2396	5181-5183	IN	denotes	of
T2397	5184-5195	NN	denotes	doxycycline
T2398	5196-5200	IN	denotes	from
T2399	5201-5204	DT	denotes	the
T2400	5205-5212	NN	denotes	culture
T2401	5213-5218	NN	denotes	media
T2402	5219-5222	IN	denotes	for
T2403	5223-5224	CD	denotes	5
T2404	5225-5229	NN	denotes	days
T2405	5230-5237	VB	denotes	results
T2406	5238-5240	IN	denotes	in
T2407	5241-5242	DT	denotes	a
T2408	5243-5253	RB	denotes	completely
T2409	5254-5258	JJ	denotes	null
T2410	5259-5262	NN	denotes	PKD
T2411	5263-5272	NN	denotes	phenotype
T2412	5273-5274	-LRB-	denotes	(
T2413	5274-5278	NN	denotes	Fig.
T2414	5279-5281	NN	denotes	1A
T2415	5281-5282	-RRB-	denotes	)
T2416	5284-5294	RB	denotes	Previously
T2417	5294-5295	-COMMA-	denotes	,
T2418	5296-5298	PRP	denotes	we
T2419	5299-5303	VB	denotes	have
T2420	5304-5316	VB	denotes	demonstrated
T2421	5317-5321	IN	denotes	that
T2422	5322-5337	NN	denotes	phosphorylation
T2423	5338-5341	CC	denotes	and
T2424	5342-5349	JJ	denotes	nuclear
T2425	5350-5359	NN	denotes	exclusion
T2426	5360-5362	IN	denotes	of
T2427	5363-5368	NN	denotes	class
T2428	5369-5371	CD	denotes	II
T2429	5372-5379	NN	denotes	histone
T2430	5380-5392	NN	denotes	deacetylases
T2431	5393-5394	-LRB-	denotes	(
T2432	5394-5399	NN	denotes	HDACs
T2433	5399-5400	-RRB-	denotes	)
T2434	5401-5407	IN	denotes	during
T2435	5408-5411	NN	denotes	BCR
T2436	5412-5422	NN	denotes	engagement
T2437	5423-5425	VB	denotes	is
T2438	5426-5435	JJ	denotes	defective
T2439	5436-5438	IN	denotes	in
T2440	5439-5448	NN	denotes	PKD1/3−/−
T2441	5449-5450	NN	denotes	B
T2442	5451-5456	NN	denotes	cells
T2443	5457-5460	CC	denotes	and
T2444	5461-5464	MD	denotes	can
T2445	5465-5473	VB	denotes	restored
T2446	5474-5478	IN	denotes	upon
T2447	5479-5492	NN	denotes	re-expression
T2448	5493-5495	IN	denotes	of
T2449	5496-5497	DT	denotes	a
T2450	5498-5504	JJ	denotes	single
T2451	5505-5508	NN	denotes	PKD
T2452	5509-5516	NN	denotes	isoform
T2453	5517-5518	-LRB-	denotes	[
T2454	5518-5519	CD	denotes	1
T2455	5519-5520	-RRB-	denotes	]
T2456	5522-5525	DT	denotes	The
T2457	5526-5531	JJ	denotes	small
T2458	5532-5536	NN	denotes	heat
T2459	5537-5542	NN	denotes	shock
T2460	5543-5550	NN	denotes	protein
T2461	5551-5556	NN	denotes	HSP27
T2462	5557-5560	VB	denotes	has
T2463	5561-5569	RB	denotes	recently
T2464	5570-5574	VB	denotes	been
T2465	5575-5583	VB	denotes	proposed
T2466	5584-5586	IN	denotes	as
T2467	5587-5588	DT	denotes	a
T2468	5589-5593	NN	denotes	PKD1
T2469	5594-5603	NN	denotes	substrate
T2470	5604-5605	-LRB-	denotes	[
T2471	5605-5607	CD	denotes	24
T2472	5607-5608	-RRB-	denotes	]
T2473	5609-5612	CC	denotes	and
T2474	5613-5615	PRP	denotes	we
T2475	5616-5627	RB	denotes	accordingly
T2476	5628-5636	VB	denotes	assessed
T2477	5637-5644	IN	denotes	whether
T2478	5645-5653	JJ	denotes	PKD-null
T2479	5654-5658	NN	denotes	DT40
T2480	5659-5664	NN	denotes	cells
T2481	5665-5669	VB	denotes	have
T2482	5670-5679	JJ	denotes	defective
T2483	5680-5695	NN	denotes	phosphorylation
T2484	5696-5698	IN	denotes	of
T2485	5699-5704	NN	denotes	HSP27
T2486	5705-5707	IN	denotes	on
T2487	5708-5714	NN	denotes	serine
T2488	5715-5717	CD	denotes	82
T2489	5717-5718	-COMMA-	denotes	,
T2490	5719-5722	DT	denotes	the
T2491	5723-5731	VB	denotes	proposed
T2492	5732-5736	NN	denotes	PKD1
T2493	5737-5746	NN	denotes	substrate
T2494	5747-5755	NN	denotes	sequence
T2495	5757-5759	PRP	denotes	We
T2496	5760-5769	RB	denotes	initially
T2497	5770-5782	VB	denotes	investigated
T2498	5783-5786	DT	denotes	the
T2499	5787-5797	NN	denotes	regulation
T2500	5798-5800	IN	denotes	of
T2501	5801-5806	NN	denotes	HSP27
T2502	5807-5822	NN	denotes	phosphorylation
T2503	5823-5825	IN	denotes	in
T2504	5826-5832	JJ	denotes	single
T2505	5833-5841	NN	denotes	knockout
T2506	5842-5846	NN	denotes	DT40
T2507	5847-5848	NN	denotes	B
T2508	5849-5854	NN	denotes	cells
T2509	5855-5862	VB	denotes	lacking
T2510	5863-5869	CC	denotes	either
T2511	5870-5874	NN	denotes	PKD1
T2512	5875-5877	CC	denotes	or
T2513	5878-5882	NN	denotes	PKD3
T2514	5884-5886	IN	denotes	As
T2515	5887-5892	VB	denotes	shown
T2516	5893-5895	IN	denotes	in
T2517	5896-5900	NNP	denotes	Fig.
T2518	5901-5903	NN	denotes	1B
T2519	5903-5904	-COMMA-	denotes	,
T2520	5905-5915	NN	denotes	activation
T2521	5916-5918	IN	denotes	of
T2522	5919-5922	DT	denotes	the
T2523	5923-5926	NN	denotes	BCR
T2524	5927-5929	CC	denotes	or
T2525	5930-5939	NN	denotes	treatment
T2526	5940-5944	IN	denotes	with
T2527	5945-5948	DT	denotes	the
T2528	5949-5960	JJ	denotes	DAG-mimetic
T2529	5961-5965	NN	denotes	PdBu
T2530	5966-5975	VB	denotes	increased
T2531	5976-5979	DT	denotes	the
T2532	5980-5986	NN	denotes	levels
T2533	5987-5989	IN	denotes	of
T2534	5990-5995	NN	denotes	HSP27
T2535	5996-6011	NN	denotes	phosphorylation
T2536	6012-6014	IN	denotes	at
T2537	6015-6018	NN	denotes	S82
T2538	6019-6021	IN	denotes	in
T2539	6022-6031	JJ	denotes	wild-type
T2540	6032-6036	NN	denotes	DT40
T2541	6037-6038	NN	denotes	B
T2542	6039-6044	NN	denotes	cells
T2543	6046-6049	NN	denotes	BCR
T2544	6050-6053	CC	denotes	and
T2545	6054-6061	NN	denotes	phorbol
T2546	6062-6067	NN	denotes	ester
T2547	6068-6075	NN	denotes	signals
T2548	6076-6080	VB	denotes	were
T2549	6081-6085	RB	denotes	also
T2550	6086-6090	JJ	denotes	able
T2551	6091-6093	TO	denotes	to
T2552	6094-6102	VB	denotes	increase
T2553	6103-6108	NN	denotes	HSP27
T2554	6109-6124	NN	denotes	phosphorylation
T2555	6125-6127	IN	denotes	in
T2556	6128-6132	NN	denotes	PKD1
T2557	6133-6135	CC	denotes	or
T2558	6136-6140	NN	denotes	PKD3
T2559	6141-6147	JJ	denotes	single
T2560	6148-6156	NN	denotes	knockout
T2561	6157-6161	NN	denotes	DT40
T2562	6162-6163	NN	denotes	B
T2563	6164-6169	NN	denotes	cells
T2564	6170-6171	-LRB-	denotes	(
T2565	6171-6175	NN	denotes	Fig.
T2566	6176-6178	NN	denotes	1B
T2567	6178-6179	-RRB-	denotes	)
T2568	6181-6188	RB	denotes	However
T2569	6188-6189	-COMMA-	denotes	,
T2570	6190-6194	NN	denotes	BCR-
T2571	6195-6198	CC	denotes	and
T2572	6199-6206	NN	denotes	phorbol
T2573	6207-6220	JJ	denotes	ester-induced
T2574	6221-6236	NN	denotes	phosphorylation
T2575	6237-6239	IN	denotes	of
T2576	6240-6245	NN	denotes	HSP27
T2577	6246-6248	IN	denotes	on
T2578	6249-6252	NN	denotes	S82
T2579	6253-6256	VB	denotes	was
T2580	6257-6266	VB	denotes	abolished
T2581	6267-6269	IN	denotes	in
T2582	6270-6271	NN	denotes	B
T2583	6272-6277	NN	denotes	cells
T2584	6278-6282	WDT	denotes	that
T2585	6283-6289	VB	denotes	lacked
T2586	6290-6294	CC	denotes	both
T2587	6295-6299	NN	denotes	PKD1
T2588	6300-6303	CC	denotes	and
T2589	6304-6308	NN	denotes	PKD3
T2590	6309-6310	-LRB-	denotes	(
T2591	6310-6314	NN	denotes	Fig.
T2592	6315-6317	NN	denotes	1C
T2593	6317-6318	-RRB-	denotes	)
T2594	6320-6333	RB	denotes	Significantly
T2595	6333-6334	-COMMA-	denotes	,
T2596	6335-6354	JJ	denotes	doxycycline-induced
T2597	6355-6365	NN	denotes	expression
T2598	6366-6368	IN	denotes	of
T2599	6369-6372	DT	denotes	the
T2600	6373-6382	NN	denotes	Flag-PKD3
T2601	6383-6392	NN	denotes	transgene
T2602	6393-6395	IN	denotes	in
T2603	6396-6399	DT	denotes	the
T2604	6400-6406	JJ	denotes	double
T2605	6407-6415	NN	denotes	knockout
T2606	6416-6421	NN	denotes	cells
T2607	6422-6425	VB	denotes	was
T2608	6426-6436	JJ	denotes	sufficient
T2609	6437-6439	TO	denotes	to
T2610	6440-6447	VB	denotes	restore
T2611	6448-6454	JJ	denotes	normal
T2612	6455-6465	NN	denotes	regulation
T2613	6466-6468	IN	denotes	of
T2614	6469-6474	NN	denotes	HSP27
T2615	6475-6490	NN	denotes	phosphorylation
T2616	6491-6492	-LRB-	denotes	(
T2617	6492-6496	NN	denotes	Fig.
T2618	6497-6499	NN	denotes	1C
T2619	6499-6500	-RRB-	denotes	)
T2620	6502-6504	IN	denotes	In
T2621	6505-6513	NN	denotes	contrast
T2622	6513-6514	-COMMA-	denotes	,
T2623	6515-6525	NN	denotes	expression
T2624	6526-6528	IN	denotes	of
T2625	6529-6530	DT	denotes	a
T2626	6531-6547	JJ	denotes	kinase-deficient
T2627	6548-6552	NN	denotes	PKD3
T2628	6553-6559	JJ	denotes	mutant
T2629	6560-6567	NN	denotes	protein
T2630	6568-6570	IN	denotes	in
T2631	6571-6574	DT	denotes	the
T2632	6575-6581	JJ	denotes	double
T2633	6582-6590	NN	denotes	knockout
T2634	6591-6596	NN	denotes	cells
T2635	6597-6600	VB	denotes	was
T2636	6601-6604	RB	denotes	not
T2637	6605-6609	JJ	denotes	able
T2638	6610-6612	TO	denotes	to
T2639	6613-6620	VB	denotes	restore
T2640	6621-6625	NN	denotes	BCR-
T2641	6626-6628	CC	denotes	or
T2642	6629-6636	NN	denotes	phorbol
T2643	6637-6650	JJ	denotes	ester-induced
T2644	6651-6656	NN	denotes	HSP27
T2645	6657-6672	NN	denotes	phosphorylation
T2646	6673-6674	-LRB-	denotes	(
T2647	6674-6678	NN	denotes	Fig.
T2648	6679-6681	NN	denotes	1D
T2649	6681-6682	-RRB-	denotes	)
T2650	6684-6689	RB	denotes	Hence
T2651	6689-6690	-COMMA-	denotes	,
T2652	6691-6695	NN	denotes	PKD3
T2653	6696-6698	RB	denotes	as
T2654	6699-6703	RB	denotes	well
T2655	6704-6706	IN	denotes	as
T2656	6707-6711	NN	denotes	PKD1
T2657	6712-6715	MD	denotes	can
T2658	6716-6724	VB	denotes	regulate
T2659	6725-6730	NN	denotes	HSP27
T2660	6731-6746	NN	denotes	phosphorylation
T2661	6747-6750	CC	denotes	and
T2662	6751-6753	IN	denotes	in
T2663	6754-6758	NN	denotes	DT40
T2664	6759-6760	NN	denotes	B
T2665	6761-6766	NN	denotes	cells
T2666	6767-6771	PRP	denotes	they
T2667	6772-6775	VB	denotes	are
T2668	6776-6788	RB	denotes	functionally
T2669	6789-6798	JJ	denotes	redundant
T2670	6799-6801	IN	denotes	as
T2671	6802-6807	NN	denotes	HSP27
T2672	6808-6815	NN	denotes	kinases
T3836	0-6831	JJ	denotes	Protein kinase D enzymes are dispensable for proliferation, survival and antigen receptor-regulated NFκB activity in vertebrate B-cells Abstract To investigate the importance of protein kinase D (PKD) enzymes we generated a PKD-null DT40 B-lymphocyte cell line. Previously we have shown that PKDs have an essential role in regulating class II histone deacetylases in DT40 B-cells [Matthews, S.A., Liu, P., Spitaler, M., Olson, E.N., McKinsey, T.A., Cantrell, D.A. and Scharenberg, A.M. (2006) Essential role for protein kinase D family kinases in the regulation of class II histone deacetylases in B lymphocytes. Mol. Cell Biol. 26, 1569–1577]. We now show that PKDs are also required to regulate HSP27 phosphorylation in DT40 B-cells. However, in contrast to previous observations in other cell types, PKD enzymes do not regulate basic cellular processes such as proliferation or survival responses, nor NFκB transcriptional activity downstream of the B cell antigen receptor. Thus, PKDs have a selective role in DT40 B-cell biology. 1 Introduction The protein kinase D (PKD) serine/threonine kinase family has three members: PKD1, PKD2 and PKD3. Most cell types express at least two PKD isoforms but PKD enzymes are especially highly expressed in haematopoietic cells, where they are activated in response to antigen receptors stimulation [2,3]. A conserved signalling pathway linking antigen receptors to PKDs involves the activation of PLCγ and the subsequent production of diacylglycerol (DAG) which stimulates classical and/or novel protein kinase Cs (PKC) that phosphorylate two key regulatory serine residues in the activation loop of PKD kinases [3–6]. The N-terminal regulatory region of PKD enzymes contains a DAG binding domain and direct binding of DAG also contributes to PKD1 activation [7] as well as regulating the spatial location of PKD enzymes within cells [8–12]. PKD enzymes have been proposed to regulate numerous cellular functions, including cell proliferation [13–16], anti-apoptotic signals [17,18] and thymocyte development [19]. Expression of mutant catalytically inactive and constitutively activated PKDs can also modify Golgi function, cell adhesion and cell motility (reviewed in [20]). In particular, PKDs have been widely linked to the activation of the NFκB transcription factor and in regulating cell survival during oxidative stress [17,21–23]. Another recently proposed PKD1 substrate is HSP27 [24], a small heat shock protein involved in regulating cell migration and cell survival [25]. An essential role for PKD enzymes in regulating class II histone deacetylases (HDACs), enzymes that repress MEF2-dependent gene transcription, has also been demonstrated [1,26–28]. To investigate the biological role of PKDs we have generated DT40 B cell lines that lack expression of one or more members of the PKD family [1], allowing us to investigate the function(s) of PKD isoforms following B cell antigen receptor (BCR) stimulation, as well addressing the issue of functional redundancy between the different PKD family members. Previous studies have shown that PKDs are indispensable for HDAC regulation in B cells [1]. Herein we show that PKDs are also indispensable for HSP27 phosphorylation in B cells. However, PKD-null DT40 B cells are viable and proliferate normally. Moreover, loss of the entire cellular pool of PKD does not critically affect oxidative stress responses in B cells nor do PKD kinases play an essential role in regulating NFκB transcriptional activity. Together, these findings reveal that in B lymphocytes, PKD kinases are not critical regulators of many of the cellular processes previously ascribed to them in other cellular systems. 2 Materials and methods 2.1 Cell culture, transient transfections and cell stimulation The generation, culture and activation of PKD1−/−, PKD3−/− and PKD1/3−/− knockout DT40 B cell lines have been described previously [1]. Cells were lysed and protein extracts were analysed in Western blotting experiments as previously described [1]. Chloramphenicol acetyl transferase assays have been described previously [29]. 2.2 sIgM staining DT40 B cells (2 × 106 cells per point) were resuspended in 200 μl buffer (RPMI 1640 media, 1% foetal calf serum) containing anti-chicken M1 monoclonal antibody conjugated to FITC for 20 min on ice. The cells were washed twice and fluorescent intensity was analysed by flow cytometry. All results shown are representative of at two to four independent experiments unless otherwise indicated. 3 Results 3.1 Loss of HSP27 phosphorylation in DT40 B cells lacking expression of PKD family kinases DT40 B cells express two PKD isoforms, PKD1 and PKD3, and as previously described we have recently generated DT40 B cell lines that lack expression of either PKD1 or PKD3 or both enzymes [1]. In generating the double knockout cell lines we targeted the PKD1 loci in a PKD3−/− cell line that expressed a Flag-PKD3 transgene under the control of a doxycycline-inducible promoter. Hence, in the presence of doxycycline, Flag-PKD3 expression in PKD1/3 double knockout cells is comparable to endogenous PKD3 present in wild-type DT40 cells and removal of doxycycline from the culture media for 5 days results in a completely null PKD phenotype (Fig. 1A). Previously, we have demonstrated that phosphorylation and nuclear exclusion of class II histone deacetylases (HDACs) during BCR engagement is defective in PKD1/3−/− B cells and can restored upon re-expression of a single PKD isoform [1]. The small heat shock protein HSP27 has recently been proposed as a PKD1 substrate [24] and we accordingly assessed whether PKD-null DT40 cells have defective phosphorylation of HSP27 on serine 82, the proposed PKD1 substrate sequence. We initially investigated the regulation of HSP27 phosphorylation in single knockout DT40 B cells lacking either PKD1 or PKD3. As shown in Fig. 1B, activation of the BCR or treatment with the DAG-mimetic PdBu increased the levels of HSP27 phosphorylation at S82 in wild-type DT40 B cells. BCR and phorbol ester signals were also able to increase HSP27 phosphorylation in PKD1 or PKD3 single knockout DT40 B cells (Fig. 1B). However, BCR- and phorbol ester-induced phosphorylation of HSP27 on S82 was abolished in B cells that lacked both PKD1 and PKD3 (Fig. 1C). Significantly, doxycycline-induced expression of the Flag-PKD3 transgene in the double knockout cells was sufficient to restore normal regulation of HSP27 phosphorylation (Fig. 1C). In contrast, expression of a kinase-deficient PKD3 mutant protein in the double knockout cells was not able to restore BCR- or phorbol ester-induced HSP27 phosphorylation (Fig. 1D). Hence, PKD3 as well as PKD1 can regulate HSP27 phosphorylation and in DT40 B cells they are functionally redundant as HSP27 kinases. 3.2 Cellular
T3837	6832-6845	NN	denotes	proliferation
T3838	6846-6849	CC	denotes	and
T3839	6850-6858	NN	denotes	survival
T3840	6859-6861	IN	denotes	in
T3841	6862-6866	NN	denotes	DT40
T3842	6867-6868	NN	denotes	B
T3843	6869-6874	NN	denotes	cells
T3844	6875-6882	VB	denotes	lacking
T3845	6883-6893	NN	denotes	expression
T3846	6894-6896	IN	denotes	of
T3847	6897-6900	NN	denotes	PKD
T3848	6901-6907	NN	denotes	family
T3849	6908-6915	NN	denotes	kinases
T3850	6916-6919	NN	denotes	PKD
T3851	6920-6927	NN	denotes	enzymes
T3852	6928-6932	VB	denotes	have
T3853	6933-6943	RB	denotes	previously
T3854	6944-6948	VB	denotes	been
T3855	6949-6955	VB	denotes	linked
T3856	6956-6958	TO	denotes	to
T3857	6959-6962	DT	denotes	the
T3858	6963-6973	NN	denotes	regulation
T3859	6974-6976	IN	denotes	of
T3860	6977-6981	NN	denotes	cell
T3861	6982-6995	NN	denotes	proliferation
T3862	6996-6999	CC	denotes	and
T3863	7000-7008	NN	denotes	survival
T3864	7009-7010	-LRB-	denotes	(
T3865	7010-7018	VB	denotes	reviewed
T3866	7019-7021	IN	denotes	in
T3867	7022-7023	-LRB-	denotes	[
T3868	7023-7025	CD	denotes	20
T3869	7025-7026	-RRB-	denotes	]
T3870	7026-7027	-RRB-	denotes	)
T3871	7029-7031	TO	denotes	To
T3872	7032-7043	VB	denotes	investigate
T3873	7044-7047	DT	denotes	the
T3874	7048-7054	NN	denotes	effect
T3875	7055-7059	IN	denotes	that
T3876	7060-7064	NN	denotes	loss
T3877	7065-7067	IN	denotes	of
T3878	7068-7071	NN	denotes	PKD
T3879	7072-7079	NN	denotes	kinases
T3880	7080-7083	VB	denotes	had
T3881	7084-7086	IN	denotes	on
T3882	7087-7088	NN	denotes	B
T3883	7089-7093	NN	denotes	cell
T3884	7094-7102	NN	denotes	survival
T3885	7103-7109	CC	denotes	and/or
T3886	7110-7123	NN	denotes	proliferation
T3887	7124-7126	PRP	denotes	we
T3888	7127-7135	VB	denotes	cultured
T3889	7136-7145	JJ	denotes	wild-type
T3890	7146-7149	CC	denotes	and
T3891	7150-7158	JJ	denotes	PKD-null
T3892	7159-7164	NN	denotes	cells
T3893	7165-7167	IN	denotes	in
T3894	7168-7171	DT	denotes	the
T3895	7172-7180	NN	denotes	presence
T3896	7181-7182	-LRB-	denotes	(
T3897	7182-7191	NN	denotes	PKD1/3−/−
T3898	7191-7192	-COLON-	denotes	:
T3899	7193-7205	NN	denotes	Flag-PKD3+ve
T3900	7205-7206	-RRB-	denotes	)
T3901	7207-7209	CC	denotes	or
T3902	7210-7217	NN	denotes	absence
T3903	7218-7219	-LRB-	denotes	(
T3904	7219-7228	NN	denotes	PKD1/3−/−
T3905	7228-7229	-RRB-	denotes	)
T3906	7230-7232	IN	denotes	of
T3907	7233-7244	NN	denotes	doxycycline
T3908	7245-7248	CC	denotes	and
T3909	7249-7258	VB	denotes	monitored
T3910	7259-7270	JJ	denotes	exponential
T3911	7271-7277	NN	denotes	growth
T3912	7279-7281	IN	denotes	As
T3913	7282-7287	VB	denotes	shown
T3914	7288-7290	IN	denotes	in
T3915	7291-7295	NNP	denotes	Fig.
T3916	7296-7298	NN	denotes	2A
T3917	7298-7299	-COMMA-	denotes	,
T3918	7300-7309	NN	denotes	PKD1/3−/−
T3919	7310-7315	NN	denotes	cells
T3920	7316-7328	VB	denotes	proliferated
T3921	7329-7342	RB	denotes	exponentially
T3922	7343-7346	CC	denotes	and
T3923	7347-7360	NN	denotes	re-expression
T3924	7361-7363	IN	denotes	of
T3925	7364-7373	NN	denotes	Flag-PKD3
T3926	7374-7376	IN	denotes	in
T3927	7377-7382	DT	denotes	these
T3928	7383-7388	NN	denotes	cells
T3929	7389-7392	VB	denotes	had
T3930	7393-7395	DT	denotes	no
T3931	7396-7402	NN	denotes	impact
T3932	7403-7405	IN	denotes	on
T3933	7406-7409	DT	denotes	the
T3934	7410-7414	NN	denotes	rate
T3935	7415-7417	IN	denotes	of
T3936	7418-7431	NN	denotes	proliferation
T3937	7433-7444	RB	denotes	Furthermore
T3938	7444-7445	-COMMA-	denotes	,
T3939	7446-7449	DT	denotes	the
T3940	7450-7459	NN	denotes	viability
T3941	7460-7462	IN	denotes	of
T3942	7463-7472	NN	denotes	PKD1/3−/−
T3943	7473-7474	NN	denotes	B
T3944	7475-7480	NN	denotes	cells
T3945	7481-7487	IN	denotes	during
T3946	7488-7495	JJ	denotes	routine
T3947	7496-7505	NN	denotes	culturing
T3948	7506-7509	VB	denotes	was
T3949	7510-7513	RB	denotes	not
T3950	7514-7527	RB	denotes	significantly
T3951	7528-7537	JJ	denotes	different
T3952	7538-7542	IN	denotes	from
T3953	7543-7547	DT	denotes	that
T3954	7548-7550	IN	denotes	of
T3955	7551-7560	JJ	denotes	wild-type
T3956	7561-7562	NN	denotes	B
T3957	7563-7568	NN	denotes	cells
T3958	7569-7570	-LRB-	denotes	(
T3959	7570-7574	NN	denotes	data
T3960	7575-7578	RB	denotes	not
T3961	7579-7584	VB	denotes	shown
T3962	7584-7585	-RRB-	denotes	)
T3963	7587-7589	PRP	denotes	It
T3964	7590-7593	VB	denotes	was
T3965	7594-7599	VB	denotes	noted
T3966	7600-7604	IN	denotes	that
T3967	7605-7608	DT	denotes	the
T3968	7609-7619	NN	denotes	population
T3969	7620-7628	NN	denotes	doubling
T3970	7629-7633	NN	denotes	time
T3971	7634-7636	IN	denotes	of
T3972	7637-7646	NN	denotes	PKD1/3−/−
T3973	7647-7652	NN	denotes	cells
T3974	7653-7656	VB	denotes	was
T3975	7657-7665	RB	denotes	slightly
T3976	7666-7672	JJ	denotes	slower
T3977	7673-7677	IN	denotes	than
T3978	7678-7682	DT	denotes	that
T3979	7683-7685	IN	denotes	of
T3980	7686-7690	JJ	denotes	wild
T3981	7691-7695	NN	denotes	type
T3982	7696-7700	NN	denotes	DT40
T3983	7701-7706	NN	denotes	cells
T3984	7707-7708	-LRB-	denotes	(
T3985	7708-7720	CD	denotes	12.7 ± 2.8 h
T3986	7721-7727	IN	denotes	versus
T3987	7728-7740	CD	denotes	10.2 ± 0.4 h
T3988	7740-7741	-RRB-	denotes	)
T3989	7742-7745	CC	denotes	but
T3990	7746-7749	DT	denotes	the
T3991	7750-7757	NN	denotes	failure
T3992	7758-7760	IN	denotes	of
T3993	7761-7765	NN	denotes	PKD3
T3994	7766-7779	NN	denotes	re-expression
T3995	7780-7782	TO	denotes	to
T3996	7783-7789	VB	denotes	modify
T3997	7790-7793	DT	denotes	the
T3998	7794-7807	NN	denotes	proliferation
T3999	7808-7812	NN	denotes	rate
T4000	7813-7815	IN	denotes	of
T4001	7816-7825	NN	denotes	PKD1/3−/−
T4002	7826-7831	NN	denotes	cells
T4003	7832-7840	VB	denotes	suggests
T4004	7841-7845	IN	denotes	that
T4005	7846-7851	DT	denotes	these
T4006	7852-7857	JJ	denotes	small
T4007	7858-7869	NN	denotes	differences
T4008	7870-7874	VB	denotes	were
T4009	7875-7879	RB	denotes	most
T4010	7880-7886	RB	denotes	likely
T4011	7887-7890	DT	denotes	the
T4012	7891-7897	NN	denotes	result
T4013	7898-7900	IN	denotes	of
T4014	7901-7907	JJ	denotes	clonal
T4015	7908-7917	NN	denotes	variation
T4016	7918-7921	CC	denotes	and
T4017	7922-7926	VB	denotes	were
T4018	7927-7930	RB	denotes	not
T4019	7931-7937	VB	denotes	caused
T4020	7938-7950	RB	denotes	specifically
T4021	7951-7953	IN	denotes	by
T4022	7954-7958	NN	denotes	loss
T4023	7959-7961	IN	denotes	of
T4024	7962-7965	NN	denotes	PKD
T4025	7966-7973	NN	denotes	enzymes
T4026	7975-7979	RB	denotes	Thus
T4027	7979-7980	-COMMA-	denotes	,
T4028	7981-7984	NN	denotes	PKD
T4029	7985-7991	NN	denotes	family
T4030	7992-7999	NN	denotes	enzymes
T4031	8000-8003	VB	denotes	are
T4032	8004-8007	RB	denotes	not
T4033	8008-8017	JJ	denotes	essential
T4034	8018-8021	IN	denotes	for
T4035	8022-8032	VB	denotes	regulating
T4036	8033-8038	JJ	denotes	basal
T4037	8039-8047	NN	denotes	survival
T4038	8048-8051	CC	denotes	and
T4039	8052-8065	NN	denotes	proliferation
T4040	8066-8068	IN	denotes	of
T4041	8069-8073	NN	denotes	DT40
T4042	8074-8075	NN	denotes	B
T4043	8076-8081	NN	denotes	cells
T4044	8083-8086	NN	denotes	PKD
T4045	8087-8094	NN	denotes	enzymes
T4046	8094-8095	-COMMA-	denotes	,
T4047	8096-8108	RB	denotes	specifically
T4048	8109-8113	NN	denotes	PKD1
T4049	8114-8117	CC	denotes	and
T4050	8118-8122	NN	denotes	PKD2
T4051	8122-8123	-COMMA-	denotes	,
T4052	8124-8128	VB	denotes	have
T4053	8129-8139	RB	denotes	previously
T4054	8140-8144	VB	denotes	been
T4055	8145-8151	VB	denotes	linked
T4056	8152-8154	TO	denotes	to
T4057	8155-8156	DT	denotes	a
T4058	8157-8167	JJ	denotes	protective
T4059	8168-8172	NN	denotes	role
T4060	8173-8180	IN	denotes	against
T4061	8181-8190	JJ	denotes	oxidative
T4062	8191-8205	JJ	denotes	stress-induced
T4063	8206-8212	NN	denotes	injury
T4064	8213-8215	IN	denotes	in
T4065	8216-8219	NN	denotes	3T3
T4066	8220-8230	NN	denotes	fibroblast
T4067	8230-8231	-COMMA-	denotes	,
T4068	8232-8236	NN	denotes	HeLa
T4069	8237-8240	CC	denotes	and
T4070	8241-8251	JJ	denotes	epithelial
T4071	8252-8256	NN	denotes	cell
T4072	8257-8262	NN	denotes	lines
T4073	8263-8264	-LRB-	denotes	[
T4074	8264-8272	CD	denotes	17,30–32
T4075	8272-8273	-RRB-	denotes	]
T4076	8275-8277	PRP	denotes	We
T4077	8278-8287	RB	denotes	therefore
T4078	8288-8297	VB	denotes	addressed
T4079	8298-8301	DT	denotes	the
T4080	8302-8306	NN	denotes	role
T4081	8307-8309	IN	denotes	of
T4082	8310-8313	NN	denotes	PKD
T4083	8314-8320	NN	denotes	family
T4084	8321-8328	NN	denotes	kinases
T4085	8329-8331	IN	denotes	in
T4086	8332-8342	VB	denotes	regulating
T4087	8343-8344	NN	denotes	B
T4088	8345-8349	NN	denotes	cell
T4089	8350-8358	NN	denotes	survival
T4090	8359-8361	IN	denotes	in
T4091	8362-8370	NN	denotes	response
T4092	8371-8373	TO	denotes	to
T4093	8374-8383	JJ	denotes	oxidative
T4094	8384-8390	NN	denotes	stress
T4095	8391-8394	CC	denotes	and
T4096	8395-8400	JJ	denotes	other
T4097	8401-8407	NN	denotes	stress
T4098	8408-8415	NN	denotes	stimuli
T4099	8417-8419	IN	denotes	As
T4100	8420-8425	VB	denotes	shown
T4101	8426-8428	IN	denotes	in
T4102	8429-8433	NNP	denotes	Fig.
T4103	8434-8436	NN	denotes	2B
T4104	8436-8437	-COMMA-	denotes	,
T4105	8438-8442	NN	denotes	loss
T4106	8443-8445	IN	denotes	of
T4107	8446-8452	NN	denotes	PKD1/3
T4108	8453-8463	NN	denotes	expression
T4109	8464-8467	VB	denotes	had
T4110	8468-8470	DT	denotes	no
T4111	8471-8482	JJ	denotes	significant
T4112	8483-8489	NN	denotes	impact
T4113	8490-8492	IN	denotes	on
T4114	8493-8496	DT	denotes	the
T4115	8497-8505	NN	denotes	survival
T4116	8506-8508	IN	denotes	of
T4117	8509-8513	NN	denotes	DT40
T4118	8514-8515	NN	denotes	B
T4119	8516-8521	NN	denotes	cells
T4120	8522-8524	IN	denotes	in
T4121	8525-8533	NN	denotes	response
T4122	8534-8536	TO	denotes	to
T4123	8537-8550	JJ	denotes	mitochondrial
T4124	8551-8557	NN	denotes	stress
T4125	8558-8565	NN	denotes	stimuli
T4126	8566-8567	-LRB-	denotes	(
T4127	8567-8571	NN	denotes	H2O2
T4128	8572-8574	CC	denotes	or
T4129	8575-8580	NN	denotes	serum
T4130	8581-8592	NN	denotes	deprivation
T4131	8592-8593	-RRB-	denotes	)
T4132	8593-8594	-COLON-	denotes	;
T4133	8595-8598	NN	denotes	DNA
T4134	8599-8607	NN	denotes	damaging
T4135	8608-8614	NN	denotes	agents
T4136	8615-8616	-LRB-	denotes	(
T4137	8616-8625	NN	denotes	etoposide
T4138	8626-8628	CC	denotes	or
T4139	8629-8640	NN	denotes	doxorubicin
T4140	8640-8641	-RRB-	denotes	)
T4141	8641-8642	-COLON-	denotes	;
T4142	8643-8645	NN	denotes	ER
T4143	8646-8653	NN	denotes	pathway
T4144	8654-8660	NN	denotes	stress
T4145	8661-8664	IN	denotes	due
T4146	8665-8667	TO	denotes	to
T4147	8668-8675	NN	denotes	calcium
T4148	8676-8684	NN	denotes	overload
T4149	8685-8686	-LRB-	denotes	(
T4150	8686-8698	NN	denotes	thapsigargin
T4151	8698-8699	-RRB-	denotes	)
T4152	8700-8702	CC	denotes	or
T4153	8703-8712	VB	denotes	following
T4154	8713-8722	VB	denotes	prolonged
T4155	8723-8732	NN	denotes	treatment
T4156	8733-8737	IN	denotes	with
T4157	8738-8745	NN	denotes	phorbol
T4158	8746-8752	NN	denotes	esters
T4159	8753-8755	CC	denotes	or
T4160	8756-8768	NN	denotes	Trichostatin
T4161	8769-8770	NN	denotes	A
T4162	8770-8771	-COMMA-	denotes	,
T4163	8772-8774	DT	denotes	an
T4164	8775-8784	NN	denotes	inhibitor
T4165	8785-8787	IN	denotes	of
T4166	8788-8793	NN	denotes	class
T4167	8794-8798	NN	denotes	I/II
T4168	8799-8804	NN	denotes	HDACs
T4169	8806-8810	RB	denotes	Thus
T4170	8810-8811	-COMMA-	denotes	,
T4171	8812-8815	NN	denotes	PKD
T4172	8816-8823	NN	denotes	kinases
T4173	8824-8826	VB	denotes	do
T4174	8827-8830	RB	denotes	not
T4175	8831-8835	VB	denotes	play
T4176	8836-8838	DT	denotes	an
T4177	8839-8848	JJ	denotes	essential
T4178	8849-8853	NN	denotes	role
T4179	8854-8856	IN	denotes	in
T4180	8857-8867	VB	denotes	regulating
T4181	8868-8869	NN	denotes	B
T4182	8870-8874	NN	denotes	cell
T4183	8875-8883	NN	denotes	survival
T4184	8884-8886	IN	denotes	in
T4185	8887-8895	NN	denotes	response
T4186	8896-8898	TO	denotes	to
T4187	8899-8900	DT	denotes	a
T4188	8901-8906	NN	denotes	range
T4189	8907-8909	IN	denotes	of
T4190	8910-8919	JJ	denotes	different
T4191	8920-8926	NN	denotes	stress
T4192	8927-8934	NN	denotes	stimuli
T26	0-7	NN	denotes	Protein
T27	8-14	NN	denotes	kinase
T28	15-16	NN	denotes	D
T29	17-24	NN	denotes	enzymes
T30	25-28	VB	denotes	are
T31	29-40	JJ	denotes	dispensable
T32	41-44	IN	denotes	for
T33	45-58	NN	denotes	proliferation
T34	58-59	-COMMA-	denotes	,
T35	60-68	NN	denotes	survival
T36	69-72	CC	denotes	and
T37	73-80	NN	denotes	antigen
T38	81-99	JJ	denotes	receptor-regulated
T39	100-104	NN	denotes	NFκB
T40	105-113	NN	denotes	activity
T41	114-116	IN	denotes	in
T42	117-127	NN	denotes	vertebrate
T43	128-135	NN	denotes	B-cells
T44	147-149	TO	denotes	To
T45	150-161	VB	denotes	investigate
T46	162-165	DT	denotes	the
T47	166-176	NN	denotes	importance
T48	177-179	IN	denotes	of
T1722	4035-4036	CD	denotes	1
T1723	4036-4037	-RRB-	denotes	]
T1724	4039-4054	NN	denotes	Chloramphenicol
T1725	4055-4061	NN	denotes	acetyl
T1726	4062-4073	NN	denotes	transferase
T1727	4074-4080	NN	denotes	assays
T1728	4081-4085	VB	denotes	have
T1729	4086-4090	VB	denotes	been
T1730	4091-4100	VB	denotes	described
T1731	4101-4111	RB	denotes	previously
T1732	4112-4113	-LRB-	denotes	[
T1733	4113-4115	CD	denotes	29
T1734	4115-4116	-RRB-	denotes	]
T1868	0-4128	NN	denotes	Protein kinase D enzymes are dispensable for proliferation, survival and antigen receptor-regulated NFκB activity in vertebrate B-cells Abstract To investigate the importance of protein kinase D (PKD) enzymes we generated a PKD-null DT40 B-lymphocyte cell line. Previously we have shown that PKDs have an essential role in regulating class II histone deacetylases in DT40 B-cells [Matthews, S.A., Liu, P., Spitaler, M., Olson, E.N., McKinsey, T.A., Cantrell, D.A. and Scharenberg, A.M. (2006) Essential role for protein kinase D family kinases in the regulation of class II histone deacetylases in B lymphocytes. Mol. Cell Biol. 26, 1569–1577]. We now show that PKDs are also required to regulate HSP27 phosphorylation in DT40 B-cells. However, in contrast to previous observations in other cell types, PKD enzymes do not regulate basic cellular processes such as proliferation or survival responses, nor NFκB transcriptional activity downstream of the B cell antigen receptor. Thus, PKDs have a selective role in DT40 B-cell biology. 1 Introduction The protein kinase D (PKD) serine/threonine kinase family has three members: PKD1, PKD2 and PKD3. Most cell types express at least two PKD isoforms but PKD enzymes are especially highly expressed in haematopoietic cells, where they are activated in response to antigen receptors stimulation [2,3]. A conserved signalling pathway linking antigen receptors to PKDs involves the activation of PLCγ and the subsequent production of diacylglycerol (DAG) which stimulates classical and/or novel protein kinase Cs (PKC) that phosphorylate two key regulatory serine residues in the activation loop of PKD kinases [3–6]. The N-terminal regulatory region of PKD enzymes contains a DAG binding domain and direct binding of DAG also contributes to PKD1 activation [7] as well as regulating the spatial location of PKD enzymes within cells [8–12]. PKD enzymes have been proposed to regulate numerous cellular functions, including cell proliferation [13–16], anti-apoptotic signals [17,18] and thymocyte development [19]. Expression of mutant catalytically inactive and constitutively activated PKDs can also modify Golgi function, cell adhesion and cell motility (reviewed in [20]). In particular, PKDs have been widely linked to the activation of the NFκB transcription factor and in regulating cell survival during oxidative stress [17,21–23]. Another recently proposed PKD1 substrate is HSP27 [24], a small heat shock protein involved in regulating cell migration and cell survival [25]. An essential role for PKD enzymes in regulating class II histone deacetylases (HDACs), enzymes that repress MEF2-dependent gene transcription, has also been demonstrated [1,26–28]. To investigate the biological role of PKDs we have generated DT40 B cell lines that lack expression of one or more members of the PKD family [1], allowing us to investigate the function(s) of PKD isoforms following B cell antigen receptor (BCR) stimulation, as well addressing the issue of functional redundancy between the different PKD family members. Previous studies have shown that PKDs are indispensable for HDAC regulation in B cells [1]. Herein we show that PKDs are also indispensable for HSP27 phosphorylation in B cells. However, PKD-null DT40 B cells are viable and proliferate normally. Moreover, loss of the entire cellular pool of PKD does not critically affect oxidative stress responses in B cells nor do PKD kinases play an essential role in regulating NFκB transcriptional activity. Together, these findings reveal that in B lymphocytes, PKD kinases are not critical regulators of many of the cellular processes previously ascribed to them in other cellular systems. 2 Materials and methods 2.1 Cell culture, transient transfections and cell stimulation The generation, culture and activation of PKD1−/−, PKD3−/− and PKD1/3−/− knockout DT40 B cell lines have been described previously [1]. Cells were lysed and protein extracts were analysed in Western blotting experiments as previously described [1]. Chloramphenicol acetyl transferase assays have been described previously [29]. 2.2 sIgM
T1869	4129-4137	NN	denotes	staining
T1870	4138-4142	NN	denotes	DT40
T1871	4143-4144	NN	denotes	B
T1872	4145-4150	NN	denotes	cells
R9	T51	T52	arg1Of	D,(
R10	T53	T52	arg2Of	PKD,(
R11	T54	T52	arg3Of	),(
R15	T56	T57	arg1Of	we,generated
R17	T63	T57	arg2Of	line,generated
R18	T63	T58	arg1Of	line,a
R19	T63	T59	arg1Of	line,PKD-null
R20	T63	T60	arg1Of	line,DT40
R21	T63	T61	arg1Of	line,B-lymphocyte
R22	T63	T62	arg1Of	line,cell
R23	T65	T66	arg1Of	we,have
R24	T65	T67	arg1Of	we,shown
R25	T67	T64	arg1Of	shown,Previously
R26	T67	T66	arg2Of	shown,have
R27	T69	T70	arg1Of	PKDs,have
R28	T70	T67	arg2Of	have,shown
R29	T70	T68	arg1Of	have,that
R30	T70	T74	arg1Of	have,in
R31	T73	T70	arg2Of	role,have
R32	T73	T71	arg1Of	role,an
R33	T73	T72	arg1Of	role,essential
R34	T75	T74	arg2Of	regulating,in
R35	T75	T80	arg1Of	regulating,in
R36	T79	T75	arg2Of	deacetylases,regulating
R37	T79	T76	arg1Of	deacetylases,class
R38	T79	T77	arg1Of	deacetylases,II
R39	T79	T78	arg1Of	deacetylases,histone
R40	T82	T80	arg2Of	B-cells,in
R41	T82	T81	arg1Of	B-cells,DT40
R42	T84	T85	arg1Of	Matthews,","
R43	T85	T87	arg1Of	",",","
R44	T86	T85	arg2Of	S.A.,","
R45	T87	T89	arg1Of	",",","
R46	T88	T87	arg2Of	Liu,","
R47	T89	T91	arg1Of	",",","
R48	T90	T89	arg2Of	P.,","
R49	T91	T93	arg1Of	",",","
R50	T92	T91	arg2Of	Spitaler,","
R51	T93	T95	arg1Of	",",","
R52	T94	T93	arg2Of	M.,","
R53	T95	T105	arg1Of	",",","
R54	T96	T97	arg1Of	Olson,","
R55	T97	T99	arg1Of	",",","
R56	T98	T97	arg2Of	E.N.,","
R57	T99	T101	arg1Of	",",","
R58	T100	T99	arg2Of	McKinsey,","
R59	T101	T103	arg1Of	",",","
R60	T102	T101	arg2Of	T.A.,","
R61	T103	T95	arg2Of	",",","
R62	T104	T103	arg2Of	Cantrell,","
R63	T105	T107	arg1Of	",",and
R64	T106	T105	arg2Of	D.A.,","
R65	T108	T109	arg1Of	Scharenberg,","
R66	T109	T107	arg2Of	",",and
R67	T110	T109	arg2Of	A.M.,","
R68	T112	T111	arg2Of	2006,(
R69	T113	T111	arg3Of	),(
R70	T115	T80	arg3Of	role,in
R71	T115	T83	arg1Of	role,[
R72	T115	T84	arg1Of	role,Matthews
R73	T115	T86	arg1Of	role,S.A.
R74	T115	T88	arg1Of	role,Liu
R75	T115	T90	arg1Of	role,P.
R76	T115	T92	arg1Of	role,Spitaler
R77	T115	T94	arg1Of	role,M.
R78	T115	T96	arg1Of	role,Olson
R79	T115	T98	arg1Of	role,E.N.
R80	T115	T100	arg1Of	role,McKinsey
R81	T115	T102	arg1Of	role,T.A.
R82	T115	T104	arg1Of	role,Cantrell
R83	T115	T106	arg1Of	role,D.A.
R84	T115	T108	arg1Of	role,Scharenberg
R85	T115	T110	arg1Of	role,A.M.
R86	T115	T111	arg1Of	role,(
R87	T115	T114	arg1Of	role,Essential
R88	T115	T116	arg1Of	role,for
R89	T115	T122	arg1Of	role,in
R90	T121	T116	arg2Of	kinases,for
R91	T121	T117	arg1Of	kinases,protein
R92	T121	T118	arg1Of	kinases,kinase
R93	T121	T119	arg1Of	kinases,D
R94	T121	T120	arg1Of	kinases,family
R95	T124	T122	arg2Of	regulation,in
R96	T124	T123	arg1Of	regulation,the
R97	T124	T125	arg1Of	regulation,of
R98	T129	T125	arg2Of	deacetylases,of
R99	T129	T126	arg1Of	deacetylases,class
R100	T129	T127	arg1Of	deacetylases,II
R101	T129	T128	arg1Of	deacetylases,histone
R102	T129	T130	arg1Of	deacetylases,in
R103	T132	T130	arg2Of	lymphocytes,in
R104	T132	T131	arg1Of	lymphocytes,B
R105	T135	T133	arg1Of	Biol.,Mol.
R106	T135	T134	arg1Of	Biol.,Cell
R107	T135	T136	arg1Of	Biol.,26
R108	T135	T137	arg1Of	Biol.,","
R109	T138	T137	arg2Of	1569–1577,","
R110	T138	T139	arg1Of	1569–1577,]
R111	T140	T142	arg1Of	We,show
R112	T142	T141	arg1Of	show,now
R113	T144	T145	arg1Of	PKDs,are
R114	T144	T147	arg2Of	PKDs,required
R115	T144	T149	arg1Of	PKDs,regulate
R116	T147	T142	arg2Of	required,show
R117	T147	T143	arg1Of	required,that
R118	T147	T145	arg2Of	required,are
R119	T147	T146	arg1Of	required,also
R120	T149	T147	arg3Of	regulate,required
R121	T149	T148	arg1Of	regulate,to
R122	T149	T152	arg1Of	regulate,in
R123	T151	T149	arg2Of	phosphorylation,regulate
R124	T151	T150	arg1Of	phosphorylation,HSP27
R125	T154	T152	arg2Of	B-cells,in
R126	T154	T153	arg1Of	B-cells,DT40
R127	T158	T157	arg2Of	contrast,in
R128	T158	T159	arg1Of	contrast,to
R129	T161	T159	arg2Of	observations,to
R130	T161	T160	arg1Of	observations,previous
R131	T161	T162	arg1Of	observations,in
R132	T165	T162	arg2Of	types,in
R133	T165	T163	arg1Of	types,other
R134	T165	T164	arg1Of	types,cell
R135	T168	T167	arg1Of	enzymes,PKD
R136	T168	T169	arg1Of	enzymes,do
R137	T168	T171	arg1Of	enzymes,regulate
R138	T171	T155	arg1Of	regulate,However
R139	T171	T156	arg1Of	regulate,","
R140	T171	T157	arg1Of	regulate,in
R141	T171	T166	arg1Of	regulate,","
R142	T171	T169	arg2Of	regulate,do
R143	T171	T170	arg1Of	regulate,not
R144	T174	T171	arg2Of	processes,regulate
R145	T174	T172	arg1Of	processes,basic
R146	T174	T173	arg1Of	processes,cellular
R147	T174	T176	arg1Of	processes,as
R148	T176	T175	arg1Of	as,such
R149	T177	T178	arg1Of	proliferation,or
R150	T178	T182	arg1Of	or,nor
R151	T180	T178	arg2Of	responses,or
R152	T180	T179	arg1Of	responses,survival
R153	T182	T176	arg2Of	nor,as
R154	T182	T181	arg1Of	nor,","
R155	T185	T182	arg2Of	activity,nor
R156	T185	T183	arg1Of	activity,NFκB
R157	T185	T184	arg1Of	activity,transcriptional
R158	T185	T186	arg1Of	activity,downstream
R159	T186	T187	arg1Of	downstream,of
R160	T192	T187	arg2Of	receptor,of
R161	T192	T188	arg1Of	receptor,the
R162	T192	T189	arg1Of	receptor,B
R163	T192	T190	arg1Of	receptor,cell
R164	T192	T191	arg1Of	receptor,antigen
R165	T195	T196	arg1Of	PKDs,have
R166	T196	T193	arg1Of	have,Thus
R167	T196	T194	arg1Of	have,","
R168	T196	T200	arg1Of	have,in
R169	T199	T196	arg2Of	role,have
R170	T199	T197	arg1Of	role,a
R171	T199	T198	arg1Of	role,selective
R172	T203	T200	arg2Of	biology,in
R173	T203	T201	arg1Of	biology,DT40
R174	T203	T202	arg1Of	biology,B-cell
R430	T535	T532	arg1Of	D,"1569–1577]. We now show that PKDs are also required to regulate HSP27 phosphorylation in DT40 B-cells. However, in contrast to previous observations in other cell types, PKD enzymes do not regulate basic cellular processes such as proliferation or survival responses, nor NFκB transcriptional activity downstream of the B cell antigen receptor. Thus, PKDs have a selective role in DT40 B-cell biology. 1 Introduction The"
R431	T535	T533	arg1Of	D,protein
R432	T535	T534	arg1Of	D,kinase
R433	T535	T536	arg1Of	D,(
R434	T537	T536	arg2Of	PKD,(
R435	T538	T536	arg3Of	),(
R436	T541	T535	arg1Of	family,D
R437	T541	T539	arg1Of	family,serine/threonine
R438	T541	T540	arg1Of	family,kinase
R439	T541	T542	arg1Of	family,has
R440	T544	T542	arg2Of	members,has
R441	T544	T543	arg1Of	members,three
R442	T544	T545	arg1Of	members,:
R443	T546	T547	arg1Of	PKD1,","
R444	T547	T549	arg1Of	",",and
R445	T548	T547	arg2Of	PKD2,","
R446	T549	T545	arg2Of	and,:
R447	T550	T549	arg2Of	PKD3,and
R448	T553	T551	arg1Of	types,Most
R449	T553	T552	arg1Of	types,cell
R450	T553	T554	arg1Of	types,express
R451	T554	T560	arg1Of	express,but
R452	T557	T555	arg1Of	two,at
R453	T557	T556	arg1Of	two,least
R454	T559	T554	arg2Of	isoforms,express
R455	T559	T557	arg1Of	isoforms,two
R456	T559	T558	arg1Of	isoforms,PKD
R457	T562	T561	arg1Of	enzymes,PKD
R458	T562	T563	arg1Of	enzymes,are
R459	T562	T566	arg2Of	enzymes,expressed
R460	T565	T564	arg1Of	highly,especially
R461	T566	T560	arg2Of	expressed,but
R462	T566	T563	arg2Of	expressed,are
R463	T566	T565	arg1Of	expressed,highly
R464	T566	T567	arg1Of	expressed,in
R465	T569	T567	arg2Of	cells,in
R466	T569	T568	arg1Of	cells,haematopoietic
R467	T569	T570	arg1Of	cells,","
R468	T569	T571	arg1Of	cells,where
R469	T572	T573	arg1Of	they,are
R470	T572	T574	arg2Of	they,activated
R471	T574	T571	arg2Of	activated,where
R472	T574	T573	arg2Of	activated,are
R473	T574	T575	arg1Of	activated,in
R474	T574	T581	arg1Of	activated,[
R475	T575	T577	arg1Of	in,to
R476	T576	T575	arg2Of	response,in
R477	T580	T575	arg3Of	stimulation,in
R478	T580	T578	arg1Of	stimulation,antigen
R479	T580	T579	arg1Of	stimulation,receptors
R480	T582	T581	arg2Of	"2,3",[
R481	T583	T581	arg3Of	],[
R482	T587	T584	arg1Of	pathway,A
R483	T587	T585	arg2Of	pathway,conserved
R484	T587	T586	arg1Of	pathway,signalling
R485	T587	T588	arg1Of	pathway,linking
R486	T587	T593	arg1Of	pathway,involves
R487	T590	T588	arg2Of	receptors,linking
R488	T590	T589	arg1Of	receptors,antigen
R489	T590	T591	arg1Of	receptors,to
R490	T592	T591	arg2Of	PKDs,to
R491	T595	T594	arg1Of	activation,the
R492	T595	T596	arg1Of	activation,of
R493	T595	T598	arg1Of	activation,and
R494	T597	T596	arg2Of	PLCγ,of
R495	T598	T593	arg2Of	and,involves
R496	T601	T598	arg2Of	production,and
R497	T601	T599	arg1Of	production,the
R498	T601	T600	arg1Of	production,subsequent
R499	T601	T602	arg1Of	production,of
R500	T601	T607	arg1Of	production,which
R501	T601	T608	arg1Of	production,stimulates
R502	T603	T602	arg2Of	diacylglycerol,of
R503	T603	T604	arg1Of	diacylglycerol,(
R504	T605	T604	arg2Of	DAG,(
R505	T606	T604	arg3Of	),(
R506	T609	T610	arg1Of	classical,and/or
R507	T611	T610	arg2Of	novel,and/or
R508	T614	T608	arg2Of	Cs,stimulates
R509	T614	T609	arg1Of	Cs,classical
R510	T614	T611	arg1Of	Cs,novel
R511	T614	T612	arg1Of	Cs,protein
R512	T614	T613	arg1Of	Cs,kinase
R513	T614	T615	arg1Of	Cs,(
R514	T614	T618	arg1Of	Cs,that
R515	T614	T619	arg1Of	Cs,phosphorylate
R516	T616	T615	arg2Of	PKC,(
R517	T617	T615	arg3Of	),(
R518	T619	T625	arg1Of	phosphorylate,in
R519	T624	T619	arg2Of	residues,phosphorylate
R520	T624	T620	arg1Of	residues,two
R521	T624	T621	arg1Of	residues,key
R522	T624	T622	arg1Of	residues,regulatory
R523	T624	T623	arg1Of	residues,serine
R524	T628	T625	arg2Of	loop,in
R525	T628	T626	arg1Of	loop,the
R526	T628	T627	arg1Of	loop,activation
R527	T628	T629	arg1Of	loop,of
R528	T628	T632	arg1Of	loop,[
R529	T631	T629	arg2Of	kinases,of
R530	T631	T630	arg1Of	kinases,PKD
R531	T633	T632	arg2Of	3–6,[
R532	T634	T632	arg3Of	],[
R533	T638	T635	arg1Of	region,The
R534	T638	T636	arg1Of	region,N-terminal
R535	T638	T637	arg1Of	region,regulatory
R536	T638	T639	arg1Of	region,of
R537	T638	T642	arg1Of	region,contains
R538	T641	T639	arg2Of	enzymes,of
R539	T641	T640	arg1Of	enzymes,PKD
R540	T642	T647	arg1Of	contains,and
R541	T646	T642	arg2Of	domain,contains
R542	T646	T643	arg1Of	domain,a
R543	T646	T644	arg1Of	domain,DAG
R544	T646	T645	arg1Of	domain,binding
R545	T649	T648	arg1Of	binding,direct
R546	T649	T650	arg1Of	binding,of
R547	T649	T653	arg1Of	binding,contributes
R548	T651	T650	arg2Of	DAG,of
R549	T653	T647	arg2Of	contributes,and
R550	T653	T652	arg1Of	contributes,also
R551	T653	T654	arg1Of	contributes,to
R552	T653	T657	arg1Of	contributes,[
R553	T653	T661	arg1Of	contributes,well
R554	T653	T662	arg1Of	contributes,as
R555	T656	T654	arg2Of	activation,to
R556	T656	T655	arg1Of	activation,PKD1
R557	T658	T657	arg2Of	7,[
R558	T659	T657	arg3Of	],[
R559	T661	T660	arg1Of	well,as
R560	T663	T662	arg2Of	regulating,as
R561	T666	T663	arg2Of	location,regulating
R562	T666	T664	arg1Of	location,the
R563	T666	T665	arg1Of	location,spatial
R564	T666	T667	arg1Of	location,of
R565	T666	T670	arg1Of	location,within
R566	T669	T667	arg2Of	enzymes,of
R567	T669	T668	arg1Of	enzymes,PKD
R568	T671	T670	arg2Of	cells,within
R569	T671	T672	arg1Of	cells,[
R570	T673	T672	arg2Of	8–12,[
R571	T674	T672	arg3Of	],[
R572	T676	T675	arg1Of	enzymes,PKD
R573	T676	T677	arg1Of	enzymes,have
R574	T676	T678	arg1Of	enzymes,been
R575	T676	T679	arg2Of	enzymes,proposed
R576	T676	T681	arg1Of	enzymes,regulate
R577	T679	T677	arg2Of	proposed,have
R578	T679	T678	arg2Of	proposed,been
R579	T681	T679	arg3Of	regulate,proposed
R580	T681	T680	arg1Of	regulate,to
R581	T684	T681	arg2Of	functions,regulate
R582	T684	T682	arg1Of	functions,numerous
R583	T684	T683	arg1Of	functions,cellular
R584	T684	T685	arg1Of	functions,","
R585	T684	T686	arg1Of	functions,including
R586	T688	T687	arg1Of	proliferation,cell
R587	T688	T689	arg1Of	proliferation,[
R588	T688	T692	arg1Of	proliferation,","
R589	T690	T689	arg2Of	13–16,[
R590	T691	T689	arg3Of	],[
R591	T692	T698	arg1Of	",",and
R592	T694	T692	arg2Of	signals,","
R593	T694	T693	arg1Of	signals,anti-apoptotic
R594	T694	T695	arg1Of	signals,[
R595	T696	T695	arg2Of	"17,18",[
R596	T697	T695	arg3Of	],[
R597	T698	T686	arg2Of	and,including
R598	T700	T698	arg2Of	development,and
R599	T700	T699	arg1Of	development,thymocyte
R600	T700	T701	arg1Of	development,[
R601	T702	T701	arg2Of	19,[
R602	T703	T701	arg3Of	],[
R603	T704	T705	arg1Of	Expression,of
R604	T704	T713	arg1Of	Expression,can
R605	T704	T715	arg1Of	Expression,modify
R606	T708	T709	arg1Of	inactive,and
R607	T710	T709	arg2Of	constitutively,and
R608	T712	T705	arg2Of	PKDs,of
R609	T712	T706	arg1Of	PKDs,mutant
R610	T712	T707	arg1Of	PKDs,catalytically
R611	T712	T708	arg1Of	PKDs,inactive
R612	T712	T710	arg1Of	PKDs,constitutively
R613	T712	T711	arg2Of	PKDs,activated
R614	T715	T713	arg2Of	modify,can
R615	T715	T714	arg1Of	modify,also
R616	T717	T716	arg1Of	function,Golgi
R617	T717	T718	arg1Of	function,","
R618	T718	T721	arg1Of	",",and
R619	T720	T718	arg2Of	adhesion,","
R620	T720	T719	arg1Of	adhesion,cell
R621	T721	T715	arg2Of	and,modify
R622	T723	T721	arg2Of	motility,and
R623	T723	T722	arg1Of	motility,cell
R624	T723	T724	arg1Of	motility,(
R625	T725	T724	arg2Of	reviewed,(
R626	T725	T726	arg1Of	reviewed,in
R627	T725	T727	arg1Of	reviewed,[
R628	T728	T727	arg2Of	20,[
R629	T729	T727	arg3Of	],[
R630	T730	T724	arg3Of	),(
R631	T732	T731	arg2Of	particular,In
R632	T734	T735	arg1Of	PKDs,have
R633	T734	T736	arg1Of	PKDs,been
R634	T734	T738	arg2Of	PKDs,linked
R635	T734	T749	arg1Of	PKDs,regulating
R636	T738	T731	arg1Of	linked,In
R637	T738	T733	arg1Of	linked,","
R638	T738	T735	arg2Of	linked,have
R639	T738	T736	arg2Of	linked,been
R640	T738	T737	arg1Of	linked,widely
R641	T738	T739	arg1Of	linked,to
R642	T738	T748	arg1Of	linked,in
R643	T739	T747	arg1Of	to,and
R644	T741	T739	arg2Of	activation,to
R645	T741	T740	arg1Of	activation,the
R646	T741	T742	arg1Of	activation,of
R647	T746	T742	arg2Of	factor,of
R648	T746	T743	arg1Of	factor,the
R649	T746	T744	arg1Of	factor,NFκB
R650	T746	T745	arg1Of	factor,transcription
R651	T748	T747	arg2Of	in,and
R652	T749	T748	arg2Of	regulating,in
R653	T749	T752	arg1Of	regulating,during
R654	T751	T749	arg2Of	survival,regulating
R655	T751	T750	arg1Of	survival,cell
R656	T754	T752	arg2Of	stress,during
R657	T754	T753	arg1Of	stress,oxidative
R658	T754	T755	arg1Of	stress,[
R659	T756	T755	arg2Of	"17,21–23",[
R660	T757	T755	arg3Of	],[
R661	T760	T759	arg1Of	proposed,recently
R662	T762	T758	arg1Of	substrate,Another
R663	T762	T760	arg1Of	substrate,proposed
R664	T762	T761	arg1Of	substrate,PKD1
R665	T762	T763	arg1Of	substrate,is
R666	T764	T763	arg2Of	HSP27,is
R667	T764	T765	arg1Of	HSP27,[
R668	T764	T768	arg1Of	HSP27,","
R669	T766	T765	arg2Of	24,[
R670	T767	T765	arg3Of	],[
R671	T773	T768	arg2Of	protein,","
R672	T773	T769	arg1Of	protein,a
R673	T773	T770	arg1Of	protein,small
R674	T773	T771	arg1Of	protein,heat
R675	T773	T772	arg1Of	protein,shock
R676	T773	T774	arg2Of	protein,involved
R677	T774	T775	arg1Of	involved,in
R678	T776	T775	arg2Of	regulating,in
R679	T776	T782	arg1Of	regulating,[
R680	T778	T777	arg1Of	migration,cell
R681	T778	T779	arg1Of	migration,and
R682	T779	T776	arg2Of	and,regulating
R683	T781	T779	arg2Of	survival,and
R684	T781	T780	arg1Of	survival,cell
R685	T783	T782	arg2Of	25,[
R686	T784	T782	arg3Of	],[
R687	T787	T785	arg1Of	role,An
R688	T787	T786	arg1Of	role,essential
R689	T787	T788	arg1Of	role,for
R690	T787	T808	arg1Of	role,has
R691	T787	T810	arg1Of	role,been
R692	T787	T811	arg2Of	role,demonstrated
R693	T790	T788	arg2Of	enzymes,for
R694	T790	T789	arg1Of	enzymes,PKD
R695	T790	T791	arg1Of	enzymes,in
R696	T792	T791	arg2Of	regulating,in
R697	T796	T792	arg2Of	deacetylases,regulating
R698	T796	T793	arg1Of	deacetylases,class
R699	T796	T794	arg1Of	deacetylases,II
R700	T796	T795	arg1Of	deacetylases,histone
R701	T796	T797	arg1Of	deacetylases,(
R702	T796	T800	arg1Of	deacetylases,","
R703	T798	T797	arg2Of	HDACs,(
R704	T799	T797	arg3Of	),(
R705	T801	T800	arg2Of	enzymes,","
R706	T801	T802	arg1Of	enzymes,that
R707	T801	T803	arg1Of	enzymes,repress
R708	T806	T803	arg2Of	transcription,repress
R709	T806	T804	arg1Of	transcription,MEF2-dependent
R710	T806	T805	arg1Of	transcription,gene
R711	T811	T807	arg1Of	demonstrated,","
R712	T811	T808	arg2Of	demonstrated,has
R713	T811	T809	arg1Of	demonstrated,also
R714	T811	T810	arg2Of	demonstrated,been
R715	T811	T812	arg1Of	demonstrated,[
R716	T813	T812	arg2Of	"1,26–28",[
R717	T814	T812	arg3Of	],[
R718	T816	T815	arg1Of	investigate,To
R719	T819	T816	arg2Of	role,investigate
R720	T819	T817	arg1Of	role,the
R721	T819	T818	arg1Of	role,biological
R722	T819	T820	arg1Of	role,of
R723	T821	T820	arg2Of	PKDs,of
R724	T822	T816	arg1Of	we,investigate
R725	T822	T823	arg1Of	we,have
R726	T822	T824	arg1Of	we,generated
R727	T824	T815	modOf	generated,To
R728	T824	T823	arg2Of	generated,have
R729	T828	T824	arg2Of	lines,generated
R730	T828	T825	arg1Of	lines,DT40
R731	T828	T826	arg1Of	lines,B
R732	T828	T827	arg1Of	lines,cell
R733	T828	T829	arg1Of	lines,that
R734	T828	T830	arg1Of	lines,lack
R735	T830	T844	arg1Of	lack,","
R736	T830	T845	modOf	lack,allowing
R737	T830	T866	arg1Of	lack,","
R738	T830	T869	modOf	lack,addressing
R739	T831	T830	arg2Of	expression,lack
R740	T831	T832	arg1Of	expression,of
R741	T833	T834	arg1Of	one,or
R742	T833	T835	arg1Of	one,more
R743	T836	T832	arg2Of	members,of
R744	T836	T833	arg1Of	members,one
R745	T836	T837	arg1Of	members,of
R746	T840	T837	arg2Of	family,of
R747	T840	T838	arg1Of	family,the
R748	T840	T839	arg1Of	family,PKD
R749	T840	T841	arg1Of	family,[
R750	T842	T841	arg2Of	1,[
R751	T843	T841	arg3Of	],[
R752	T846	T845	arg2Of	us,allowing
R753	T846	T848	arg1Of	us,investigate
R754	T848	T845	arg3Of	investigate,allowing
R755	T848	T847	arg1Of	investigate,to
R756	T850	T848	arg2Of	function,investigate
R757	T850	T849	arg1Of	function,the
R758	T850	T851	arg1Of	function,(
R759	T850	T854	arg1Of	function,of
R760	T850	T857	arg1Of	function,following
R761	T852	T851	arg2Of	s,(
R762	T853	T851	arg3Of	),(
R763	T856	T854	arg2Of	isoforms,of
R764	T856	T855	arg1Of	isoforms,PKD
R765	T863	T862	arg2Of	BCR,(
R766	T864	T862	arg3Of	),(
R767	T865	T857	arg2Of	stimulation,following
R768	T865	T858	arg1Of	stimulation,B
R769	T865	T859	arg1Of	stimulation,cell
R770	T865	T860	arg1Of	stimulation,antigen
R771	T865	T861	arg1Of	stimulation,receptor
R772	T865	T862	arg1Of	stimulation,(
R773	T869	T867	arg1Of	addressing,as
R774	T869	T868	arg1Of	addressing,well
R775	T871	T869	arg2Of	issue,addressing
R776	T871	T870	arg1Of	issue,the
R777	T871	T872	arg1Of	issue,of
R778	T874	T872	arg2Of	redundancy,of
R779	T874	T873	arg1Of	redundancy,functional
R780	T874	T875	arg1Of	redundancy,between
R781	T880	T875	arg2Of	members,between
R782	T880	T876	arg1Of	members,the
R783	T880	T877	arg1Of	members,different
R784	T880	T878	arg1Of	members,PKD
R785	T880	T879	arg1Of	members,family
R786	T882	T881	arg1Of	studies,Previous
R787	T882	T883	arg1Of	studies,have
R788	T882	T884	arg1Of	studies,shown
R789	T884	T883	arg2Of	shown,have
R790	T886	T887	arg1Of	PKDs,are
R791	T886	T888	arg1Of	PKDs,indispensable
R792	T887	T884	arg2Of	are,shown
R793	T887	T885	arg1Of	are,that
R794	T887	T895	arg1Of	are,[
R795	T888	T887	arg2Of	indispensable,are
R796	T888	T889	arg1Of	indispensable,for
R797	T891	T889	arg2Of	regulation,for
R798	T891	T890	arg1Of	regulation,HDAC
R799	T891	T892	arg1Of	regulation,in
R800	T894	T892	arg2Of	cells,in
R801	T894	T893	arg1Of	cells,B
R802	T896	T895	arg2Of	1,[
R803	T897	T895	arg3Of	],[
R804	T899	T900	arg1Of	we,show
R805	T900	T898	arg1Of	show,Herein
R806	T902	T903	arg1Of	PKDs,are
R807	T902	T905	arg1Of	PKDs,indispensable
R808	T903	T900	arg2Of	are,show
R809	T903	T901	arg1Of	are,that
R810	T903	T904	arg1Of	are,also
R811	T905	T903	arg2Of	indispensable,are
R812	T905	T906	arg1Of	indispensable,for
R813	T908	T906	arg2Of	phosphorylation,for
R814	T908	T907	arg1Of	phosphorylation,HSP27
R815	T908	T909	arg1Of	phosphorylation,in
R816	T911	T909	arg2Of	cells,in
R817	T911	T910	arg1Of	cells,B
R818	T917	T914	arg1Of	cells,PKD-null
R819	T917	T915	arg1Of	cells,DT40
R820	T917	T916	arg1Of	cells,B
R821	T917	T918	arg1Of	cells,are
R822	T917	T919	arg1Of	cells,viable
R823	T917	T921	arg1Of	cells,proliferate
R824	T918	T920	arg1Of	are,and
R825	T919	T918	arg2Of	viable,are
R826	T920	T912	arg1Of	and,However
R827	T920	T913	arg1Of	and,","
R828	T921	T920	arg2Of	proliferate,and
R829	T921	T922	arg1Of	proliferate,normally
R830	T925	T926	arg1Of	loss,of
R831	T925	T933	arg1Of	loss,does
R832	T925	T936	arg1Of	loss,affect
R833	T925	T944	arg1Of	loss,do
R834	T930	T926	arg2Of	pool,of
R835	T930	T927	arg1Of	pool,the
R836	T930	T928	arg1Of	pool,entire
R837	T930	T929	arg1Of	pool,cellular
R838	T930	T931	arg1Of	pool,of
R839	T932	T931	arg2Of	PKD,of
R840	T936	T933	arg2Of	affect,does
R841	T936	T934	arg1Of	affect,not
R842	T936	T935	arg1Of	affect,critically
R843	T936	T943	arg1Of	affect,nor
R844	T939	T936	arg2Of	responses,affect
R845	T939	T937	arg1Of	responses,oxidative
R846	T939	T938	arg1Of	responses,stress
R847	T939	T940	arg1Of	responses,in
R848	T942	T940	arg2Of	cells,in
R849	T942	T941	arg1Of	cells,B
R850	T943	T923	arg1Of	nor,Moreover
R851	T943	T924	arg1Of	nor,","
R852	T944	T943	arg2Of	do,nor
R853	T945	T944	arg2Of	PKD,do
R854	T946	T947	arg1Of	kinases,play
R855	T946	T952	arg1Of	kinases,regulating
R856	T947	T944	arg3Of	play,do
R857	T947	T951	arg1Of	play,in
R858	T950	T947	arg2Of	role,play
R859	T950	T948	arg1Of	role,an
R860	T950	T949	arg1Of	role,essential
R861	T952	T951	arg2Of	regulating,in
R862	T955	T952	arg2Of	activity,regulating
R863	T955	T953	arg1Of	activity,NFκB
R864	T955	T954	arg1Of	activity,transcriptional
R865	T959	T958	arg1Of	findings,these
R866	T959	T960	arg1Of	findings,reveal
R867	T960	T956	arg1Of	reveal,Together
R868	T960	T957	arg1Of	reveal,","
R869	T964	T962	arg2Of	lymphocytes,in
R870	T964	T963	arg1Of	lymphocytes,B
R871	T967	T966	arg1Of	kinases,PKD
R872	T967	T968	arg1Of	kinases,are
R873	T968	T960	arg2Of	are,reveal
R874	T968	T961	arg1Of	are,that
R875	T968	T962	arg1Of	are,in
R876	T968	T965	arg1Of	are,","
R877	T968	T969	arg1Of	are,not
R878	T971	T968	arg2Of	regulators,are
R879	T971	T970	arg1Of	regulators,critical
R880	T971	T972	arg1Of	regulators,of
R881	T973	T972	arg2Of	many,of
R882	T973	T974	arg1Of	many,of
R883	T977	T974	arg2Of	processes,of
R884	T977	T975	arg1Of	processes,the
R885	T977	T976	arg1Of	processes,cellular
R886	T977	T979	arg2Of	processes,ascribed
R887	T979	T978	arg1Of	ascribed,previously
R888	T979	T980	arg1Of	ascribed,to
R889	T979	T982	arg1Of	ascribed,in
R890	T981	T980	arg2Of	them,to
R891	T985	T982	arg2Of	systems,in
R892	T985	T983	arg1Of	systems,other
R893	T985	T984	arg1Of	systems,cellular
R1437	T1675	T1674	arg1Of	culture,"Protein kinase D enzymes are dispensable for proliferation, survival and antigen receptor-regulated NFκB activity in vertebrate B-cells Abstract To investigate the importance of protein kinase D (PKD) enzymes we generated a PKD-null DT40 B-lymphocyte cell line. Previously we have shown that PKDs have an essential role in regulating class II histone deacetylases in DT40 B-cells [Matthews, S.A., Liu, P., Spitaler, M., Olson, E.N., McKinsey, T.A., Cantrell, D.A. and Scharenberg, A.M. (2006) Essential role for protein kinase D family kinases in the regulation of class II histone deacetylases in B lymphocytes. Mol. Cell Biol. 26, 1569–1577]. We now show that PKDs are also required to regulate HSP27 phosphorylation in DT40 B-cells. However, in contrast to previous observations in other cell types, PKD enzymes do not regulate basic cellular processes such as proliferation or survival responses, nor NFκB transcriptional activity downstream of the B cell antigen receptor. Thus, PKDs have a selective role in DT40 B-cell biology. 1 Introduction The protein kinase D (PKD) serine/threonine kinase family has three members: PKD1, PKD2 and PKD3. Most cell types express at least two PKD isoforms but PKD enzymes are especially highly expressed in haematopoietic cells, where they are activated in response to antigen receptors stimulation [2,3]. A conserved signalling pathway linking antigen receptors to PKDs involves the activation of PLCγ and the subsequent production of diacylglycerol (DAG) which stimulates classical and/or novel protein kinase Cs (PKC) that phosphorylate two key regulatory serine residues in the activation loop of PKD kinases [3–6]. The N-terminal regulatory region of PKD enzymes contains a DAG binding domain and direct binding of DAG also contributes to PKD1 activation [7] as well as regulating the spatial location of PKD enzymes within cells [8–12]. PKD enzymes have been proposed to regulate numerous cellular functions, including cell proliferation [13–16], anti-apoptotic signals [17,18] and thymocyte development [19]. Expression of mutant catalytically inactive and constitutively activated PKDs can also modify Golgi function, cell adhesion and cell motility (reviewed in [20]). In particular, PKDs have been widely linked to the activation of the NFκB transcription factor and in regulating cell survival during oxidative stress [17,21–23]. Another recently proposed PKD1 substrate is HSP27 [24], a small heat shock protein involved in regulating cell migration and cell survival [25]. An essential role for PKD enzymes in regulating class II histone deacetylases (HDACs), enzymes that repress MEF2-dependent gene transcription, has also been demonstrated [1,26–28]. To investigate the biological role of PKDs we have generated DT40 B cell lines that lack expression of one or more members of the PKD family [1], allowing us to investigate the function(s) of PKD isoforms following B cell antigen receptor (BCR) stimulation, as well addressing the issue of functional redundancy between the different PKD family members. Previous studies have shown that PKDs are indispensable for HDAC regulation in B cells [1]. Herein we show that PKDs are also indispensable for HSP27 phosphorylation in B cells. However, PKD-null DT40 B cells are viable and proliferate normally. Moreover, loss of the entire cellular pool of PKD does not critically affect oxidative stress responses in B cells nor do PKD kinases play an essential role in regulating NFκB transcriptional activity. Together, these findings reveal that in B lymphocytes, PKD kinases are not critical regulators of many of the cellular processes previously ascribed to them in other cellular systems. 2 Materials and methods 2.1 Cell"
R1438	T1675	T1676	arg1Of	culture,","
R1439	T1676	T1679	arg1Of	",",and
R1440	T1678	T1676	arg2Of	transfections,","
R1441	T1678	T1677	arg1Of	transfections,transient
R1442	T1681	T1679	arg2Of	stimulation,and
R1443	T1681	T1680	arg1Of	stimulation,cell
R1444	T1683	T1684	arg1Of	generation,","
R1445	T1684	T1686	arg1Of	",",and
R1446	T1685	T1684	arg2Of	culture,","
R1447	T1686	T1682	arg1Of	and,The
R1448	T1686	T1688	arg1Of	and,of
R1449	T1686	T1699	arg1Of	and,have
R1450	T1686	T1700	arg1Of	and,been
R1451	T1686	T1701	arg2Of	and,described
R1452	T1687	T1686	arg2Of	activation,and
R1453	T1689	T1690	arg1Of	PKD1−/−,","
R1454	T1690	T1692	arg1Of	",",and
R1455	T1691	T1690	arg2Of	PKD3−/−,","
R1456	T1693	T1692	arg2Of	PKD1/3−/−,and
R1457	T1698	T1688	arg2Of	lines,of
R1458	T1698	T1689	arg1Of	lines,PKD1−/−
R1459	T1698	T1691	arg1Of	lines,PKD3−/−
R1460	T1698	T1693	arg1Of	lines,PKD1/3−/−
R1461	T1698	T1694	arg1Of	lines,knockout
R1462	T1698	T1695	arg1Of	lines,DT40
R1463	T1698	T1696	arg1Of	lines,B
R1464	T1698	T1697	arg1Of	lines,cell
R1465	T1701	T1699	arg2Of	described,have
R1466	T1701	T1700	arg2Of	described,been
R1467	T1701	T1702	arg1Of	described,previously
R1468	T1701	T1703	arg1Of	described,[
R1469	T1704	T1703	arg2Of	1,[
R1470	T1705	T1703	arg3Of	],[
R1471	T1706	T1707	arg1Of	Cells,were
R1472	T1706	T1708	arg2Of	Cells,lysed
R1473	T1708	T1707	arg2Of	lysed,were
R1474	T1708	T1709	arg1Of	lysed,and
R1475	T1711	T1710	arg1Of	extracts,protein
R1476	T1711	T1712	arg1Of	extracts,were
R1477	T1711	T1713	arg2Of	extracts,analysed
R1478	T1713	T1709	arg2Of	analysed,and
R1479	T1713	T1712	arg2Of	analysed,were
R1480	T1713	T1714	arg1Of	analysed,in
R1481	T1713	T1718	arg1Of	analysed,as
R1482	T1717	T1714	arg2Of	experiments,in
R1483	T1717	T1715	arg1Of	experiments,Western
R1484	T1717	T1716	arg1Of	experiments,blotting
R1485	T1720	T1719	arg1Of	described,previously
R1486	T1722	T1718	arg2Of	1,as
R1487	T1722	T1720	arg1Of	1,described
R1488	T1722	T1721	arg2Of	1,[
R1489	T1723	T1721	arg3Of	],[
R1490	T1727	T1724	arg1Of	assays,Chloramphenicol
R1491	T1727	T1725	arg1Of	assays,acetyl
R1492	T1727	T1726	arg1Of	assays,transferase
R1493	T1727	T1728	arg1Of	assays,have
R1494	T1727	T1729	arg1Of	assays,been
R1495	T1727	T1730	arg2Of	assays,described
R1496	T1730	T1728	arg2Of	described,have
R1497	T1730	T1729	arg2Of	described,been
R1498	T1730	T1731	arg1Of	described,previously
R1499	T1730	T1732	arg1Of	described,[
R1500	T1733	T1732	arg2Of	29,[
R1501	T1734	T1732	arg3Of	],[
R1596	T1869	T1868	arg1Of	staining,"Protein kinase D enzymes are dispensable for proliferation, survival and antigen receptor-regulated NFκB activity in vertebrate B-cells Abstract To investigate the importance of protein kinase D (PKD) enzymes we generated a PKD-null DT40 B-lymphocyte cell line. Previously we have shown that PKDs have an essential role in regulating class II histone deacetylases in DT40 B-cells [Matthews, S.A., Liu, P., Spitaler, M., Olson, E.N., McKinsey, T.A., Cantrell, D.A. and Scharenberg, A.M. (2006) Essential role for protein kinase D family kinases in the regulation of class II histone deacetylases in B lymphocytes. Mol. Cell Biol. 26, 1569–1577]. We now show that PKDs are also required to regulate HSP27 phosphorylation in DT40 B-cells. However, in contrast to previous observations in other cell types, PKD enzymes do not regulate basic cellular processes such as proliferation or survival responses, nor NFκB transcriptional activity downstream of the B cell antigen receptor. Thus, PKDs have a selective role in DT40 B-cell biology. 1 Introduction The protein kinase D (PKD) serine/threonine kinase family has three members: PKD1, PKD2 and PKD3. Most cell types express at least two PKD isoforms but PKD enzymes are especially highly expressed in haematopoietic cells, where they are activated in response to antigen receptors stimulation [2,3]. A conserved signalling pathway linking antigen receptors to PKDs involves the activation of PLCγ and the subsequent production of diacylglycerol (DAG) which stimulates classical and/or novel protein kinase Cs (PKC) that phosphorylate two key regulatory serine residues in the activation loop of PKD kinases [3–6]. The N-terminal regulatory region of PKD enzymes contains a DAG binding domain and direct binding of DAG also contributes to PKD1 activation [7] as well as regulating the spatial location of PKD enzymes within cells [8–12]. PKD enzymes have been proposed to regulate numerous cellular functions, including cell proliferation [13–16], anti-apoptotic signals [17,18] and thymocyte development [19]. Expression of mutant catalytically inactive and constitutively activated PKDs can also modify Golgi function, cell adhesion and cell motility (reviewed in [20]). In particular, PKDs have been widely linked to the activation of the NFκB transcription factor and in regulating cell survival during oxidative stress [17,21–23]. Another recently proposed PKD1 substrate is HSP27 [24], a small heat shock protein involved in regulating cell migration and cell survival [25]. An essential role for PKD enzymes in regulating class II histone deacetylases (HDACs), enzymes that repress MEF2-dependent gene transcription, has also been demonstrated [1,26–28]. To investigate the biological role of PKDs we have generated DT40 B cell lines that lack expression of one or more members of the PKD family [1], allowing us to investigate the function(s) of PKD isoforms following B cell antigen receptor (BCR) stimulation, as well addressing the issue of functional redundancy between the different PKD family members. Previous studies have shown that PKDs are indispensable for HDAC regulation in B cells [1]. Herein we show that PKDs are also indispensable for HSP27 phosphorylation in B cells. However, PKD-null DT40 B cells are viable and proliferate normally. Moreover, loss of the entire cellular pool of PKD does not critically affect oxidative stress responses in B cells nor do PKD kinases play an essential role in regulating NFκB transcriptional activity. Together, these findings reveal that in B lymphocytes, PKD kinases are not critical regulators of many of the cellular processes previously ascribed to them in other cellular systems. 2 Materials and methods 2.1 Cell culture, transient transfections and cell stimulation The generation, culture and activation of PKD1−/−, PKD3−/− and PKD1/3−/− knockout DT40 B cell lines have been described previously [1]. Cells were lysed and protein extracts were analysed in Western blotting experiments as previously described [1]. Chloramphenicol acetyl transferase assays have been described previously [29]. 2.2 sIgM"
R1598	T1872	T1870	arg1Of	cells,DT40
R1599	T1872	T1871	arg1Of	cells,B
R1600	T1872	T1873	arg1Of	cells,(
R1601	T1872	T1880	arg2Of	cells,resuspended
R1604	T1875	T1873	arg2Of	cells,(
R1606	T1878	T1873	arg3Of	),(
R1607	T1880	T1879	arg2Of	resuspended,were
R1608	T1880	T1881	arg1Of	resuspended,in
R1609	T1883	T1881	arg2Of	buffer,in
R1610	T1883	T1882	arg1Of	buffer,200 μl
R1611	T1883	T1884	arg1Of	buffer,(
R1613	T1887	T1884	arg2Of	media,(
R1614	T1887	T1885	arg1Of	media,RPMI
R1615	T1887	T1886	arg1Of	media,1640
R1616	T1887	T1888	arg1Of	media,","
R1618	T1893	T1888	arg2Of	serum,","
R1619	T1893	T1889	arg1Of	serum,1
R1622	T1894	T1884	arg3Of	),(
R1653	T1924	T1923	arg2Of	representative,are
R1654	T1924	T1926	arg1Of	representative,at
R1655	T1926	T1925	arg1Of	at,of
R1656	T1927	T1928	arg1Of	two,to
R1657	T1929	T1928	arg2Of	four,to
R1658	T1931	T1926	arg2Of	experiments,at
R1659	T1931	T1927	arg1Of	experiments,two
R1660	T1931	T1930	arg1Of	experiments,independent
R1661	T1934	T1932	arg2Of	indicated,unless
R1662	T1934	T1933	arg1Of	indicated,otherwise
R1887	T2288	T2289	arg1Of	"Protein kinase D enzymes are dispensable for proliferation, survival and antigen receptor-regulated NFκB activity in vertebrate B-cells Abstract To investigate the importance of protein kinase D (PKD) enzymes we generated a PKD-null DT40 B-lymphocyte cell line. Previously we have shown that PKDs have an essential role in regulating class II histone deacetylases in DT40 B-cells [Matthews, S.A., Liu, P., Spitaler, M., Olson, E.N., McKinsey, T.A., Cantrell, D.A. and Scharenberg, A.M. (2006) Essential role for protein kinase D family kinases in the regulation of class II histone deacetylases in B lymphocytes. Mol. Cell Biol. 26, 1569–1577]. We now show that PKDs are also required to regulate HSP27 phosphorylation in DT40 B-cells. However, in contrast to previous observations in other cell types, PKD enzymes do not regulate basic cellular processes such as proliferation or survival responses, nor NFκB transcriptional activity downstream of the B cell antigen receptor. Thus, PKDs have a selective role in DT40 B-cell biology. 1 Introduction The protein kinase D (PKD) serine/threonine kinase family has three members: PKD1, PKD2 and PKD3. Most cell types express at least two PKD isoforms but PKD enzymes are especially highly expressed in haematopoietic cells, where they are activated in response to antigen receptors stimulation [2,3]. A conserved signalling pathway linking antigen receptors to PKDs involves the activation of PLCγ and the subsequent production of diacylglycerol (DAG) which stimulates classical and/or novel protein kinase Cs (PKC) that phosphorylate two key regulatory serine residues in the activation loop of PKD kinases [3–6]. The N-terminal regulatory region of PKD enzymes contains a DAG binding domain and direct binding of DAG also contributes to PKD1 activation [7] as well as regulating the spatial location of PKD enzymes within cells [8–12]. PKD enzymes have been proposed to regulate numerous cellular functions, including cell proliferation [13–16], anti-apoptotic signals [17,18] and thymocyte development [19]. Expression of mutant catalytically inactive and constitutively activated PKDs can also modify Golgi function, cell adhesion and cell motility (reviewed in [20]). In particular, PKDs have been widely linked to the activation of the NFκB transcription factor and in regulating cell survival during oxidative stress [17,21–23]. Another recently proposed PKD1 substrate is HSP27 [24], a small heat shock protein involved in regulating cell migration and cell survival [25]. An essential role for PKD enzymes in regulating class II histone deacetylases (HDACs), enzymes that repress MEF2-dependent gene transcription, has also been demonstrated [1,26–28]. To investigate the biological role of PKDs we have generated DT40 B cell lines that lack expression of one or more members of the PKD family [1], allowing us to investigate the function(s) of PKD isoforms following B cell antigen receptor (BCR) stimulation, as well addressing the issue of functional redundancy between the different PKD family members. Previous studies have shown that PKDs are indispensable for HDAC regulation in B cells [1]. Herein we show that PKDs are also indispensable for HSP27 phosphorylation in B cells. However, PKD-null DT40 B cells are viable and proliferate normally. Moreover, loss of the entire cellular pool of PKD does not critically affect oxidative stress responses in B cells nor do PKD kinases play an essential role in regulating NFκB transcriptional activity. Together, these findings reveal that in B lymphocytes, PKD kinases are not critical regulators of many of the cellular processes previously ascribed to them in other cellular systems. 2 Materials and methods 2.1 Cell culture, transient transfections and cell stimulation The generation, culture and activation of PKD1−/−, PKD3−/− and PKD1/3−/− knockout DT40 B cell lines have been described previously [1]. Cells were lysed and protein extracts were analysed in Western blotting experiments as previously described [1]. Chloramphenicol acetyl transferase assays have been described previously [29]. 2.2 sIgM staining DT40 B cells (2 × 106 cells per point) were resuspended in 200 μl buffer (RPMI 1640 media, 1% foetal calf serum) containing anti-chicken M1 monoclonal antibody conjugated to FITC for 20 min on ice. The cells were washed twice and fluorescent intensity was analysed by flow cytometry. All results shown are representative of at two to four independent experiments unless otherwise indicated. 3 Results 3.1 Loss",of
R1888	T2288	T2292	arg1Of	"Protein kinase D enzymes are dispensable for proliferation, survival and antigen receptor-regulated NFκB activity in vertebrate B-cells Abstract To investigate the importance of protein kinase D (PKD) enzymes we generated a PKD-null DT40 B-lymphocyte cell line. Previously we have shown that PKDs have an essential role in regulating class II histone deacetylases in DT40 B-cells [Matthews, S.A., Liu, P., Spitaler, M., Olson, E.N., McKinsey, T.A., Cantrell, D.A. and Scharenberg, A.M. (2006) Essential role for protein kinase D family kinases in the regulation of class II histone deacetylases in B lymphocytes. Mol. Cell Biol. 26, 1569–1577]. We now show that PKDs are also required to regulate HSP27 phosphorylation in DT40 B-cells. However, in contrast to previous observations in other cell types, PKD enzymes do not regulate basic cellular processes such as proliferation or survival responses, nor NFκB transcriptional activity downstream of the B cell antigen receptor. Thus, PKDs have a selective role in DT40 B-cell biology. 1 Introduction The protein kinase D (PKD) serine/threonine kinase family has three members: PKD1, PKD2 and PKD3. Most cell types express at least two PKD isoforms but PKD enzymes are especially highly expressed in haematopoietic cells, where they are activated in response to antigen receptors stimulation [2,3]. A conserved signalling pathway linking antigen receptors to PKDs involves the activation of PLCγ and the subsequent production of diacylglycerol (DAG) which stimulates classical and/or novel protein kinase Cs (PKC) that phosphorylate two key regulatory serine residues in the activation loop of PKD kinases [3–6]. The N-terminal regulatory region of PKD enzymes contains a DAG binding domain and direct binding of DAG also contributes to PKD1 activation [7] as well as regulating the spatial location of PKD enzymes within cells [8–12]. PKD enzymes have been proposed to regulate numerous cellular functions, including cell proliferation [13–16], anti-apoptotic signals [17,18] and thymocyte development [19]. Expression of mutant catalytically inactive and constitutively activated PKDs can also modify Golgi function, cell adhesion and cell motility (reviewed in [20]). In particular, PKDs have been widely linked to the activation of the NFκB transcription factor and in regulating cell survival during oxidative stress [17,21–23]. Another recently proposed PKD1 substrate is HSP27 [24], a small heat shock protein involved in regulating cell migration and cell survival [25]. An essential role for PKD enzymes in regulating class II histone deacetylases (HDACs), enzymes that repress MEF2-dependent gene transcription, has also been demonstrated [1,26–28]. To investigate the biological role of PKDs we have generated DT40 B cell lines that lack expression of one or more members of the PKD family [1], allowing us to investigate the function(s) of PKD isoforms following B cell antigen receptor (BCR) stimulation, as well addressing the issue of functional redundancy between the different PKD family members. Previous studies have shown that PKDs are indispensable for HDAC regulation in B cells [1]. Herein we show that PKDs are also indispensable for HSP27 phosphorylation in B cells. However, PKD-null DT40 B cells are viable and proliferate normally. Moreover, loss of the entire cellular pool of PKD does not critically affect oxidative stress responses in B cells nor do PKD kinases play an essential role in regulating NFκB transcriptional activity. Together, these findings reveal that in B lymphocytes, PKD kinases are not critical regulators of many of the cellular processes previously ascribed to them in other cellular systems. 2 Materials and methods 2.1 Cell culture, transient transfections and cell stimulation The generation, culture and activation of PKD1−/−, PKD3−/− and PKD1/3−/− knockout DT40 B cell lines have been described previously [1]. Cells were lysed and protein extracts were analysed in Western blotting experiments as previously described [1]. Chloramphenicol acetyl transferase assays have been described previously [29]. 2.2 sIgM staining DT40 B cells (2 × 106 cells per point) were resuspended in 200 μl buffer (RPMI 1640 media, 1% foetal calf serum) containing anti-chicken M1 monoclonal antibody conjugated to FITC for 20 min on ice. The cells were washed twice and fluorescent intensity was analysed by flow cytometry. All results shown are representative of at two to four independent experiments unless otherwise indicated. 3 Results 3.1 Loss",in
R1889	T2291	T2289	arg2Of	phosphorylation,of
R1890	T2291	T2290	arg1Of	phosphorylation,HSP27
R1891	T2295	T2292	arg2Of	cells,in
R1892	T2295	T2293	arg1Of	cells,DT40
R1893	T2295	T2294	arg1Of	cells,B
R1894	T2295	T2296	arg1Of	cells,lacking
R1895	T2297	T2296	arg2Of	expression,lacking
R1896	T2297	T2298	arg1Of	expression,of
R1897	T2301	T2298	arg2Of	kinases,of
R1898	T2301	T2299	arg1Of	kinases,PKD
R1899	T2301	T2300	arg1Of	kinases,family
R1900	T2304	T2302	arg1Of	cells,DT40
R1901	T2304	T2303	arg1Of	cells,B
R1902	T2304	T2305	arg1Of	cells,express
R1903	T2308	T2305	arg2Of	isoforms,express
R1904	T2308	T2306	arg1Of	isoforms,two
R1905	T2308	T2307	arg1Of	isoforms,PKD
R1906	T2308	T2309	arg1Of	isoforms,","
R1907	T2310	T2311	arg1Of	PKD1,and
R1908	T2311	T2309	arg2Of	and,","
R1909	T2311	T2314	arg1Of	and,and
R1910	T2311	T2315	arg1Of	and,as
R1911	T2312	T2311	arg2Of	PKD3,and
R1912	T2314	T2313	arg1Of	and,","
R1913	T2317	T2315	arg2Of	described,as
R1914	T2317	T2316	arg1Of	described,previously
R1915	T2318	T2319	arg1Of	we,have
R1916	T2318	T2321	arg1Of	we,generated
R1917	T2321	T2317	arg3Of	generated,described
R1918	T2321	T2319	arg2Of	generated,have
R1919	T2321	T2320	arg1Of	generated,recently
R1920	T2321	T2337	arg1Of	generated,[
R1921	T2325	T2321	arg2Of	lines,generated
R1922	T2325	T2322	arg1Of	lines,DT40
R1923	T2325	T2323	arg1Of	lines,B
R1924	T2325	T2324	arg1Of	lines,cell
R1925	T2325	T2326	arg1Of	lines,that
R1926	T2325	T2327	arg1Of	lines,lack
R1927	T2328	T2329	arg1Of	expression,of
R1928	T2328	T2334	arg1Of	expression,or
R1929	T2331	T2332	arg1Of	PKD1,or
R1930	T2332	T2329	arg2Of	or,of
R1931	T2332	T2330	arg1Of	or,either
R1932	T2333	T2332	arg2Of	PKD3,or
R1933	T2334	T2327	arg2Of	or,lack
R1934	T2336	T2334	arg2Of	enzymes,or
R1935	T2336	T2335	arg1Of	enzymes,both
R1936	T2338	T2337	arg2Of	1,[
R1937	T2339	T2337	arg3Of	],[
R1938	T2341	T2340	arg2Of	generating,In
R1939	T2346	T2341	arg2Of	lines,generating
R1940	T2346	T2342	arg1Of	lines,the
R1941	T2346	T2343	arg1Of	lines,double
R1942	T2346	T2344	arg1Of	lines,knockout
R1943	T2346	T2345	arg1Of	lines,cell
R1944	T2347	T2348	arg1Of	we,targeted
R1945	T2348	T2340	arg1Of	targeted,In
R1946	T2348	T2352	arg1Of	targeted,in
R1947	T2351	T2348	arg2Of	loci,targeted
R1948	T2351	T2349	arg1Of	loci,the
R1949	T2351	T2350	arg1Of	loci,PKD1
R1950	T2356	T2352	arg2Of	line,in
R1951	T2356	T2353	arg1Of	line,a
R1952	T2356	T2354	arg1Of	line,PKD3−/−
R1953	T2356	T2355	arg1Of	line,cell
R1954	T2356	T2357	arg1Of	line,that
R1955	T2356	T2358	arg1Of	line,expressed
R1956	T2358	T2362	arg1Of	expressed,under
R1957	T2361	T2358	arg2Of	transgene,expressed
R1958	T2361	T2359	arg1Of	transgene,a
R1959	T2361	T2360	arg1Of	transgene,Flag-PKD3
R1960	T2364	T2362	arg2Of	control,under
R1961	T2364	T2363	arg1Of	control,the
R1962	T2364	T2365	arg1Of	control,of
R1963	T2368	T2365	arg2Of	promoter,of
R1964	T2368	T2366	arg1Of	promoter,a
R1965	T2368	T2367	arg1Of	promoter,doxycycline-inducible
R1966	T2373	T2371	arg2Of	presence,in
R1967	T2373	T2372	arg1Of	presence,the
R1968	T2373	T2374	arg1Of	presence,of
R1969	T2375	T2374	arg2Of	doxycycline,of
R1970	T2378	T2377	arg1Of	expression,Flag-PKD3
R1971	T2378	T2379	arg1Of	expression,in
R1972	T2378	T2384	arg1Of	expression,is
R1973	T2378	T2385	arg1Of	expression,comparable
R1974	T2383	T2379	arg2Of	cells,in
R1975	T2383	T2380	arg1Of	cells,PKD1/3
R1976	T2383	T2381	arg1Of	cells,double
R1977	T2383	T2382	arg1Of	cells,knockout
R1978	T2384	T2394	arg1Of	is,and
R1979	T2385	T2384	arg2Of	comparable,is
R1980	T2385	T2386	arg1Of	comparable,to
R1981	T2385	T2390	arg1Of	comparable,in
R1982	T2388	T2386	arg2Of	PKD3,to
R1983	T2388	T2387	arg1Of	PKD3,endogenous
R1984	T2388	T2389	arg1Of	PKD3,present
R1985	T2393	T2390	arg2Of	cells,in
R1986	T2393	T2391	arg1Of	cells,wild-type
R1987	T2393	T2392	arg1Of	cells,DT40
R1988	T2394	T2369	arg1Of	and,Hence
R1989	T2394	T2370	arg1Of	and,","
R1990	T2394	T2371	arg1Of	and,in
R1991	T2394	T2376	arg1Of	and,","
R1992	T2395	T2396	arg1Of	removal,of
R1993	T2395	T2405	arg1Of	removal,results
R1994	T2397	T2396	arg2Of	doxycycline,of
R1995	T2397	T2398	arg1Of	doxycycline,from
R1996	T2397	T2402	arg1Of	doxycycline,for
R1997	T2401	T2398	arg2Of	media,from
R1998	T2401	T2399	arg1Of	media,the
R1999	T2401	T2400	arg1Of	media,culture
R2000	T2404	T2402	arg2Of	days,for
R2001	T2404	T2403	arg1Of	days,5
R2002	T2405	T2394	arg2Of	results,and
R2003	T2405	T2406	arg1Of	results,in
R2004	T2409	T2408	arg1Of	null,completely
R2005	T2411	T2406	arg2Of	phenotype,in
R2006	T2411	T2407	arg1Of	phenotype,a
R2007	T2411	T2409	arg1Of	phenotype,null
R2008	T2411	T2410	arg1Of	phenotype,PKD
R2009	T2411	T2412	arg1Of	phenotype,(
R2010	T2414	T2412	arg2Of	1A,(
R2011	T2414	T2413	arg1Of	1A,Fig.
R2012	T2415	T2412	arg3Of	),(
R2013	T2418	T2419	arg1Of	we,have
R2014	T2418	T2420	arg1Of	we,demonstrated
R2015	T2420	T2416	arg1Of	demonstrated,Previously
R2016	T2420	T2417	arg1Of	demonstrated,","
R2017	T2420	T2419	arg2Of	demonstrated,have
R2018	T2422	T2423	arg1Of	phosphorylation,and
R2019	T2423	T2426	arg1Of	and,of
R2020	T2423	T2437	arg1Of	and,is
R2021	T2423	T2438	arg1Of	and,defective
R2022	T2423	T2444	arg1Of	and,can
R2023	T2423	T2445	arg1Of	and,restored
R2024	T2425	T2423	arg2Of	exclusion,and
R2025	T2425	T2424	arg1Of	exclusion,nuclear
R2026	T2430	T2426	arg2Of	deacetylases,of
R2027	T2430	T2427	arg1Of	deacetylases,class
R2028	T2430	T2428	arg1Of	deacetylases,II
R2029	T2430	T2429	arg1Of	deacetylases,histone
R2030	T2430	T2431	arg1Of	deacetylases,(
R2031	T2430	T2434	arg1Of	deacetylases,during
R2032	T2432	T2431	arg2Of	HDACs,(
R2033	T2433	T2431	arg3Of	),(
R2034	T2436	T2434	arg2Of	engagement,during
R2035	T2436	T2435	arg1Of	engagement,BCR
R2036	T2437	T2443	arg1Of	is,and
R2037	T2438	T2437	arg2Of	defective,is
R2038	T2438	T2439	arg1Of	defective,in
R2039	T2442	T2439	arg2Of	cells,in
R2040	T2442	T2440	arg1Of	cells,PKD1/3−/−
R2041	T2442	T2441	arg1Of	cells,B
R2042	T2443	T2420	arg2Of	and,demonstrated
R2043	T2443	T2421	arg1Of	and,that
R2044	T2445	T2443	arg2Of	restored,and
R2045	T2445	T2444	arg2Of	restored,can
R2046	T2445	T2446	arg1Of	restored,upon
R2047	T2447	T2446	arg2Of	re-expression,upon
R2048	T2447	T2448	arg1Of	re-expression,of
R2049	T2452	T2448	arg2Of	isoform,of
R2050	T2452	T2449	arg1Of	isoform,a
R2051	T2452	T2450	arg1Of	isoform,single
R2052	T2452	T2451	arg1Of	isoform,PKD
R2053	T2452	T2453	arg1Of	isoform,[
R2054	T2454	T2453	arg2Of	1,[
R2055	T2455	T2453	arg3Of	],[
R2056	T2461	T2456	arg1Of	HSP27,The
R2057	T2461	T2457	arg1Of	HSP27,small
R2058	T2461	T2458	arg1Of	HSP27,heat
R2059	T2461	T2459	arg1Of	HSP27,shock
R2060	T2461	T2460	arg1Of	HSP27,protein
R2061	T2461	T2462	arg1Of	HSP27,has
R2062	T2461	T2464	arg1Of	HSP27,been
R2063	T2461	T2465	arg2Of	HSP27,proposed
R2064	T2465	T2462	arg2Of	proposed,has
R2065	T2465	T2463	arg1Of	proposed,recently
R2066	T2465	T2464	arg2Of	proposed,been
R2067	T2465	T2466	arg1Of	proposed,as
R2068	T2465	T2470	arg1Of	proposed,[
R2069	T2465	T2473	arg1Of	proposed,and
R2070	T2469	T2466	arg2Of	substrate,as
R2071	T2469	T2467	arg1Of	substrate,a
R2072	T2469	T2468	arg1Of	substrate,PKD1
R2073	T2471	T2470	arg2Of	24,[
R2074	T2472	T2470	arg3Of	],[
R2075	T2474	T2476	arg1Of	we,assessed
R2076	T2476	T2473	arg2Of	assessed,and
R2077	T2476	T2475	arg1Of	assessed,accordingly
R2078	T2480	T2478	arg1Of	cells,PKD-null
R2079	T2480	T2479	arg1Of	cells,DT40
R2080	T2480	T2481	arg1Of	cells,have
R2081	T2481	T2476	arg2Of	have,assessed
R2082	T2481	T2477	arg1Of	have,whether
R2083	T2483	T2481	arg2Of	phosphorylation,have
R2084	T2483	T2482	arg1Of	phosphorylation,defective
R2085	T2483	T2484	arg1Of	phosphorylation,of
R2086	T2485	T2484	arg2Of	HSP27,of
R2087	T2485	T2486	arg1Of	HSP27,on
R2088	T2485	T2489	arg1Of	HSP27,","
R2089	T2487	T2486	arg2Of	serine,on
R2090	T2487	T2488	arg1Of	serine,82
R2091	T2494	T2489	arg2Of	sequence,","
R2092	T2494	T2490	arg1Of	sequence,the
R2093	T2494	T2491	arg2Of	sequence,proposed
R2094	T2494	T2492	arg1Of	sequence,PKD1
R2095	T2494	T2493	arg1Of	sequence,substrate
R2096	T2495	T2497	arg1Of	We,investigated
R2097	T2497	T2496	arg1Of	investigated,initially
R2098	T2499	T2497	arg2Of	regulation,investigated
R2099	T2499	T2498	arg1Of	regulation,the
R2100	T2499	T2500	arg1Of	regulation,of
R2101	T2499	T2503	arg1Of	regulation,in
R2102	T2502	T2500	arg2Of	phosphorylation,of
R2103	T2502	T2501	arg1Of	phosphorylation,HSP27
R2104	T2508	T2503	arg2Of	cells,in
R2105	T2508	T2504	arg1Of	cells,single
R2106	T2508	T2505	arg1Of	cells,knockout
R2107	T2508	T2506	arg1Of	cells,DT40
R2108	T2508	T2507	arg1Of	cells,B
R2109	T2508	T2509	arg1Of	cells,lacking
R2110	T2511	T2512	arg1Of	PKD1,or
R2111	T2512	T2509	arg2Of	or,lacking
R2112	T2512	T2510	arg1Of	or,either
R2113	T2513	T2512	arg2Of	PKD3,or
R2114	T2515	T2514	arg2Of	shown,As
R2115	T2515	T2516	arg1Of	shown,in
R2116	T2518	T2516	arg2Of	1B,in
R2117	T2518	T2517	arg1Of	1B,Fig.
R2118	T2520	T2521	arg1Of	activation,of
R2119	T2520	T2524	arg1Of	activation,or
R2120	T2523	T2521	arg2Of	BCR,of
R2121	T2523	T2522	arg1Of	BCR,the
R2122	T2524	T2530	arg1Of	or,increased
R2123	T2525	T2524	arg2Of	treatment,or
R2124	T2525	T2526	arg1Of	treatment,with
R2125	T2529	T2526	arg2Of	PdBu,with
R2126	T2529	T2527	arg1Of	PdBu,the
R2127	T2529	T2528	arg1Of	PdBu,DAG-mimetic
R2128	T2530	T2514	arg1Of	increased,As
R2129	T2530	T2519	arg1Of	increased,","
R2130	T2532	T2530	arg2Of	levels,increased
R2131	T2532	T2531	arg1Of	levels,the
R2132	T2532	T2533	arg1Of	levels,of
R2133	T2535	T2533	arg2Of	phosphorylation,of
R2134	T2535	T2534	arg1Of	phosphorylation,HSP27
R2135	T2535	T2536	arg1Of	phosphorylation,at
R2136	T2537	T2536	arg2Of	S82,at
R2137	T2537	T2538	arg1Of	S82,in
R2138	T2542	T2538	arg2Of	cells,in
R2139	T2542	T2539	arg1Of	cells,wild-type
R2140	T2542	T2540	arg1Of	cells,DT40
R2141	T2542	T2541	arg1Of	cells,B
R2142	T2543	T2544	arg1Of	BCR,and
R2143	T2545	T2544	arg2Of	phorbol,and
R2144	T2547	T2543	arg1Of	signals,BCR
R2145	T2547	T2545	arg1Of	signals,phorbol
R2146	T2547	T2546	arg1Of	signals,ester
R2147	T2547	T2548	arg1Of	signals,were
R2148	T2547	T2550	arg1Of	signals,able
R2149	T2547	T2552	arg1Of	signals,increase
R2150	T2548	T2549	arg1Of	were,also
R2151	T2550	T2548	arg2Of	able,were
R2152	T2552	T2550	arg2Of	increase,able
R2153	T2552	T2551	arg1Of	increase,to
R2154	T2554	T2552	arg2Of	phosphorylation,increase
R2155	T2554	T2553	arg1Of	phosphorylation,HSP27
R2156	T2554	T2555	arg1Of	phosphorylation,in
R2157	T2556	T2557	arg1Of	PKD1,or
R2158	T2558	T2557	arg2Of	PKD3,or
R2159	T2563	T2555	arg2Of	cells,in
R2160	T2563	T2556	arg1Of	cells,PKD1
R2161	T2563	T2558	arg1Of	cells,PKD3
R2162	T2563	T2559	arg1Of	cells,single
R2163	T2563	T2560	arg1Of	cells,knockout
R2164	T2563	T2561	arg1Of	cells,DT40
R2165	T2563	T2562	arg1Of	cells,B
R2166	T2563	T2564	arg1Of	cells,(
R2167	T2566	T2564	arg2Of	1B,(
R2168	T2566	T2565	arg1Of	1B,Fig.
R2169	T2567	T2564	arg3Of	),(
R2170	T2570	T2571	arg1Of	BCR-,and
R2171	T2571	T2577	arg1Of	and,on
R2172	T2571	T2579	arg1Of	and,was
R2173	T2571	T2580	arg2Of	and,abolished
R2174	T2573	T2572	arg1Of	ester-induced,phorbol
R2175	T2574	T2571	arg2Of	phosphorylation,and
R2176	T2574	T2573	arg1Of	phosphorylation,ester-induced
R2177	T2574	T2575	arg1Of	phosphorylation,of
R2178	T2576	T2575	arg2Of	HSP27,of
R2179	T2578	T2577	arg2Of	S82,on
R2180	T2580	T2568	arg1Of	abolished,However
R2181	T2580	T2569	arg1Of	abolished,","
R2182	T2580	T2579	arg2Of	abolished,was
R2183	T2580	T2581	arg1Of	abolished,in
R2184	T2583	T2581	arg2Of	cells,in
R2185	T2583	T2582	arg1Of	cells,B
R2186	T2583	T2584	arg1Of	cells,that
R2187	T2583	T2585	arg1Of	cells,lacked
R2188	T2587	T2588	arg1Of	PKD1,and
R2189	T2588	T2585	arg2Of	and,lacked
R2190	T2588	T2586	arg1Of	and,both
R2191	T2588	T2590	arg1Of	and,(
R2192	T2589	T2588	arg2Of	PKD3,and
R2193	T2592	T2590	arg2Of	1C,(
R2194	T2592	T2591	arg1Of	1C,Fig.
R2195	T2593	T2590	arg3Of	),(
R2196	T2597	T2596	arg1Of	expression,doxycycline-induced
R2197	T2597	T2598	arg1Of	expression,of
R2198	T2597	T2602	arg1Of	expression,in
R2199	T2597	T2607	arg1Of	expression,was
R2200	T2597	T2608	arg1Of	expression,sufficient
R2201	T2601	T2598	arg2Of	transgene,of
R2202	T2601	T2599	arg1Of	transgene,the
R2203	T2601	T2600	arg1Of	transgene,Flag-PKD3
R2204	T2606	T2602	arg2Of	cells,in
R2205	T2606	T2603	arg1Of	cells,the
R2206	T2606	T2604	arg1Of	cells,double
R2207	T2606	T2605	arg1Of	cells,knockout
R2208	T2607	T2594	arg1Of	was,Significantly
R2209	T2607	T2595	arg1Of	was,","
R2210	T2607	T2609	modOf	was,to
R2211	T2608	T2607	arg2Of	sufficient,was
R2212	T2610	T2609	arg1Of	restore,to
R2213	T2612	T2610	arg2Of	regulation,restore
R2214	T2612	T2611	arg1Of	regulation,normal
R2215	T2612	T2613	arg1Of	regulation,of
R2216	T2615	T2613	arg2Of	phosphorylation,of
R2217	T2615	T2614	arg1Of	phosphorylation,HSP27
R2218	T2615	T2616	arg1Of	phosphorylation,(
R2219	T2618	T2616	arg2Of	1C,(
R2220	T2618	T2617	arg1Of	1C,Fig.
R2221	T2619	T2616	arg3Of	),(
R2222	T2621	T2620	arg2Of	contrast,In
R2223	T2623	T2624	arg1Of	expression,of
R2224	T2623	T2630	arg1Of	expression,in
R2225	T2623	T2635	arg1Of	expression,was
R2226	T2623	T2637	arg1Of	expression,able
R2227	T2623	T2639	arg1Of	expression,restore
R2228	T2629	T2624	arg2Of	protein,of
R2229	T2629	T2625	arg1Of	protein,a
R2230	T2629	T2626	arg1Of	protein,kinase-deficient
R2231	T2629	T2627	arg1Of	protein,PKD3
R2232	T2629	T2628	arg1Of	protein,mutant
R2233	T2634	T2630	arg2Of	cells,in
R2234	T2634	T2631	arg1Of	cells,the
R2235	T2634	T2632	arg1Of	cells,double
R2236	T2634	T2633	arg1Of	cells,knockout
R2237	T2635	T2620	arg1Of	was,In
R2238	T2635	T2622	arg1Of	was,","
R2239	T2635	T2636	arg1Of	was,not
R2240	T2637	T2635	arg2Of	able,was
R2241	T2639	T2637	arg2Of	restore,able
R2242	T2639	T2638	arg1Of	restore,to
R2243	T2640	T2641	arg1Of	BCR-,or
R2244	T2641	T2639	arg2Of	or,restore
R2245	T2645	T2641	arg2Of	phosphorylation,or
R2246	T2645	T2642	arg1Of	phosphorylation,phorbol
R2247	T2645	T2643	arg1Of	phosphorylation,ester-induced
R2248	T2645	T2644	arg1Of	phosphorylation,HSP27
R2249	T2645	T2646	arg1Of	phosphorylation,(
R2250	T2648	T2646	arg2Of	1D,(
R2251	T2648	T2647	arg1Of	1D,Fig.
R2252	T2649	T2646	arg3Of	),(
R2253	T2652	T2655	arg1Of	PKD3,as
R2254	T2655	T2653	arg1Of	as,as
R2255	T2655	T2654	arg1Of	as,well
R2256	T2655	T2657	arg1Of	as,can
R2257	T2655	T2658	arg1Of	as,regulate
R2258	T2656	T2655	arg2Of	PKD1,as
R2259	T2658	T2657	arg2Of	regulate,can
R2260	T2658	T2661	arg1Of	regulate,and
R2261	T2660	T2658	arg2Of	phosphorylation,regulate
R2262	T2660	T2659	arg1Of	phosphorylation,HSP27
R2263	T2661	T2650	arg1Of	and,Hence
R2264	T2661	T2651	arg1Of	and,","
R2265	T2665	T2662	arg2Of	cells,in
R2266	T2665	T2663	arg1Of	cells,DT40
R2267	T2665	T2664	arg1Of	cells,B
R2268	T2666	T2667	arg1Of	they,are
R2269	T2666	T2669	arg2Of	they,redundant
R2270	T2669	T2661	arg2Of	redundant,and
R2271	T2669	T2662	arg1Of	redundant,in
R2272	T2669	T2667	arg2Of	redundant,are
R2273	T2669	T2668	arg1Of	redundant,functionally
R2274	T2669	T2670	arg1Of	redundant,as
R2275	T2672	T2670	arg2Of	kinases,as
R2276	T2672	T2671	arg1Of	kinases,HSP27
R3056	T3837	T3838	arg1Of	proliferation,and
R3057	T3838	T3836	arg1Of	and,"Protein kinase D enzymes are dispensable for proliferation, survival and antigen receptor-regulated NFκB activity in vertebrate B-cells Abstract To investigate the importance of protein kinase D (PKD) enzymes we generated a PKD-null DT40 B-lymphocyte cell line. Previously we have shown that PKDs have an essential role in regulating class II histone deacetylases in DT40 B-cells [Matthews, S.A., Liu, P., Spitaler, M., Olson, E.N., McKinsey, T.A., Cantrell, D.A. and Scharenberg, A.M. (2006) Essential role for protein kinase D family kinases in the regulation of class II histone deacetylases in B lymphocytes. Mol. Cell Biol. 26, 1569–1577]. We now show that PKDs are also required to regulate HSP27 phosphorylation in DT40 B-cells. However, in contrast to previous observations in other cell types, PKD enzymes do not regulate basic cellular processes such as proliferation or survival responses, nor NFκB transcriptional activity downstream of the B cell antigen receptor. Thus, PKDs have a selective role in DT40 B-cell biology. 1 Introduction The protein kinase D (PKD) serine/threonine kinase family has three members: PKD1, PKD2 and PKD3. Most cell types express at least two PKD isoforms but PKD enzymes are especially highly expressed in haematopoietic cells, where they are activated in response to antigen receptors stimulation [2,3]. A conserved signalling pathway linking antigen receptors to PKDs involves the activation of PLCγ and the subsequent production of diacylglycerol (DAG) which stimulates classical and/or novel protein kinase Cs (PKC) that phosphorylate two key regulatory serine residues in the activation loop of PKD kinases [3–6]. The N-terminal regulatory region of PKD enzymes contains a DAG binding domain and direct binding of DAG also contributes to PKD1 activation [7] as well as regulating the spatial location of PKD enzymes within cells [8–12]. PKD enzymes have been proposed to regulate numerous cellular functions, including cell proliferation [13–16], anti-apoptotic signals [17,18] and thymocyte development [19]. Expression of mutant catalytically inactive and constitutively activated PKDs can also modify Golgi function, cell adhesion and cell motility (reviewed in [20]). In particular, PKDs have been widely linked to the activation of the NFκB transcription factor and in regulating cell survival during oxidative stress [17,21–23]. Another recently proposed PKD1 substrate is HSP27 [24], a small heat shock protein involved in regulating cell migration and cell survival [25]. An essential role for PKD enzymes in regulating class II histone deacetylases (HDACs), enzymes that repress MEF2-dependent gene transcription, has also been demonstrated [1,26–28]. To investigate the biological role of PKDs we have generated DT40 B cell lines that lack expression of one or more members of the PKD family [1], allowing us to investigate the function(s) of PKD isoforms following B cell antigen receptor (BCR) stimulation, as well addressing the issue of functional redundancy between the different PKD family members. Previous studies have shown that PKDs are indispensable for HDAC regulation in B cells [1]. Herein we show that PKDs are also indispensable for HSP27 phosphorylation in B cells. However, PKD-null DT40 B cells are viable and proliferate normally. Moreover, loss of the entire cellular pool of PKD does not critically affect oxidative stress responses in B cells nor do PKD kinases play an essential role in regulating NFκB transcriptional activity. Together, these findings reveal that in B lymphocytes, PKD kinases are not critical regulators of many of the cellular processes previously ascribed to them in other cellular systems. 2 Materials and methods 2.1 Cell culture, transient transfections and cell stimulation The generation, culture and activation of PKD1−/−, PKD3−/− and PKD1/3−/− knockout DT40 B cell lines have been described previously [1]. Cells were lysed and protein extracts were analysed in Western blotting experiments as previously described [1]. Chloramphenicol acetyl transferase assays have been described previously [29]. 2.2 sIgM staining DT40 B cells (2 × 106 cells per point) were resuspended in 200 μl buffer (RPMI 1640 media, 1% foetal calf serum) containing anti-chicken M1 monoclonal antibody conjugated to FITC for 20 min on ice. The cells were washed twice and fluorescent intensity was analysed by flow cytometry. All results shown are representative of at two to four independent experiments unless otherwise indicated. 3 Results 3.1 Loss of HSP27 phosphorylation in DT40 B cells lacking expression of PKD family kinases DT40 B cells express two PKD isoforms, PKD1 and PKD3, and as previously described we have recently generated DT40 B cell lines that lack expression of either PKD1 or PKD3 or both enzymes [1]. In generating the double knockout cell lines we targeted the PKD1 loci in a PKD3−/− cell line that expressed a Flag-PKD3 transgene under the control of a doxycycline-inducible promoter. Hence, in the presence of doxycycline, Flag-PKD3 expression in PKD1/3 double knockout cells is comparable to endogenous PKD3 present in wild-type DT40 cells and removal of doxycycline from the culture media for 5 days results in a completely null PKD phenotype (Fig. 1A). Previously, we have demonstrated that phosphorylation and nuclear exclusion of class II histone deacetylases (HDACs) during BCR engagement is defective in PKD1/3−/− B cells and can restored upon re-expression of a single PKD isoform [1]. The small heat shock protein HSP27 has recently been proposed as a PKD1 substrate [24] and we accordingly assessed whether PKD-null DT40 cells have defective phosphorylation of HSP27 on serine 82, the proposed PKD1 substrate sequence. We initially investigated the regulation of HSP27 phosphorylation in single knockout DT40 B cells lacking either PKD1 or PKD3. As shown in Fig. 1B, activation of the BCR or treatment with the DAG-mimetic PdBu increased the levels of HSP27 phosphorylation at S82 in wild-type DT40 B cells. BCR and phorbol ester signals were also able to increase HSP27 phosphorylation in PKD1 or PKD3 single knockout DT40 B cells (Fig. 1B). However, BCR- and phorbol ester-induced phosphorylation of HSP27 on S82 was abolished in B cells that lacked both PKD1 and PKD3 (Fig. 1C). Significantly, doxycycline-induced expression of the Flag-PKD3 transgene in the double knockout cells was sufficient to restore normal regulation of HSP27 phosphorylation (Fig. 1C). In contrast, expression of a kinase-deficient PKD3 mutant protein in the double knockout cells was not able to restore BCR- or phorbol ester-induced HSP27 phosphorylation (Fig. 1D). Hence, PKD3 as well as PKD1 can regulate HSP27 phosphorylation and in DT40 B cells they are functionally redundant as HSP27 kinases. 3.2 Cellular"
R3058	T3838	T3840	arg1Of	and,in
R3059	T3839	T3838	arg2Of	survival,and
R3060	T3843	T3840	arg2Of	cells,in
R3061	T3843	T3841	arg1Of	cells,DT40
R3062	T3843	T3842	arg1Of	cells,B
R3063	T3843	T3844	arg1Of	cells,lacking
R3064	T3845	T3844	arg2Of	expression,lacking
R3065	T3845	T3846	arg1Of	expression,of
R3066	T3849	T3846	arg2Of	kinases,of
R3067	T3849	T3847	arg1Of	kinases,PKD
R3068	T3849	T3848	arg1Of	kinases,family
R3069	T3851	T3850	arg1Of	enzymes,PKD
R3070	T3851	T3852	arg1Of	enzymes,have
R3071	T3851	T3854	arg1Of	enzymes,been
R3072	T3851	T3855	arg2Of	enzymes,linked
R3073	T3855	T3852	arg2Of	linked,have
R3074	T3855	T3853	arg1Of	linked,previously
R3075	T3855	T3854	arg2Of	linked,been
R3076	T3855	T3856	arg1Of	linked,to
R3077	T3858	T3856	arg2Of	regulation,to
R3078	T3858	T3857	arg1Of	regulation,the
R3079	T3858	T3859	arg1Of	regulation,of
R3080	T3861	T3862	arg1Of	proliferation,and
R3081	T3862	T3859	arg2Of	and,of
R3082	T3862	T3860	arg1Of	and,cell
R3083	T3862	T3864	arg1Of	and,(
R3084	T3863	T3862	arg2Of	survival,and
R3085	T3865	T3864	arg2Of	reviewed,(
R3086	T3865	T3866	arg1Of	reviewed,in
R3087	T3865	T3867	arg1Of	reviewed,[
R3088	T3868	T3867	arg2Of	20,[
R3089	T3869	T3867	arg3Of	],[
R3090	T3870	T3864	arg3Of	),(
R3091	T3872	T3871	arg1Of	investigate,To
R3092	T3874	T3872	arg2Of	effect,investigate
R3093	T3874	T3873	arg1Of	effect,the
R3094	T3876	T3877	arg1Of	loss,of
R3095	T3876	T3880	arg1Of	loss,had
R3096	T3879	T3877	arg2Of	kinases,of
R3097	T3879	T3878	arg1Of	kinases,PKD
R3098	T3880	T3874	arg2Of	had,effect
R3099	T3880	T3875	arg1Of	had,that
R3100	T3880	T3881	arg1Of	had,on
R3101	T3884	T3885	arg1Of	survival,and/or
R3102	T3885	T3881	arg2Of	and/or,on
R3103	T3885	T3882	arg1Of	and/or,B
R3104	T3885	T3883	arg1Of	and/or,cell
R3105	T3886	T3885	arg2Of	proliferation,and/or
R3106	T3887	T3872	arg1Of	we,investigate
R3107	T3887	T3888	arg1Of	we,cultured
R3108	T3888	T3871	modOf	cultured,To
R3109	T3888	T3893	arg1Of	cultured,in
R3110	T3889	T3890	arg1Of	wild-type,and
R3111	T3891	T3890	arg2Of	PKD-null,and
R3112	T3892	T3888	arg2Of	cells,cultured
R3113	T3892	T3889	arg1Of	cells,wild-type
R3114	T3892	T3891	arg1Of	cells,PKD-null
R3115	T3895	T3893	arg2Of	presence,in
R3116	T3895	T3894	arg1Of	presence,the
R3117	T3895	T3896	arg1Of	presence,(
R3118	T3897	T3896	arg2Of	PKD1/3−/−,(
R3119	T3898	T3896	arg3Of	:,(
R3120	T3899	T3900	arg1Of	Flag-PKD3+ve,)
R3121	T3899	T3901	arg1Of	Flag-PKD3+ve,or
R3122	T3901	T3909	arg1Of	or,monitored
R3123	T3902	T3901	arg2Of	absence,or
R3124	T3902	T3903	arg1Of	absence,(
R3125	T3902	T3906	arg1Of	absence,of
R3126	T3904	T3903	arg2Of	PKD1/3−/−,(
R3127	T3905	T3903	arg3Of	),(
R3128	T3907	T3906	arg2Of	doxycycline,of
R3129	T3909	T3908	arg1Of	monitored,and
R3130	T3911	T3909	arg2Of	growth,monitored
R3131	T3911	T3910	arg1Of	growth,exponential
R3132	T3913	T3912	arg2Of	shown,As
R3133	T3913	T3914	arg1Of	shown,in
R3134	T3916	T3914	arg2Of	2A,in
R3135	T3916	T3915	arg1Of	2A,Fig.
R3136	T3919	T3918	arg1Of	cells,PKD1/3−/−
R3137	T3919	T3920	arg1Of	cells,proliferated
R3138	T3920	T3921	arg1Of	proliferated,exponentially
R3139	T3920	T3922	arg1Of	proliferated,and
R3140	T3922	T3912	arg1Of	and,As
R3141	T3922	T3917	arg1Of	and,","
R3142	T3923	T3924	arg1Of	re-expression,of
R3143	T3923	T3926	arg1Of	re-expression,in
R3144	T3923	T3929	arg1Of	re-expression,had
R3145	T3925	T3924	arg2Of	Flag-PKD3,of
R3146	T3928	T3926	arg2Of	cells,in
R3147	T3928	T3927	arg1Of	cells,these
R3148	T3929	T3922	arg2Of	had,and
R3149	T3931	T3929	arg2Of	impact,had
R3150	T3931	T3930	arg1Of	impact,no
R3151	T3931	T3932	arg1Of	impact,on
R3152	T3934	T3932	arg2Of	rate,on
R3153	T3934	T3933	arg1Of	rate,the
R3154	T3934	T3935	arg1Of	rate,of
R3155	T3936	T3935	arg2Of	proliferation,of
R3156	T3940	T3939	arg1Of	viability,the
R3157	T3940	T3941	arg1Of	viability,of
R3158	T3940	T3945	arg1Of	viability,during
R3159	T3940	T3948	arg1Of	viability,was
R3160	T3940	T3951	arg1Of	viability,different
R3161	T3944	T3941	arg2Of	cells,of
R3162	T3944	T3942	arg1Of	cells,PKD1/3−/−
R3163	T3944	T3943	arg1Of	cells,B
R3164	T3947	T3945	arg2Of	culturing,during
R3165	T3947	T3946	arg1Of	culturing,routine
R3166	T3948	T3937	arg1Of	was,Furthermore
R3167	T3948	T3938	arg1Of	was,","
R3168	T3948	T3949	arg1Of	was,not
R3169	T3951	T3948	arg2Of	different,was
R3170	T3951	T3950	arg1Of	different,significantly
R3171	T3951	T3952	arg1Of	different,from
R3172	T3953	T3952	arg2Of	that,from
R3173	T3953	T3954	arg1Of	that,of
R3174	T3957	T3954	arg2Of	cells,of
R3175	T3957	T3955	arg1Of	cells,wild-type
R3176	T3957	T3956	arg1Of	cells,B
R3177	T3957	T3958	arg1Of	cells,(
R3178	T3959	T3958	arg2Of	data,(
R3179	T3959	T3961	arg2Of	data,shown
R3180	T3961	T3960	arg1Of	shown,not
R3181	T3962	T3958	arg3Of	),(
R3182	T3963	T3964	arg1Of	It,was
R3183	T3963	T3965	arg2Of	It,noted
R3184	T3965	T3964	arg2Of	noted,was
R3185	T3970	T3967	arg1Of	time,the
R3186	T3970	T3968	arg1Of	time,population
R3187	T3970	T3969	arg1Of	time,doubling
R3188	T3970	T3971	arg1Of	time,of
R3189	T3970	T3974	arg1Of	time,was
R3190	T3970	T3976	arg1Of	time,slower
R3191	T3973	T3971	arg2Of	cells,of
R3192	T3973	T3972	arg1Of	cells,PKD1/3−/−
R3193	T3974	T3989	arg1Of	was,but
R3194	T3976	T3974	arg2Of	slower,was
R3195	T3976	T3975	arg1Of	slower,slightly
R3196	T3976	T3977	arg1Of	slower,than
R3197	T3978	T3977	arg2Of	that,than
R3198	T3978	T3979	arg1Of	that,of
R3199	T3983	T3979	arg2Of	cells,of
R3200	T3983	T3980	arg1Of	cells,wild
R3201	T3983	T3981	arg1Of	cells,type
R3202	T3983	T3982	arg1Of	cells,DT40
R3203	T3983	T3984	arg1Of	cells,(
R3204	T3985	T3984	arg2Of	12.7 ± 2.8 h,(
R3205	T3985	T3986	arg1Of	12.7 ± 2.8 h,versus
R3206	T3987	T3986	arg2Of	10.2 ± 0.4 h,versus
R3207	T3988	T3984	arg3Of	),(
R3208	T3989	T3965	arg3Of	but,noted
R3209	T3989	T3966	arg1Of	but,that
R3210	T3991	T3990	arg1Of	failure,the
R3211	T3991	T3992	arg1Of	failure,of
R3212	T3991	T3995	modOf	failure,to
R3213	T3991	T4003	arg1Of	failure,suggests
R3214	T3994	T3992	arg2Of	re-expression,of
R3215	T3994	T3993	arg1Of	re-expression,PKD3
R3216	T3996	T3995	arg1Of	modify,to
R3217	T3999	T3996	arg2Of	rate,modify
R3218	T3999	T3997	arg1Of	rate,the
R3219	T3999	T3998	arg1Of	rate,proliferation
R3220	T3999	T4000	arg1Of	rate,of
R3221	T4002	T4000	arg2Of	cells,of
R3222	T4002	T4001	arg1Of	cells,PKD1/3−/−
R3223	T4003	T3989	arg2Of	suggests,but
R3224	T4007	T4005	arg1Of	differences,these
R3225	T4007	T4006	arg1Of	differences,small
R3226	T4007	T4008	arg1Of	differences,were
R3227	T4007	T4017	arg1Of	differences,were
R3228	T4007	T4019	arg2Of	differences,caused
R3229	T4008	T4010	arg1Of	were,likely
R3230	T4008	T4016	arg1Of	were,and
R3231	T4010	T4009	arg1Of	likely,most
R3232	T4012	T4008	arg2Of	result,were
R3233	T4012	T4011	arg1Of	result,the
R3234	T4012	T4013	arg1Of	result,of
R3235	T4015	T4013	arg2Of	variation,of
R3236	T4015	T4014	arg1Of	variation,clonal
R3237	T4016	T4003	arg2Of	and,suggests
R3238	T4016	T4004	arg1Of	and,that
R3239	T4019	T4016	arg2Of	caused,and
R3240	T4019	T4017	arg2Of	caused,were
R3241	T4019	T4018	arg1Of	caused,not
R3242	T4019	T4020	arg1Of	caused,specifically
R3243	T4022	T4019	arg1Of	loss,caused
R3244	T4022	T4021	arg2Of	loss,by
R3245	T4022	T4023	arg1Of	loss,of
R3246	T4025	T4023	arg2Of	enzymes,of
R3247	T4025	T4024	arg1Of	enzymes,PKD
R3248	T4030	T4028	arg1Of	enzymes,PKD
R3249	T4030	T4029	arg1Of	enzymes,family
R3250	T4030	T4031	arg1Of	enzymes,are
R3251	T4030	T4033	arg1Of	enzymes,essential
R3252	T4031	T4026	arg1Of	are,Thus
R3253	T4031	T4027	arg1Of	are,","
R3254	T4031	T4032	arg1Of	are,not
R3255	T4033	T4031	arg2Of	essential,are
R3256	T4033	T4034	arg1Of	essential,for
R3257	T4035	T4034	arg2Of	regulating,for
R3258	T4037	T4036	arg1Of	survival,basal
R3259	T4037	T4038	arg1Of	survival,and
R3260	T4038	T4035	arg2Of	and,regulating
R3261	T4039	T4038	arg2Of	proliferation,and
R3262	T4039	T4040	arg1Of	proliferation,of
R3263	T4043	T4040	arg2Of	cells,of
R3264	T4043	T4041	arg1Of	cells,DT40
R3265	T4043	T4042	arg1Of	cells,B
R3266	T4045	T4044	arg1Of	enzymes,PKD
R3267	T4045	T4046	arg1Of	enzymes,","
R3268	T4045	T4052	arg1Of	enzymes,have
R3269	T4045	T4054	arg1Of	enzymes,been
R3270	T4045	T4055	arg2Of	enzymes,linked
R3271	T4048	T4049	arg1Of	PKD1,and
R3272	T4049	T4046	arg2Of	and,","
R3273	T4049	T4047	arg1Of	and,specifically
R3274	T4050	T4049	arg2Of	PKD2,and
R3275	T4055	T4051	arg1Of	linked,","
R3276	T4055	T4052	arg2Of	linked,have
R3277	T4055	T4053	arg1Of	linked,previously
R3278	T4055	T4054	arg2Of	linked,been
R3279	T4055	T4056	arg1Of	linked,to
R3280	T4059	T4056	arg2Of	role,to
R3281	T4059	T4057	arg1Of	role,a
R3282	T4059	T4058	arg1Of	role,protective
R3283	T4059	T4060	arg1Of	role,against
R3284	T4062	T4061	arg1Of	stress-induced,oxidative
R3285	T4063	T4060	arg2Of	injury,against
R3286	T4063	T4062	arg1Of	injury,stress-induced
R3287	T4063	T4064	arg1Of	injury,in
R3288	T4066	T4067	arg1Of	fibroblast,","
R3289	T4068	T4067	arg2Of	HeLa,","
R3290	T4072	T4064	arg2Of	lines,in
R3291	T4072	T4065	arg1Of	lines,3T3
R3292	T4072	T4066	arg1Of	lines,fibroblast
R3293	T4072	T4068	arg1Of	lines,HeLa
R3294	T4072	T4069	arg1Of	lines,and
R3295	T4072	T4070	arg1Of	lines,epithelial
R3296	T4072	T4071	arg1Of	lines,cell
R3297	T4072	T4073	arg1Of	lines,[
R3298	T4074	T4073	arg2Of	"17,30–32",[
R3299	T4075	T4073	arg3Of	],[
R3300	T4076	T4078	arg1Of	We,addressed
R3301	T4078	T4077	arg1Of	addressed,therefore
R3302	T4080	T4078	arg2Of	role,addressed
R3303	T4080	T4079	arg1Of	role,the
R3304	T4080	T4081	arg1Of	role,of
R3305	T4080	T4085	arg1Of	role,in
R3306	T4084	T4081	arg2Of	kinases,of
R3307	T4084	T4082	arg1Of	kinases,PKD
R3308	T4084	T4083	arg1Of	kinases,family
R3309	T4086	T4085	arg2Of	regulating,in
R3310	T4086	T4090	arg1Of	regulating,in
R3311	T4089	T4086	arg2Of	survival,regulating
R3312	T4089	T4087	arg1Of	survival,B
R3313	T4089	T4088	arg1Of	survival,cell
R3314	T4090	T4092	arg1Of	in,to
R3315	T4091	T4090	arg2Of	response,in
R3316	T4094	T4093	arg1Of	stress,oxidative
R3317	T4094	T4095	arg1Of	stress,and
R3318	T4095	T4090	arg3Of	and,in
R3319	T4098	T4095	arg2Of	stimuli,and
R3320	T4098	T4096	arg1Of	stimuli,other
R3321	T4098	T4097	arg1Of	stimuli,stress
R3322	T4100	T4099	arg2Of	shown,As
R3323	T4100	T4101	arg1Of	shown,in
R3324	T4103	T4101	arg2Of	2B,in
R3325	T4103	T4102	arg1Of	2B,Fig.
R3326	T4105	T4106	arg1Of	loss,of
R3327	T4105	T4109	arg1Of	loss,had
R3328	T4108	T4106	arg2Of	expression,of
R3329	T4108	T4107	arg1Of	expression,PKD1/3
R3330	T4109	T4099	arg1Of	had,As
R3331	T4109	T4104	arg1Of	had,","
R3332	T4109	T4113	arg1Of	had,on
R3333	T4109	T4120	arg1Of	had,in
R3334	T4109	T4153	arg1Of	had,following
R3335	T4112	T4109	arg2Of	impact,had
R3336	T4112	T4110	arg1Of	impact,no
R3337	T4112	T4111	arg1Of	impact,significant
R3338	T4115	T4113	arg2Of	survival,on
R3339	T4115	T4114	arg1Of	survival,the
R3340	T4115	T4116	arg1Of	survival,of
R3341	T4119	T4116	arg2Of	cells,of
R3342	T4119	T4117	arg1Of	cells,DT40
R3343	T4119	T4118	arg1Of	cells,B
R3344	T4120	T4122	arg1Of	in,to
R3345	T4120	T4152	arg1Of	in,or
R3346	T4121	T4120	arg2Of	response,in
R3347	T4125	T4123	arg1Of	stimuli,mitochondrial
R3348	T4125	T4124	arg1Of	stimuli,stress
R3349	T4125	T4126	arg1Of	stimuli,(
R3350	T4125	T4132	arg1Of	stimuli,;
R3351	T4127	T4128	arg1Of	H2O2,or
R3352	T4128	T4126	arg2Of	or,(
R3353	T4130	T4128	arg2Of	deprivation,or
R3354	T4130	T4129	arg1Of	deprivation,serum
R3355	T4131	T4126	arg3Of	),(
R3356	T4132	T4141	arg1Of	;,;
R3357	T4135	T4132	arg2Of	agents,;
R3358	T4135	T4133	arg1Of	agents,DNA
R3359	T4135	T4134	arg1Of	agents,damaging
R3360	T4135	T4136	arg1Of	agents,(
R3361	T4137	T4138	arg1Of	etoposide,or
R3362	T4138	T4136	arg2Of	or,(
R3363	T4139	T4138	arg2Of	doxorubicin,or
R3364	T4140	T4136	arg3Of	),(
R3365	T4141	T4120	arg3Of	;,in
R3366	T4144	T4141	arg2Of	stress,;
R3367	T4144	T4142	arg1Of	stress,ER
R3368	T4144	T4143	arg1Of	stress,pathway
R3369	T4144	T4145	arg1Of	stress,due
R3370	T4145	T4146	arg1Of	due,to
R3371	T4148	T4145	arg2Of	overload,due
R3372	T4148	T4147	arg1Of	overload,calcium
R3373	T4148	T4149	arg1Of	overload,(
R3374	T4150	T4149	arg2Of	thapsigargin,(
R3375	T4151	T4149	arg3Of	),(
R3376	T4153	T4152	arg2Of	following,or
R3377	T4155	T4153	arg2Of	treatment,following
R3378	T4155	T4154	arg2Of	treatment,prolonged
R3379	T4155	T4156	arg1Of	treatment,with
R3380	T4158	T4157	arg1Of	esters,phorbol
R3381	T4158	T4159	arg1Of	esters,or
R3382	T4159	T4156	arg2Of	or,with
R3383	T4159	T4162	arg1Of	or,","
R3384	T4161	T4159	arg2Of	A,or
R3385	T4161	T4160	arg1Of	A,Trichostatin
R3386	T4164	T4162	arg2Of	inhibitor,","
R3387	T4164	T4163	arg1Of	inhibitor,an
R3388	T4164	T4165	arg1Of	inhibitor,of
R3389	T4168	T4165	arg2Of	HDACs,of
R3390	T4168	T4166	arg1Of	HDACs,class
R3391	T4168	T4167	arg1Of	HDACs,I/II
R3392	T4172	T4171	arg1Of	kinases,PKD
R3393	T4172	T4173	arg1Of	kinases,do
R3394	T4172	T4175	arg1Of	kinases,play
R3395	T4175	T4169	arg1Of	play,Thus
R3396	T4175	T4170	arg1Of	play,","
R3397	T4175	T4173	arg2Of	play,do
R3398	T4175	T4174	arg1Of	play,not
R3399	T4175	T4179	arg1Of	play,in
R3400	T4178	T4175	arg2Of	role,play
R3401	T4178	T4176	arg1Of	role,an
R3402	T4178	T4177	arg1Of	role,essential
R3403	T4180	T4179	arg2Of	regulating,in
R3404	T4180	T4184	arg1Of	regulating,in
R3405	T4183	T4180	arg2Of	survival,regulating
R3406	T4183	T4181	arg1Of	survival,B
R3407	T4183	T4182	arg1Of	survival,cell
R3408	T4184	T4186	arg1Of	in,to
R3409	T4185	T4184	arg2Of	response,in
R3410	T4188	T4184	arg3Of	range,in
R3411	T4188	T4187	arg1Of	range,a
R3412	T4188	T4189	arg1Of	range,of
R3413	T4192	T4189	arg2Of	stimuli,of
R3414	T4192	T4190	arg1Of	stimuli,different
R3415	T4192	T4191	arg1Of	stimuli,stress
R1597	T1872	T1879	arg1Of	cells,were
R1602	T1875	T1874	arg1Of	cells,2 × 106
R1603	T1875	T1876	arg1Of	cells,per
R1605	T1877	T1876	arg2Of	point,per
R7	T51	T49	arg1Of	D,protein
R8	T51	T50	arg1Of	D,kinase
R12	T55	T48	arg2Of	enzymes,of
R13	T55	T51	arg1Of	enzymes,D
R14	T56	T45	arg1Of	we,investigate
R16	T57	T44	modOf	generated,To
R374	T29	T26	arg1Of	enzymes,Protein
R375	T29	T27	arg1Of	enzymes,kinase
R376	T29	T28	arg1Of	enzymes,D
R377	T29	T30	arg1Of	enzymes,are
R378	T29	T31	arg1Of	enzymes,dispensable
R379	T31	T30	arg2Of	dispensable,are
R380	T31	T32	arg1Of	dispensable,for
R381	T33	T34	arg1Of	proliferation,","
R382	T34	T36	arg1Of	",",and
R383	T35	T34	arg2Of	survival,","
R384	T36	T32	arg2Of	and,for
R385	T36	T41	arg1Of	and,in
R386	T40	T36	arg2Of	activity,and
R387	T40	T37	arg1Of	activity,antigen
R388	T40	T38	arg1Of	activity,receptor-regulated
R389	T40	T39	arg1Of	activity,NFκB
R390	T43	T41	arg2Of	B-cells,in
R391	T43	T42	arg1Of	B-cells,vertebrate
R392	T45	T44	arg1Of	investigate,To
R393	T47	T45	arg2Of	importance,investigate
R394	T47	T46	arg1Of	importance,the
R395	T47	T48	arg1Of	importance,of
R1612	T1883	T1895	arg1Of	buffer,containing
R1617	T1889	T1890	arg1Of	1,%
R1620	T1893	T1891	arg1Of	serum,foetal
R1621	T1893	T1892	arg1Of	serum,calf
R1623	T1899	T1895	arg2Of	antibody,containing
R1624	T1899	T1896	arg1Of	antibody,anti-chicken
R1625	T1899	T1897	arg1Of	antibody,M1
R1626	T1899	T1898	arg1Of	antibody,monoclonal
R1627	T1899	T1900	arg2Of	antibody,conjugated
R1628	T1900	T1901	arg1Of	conjugated,to
R1629	T1902	T1901	arg2Of	FITC,to
R1630	T1902	T1903	arg1Of	FITC,for
R1631	T1904	T1903	arg2Of	20 min,for
R1632	T1904	T1905	arg1Of	20 min,on
R1633	T1906	T1905	arg2Of	ice,on
R1634	T1908	T1907	arg1Of	cells,The
R1635	T1908	T1909	arg1Of	cells,were
R1636	T1908	T1910	arg2Of	cells,washed
R1637	T1910	T1909	arg2Of	washed,were
R1638	T1910	T1911	arg1Of	washed,twice
R1639	T1910	T1912	arg1Of	washed,and
R1640	T1914	T1913	arg1Of	intensity,fluorescent
R1641	T1914	T1915	arg1Of	intensity,was
R1642	T1914	T1916	arg2Of	intensity,analysed
R1643	T1916	T1912	arg2Of	analysed,and
R1644	T1916	T1915	arg2Of	analysed,was
R1645	T1919	T1916	arg1Of	cytometry,analysed
R1646	T1919	T1917	arg2Of	cytometry,by
R1647	T1919	T1918	arg1Of	cytometry,flow
R1648	T1921	T1920	arg1Of	results,All
R1649	T1921	T1922	arg2Of	results,shown
R1650	T1921	T1923	arg1Of	results,are
R1651	T1921	T1924	arg1Of	results,representative
R1652	T1923	T1932	arg1Of	are,unless

bionlp-st-ge-2016-coref

Id	Subject	Object	Predicate	Lexical cue
T2673	4655-4671	Anaphor	denotes	two PKD isoforms
T2674	4673-4677	Antecedent	denotes	PKD1
T2675	4682-4686	Antecedent	denotes	PKD3
R2277	T2673	T2674	boundBy	two PKD isoforms,PKD1
R2278	T2673	T2675	boundBy	two PKD isoforms,PKD3

bionlp-st-ge-2016-spacy-parsed

Id	Subject	Object	Predicate	Lexical cue
T212	0-7	NN	denotes	Protein
T213	8-14	NN	denotes	kinase
T214	15-16	NNP	denotes	D
T215	17-24	NNS	denotes	enzymes
T216	25-28	VBP	denotes	are
T217	29-40	JJ	denotes	dispensable
T218	41-44	IN	denotes	for
T219	45-58	NN	denotes	proliferation
T220	58-59	,	denotes	,
T221	60-68	NN	denotes	survival
T222	69-72	CC	denotes	and
T223	73-80	NN	denotes	antigen
T224	81-99	JJ	denotes	receptor-regulated
T225	100-104	NN	denotes	NFκB
T226	105-113	NN	denotes	activity
T227	114-116	IN	denotes	in
T228	117-127	JJ	denotes	vertebrate
T229	128-135	NNPS	denotes	B-cells
T230	147-149	TO	denotes	To
T231	150-161	VB	denotes	investigate
T232	162-165	DT	denotes	the
T233	166-176	NN	denotes	importance
T234	177-179	IN	denotes	of
T235	180-187	NN	denotes	protein
T236	188-194	NN	denotes	kinase
T237	195-196	NNP	denotes	D
T238	197-198	-LRB-	denotes	(
T239	198-201	NNP	denotes	PKD
T240	201-202	-RRB-	denotes	)
T241	203-210	VBZ	denotes	enzymes
T242	211-213	PRP	denotes	we
T243	214-223	VBD	denotes	generated
T244	224-225	DT	denotes	a
T245	226-234	NNP	denotes	PKD-null
T246	235-239	NNP	denotes	DT40
T247	240-252	NNP	denotes	B-lymphocyte
T248	253-257	NN	denotes	cell
T249	258-262	NN	denotes	line
T250	262-263	.	denotes	.
T251	264-274	RB	denotes	Previously
T252	275-277	PRP	denotes	we
T253	278-282	VBP	denotes	have
T254	283-288	VBN	denotes	shown
T255	289-293	IN	denotes	that
T256	294-298	NNS	denotes	PKDs
T257	299-303	VBP	denotes	have
T258	304-306	DT	denotes	an
T259	307-316	JJ	denotes	essential
T260	317-321	NN	denotes	role
T261	322-324	IN	denotes	in
T262	325-335	VBG	denotes	regulating
T263	336-341	NN	denotes	class
T264	342-344	NNP	denotes	II
T265	345-352	NN	denotes	histone
T266	353-365	NNS	denotes	deacetylases
T267	366-368	IN	denotes	in
T268	369-373	NNP	denotes	DT40
T269	374-381	NNPS	denotes	B-cells
T270	382-383	NNP	denotes	[
T271	383-391	NNP	denotes	Matthews
T272	391-392	,	denotes	,
T273	393-397	NNP	denotes	S.A.
T274	397-398	,	denotes	,
T275	399-402	NNP	denotes	Liu
T276	402-403	,	denotes	,
T277	404-406	NNP	denotes	P.
T278	406-407	,	denotes	,
T279	408-416	NNP	denotes	Spitaler
T280	416-417	,	denotes	,
T281	418-420	NNP	denotes	M.
T282	420-421	,	denotes	,
T283	422-427	NNP	denotes	Olson
T284	427-428	,	denotes	,
T285	429-433	NNP	denotes	E.N.
T286	433-434	,	denotes	,
T287	435-443	NNP	denotes	McKinsey
T288	443-444	,	denotes	,
T289	445-449	NNP	denotes	T.A.
T290	449-450	,	denotes	,
T291	451-459	NNP	denotes	Cantrell
T292	459-460	,	denotes	,
T293	461-465	NNP	denotes	D.A.
T294	466-469	CC	denotes	and
T295	470-481	NNP	denotes	Scharenberg
T296	481-482	,	denotes	,
T297	483-487	NNP	denotes	A.M.
T298	488-489	-LRB-	denotes	(
T299	489-493	CD	denotes	2006
T300	493-494	-RRB-	denotes	)
T301	495-504	JJ	denotes	Essential
T302	505-509	NN	denotes	role
T303	510-513	IN	denotes	for
T304	514-521	NN	denotes	protein
T305	522-528	NN	denotes	kinase
T306	529-530	NNP	denotes	D
T307	531-537	NN	denotes	family
T308	538-545	NNS	denotes	kinases
T309	546-548	IN	denotes	in
T310	549-552	DT	denotes	the
T311	553-563	NN	denotes	regulation
T312	564-566	IN	denotes	of
T313	567-572	NN	denotes	class
T314	573-575	NNP	denotes	II
T315	576-583	NN	denotes	histone
T316	584-596	NNS	denotes	deacetylases
T317	597-599	IN	denotes	in
T318	600-601	NNP	denotes	B
T319	602-613	NNS	denotes	lymphocytes
T320	613-614	.	denotes	.
T321	615-618	NNP	denotes	Mol
T322	618-619	.	denotes	.
T323	620-624	NNP	denotes	Cell
T324	625-629	NNP	denotes	Biol
T325	629-630	.	denotes	.
T326	631-633	CD	denotes	26
T327	633-634	,	denotes	,
T328	635-639	CD	denotes	1569
T329	640-644	CD	denotes	1577
T330	644-645	NNP	denotes	]
T331	645-646	.	denotes	.
T332	647-649	PRP	denotes	We
T333	650-653	RB	denotes	now
T334	654-658	VBP	denotes	show
T335	659-663	IN	denotes	that
T336	664-668	NNS	denotes	PKDs
T337	669-672	VBP	denotes	are
T338	673-677	RB	denotes	also
T339	678-686	VBN	denotes	required
T340	687-689	TO	denotes	to
T341	690-698	VB	denotes	regulate
T342	699-704	NNP	denotes	HSP27
T343	705-720	NN	denotes	phosphorylation
T344	721-723	IN	denotes	in
T345	724-728	NNP	denotes	DT40
T346	729-736	NNPS	denotes	B-cells
T347	736-737	.	denotes	.
T348	738-745	RB	denotes	However
T349	745-746	,	denotes	,
T350	747-749	IN	denotes	in
T351	750-758	NN	denotes	contrast
T352	759-761	TO	denotes	to
T353	762-770	JJ	denotes	previous
T354	771-783	NNS	denotes	observations
T355	784-786	IN	denotes	in
T356	787-792	JJ	denotes	other
T357	793-797	NN	denotes	cell
T358	798-803	NNS	denotes	types
T359	803-804	,	denotes	,
T360	805-808	NNP	denotes	PKD
T361	809-816	VBZ	denotes	enzymes
T362	817-819	VB	denotes	do
T363	820-823	RB	denotes	not
T364	824-832	VB	denotes	regulate
T365	833-838	JJ	denotes	basic
T366	839-847	JJ	denotes	cellular
T367	848-857	NNS	denotes	processes
T368	858-862	JJ	denotes	such
T369	863-865	IN	denotes	as
T370	866-879	NN	denotes	proliferation
T371	880-882	CC	denotes	or
T372	883-891	NN	denotes	survival
T373	892-901	NNS	denotes	responses
T374	901-902	,	denotes	,
T375	903-906	CC	denotes	nor
T376	907-911	NNP	denotes	NFκB
T377	912-927	JJ	denotes	transcriptional
T378	928-936	NN	denotes	activity
T379	937-947	JJ	denotes	downstream
T380	948-950	IN	denotes	of
T381	951-954	DT	denotes	the
T382	955-956	NNP	denotes	B
T383	957-961	NN	denotes	cell
T384	962-969	NN	denotes	antigen
T385	970-978	NN	denotes	receptor
T386	978-979	.	denotes	.
T387	980-984	RB	denotes	Thus
T388	984-985	,	denotes	,
T389	986-990	NNS	denotes	PKDs
T390	991-995	VBP	denotes	have
T391	996-997	DT	denotes	a
T392	998-1007	JJ	denotes	selective
T393	1008-1012	NN	denotes	role
T394	1013-1015	IN	denotes	in
T395	1016-1020	NNP	denotes	DT40
T396	1021-1027	NNP	denotes	B-cell
T397	1028-1035	NN	denotes	biology
T398	1035-1036	.	denotes	.
T1008	635-1057	JJ	denotes	1569–1577]. We now show that PKDs are also required to regulate HSP27 phosphorylation in DT40 B-cells. However, in contrast to previous observations in other cell types, PKD enzymes do not regulate basic cellular processes such as proliferation or survival responses, nor NFκB transcriptional activity downstream of the B cell antigen receptor. Thus, PKDs have a selective role in DT40 B-cell biology. 1 Introduction The
T1009	1058-1065	NN	denotes	protein
T1010	1066-1072	NN	denotes	kinase
T1011	1073-1074	NNP	denotes	D
T1012	1075-1076	-LRB-	denotes	(
T1013	1076-1079	NNP	denotes	PKD
T1014	1079-1080	-RRB-	denotes	)
T1015	1081-1097	JJ	denotes	serine/threonine
T1016	1098-1104	NN	denotes	kinase
T1017	1105-1111	NN	denotes	family
T1018	1112-1115	VBZ	denotes	has
T1019	1116-1121	CD	denotes	three
T1020	1122-1129	NNS	denotes	members
T1021	1129-1130	:	denotes	:
T1022	1131-1135	NNP	denotes	PKD1
T1023	1135-1136	,	denotes	,
T1024	1137-1141	NNP	denotes	PKD2
T1025	1142-1145	CC	denotes	and
T1026	1146-1150	NNP	denotes	PKD3
T1027	1150-1151	.	denotes	.
T1028	1152-1156	JJS	denotes	Most
T1029	1157-1161	NN	denotes	cell
T1030	1162-1167	NNS	denotes	types
T1031	1168-1175	VBP	denotes	express
T1032	1176-1178	IN	denotes	at
T1033	1179-1184	JJS	denotes	least
T1034	1185-1188	CD	denotes	two
T1035	1189-1192	NNP	denotes	PKD
T1036	1193-1201	NNS	denotes	isoforms
T1037	1202-1205	CC	denotes	but
T1038	1206-1209	NNP	denotes	PKD
T1039	1210-1217	NNS	denotes	enzymes
T1040	1218-1221	VBP	denotes	are
T1041	1222-1232	RB	denotes	especially
T1042	1233-1239	RB	denotes	highly
T1043	1240-1249	VBN	denotes	expressed
T1044	1250-1252	IN	denotes	in
T1045	1253-1267	JJ	denotes	haematopoietic
T1046	1268-1273	NNS	denotes	cells
T1047	1273-1274	,	denotes	,
T1048	1275-1280	WRB	denotes	where
T1049	1281-1285	PRP	denotes	they
T1050	1286-1289	VBP	denotes	are
T1051	1290-1299	VBN	denotes	activated
T1052	1300-1302	IN	denotes	in
T1053	1303-1311	NN	denotes	response
T1054	1312-1314	TO	denotes	to
T1055	1315-1322	NN	denotes	antigen
T1056	1323-1332	NNS	denotes	receptors
T1057	1333-1344	NN	denotes	stimulation
T1058	1345-1346	NNP	denotes	[
T1059	1346-1349	CD	denotes	2,3
T1060	1349-1350	NNP	denotes	]
T1061	1350-1351	.	denotes	.
T1062	1352-1353	DT	denotes	A
T1063	1354-1363	JJ	denotes	conserved
T1064	1364-1374	JJ	denotes	signalling
T1065	1375-1382	NN	denotes	pathway
T1066	1383-1390	VBG	denotes	linking
T1067	1391-1398	NN	denotes	antigen
T1068	1399-1408	NNS	denotes	receptors
T1069	1409-1411	TO	denotes	to
T1070	1412-1416	NNS	denotes	PKDs
T1071	1417-1425	VBZ	denotes	involves
T1072	1426-1429	DT	denotes	the
T1073	1430-1440	NN	denotes	activation
T1074	1441-1443	IN	denotes	of
T1075	1444-1448	NNP	denotes	PLCγ
T1076	1449-1452	CC	denotes	and
T1077	1453-1456	DT	denotes	the
T1078	1457-1467	JJ	denotes	subsequent
T1079	1468-1478	NN	denotes	production
T1080	1479-1481	IN	denotes	of
T1081	1482-1496	NN	denotes	diacylglycerol
T1082	1497-1498	-LRB-	denotes	(
T1083	1498-1501	NNP	denotes	DAG
T1084	1501-1502	-RRB-	denotes	)
T1085	1503-1508	WDT	denotes	which
T1086	1509-1519	VBZ	denotes	stimulates
T1087	1520-1529	JJ	denotes	classical
T1088	1530-1536	CC	denotes	and/or
T1089	1537-1542	JJ	denotes	novel
T1090	1543-1550	NN	denotes	protein
T1091	1551-1557	NN	denotes	kinase
T1092	1558-1560	NNS	denotes	Cs
T1093	1561-1562	-LRB-	denotes	(
T1094	1562-1565	NNP	denotes	PKC
T1095	1565-1566	-RRB-	denotes	)
T1096	1567-1571	WDT	denotes	that
T1097	1572-1585	VBP	denotes	phosphorylate
T1098	1586-1589	CD	denotes	two
T1099	1590-1593	JJ	denotes	key
T1100	1594-1604	JJ	denotes	regulatory
T1101	1605-1611	NN	denotes	serine
T1102	1612-1620	NNS	denotes	residues
T1103	1621-1623	IN	denotes	in
T1104	1624-1627	DT	denotes	the
T1105	1628-1638	NN	denotes	activation
T1106	1639-1643	NN	denotes	loop
T1107	1644-1646	IN	denotes	of
T1108	1647-1650	NNP	denotes	PKD
T1109	1651-1658	VBZ	denotes	kinases
T1110	1659-1660	NNP	denotes	[
T1111	1660-1661	CD	denotes	3
T1112	1662-1663	CD	denotes	6
T1113	1663-1664	NNP	denotes	]
T1114	1664-1665	.	denotes	.
T1115	1666-1669	DT	denotes	The
T1116	1670-1680	JJ	denotes	N-terminal
T1117	1681-1691	JJ	denotes	regulatory
T1118	1692-1698	NN	denotes	region
T1119	1699-1701	IN	denotes	of
T1120	1702-1705	NNP	denotes	PKD
T1121	1706-1713	VBZ	denotes	enzymes
T1122	1714-1722	VBZ	denotes	contains
T1123	1723-1724	DT	denotes	a
T1124	1725-1728	NNP	denotes	DAG
T1125	1729-1736	JJ	denotes	binding
T1126	1737-1743	NN	denotes	domain
T1127	1744-1747	CC	denotes	and
T1128	1748-1754	JJ	denotes	direct
T1129	1755-1762	JJ	denotes	binding
T1130	1763-1765	IN	denotes	of
T1131	1766-1769	NNP	denotes	DAG
T1132	1770-1774	RB	denotes	also
T1133	1775-1786	VBZ	denotes	contributes
T1134	1787-1789	TO	denotes	to
T1135	1790-1794	CD	denotes	PKD1
T1136	1795-1805	NN	denotes	activation
T1137	1806-1807	NN	denotes	[
T1138	1807-1808	CD	denotes	7
T1139	1808-1809	NNP	denotes	]
T1140	1810-1812	RB	denotes	as
T1141	1813-1817	RB	denotes	well
T1142	1818-1820	IN	denotes	as
T1143	1821-1831	VBG	denotes	regulating
T1144	1832-1835	DT	denotes	the
T1145	1836-1843	JJ	denotes	spatial
T1146	1844-1852	NN	denotes	location
T1147	1853-1855	IN	denotes	of
T1148	1856-1859	NNP	denotes	PKD
T1149	1860-1867	VBZ	denotes	enzymes
T1150	1868-1874	IN	denotes	within
T1151	1875-1880	NNS	denotes	cells
T1152	1881-1882	VBP	denotes	[
T1153	1882-1883	CD	denotes	8
T1154	1884-1886	CD	denotes	12
T1155	1886-1887	NN	denotes	]
T1156	1887-1888	.	denotes	.
T1157	1889-1892	NNP	denotes	PKD
T1158	1893-1900	NNS	denotes	enzymes
T1159	1901-1905	VBP	denotes	have
T1160	1906-1910	VBN	denotes	been
T1161	1911-1919	VBN	denotes	proposed
T1162	1920-1922	TO	denotes	to
T1163	1923-1931	VB	denotes	regulate
T1164	1932-1940	JJ	denotes	numerous
T1165	1941-1949	JJ	denotes	cellular
T1166	1950-1959	NNS	denotes	functions
T1167	1959-1960	,	denotes	,
T1168	1961-1970	VBG	denotes	including
T1169	1971-1975	NN	denotes	cell
T1170	1976-1989	NN	denotes	proliferation
T1171	1990-1991	NNP	denotes	[
T1172	1991-1993	CD	denotes	13
T1173	1994-1996	CD	denotes	16
T1174	1996-1997	NNP	denotes	]
T1175	1997-1998	,	denotes	,
T1176	1999-2013	JJ	denotes	anti-apoptotic
T1177	2014-2021	NNS	denotes	signals
T1178	2022-2023	NNP	denotes	[
T1179	2023-2028	CD	denotes	17,18
T1180	2028-2029	NNP	denotes	]
T1181	2030-2033	CC	denotes	and
T1182	2034-2043	JJ	denotes	thymocyte
T1183	2044-2055	NN	denotes	development
T1184	2056-2057	NNP	denotes	[
T1185	2057-2059	CD	denotes	19
T1186	2059-2060	NNP	denotes	]
T1187	2060-2061	.	denotes	.
T1188	2062-2072	NN	denotes	Expression
T1189	2073-2075	IN	denotes	of
T1190	2076-2082	JJ	denotes	mutant
T1191	2083-2096	RB	denotes	catalytically
T1192	2097-2105	JJ	denotes	inactive
T1193	2106-2109	CC	denotes	and
T1194	2110-2124	RB	denotes	constitutively
T1195	2125-2134	VBN	denotes	activated
T1196	2135-2139	NNS	denotes	PKDs
T1197	2140-2143	MD	denotes	can
T1198	2144-2148	RB	denotes	also
T1199	2149-2155	VB	denotes	modify
T1200	2156-2161	NNP	denotes	Golgi
T1201	2162-2170	NN	denotes	function
T1202	2170-2171	,	denotes	,
T1203	2172-2176	NN	denotes	cell
T1204	2177-2185	NN	denotes	adhesion
T1205	2186-2189	CC	denotes	and
T1206	2190-2194	NN	denotes	cell
T1207	2195-2203	NN	denotes	motility
T1208	2204-2205	-LRB-	denotes	(
T1209	2205-2213	VBN	denotes	reviewed
T1210	2214-2216	IN	denotes	in
T1211	2217-2218	NNP	denotes	[
T1212	2218-2220	CD	denotes	20
T1213	2220-2221	NNP	denotes	]
T1214	2221-2222	-RRB-	denotes	)
T1215	2222-2223	.	denotes	.
T1216	2224-2226	IN	denotes	In
T1217	2227-2237	JJ	denotes	particular
T1218	2237-2238	,	denotes	,
T1219	2239-2243	NNS	denotes	PKDs
T1220	2244-2248	VBP	denotes	have
T1221	2249-2253	VBN	denotes	been
T1222	2254-2260	RB	denotes	widely
T1223	2261-2267	VBN	denotes	linked
T1224	2268-2270	TO	denotes	to
T1225	2271-2274	DT	denotes	the
T1226	2275-2285	NN	denotes	activation
T1227	2286-2288	IN	denotes	of
T1228	2289-2292	DT	denotes	the
T1229	2293-2297	NNP	denotes	NFκB
T1230	2298-2311	NN	denotes	transcription
T1231	2312-2318	NN	denotes	factor
T1232	2319-2322	CC	denotes	and
T1233	2323-2325	IN	denotes	in
T1234	2326-2336	VBG	denotes	regulating
T1235	2337-2341	NN	denotes	cell
T1236	2342-2350	NN	denotes	survival
T1237	2351-2357	IN	denotes	during
T1238	2358-2367	JJ	denotes	oxidative
T1239	2368-2374	NN	denotes	stress
T1240	2375-2376	NNP	denotes	[
T1241	2376-2381	CD	denotes	17,21
T1242	2382-2384	CD	denotes	23
T1243	2384-2385	NNP	denotes	]
T1244	2385-2386	.	denotes	.
T1245	2387-2394	DT	denotes	Another
T1246	2395-2403	RB	denotes	recently
T1247	2404-2412	VBN	denotes	proposed
T1248	2413-2417	NNP	denotes	PKD1
T1249	2418-2427	NN	denotes	substrate
T1250	2428-2430	VBZ	denotes	is
T1251	2431-2436	NNP	denotes	HSP27
T1252	2437-2438	NNP	denotes	[
T1253	2438-2440	CD	denotes	24
T1254	2440-2441	NNP	denotes	]
T1255	2441-2442	,	denotes	,
T1256	2443-2444	DT	denotes	a
T1257	2445-2450	JJ	denotes	small
T1258	2451-2455	NN	denotes	heat
T1259	2456-2461	NN	denotes	shock
T1260	2462-2469	NN	denotes	protein
T1261	2470-2478	VBN	denotes	involved
T1262	2479-2481	IN	denotes	in
T1263	2482-2492	VBG	denotes	regulating
T1264	2493-2497	NN	denotes	cell
T1265	2498-2507	NN	denotes	migration
T1266	2508-2511	CC	denotes	and
T1267	2512-2516	NN	denotes	cell
T1268	2517-2525	NN	denotes	survival
T1269	2526-2527	NNP	denotes	[
T1270	2527-2529	CD	denotes	25
T1271	2529-2530	NNP	denotes	]
T1272	2530-2531	.	denotes	.
T1273	2532-2534	DT	denotes	An
T1274	2535-2544	JJ	denotes	essential
T1275	2545-2549	NN	denotes	role
T1276	2550-2553	IN	denotes	for
T1277	2554-2557	NNP	denotes	PKD
T1278	2558-2565	VBZ	denotes	enzymes
T1279	2566-2568	IN	denotes	in
T1280	2569-2579	VBG	denotes	regulating
T1281	2580-2585	NN	denotes	class
T1282	2586-2588	NNP	denotes	II
T1283	2589-2596	NN	denotes	histone
T1284	2597-2609	NNS	denotes	deacetylases
T1285	2610-2611	-LRB-	denotes	(
T1286	2611-2616	NNS	denotes	HDACs
T1287	2616-2617	-RRB-	denotes	)
T1288	2617-2618	,	denotes	,
T1289	2619-2626	VBZ	denotes	enzymes
T1290	2627-2631	IN	denotes	that
T1291	2632-2639	VBZ	denotes	repress
T1292	2640-2654	JJ	denotes	MEF2-dependent
T1293	2655-2659	NN	denotes	gene
T1294	2660-2673	NN	denotes	transcription
T1295	2673-2674	,	denotes	,
T1296	2675-2678	VBZ	denotes	has
T1297	2679-2683	RB	denotes	also
T1298	2684-2688	VBN	denotes	been
T1299	2689-2701	VBN	denotes	demonstrated
T1300	2702-2703	NNP	denotes	[
T1301	2703-2707	CD	denotes	1,26
T1302	2708-2710	CD	denotes	28
T1303	2710-2711	NN	denotes	]
T1304	2711-2712	.	denotes	.
T1305	2713-2715	TO	denotes	To
T1306	2716-2727	VB	denotes	investigate
T1307	2728-2731	DT	denotes	the
T1308	2732-2742	JJ	denotes	biological
T1309	2743-2747	NN	denotes	role
T1310	2748-2750	IN	denotes	of
T1311	2751-2755	NNS	denotes	PKDs
T1312	2756-2758	PRP	denotes	we
T1313	2759-2763	VBP	denotes	have
T1314	2764-2773	VBN	denotes	generated
T1315	2774-2778	NNP	denotes	DT40
T1316	2779-2780	NNP	denotes	B
T1317	2781-2785	NN	denotes	cell
T1318	2786-2791	NNS	denotes	lines
T1319	2792-2796	WDT	denotes	that
T1320	2797-2801	VBP	denotes	lack
T1321	2802-2812	NN	denotes	expression
T1322	2813-2815	IN	denotes	of
T1323	2816-2819	CD	denotes	one
T1324	2820-2822	CC	denotes	or
T1325	2823-2827	JJR	denotes	more
T1326	2828-2835	NNS	denotes	members
T1327	2836-2838	IN	denotes	of
T1328	2839-2842	DT	denotes	the
T1329	2843-2846	NNP	denotes	PKD
T1330	2847-2853	NN	denotes	family
T1331	2854-2855	NNP	denotes	[
T1332	2855-2856	CD	denotes	1
T1333	2856-2857	NNP	denotes	]
T1334	2857-2858	,	denotes	,
T1335	2859-2867	VBG	denotes	allowing
T1336	2868-2870	PRP	denotes	us
T1337	2871-2873	TO	denotes	to
T1338	2874-2885	VB	denotes	investigate
T1339	2886-2889	DT	denotes	the
T1340	2890-2898	NN	denotes	function
T1341	2898-2899	-LRB-	denotes	(
T1342	2899-2900	PRP	denotes	s
T1343	2900-2901	-RRB-	denotes	)
T1344	2902-2904	IN	denotes	of
T1345	2905-2908	NNP	denotes	PKD
T1346	2909-2917	VBZ	denotes	isoforms
T1347	2918-2927	VBG	denotes	following
T1348	2928-2929	NNP	denotes	B
T1349	2930-2934	NN	denotes	cell
T1350	2935-2942	NN	denotes	antigen
T1351	2943-2951	NN	denotes	receptor
T1352	2952-2953	-LRB-	denotes	(
T1353	2953-2956	NNP	denotes	BCR
T1354	2956-2957	-RRB-	denotes	)
T1355	2958-2969	NN	denotes	stimulation
T1356	2969-2970	,	denotes	,
T1357	2971-2973	RB	denotes	as
T1358	2974-2978	RB	denotes	well
T1359	2979-2989	VBG	denotes	addressing
T1360	2990-2993	DT	denotes	the
T1361	2994-2999	NN	denotes	issue
T1362	3000-3002	IN	denotes	of
T1363	3003-3013	JJ	denotes	functional
T1364	3014-3024	NN	denotes	redundancy
T1365	3025-3032	IN	denotes	between
T1366	3033-3036	DT	denotes	the
T1367	3037-3046	JJ	denotes	different
T1368	3047-3050	NNP	denotes	PKD
T1369	3051-3057	NN	denotes	family
T1370	3058-3065	NNS	denotes	members
T1371	3065-3066	.	denotes	.
T1372	3067-3075	JJ	denotes	Previous
T1373	3076-3083	NNS	denotes	studies
T1374	3084-3088	VBP	denotes	have
T1375	3089-3094	VBN	denotes	shown
T1376	3095-3099	IN	denotes	that
T1377	3100-3104	NNS	denotes	PKDs
T1378	3105-3108	VBP	denotes	are
T1379	3109-3122	JJ	denotes	indispensable
T1380	3123-3126	IN	denotes	for
T1381	3127-3131	NNP	denotes	HDAC
T1382	3132-3142	NN	denotes	regulation
T1383	3143-3145	IN	denotes	in
T1384	3146-3147	NNP	denotes	B
T1385	3148-3153	NNS	denotes	cells
T1386	3154-3155	NNP	denotes	[
T1387	3155-3156	CD	denotes	1
T1388	3156-3157	NNP	denotes	]
T1389	3157-3158	.	denotes	.
T1390	3159-3165	NNP	denotes	Herein
T1391	3166-3168	PRP	denotes	we
T1392	3169-3173	VBP	denotes	show
T1393	3174-3178	IN	denotes	that
T1394	3179-3183	NNS	denotes	PKDs
T1395	3184-3187	VBP	denotes	are
T1396	3188-3192	RB	denotes	also
T1397	3193-3206	JJ	denotes	indispensable
T1398	3207-3210	IN	denotes	for
T1399	3211-3216	NNP	denotes	HSP27
T1400	3217-3232	NN	denotes	phosphorylation
T1401	3233-3235	IN	denotes	in
T1402	3236-3237	NNP	denotes	B
T1403	3238-3243	NNS	denotes	cells
T1404	3243-3244	.	denotes	.
T1405	3245-3252	RB	denotes	However
T1406	3252-3253	,	denotes	,
T1407	3254-3262	NNP	denotes	PKD-null
T1408	3263-3267	NNP	denotes	DT40
T1409	3268-3269	NNP	denotes	B
T1410	3270-3275	NNS	denotes	cells
T1411	3276-3279	VBP	denotes	are
T1412	3280-3286	JJ	denotes	viable
T1413	3287-3290	CC	denotes	and
T1414	3291-3302	VB	denotes	proliferate
T1415	3303-3311	RB	denotes	normally
T1416	3311-3312	.	denotes	.
T1417	3313-3321	RB	denotes	Moreover
T1418	3321-3322	,	denotes	,
T1419	3323-3327	NN	denotes	loss
T1420	3328-3330	IN	denotes	of
T1421	3331-3334	DT	denotes	the
T1422	3335-3341	JJ	denotes	entire
T1423	3342-3350	JJ	denotes	cellular
T1424	3351-3355	NN	denotes	pool
T1425	3356-3358	IN	denotes	of
T1426	3359-3362	NNP	denotes	PKD
T1427	3363-3367	VBZ	denotes	does
T1428	3368-3371	RB	denotes	not
T1429	3372-3382	RB	denotes	critically
T1430	3383-3389	VB	denotes	affect
T1431	3390-3399	JJ	denotes	oxidative
T1432	3400-3406	NN	denotes	stress
T1433	3407-3416	NNS	denotes	responses
T1434	3417-3419	IN	denotes	in
T1435	3420-3421	NNP	denotes	B
T1436	3422-3427	NNS	denotes	cells
T1437	3428-3431	CC	denotes	nor
T1438	3432-3434	VBP	denotes	do
T1439	3435-3438	NNP	denotes	PKD
T1440	3439-3446	VBZ	denotes	kinases
T1441	3447-3451	VB	denotes	play
T1442	3452-3454	DT	denotes	an
T1443	3455-3464	JJ	denotes	essential
T1444	3465-3469	NN	denotes	role
T1445	3470-3472	IN	denotes	in
T1446	3473-3483	VBG	denotes	regulating
T1447	3484-3488	NNP	denotes	NFκB
T1448	3489-3504	JJ	denotes	transcriptional
T1449	3505-3513	NN	denotes	activity
T1450	3513-3514	.	denotes	.
T1451	3515-3523	RB	denotes	Together
T1452	3523-3524	,	denotes	,
T1453	3525-3530	DT	denotes	these
T1454	3531-3539	NNS	denotes	findings
T1455	3540-3546	VBP	denotes	reveal
T1456	3547-3551	IN	denotes	that
T1457	3552-3554	IN	denotes	in
T1458	3555-3556	NNP	denotes	B
T1459	3557-3568	NNS	denotes	lymphocytes
T1460	3568-3569	,	denotes	,
T1461	3570-3573	NNP	denotes	PKD
T1462	3574-3581	NNS	denotes	kinases
T1463	3582-3585	VBP	denotes	are
T1464	3586-3589	RB	denotes	not
T1465	3590-3598	JJ	denotes	critical
T1466	3599-3609	NNS	denotes	regulators
T1467	3610-3612	IN	denotes	of
T1468	3613-3617	JJ	denotes	many
T1469	3618-3620	IN	denotes	of
T1470	3621-3624	DT	denotes	the
T1471	3625-3633	JJ	denotes	cellular
T1472	3634-3643	NNS	denotes	processes
T1473	3644-3654	RB	denotes	previously
T1474	3655-3663	VBD	denotes	ascribed
T1475	3664-3666	TO	denotes	to
T1476	3667-3671	PRP	denotes	them
T1477	3672-3674	IN	denotes	in
T1478	3675-3680	JJ	denotes	other
T1479	3681-3689	JJ	denotes	cellular
T1480	3690-3697	NNS	denotes	systems
T1481	3697-3698	.	denotes	.
T1742	0-3731	CD	denotes	Protein kinase D enzymes are dispensable for proliferation, survival and antigen receptor-regulated NFκB activity in vertebrate B-cells Abstract To investigate the importance of protein kinase D (PKD) enzymes we generated a PKD-null DT40 B-lymphocyte cell line. Previously we have shown that PKDs have an essential role in regulating class II histone deacetylases in DT40 B-cells [Matthews, S.A., Liu, P., Spitaler, M., Olson, E.N., McKinsey, T.A., Cantrell, D.A. and Scharenberg, A.M. (2006) Essential role for protein kinase D family kinases in the regulation of class II histone deacetylases in B lymphocytes. Mol. Cell Biol. 26, 1569–1577]. We now show that PKDs are also required to regulate HSP27 phosphorylation in DT40 B-cells. However, in contrast to previous observations in other cell types, PKD enzymes do not regulate basic cellular processes such as proliferation or survival responses, nor NFκB transcriptional activity downstream of the B cell antigen receptor. Thus, PKDs have a selective role in DT40 B-cell biology. 1 Introduction The protein kinase D (PKD) serine/threonine kinase family has three members: PKD1, PKD2 and PKD3. Most cell types express at least two PKD isoforms but PKD enzymes are especially highly expressed in haematopoietic cells, where they are activated in response to antigen receptors stimulation [2,3]. A conserved signalling pathway linking antigen receptors to PKDs involves the activation of PLCγ and the subsequent production of diacylglycerol (DAG) which stimulates classical and/or novel protein kinase Cs (PKC) that phosphorylate two key regulatory serine residues in the activation loop of PKD kinases [3–6]. The N-terminal regulatory region of PKD enzymes contains a DAG binding domain and direct binding of DAG also contributes to PKD1 activation [7] as well as regulating the spatial location of PKD enzymes within cells [8–12]. PKD enzymes have been proposed to regulate numerous cellular functions, including cell proliferation [13–16], anti-apoptotic signals [17,18] and thymocyte development [19]. Expression of mutant catalytically inactive and constitutively activated PKDs can also modify Golgi function, cell adhesion and cell motility (reviewed in [20]). In particular, PKDs have been widely linked to the activation of the NFκB transcription factor and in regulating cell survival during oxidative stress [17,21–23]. Another recently proposed PKD1 substrate is HSP27 [24], a small heat shock protein involved in regulating cell migration and cell survival [25]. An essential role for PKD enzymes in regulating class II histone deacetylases (HDACs), enzymes that repress MEF2-dependent gene transcription, has also been demonstrated [1,26–28]. To investigate the biological role of PKDs we have generated DT40 B cell lines that lack expression of one or more members of the PKD family [1], allowing us to investigate the function(s) of PKD isoforms following B cell antigen receptor (BCR) stimulation, as well addressing the issue of functional redundancy between the different PKD family members. Previous studies have shown that PKDs are indispensable for HDAC regulation in B cells [1]. Herein we show that PKDs are also indispensable for HSP27 phosphorylation in B cells. However, PKD-null DT40 B cells are viable and proliferate normally. Moreover, loss of the entire cellular pool of PKD does not critically affect oxidative stress responses in B cells nor do PKD kinases play an essential role in regulating NFκB transcriptional activity. Together, these findings reveal that in B lymphocytes, PKD kinases are not critical regulators of many of the cellular processes previously ascribed to them in other cellular systems. 2 Materials and methods 2.1
T1743	3731-3735	NNP	denotes	Cell
T1744	3736-3743	NN	denotes	culture
T1745	3743-3744	,	denotes	,
T1746	3745-3754	NN	denotes	transient
T1747	3755-3768	NNS	denotes	transfections
T1748	3769-3772	CC	denotes	and
T1749	3773-3777	NN	denotes	cell
T1750	3778-3789	NN	denotes	stimulation
T1751	3790-3793	DT	denotes	The
T1752	3794-3804	NN	denotes	generation
T1753	3804-3805	,	denotes	,
T1754	3806-3813	NN	denotes	culture
T1755	3814-3817	CC	denotes	and
T1756	3818-3828	NN	denotes	activation
T1757	3829-3831	IN	denotes	of
T1758	3832-3836	CD	denotes	PKD1
T1759	3836-3837	CD	denotes	−
T1760	3837-3838	NN	denotes	/
T1761	3838-3839	NN	denotes	−
T1762	3839-3840	,	denotes	,
T1763	3841-3845	NNP	denotes	PKD3
T1764	3845-3846	NNP	denotes	−
T1765	3846-3847	VBD	denotes	/
T1766	3847-3848	CD	denotes	−
T1767	3849-3852	CC	denotes	and
T1768	3853-3859	CD	denotes	PKD1/3
T1769	3859-3860	CD	denotes	−
T1770	3860-3861	NN	denotes	/
T1771	3861-3862	NN	denotes	−
T1772	3863-3871	NN	denotes	knockout
T1773	3872-3876	NNP	denotes	DT40
T1774	3877-3878	NNP	denotes	B
T1775	3879-3883	NN	denotes	cell
T1776	3884-3889	NNS	denotes	lines
T1777	3890-3894	VBP	denotes	have
T1778	3895-3899	VBN	denotes	been
T1779	3900-3909	VBN	denotes	described
T1780	3910-3920	RB	denotes	previously
T1781	3921-3922	NNP	denotes	[
T1782	3922-3923	CD	denotes	1
T1783	3923-3924	NNP	denotes	]
T1784	3924-3925	.	denotes	.
T1785	3926-3931	NNS	denotes	Cells
T1786	3932-3936	VBD	denotes	were
T1787	3937-3942	VBN	denotes	lysed
T1788	3943-3946	CC	denotes	and
T1789	3947-3954	NN	denotes	protein
T1790	3955-3963	NNS	denotes	extracts
T1791	3964-3968	VBD	denotes	were
T1792	3969-3977	VBN	denotes	analysed
T1793	3978-3980	IN	denotes	in
T1794	3981-3988	JJ	denotes	Western
T1795	3989-3997	VBG	denotes	blotting
T1796	3998-4009	NNS	denotes	experiments
T1797	4010-4012	IN	denotes	as
T1798	4013-4023	RB	denotes	previously
T1799	4024-4033	VBN	denotes	described
T1800	4034-4035	NNP	denotes	[
T1801	4035-4036	CD	denotes	1
T1802	4036-4037	NNP	denotes	]
T1803	4037-4038	.	denotes	.
T1804	4039-4054	NNP	denotes	Chloramphenicol
T1805	4055-4061	NN	denotes	acetyl
T1806	4062-4073	NN	denotes	transferase
T1807	4074-4080	NNS	denotes	assays
T1808	4081-4085	VBP	denotes	have
T1809	4086-4090	VBN	denotes	been
T1810	4091-4100	VBN	denotes	described
T1811	4101-4111	RB	denotes	previously
T1812	4112-4113	NNP	denotes	[
T1813	4113-4115	CD	denotes	29
T1814	4115-4116	NNP	denotes	]
T1815	4116-4117	.	denotes	.
T1939	0-4124	CD	denotes	Protein kinase D enzymes are dispensable for proliferation, survival and antigen receptor-regulated NFκB activity in vertebrate B-cells Abstract To investigate the importance of protein kinase D (PKD) enzymes we generated a PKD-null DT40 B-lymphocyte cell line. Previously we have shown that PKDs have an essential role in regulating class II histone deacetylases in DT40 B-cells [Matthews, S.A., Liu, P., Spitaler, M., Olson, E.N., McKinsey, T.A., Cantrell, D.A. and Scharenberg, A.M. (2006) Essential role for protein kinase D family kinases in the regulation of class II histone deacetylases in B lymphocytes. Mol. Cell Biol. 26, 1569–1577]. We now show that PKDs are also required to regulate HSP27 phosphorylation in DT40 B-cells. However, in contrast to previous observations in other cell types, PKD enzymes do not regulate basic cellular processes such as proliferation or survival responses, nor NFκB transcriptional activity downstream of the B cell antigen receptor. Thus, PKDs have a selective role in DT40 B-cell biology. 1 Introduction The protein kinase D (PKD) serine/threonine kinase family has three members: PKD1, PKD2 and PKD3. Most cell types express at least two PKD isoforms but PKD enzymes are especially highly expressed in haematopoietic cells, where they are activated in response to antigen receptors stimulation [2,3]. A conserved signalling pathway linking antigen receptors to PKDs involves the activation of PLCγ and the subsequent production of diacylglycerol (DAG) which stimulates classical and/or novel protein kinase Cs (PKC) that phosphorylate two key regulatory serine residues in the activation loop of PKD kinases [3–6]. The N-terminal regulatory region of PKD enzymes contains a DAG binding domain and direct binding of DAG also contributes to PKD1 activation [7] as well as regulating the spatial location of PKD enzymes within cells [8–12]. PKD enzymes have been proposed to regulate numerous cellular functions, including cell proliferation [13–16], anti-apoptotic signals [17,18] and thymocyte development [19]. Expression of mutant catalytically inactive and constitutively activated PKDs can also modify Golgi function, cell adhesion and cell motility (reviewed in [20]). In particular, PKDs have been widely linked to the activation of the NFκB transcription factor and in regulating cell survival during oxidative stress [17,21–23]. Another recently proposed PKD1 substrate is HSP27 [24], a small heat shock protein involved in regulating cell migration and cell survival [25]. An essential role for PKD enzymes in regulating class II histone deacetylases (HDACs), enzymes that repress MEF2-dependent gene transcription, has also been demonstrated [1,26–28]. To investigate the biological role of PKDs we have generated DT40 B cell lines that lack expression of one or more members of the PKD family [1], allowing us to investigate the function(s) of PKD isoforms following B cell antigen receptor (BCR) stimulation, as well addressing the issue of functional redundancy between the different PKD family members. Previous studies have shown that PKDs are indispensable for HDAC regulation in B cells [1]. Herein we show that PKDs are also indispensable for HSP27 phosphorylation in B cells. However, PKD-null DT40 B cells are viable and proliferate normally. Moreover, loss of the entire cellular pool of PKD does not critically affect oxidative stress responses in B cells nor do PKD kinases play an essential role in regulating NFκB transcriptional activity. Together, these findings reveal that in B lymphocytes, PKD kinases are not critical regulators of many of the cellular processes previously ascribed to them in other cellular systems. 2 Materials and methods 2.1 Cell culture, transient transfections and cell stimulation The generation, culture and activation of PKD1−/−, PKD3−/− and PKD1/3−/− knockout DT40 B cell lines have been described previously [1]. Cells were lysed and protein extracts were analysed in Western blotting experiments as previously described [1]. Chloramphenicol acetyl transferase assays have been described previously [29]. 2.2
T1940	4124-4128	JJ	denotes	sIgM
T1941	4129-4137	VBG	denotes	staining
T1942	4138-4142	NNP	denotes	DT40
T1943	4143-4144	NNP	denotes	B
T1944	4145-4150	NNS	denotes	cells
T1945	4151-4152	-LRB-	denotes	(
T1946	4152-4153	CD	denotes	2
T1947	4154-4155	CD	denotes	×
T1948	4156-4159	CD	denotes	106
T1949	4160-4165	NNS	denotes	cells
T1950	4166-4169	IN	denotes	per
T1951	4170-4175	NN	denotes	point
T1952	4175-4176	-RRB-	denotes	)
T1953	4177-4181	VBD	denotes	were
T1954	4182-4193	VBN	denotes	resuspended
T1955	4194-4196	IN	denotes	in
T1956	4197-4200	CD	denotes	200
T1957	4201-4203	NN	denotes	μl
T1958	4204-4210	NN	denotes	buffer
T1959	4211-4212	-LRB-	denotes	(
T1960	4212-4216	NNP	denotes	RPMI
T1961	4217-4221	CD	denotes	1640
T1962	4222-4227	NNS	denotes	media
T1963	4227-4228	,	denotes	,
T1964	4229-4230	CD	denotes	1
T1965	4230-4231	NN	denotes	%
T1966	4232-4238	JJ	denotes	foetal
T1967	4239-4243	NN	denotes	calf
T1968	4244-4249	NN	denotes	serum
T1969	4249-4250	-RRB-	denotes	)
T1970	4251-4261	VBG	denotes	containing
T1971	4262-4274	JJ	denotes	anti-chicken
T1972	4275-4277	CD	denotes	M1
T1973	4278-4288	JJ	denotes	monoclonal
T1974	4289-4297	NN	denotes	antibody
T1975	4298-4308	VBN	denotes	conjugated
T1976	4309-4311	TO	denotes	to
T1977	4312-4316	NNP	denotes	FITC
T1978	4317-4320	IN	denotes	for
T1979	4321-4323	CD	denotes	20
T1980	4324-4327	NN	denotes	min
T1981	4328-4330	IN	denotes	on
T1982	4331-4334	NN	denotes	ice
T1983	4334-4335	.	denotes	.
T1984	4336-4339	DT	denotes	The
T1985	4340-4345	NNS	denotes	cells
T1986	4346-4350	VBD	denotes	were
T1987	4351-4357	VBN	denotes	washed
T1988	4358-4363	RB	denotes	twice
T1989	4364-4367	CC	denotes	and
T1990	4368-4379	JJ	denotes	fluorescent
T1991	4380-4389	NN	denotes	intensity
T1992	4390-4393	VBD	denotes	was
T1993	4394-4402	VBN	denotes	analysed
T1994	4403-4405	IN	denotes	by
T1995	4406-4410	NN	denotes	flow
T1996	4411-4420	NN	denotes	cytometry
T1997	4420-4421	.	denotes	.
T1998	4422-4425	DT	denotes	All
T1999	4426-4433	NNS	denotes	results
T2000	4434-4439	VBN	denotes	shown
T2001	4440-4443	VBP	denotes	are
T2002	4444-4458	NN	denotes	representative
T2003	4459-4461	IN	denotes	of
T2004	4462-4464	IN	denotes	at
T2005	4465-4468	CD	denotes	two
T2006	4469-4471	TO	denotes	to
T2007	4472-4476	CD	denotes	four
T2008	4477-4488	JJ	denotes	independent
T2009	4489-4500	NNS	denotes	experiments
T2010	4501-4507	IN	denotes	unless
T2011	4508-4517	RB	denotes	otherwise
T2012	4518-4527	VBN	denotes	indicated
T2013	4527-4528	.	denotes	.
T2730	0-4547	CD	denotes	Protein kinase D enzymes are dispensable for proliferation, survival and antigen receptor-regulated NFκB activity in vertebrate B-cells Abstract To investigate the importance of protein kinase D (PKD) enzymes we generated a PKD-null DT40 B-lymphocyte cell line. Previously we have shown that PKDs have an essential role in regulating class II histone deacetylases in DT40 B-cells [Matthews, S.A., Liu, P., Spitaler, M., Olson, E.N., McKinsey, T.A., Cantrell, D.A. and Scharenberg, A.M. (2006) Essential role for protein kinase D family kinases in the regulation of class II histone deacetylases in B lymphocytes. Mol. Cell Biol. 26, 1569–1577]. We now show that PKDs are also required to regulate HSP27 phosphorylation in DT40 B-cells. However, in contrast to previous observations in other cell types, PKD enzymes do not regulate basic cellular processes such as proliferation or survival responses, nor NFκB transcriptional activity downstream of the B cell antigen receptor. Thus, PKDs have a selective role in DT40 B-cell biology. 1 Introduction The protein kinase D (PKD) serine/threonine kinase family has three members: PKD1, PKD2 and PKD3. Most cell types express at least two PKD isoforms but PKD enzymes are especially highly expressed in haematopoietic cells, where they are activated in response to antigen receptors stimulation [2,3]. A conserved signalling pathway linking antigen receptors to PKDs involves the activation of PLCγ and the subsequent production of diacylglycerol (DAG) which stimulates classical and/or novel protein kinase Cs (PKC) that phosphorylate two key regulatory serine residues in the activation loop of PKD kinases [3–6]. The N-terminal regulatory region of PKD enzymes contains a DAG binding domain and direct binding of DAG also contributes to PKD1 activation [7] as well as regulating the spatial location of PKD enzymes within cells [8–12]. PKD enzymes have been proposed to regulate numerous cellular functions, including cell proliferation [13–16], anti-apoptotic signals [17,18] and thymocyte development [19]. Expression of mutant catalytically inactive and constitutively activated PKDs can also modify Golgi function, cell adhesion and cell motility (reviewed in [20]). In particular, PKDs have been widely linked to the activation of the NFκB transcription factor and in regulating cell survival during oxidative stress [17,21–23]. Another recently proposed PKD1 substrate is HSP27 [24], a small heat shock protein involved in regulating cell migration and cell survival [25]. An essential role for PKD enzymes in regulating class II histone deacetylases (HDACs), enzymes that repress MEF2-dependent gene transcription, has also been demonstrated [1,26–28]. To investigate the biological role of PKDs we have generated DT40 B cell lines that lack expression of one or more members of the PKD family [1], allowing us to investigate the function(s) of PKD isoforms following B cell antigen receptor (BCR) stimulation, as well addressing the issue of functional redundancy between the different PKD family members. Previous studies have shown that PKDs are indispensable for HDAC regulation in B cells [1]. Herein we show that PKDs are also indispensable for HSP27 phosphorylation in B cells. However, PKD-null DT40 B cells are viable and proliferate normally. Moreover, loss of the entire cellular pool of PKD does not critically affect oxidative stress responses in B cells nor do PKD kinases play an essential role in regulating NFκB transcriptional activity. Together, these findings reveal that in B lymphocytes, PKD kinases are not critical regulators of many of the cellular processes previously ascribed to them in other cellular systems. 2 Materials and methods 2.1 Cell culture, transient transfections and cell stimulation The generation, culture and activation of PKD1−/−, PKD3−/− and PKD1/3−/− knockout DT40 B cell lines have been described previously [1]. Cells were lysed and protein extracts were analysed in Western blotting experiments as previously described [1]. Chloramphenicol acetyl transferase assays have been described previously [29]. 2.2 sIgM staining DT40 B cells (2 × 106 cells per point) were resuspended in 200 μl buffer (RPMI 1640 media, 1% foetal calf serum) containing anti-chicken M1 monoclonal antibody conjugated to FITC for 20 min on ice. The cells were washed twice and fluorescent intensity was analysed by flow cytometry. All results shown are representative of at two to four independent experiments unless otherwise indicated. 3 Results 3.1
T2731	4547-4551	NN	denotes	Loss
T2732	4552-4554	IN	denotes	of
T2733	4555-4560	NNP	denotes	HSP27
T2734	4561-4576	NN	denotes	phosphorylation
T2735	4577-4579	IN	denotes	in
T2736	4580-4584	NNP	denotes	DT40
T2737	4585-4586	NNP	denotes	B
T2738	4587-4592	NNS	denotes	cells
T2739	4593-4600	VBG	denotes	lacking
T2740	4601-4611	NN	denotes	expression
T2741	4612-4614	IN	denotes	of
T2742	4615-4618	NNP	denotes	PKD
T2743	4619-4625	NN	denotes	family
T2744	4626-4633	VBZ	denotes	kinases
T2745	4634-4638	NNP	denotes	DT40
T2746	4639-4640	NNP	denotes	B
T2747	4641-4646	NNS	denotes	cells
T2748	4647-4654	VBP	denotes	express
T2749	4655-4658	CD	denotes	two
T2750	4659-4662	NNP	denotes	PKD
T2751	4663-4671	NNS	denotes	isoforms
T2752	4671-4672	,	denotes	,
T2753	4673-4677	NNP	denotes	PKD1
T2754	4678-4681	CC	denotes	and
T2755	4682-4686	NNP	denotes	PKD3
T2756	4686-4687	,	denotes	,
T2757	4688-4691	CC	denotes	and
T2758	4692-4694	IN	denotes	as
T2759	4695-4705	RB	denotes	previously
T2760	4706-4715	VBN	denotes	described
T2761	4716-4718	PRP	denotes	we
T2762	4719-4723	VBP	denotes	have
T2763	4724-4732	RB	denotes	recently
T2764	4733-4742	VBN	denotes	generated
T2765	4743-4747	NNP	denotes	DT40
T2766	4748-4749	NNP	denotes	B
T2767	4750-4754	NN	denotes	cell
T2768	4755-4760	NNS	denotes	lines
T2769	4761-4765	WDT	denotes	that
T2770	4766-4770	VBP	denotes	lack
T2771	4771-4781	NN	denotes	expression
T2772	4782-4784	IN	denotes	of
T2773	4785-4791	DT	denotes	either
T2774	4792-4796	CD	denotes	PKD1
T2775	4797-4799	CC	denotes	or
T2776	4800-4804	CD	denotes	PKD3
T2777	4805-4807	CC	denotes	or
T2778	4808-4812	DT	denotes	both
T2779	4813-4820	NNS	denotes	enzymes
T2780	4821-4822	VBP	denotes	[
T2781	4822-4823	CD	denotes	1
T2782	4823-4824	NNP	denotes	]
T2783	4824-4825	.	denotes	.
T2784	4826-4828	IN	denotes	In
T2785	4829-4839	VBG	denotes	generating
T2786	4840-4843	DT	denotes	the
T2787	4844-4850	JJ	denotes	double
T2788	4851-4859	NN	denotes	knockout
T2789	4860-4864	NN	denotes	cell
T2790	4865-4870	NNS	denotes	lines
T2791	4871-4873	PRP	denotes	we
T2792	4874-4882	VBD	denotes	targeted
T2793	4883-4886	DT	denotes	the
T2794	4887-4891	NNP	denotes	PKD1
T2795	4892-4896	NN	denotes	loci
T2796	4897-4899	IN	denotes	in
T2797	4900-4901	DT	denotes	a
T2798	4902-4906	NNP	denotes	PKD3
T2799	4906-4907	NNP	denotes	−
T2800	4907-4908	VBD	denotes	/
T2801	4908-4909	CD	denotes	−
T2802	4910-4914	NN	denotes	cell
T2803	4915-4919	NN	denotes	line
T2804	4920-4924	WDT	denotes	that
T2805	4925-4934	VBD	denotes	expressed
T2806	4935-4936	DT	denotes	a
T2807	4937-4946	NN	denotes	Flag-PKD3
T2808	4947-4956	NN	denotes	transgene
T2809	4957-4962	IN	denotes	under
T2810	4963-4966	DT	denotes	the
T2811	4967-4974	NN	denotes	control
T2812	4975-4977	IN	denotes	of
T2813	4978-4979	DT	denotes	a
T2814	4980-5001	JJ	denotes	doxycycline-inducible
T2815	5002-5010	NN	denotes	promoter
T2816	5010-5011	.	denotes	.
T2817	5012-5017	RB	denotes	Hence
T2818	5017-5018	,	denotes	,
T2819	5019-5021	IN	denotes	in
T2820	5022-5025	DT	denotes	the
T2821	5026-5034	NN	denotes	presence
T2822	5035-5037	IN	denotes	of
T2823	5038-5049	NN	denotes	doxycycline
T2824	5049-5050	,	denotes	,
T2825	5051-5060	NN	denotes	Flag-PKD3
T2826	5061-5071	NN	denotes	expression
T2827	5072-5074	IN	denotes	in
T2828	5075-5081	CD	denotes	PKD1/3
T2829	5082-5088	JJ	denotes	double
T2830	5089-5097	NN	denotes	knockout
T2831	5098-5103	NNS	denotes	cells
T2832	5104-5106	VBZ	denotes	is
T2833	5107-5117	JJ	denotes	comparable
T2834	5118-5120	TO	denotes	to
T2835	5121-5131	JJ	denotes	endogenous
T2836	5132-5136	JJ	denotes	PKD3
T2837	5137-5144	NN	denotes	present
T2838	5145-5147	IN	denotes	in
T2839	5148-5157	JJ	denotes	wild-type
T2840	5158-5162	NNP	denotes	DT40
T2841	5163-5168	NNS	denotes	cells
T2842	5169-5172	CC	denotes	and
T2843	5173-5180	NN	denotes	removal
T2844	5181-5183	IN	denotes	of
T2845	5184-5195	NN	denotes	doxycycline
T2846	5196-5200	IN	denotes	from
T2847	5201-5204	DT	denotes	the
T2848	5205-5212	NN	denotes	culture
T2849	5213-5218	NNS	denotes	media
T2850	5219-5222	IN	denotes	for
T2851	5223-5224	CD	denotes	5
T2852	5225-5229	NNS	denotes	days
T2853	5230-5237	NNS	denotes	results
T2854	5238-5240	IN	denotes	in
T2855	5241-5242	DT	denotes	a
T2856	5243-5253	RB	denotes	completely
T2857	5254-5258	JJ	denotes	null
T2858	5259-5262	NNP	denotes	PKD
T2859	5263-5272	NN	denotes	phenotype
T2860	5273-5274	-LRB-	denotes	(
T2861	5274-5278	NNP	denotes	Fig.
T2862	5279-5281	NNP	denotes	1A
T2863	5281-5282	-RRB-	denotes	)
T2864	5282-5283	.	denotes	.
T2865	5284-5294	RB	denotes	Previously
T2866	5294-5295	,	denotes	,
T2867	5296-5298	PRP	denotes	we
T2868	5299-5303	VBP	denotes	have
T2869	5304-5316	VBN	denotes	demonstrated
T2870	5317-5321	IN	denotes	that
T2871	5322-5337	NN	denotes	phosphorylation
T2872	5338-5341	CC	denotes	and
T2873	5342-5349	JJ	denotes	nuclear
T2874	5350-5359	NN	denotes	exclusion
T2875	5360-5362	IN	denotes	of
T2876	5363-5368	NN	denotes	class
T2877	5369-5371	NNP	denotes	II
T2878	5372-5379	NN	denotes	histone
T2879	5380-5392	NNS	denotes	deacetylases
T2880	5393-5394	-LRB-	denotes	(
T2881	5394-5399	NNS	denotes	HDACs
T2882	5399-5400	-RRB-	denotes	)
T2883	5401-5407	IN	denotes	during
T2884	5408-5411	NNP	denotes	BCR
T2885	5412-5422	NN	denotes	engagement
T2886	5423-5425	VBZ	denotes	is
T2887	5426-5435	JJ	denotes	defective
T2888	5436-5438	IN	denotes	in
T2889	5439-5445	CD	denotes	PKD1/3
T2890	5445-5446	CD	denotes	−
T2891	5446-5447	NN	denotes	/
T2892	5447-5448	NN	denotes	−
T2893	5449-5450	NNP	denotes	B
T2894	5451-5456	NNS	denotes	cells
T2895	5457-5460	CC	denotes	and
T2896	5461-5464	MD	denotes	can
T2897	5465-5473	VBD	denotes	restored
T2898	5474-5478	IN	denotes	upon
T2899	5479-5492	NN	denotes	re-expression
T2900	5493-5495	IN	denotes	of
T2901	5496-5497	DT	denotes	a
T2902	5498-5504	JJ	denotes	single
T2903	5505-5508	NNP	denotes	PKD
T2904	5509-5516	NN	denotes	isoform
T2905	5517-5518	NNP	denotes	[
T2906	5518-5519	CD	denotes	1
T2907	5519-5520	NNP	denotes	]
T2908	5520-5521	.	denotes	.
T2909	5522-5525	DT	denotes	The
T2910	5526-5531	JJ	denotes	small
T2911	5532-5536	NN	denotes	heat
T2912	5537-5542	NN	denotes	shock
T2913	5543-5550	NN	denotes	protein
T2914	5551-5556	NNP	denotes	HSP27
T2915	5557-5560	VBZ	denotes	has
T2916	5561-5569	RB	denotes	recently
T2917	5570-5574	VBN	denotes	been
T2918	5575-5583	VBN	denotes	proposed
T2919	5584-5586	IN	denotes	as
T2920	5587-5588	DT	denotes	a
T2921	5589-5593	NNP	denotes	PKD1
T2922	5594-5603	VB	denotes	substrate
T2923	5604-5605	NNP	denotes	[
T2924	5605-5607	CD	denotes	24
T2925	5607-5608	NNP	denotes	]
T2926	5609-5612	CC	denotes	and
T2927	5613-5615	PRP	denotes	we
T2928	5616-5627	RB	denotes	accordingly
T2929	5628-5636	VBD	denotes	assessed
T2930	5637-5644	IN	denotes	whether
T2931	5645-5653	NNP	denotes	PKD-null
T2932	5654-5658	NNP	denotes	DT40
T2933	5659-5664	NNS	denotes	cells
T2934	5665-5669	VBP	denotes	have
T2935	5670-5679	JJ	denotes	defective
T2936	5680-5695	NN	denotes	phosphorylation
T2937	5696-5698	IN	denotes	of
T2938	5699-5704	NNP	denotes	HSP27
T2939	5705-5707	IN	denotes	on
T2940	5708-5714	NN	denotes	serine
T2941	5715-5717	CD	denotes	82
T2942	5717-5718	,	denotes	,
T2943	5719-5722	DT	denotes	the
T2944	5723-5731	VBN	denotes	proposed
T2945	5732-5736	NNP	denotes	PKD1
T2946	5737-5746	VB	denotes	substrate
T2947	5747-5755	NN	denotes	sequence
T2948	5755-5756	.	denotes	.
T2949	5757-5759	PRP	denotes	We
T2950	5760-5769	RB	denotes	initially
T2951	5770-5782	VBD	denotes	investigated
T2952	5783-5786	DT	denotes	the
T2953	5787-5797	NN	denotes	regulation
T2954	5798-5800	IN	denotes	of
T2955	5801-5806	NNP	denotes	HSP27
T2956	5807-5822	NN	denotes	phosphorylation
T2957	5823-5825	IN	denotes	in
T2958	5826-5832	JJ	denotes	single
T2959	5833-5841	NN	denotes	knockout
T2960	5842-5846	NNP	denotes	DT40
T2961	5847-5848	NNP	denotes	B
T2962	5849-5854	NNS	denotes	cells
T2963	5855-5862	VBG	denotes	lacking
T2964	5863-5869	CC	denotes	either
T2965	5870-5874	CD	denotes	PKD1
T2966	5875-5877	CC	denotes	or
T2967	5878-5882	CD	denotes	PKD3
T2968	5882-5883	.	denotes	.
T2969	5884-5886	IN	denotes	As
T2970	5887-5892	VBN	denotes	shown
T2971	5893-5895	IN	denotes	in
T2972	5896-5900	NNP	denotes	Fig.
T2973	5901-5903	NNP	denotes	1B
T2974	5903-5904	,	denotes	,
T2975	5905-5915	NN	denotes	activation
T2976	5916-5918	IN	denotes	of
T2977	5919-5922	DT	denotes	the
T2978	5923-5926	NNP	denotes	BCR
T2979	5927-5929	CC	denotes	or
T2980	5930-5939	NN	denotes	treatment
T2981	5940-5944	IN	denotes	with
T2982	5945-5948	DT	denotes	the
T2983	5949-5960	JJ	denotes	DAG-mimetic
T2984	5961-5965	NNP	denotes	PdBu
T2985	5966-5975	VBD	denotes	increased
T2986	5976-5979	DT	denotes	the
T2987	5980-5986	NNS	denotes	levels
T2988	5987-5989	IN	denotes	of
T2989	5990-5995	NNP	denotes	HSP27
T2990	5996-6011	NN	denotes	phosphorylation
T2991	6012-6014	IN	denotes	at
T2992	6015-6018	CD	denotes	S82
T2993	6019-6021	IN	denotes	in
T2994	6022-6031	JJ	denotes	wild-type
T2995	6032-6036	NNP	denotes	DT40
T2996	6037-6038	NNP	denotes	B
T2997	6039-6044	NNS	denotes	cells
T2998	6044-6045	.	denotes	.
T2999	6046-6049	NNP	denotes	BCR
T3000	6050-6053	CC	denotes	and
T3001	6054-6061	JJ	denotes	phorbol
T3002	6062-6067	NN	denotes	ester
T3003	6068-6075	NNS	denotes	signals
T3004	6076-6080	VBD	denotes	were
T3005	6081-6085	RB	denotes	also
T3006	6086-6090	JJ	denotes	able
T3007	6091-6093	TO	denotes	to
T3008	6094-6102	VB	denotes	increase
T3009	6103-6108	NNP	denotes	HSP27
T3010	6109-6124	NN	denotes	phosphorylation
T3011	6125-6127	IN	denotes	in
T3012	6128-6132	CD	denotes	PKD1
T3013	6133-6135	CC	denotes	or
T3014	6136-6140	CD	denotes	PKD3
T3015	6141-6147	JJ	denotes	single
T3016	6148-6156	NN	denotes	knockout
T3017	6157-6161	NNP	denotes	DT40
T3018	6162-6163	NNP	denotes	B
T3019	6164-6169	NNS	denotes	cells
T3020	6170-6171	-LRB-	denotes	(
T3021	6171-6175	NNP	denotes	Fig.
T3022	6176-6178	NNP	denotes	1B
T3023	6178-6179	-RRB-	denotes	)
T3024	6179-6180	.	denotes	.
T3025	6181-6188	RB	denotes	However
T3026	6188-6189	,	denotes	,
T3027	6190-6193	NNP	denotes	BCR
T3028	6193-6194	:	denotes	-
T3029	6195-6198	CC	denotes	and
T3030	6199-6206	VB	denotes	phorbol
T3031	6207-6220	JJ	denotes	ester-induced
T3032	6221-6236	NN	denotes	phosphorylation
T3033	6237-6239	IN	denotes	of
T3034	6240-6245	NNP	denotes	HSP27
T3035	6246-6248	IN	denotes	on
T3036	6249-6252	NNP	denotes	S82
T3037	6253-6256	VBD	denotes	was
T3038	6257-6266	VBN	denotes	abolished
T3039	6267-6269	IN	denotes	in
T3040	6270-6271	NNP	denotes	B
T3041	6272-6277	NNS	denotes	cells
T3042	6278-6282	WDT	denotes	that
T3043	6283-6289	VBD	denotes	lacked
T3044	6290-6294	DT	denotes	both
T3045	6295-6299	NNP	denotes	PKD1
T3046	6300-6303	CC	denotes	and
T3047	6304-6308	NNP	denotes	PKD3
T3048	6309-6310	-LRB-	denotes	(
T3049	6310-6314	NNP	denotes	Fig.
T3050	6315-6317	NNP	denotes	1C
T3051	6317-6318	-RRB-	denotes	)
T3052	6318-6319	.	denotes	.
T3053	6320-6333	RB	denotes	Significantly
T3054	6333-6334	,	denotes	,
T3055	6335-6354	JJ	denotes	doxycycline-induced
T3056	6355-6365	NN	denotes	expression
T3057	6366-6368	IN	denotes	of
T3058	6369-6372	DT	denotes	the
T3059	6373-6382	NN	denotes	Flag-PKD3
T3060	6383-6392	NN	denotes	transgene
T3061	6393-6395	IN	denotes	in
T3062	6396-6399	DT	denotes	the
T3063	6400-6406	JJ	denotes	double
T3064	6407-6415	NN	denotes	knockout
T3065	6416-6421	NNS	denotes	cells
T3066	6422-6425	VBD	denotes	was
T3067	6426-6436	JJ	denotes	sufficient
T3068	6437-6439	TO	denotes	to
T3069	6440-6447	VB	denotes	restore
T3070	6448-6454	JJ	denotes	normal
T3071	6455-6465	NN	denotes	regulation
T3072	6466-6468	IN	denotes	of
T3073	6469-6474	NNP	denotes	HSP27
T3074	6475-6490	NN	denotes	phosphorylation
T3075	6491-6492	-LRB-	denotes	(
T3076	6492-6496	NNP	denotes	Fig.
T3077	6497-6499	NNP	denotes	1C
T3078	6499-6500	-RRB-	denotes	)
T3079	6500-6501	.	denotes	.
T3080	6502-6504	IN	denotes	In
T3081	6505-6513	NN	denotes	contrast
T3082	6513-6514	,	denotes	,
T3083	6515-6525	NN	denotes	expression
T3084	6526-6528	IN	denotes	of
T3085	6529-6530	DT	denotes	a
T3086	6531-6547	JJ	denotes	kinase-deficient
T3087	6548-6552	NN	denotes	PKD3
T3088	6553-6559	JJ	denotes	mutant
T3089	6560-6567	NN	denotes	protein
T3090	6568-6570	IN	denotes	in
T3091	6571-6574	DT	denotes	the
T3092	6575-6581	JJ	denotes	double
T3093	6582-6590	NN	denotes	knockout
T3094	6591-6596	NNS	denotes	cells
T3095	6597-6600	VBD	denotes	was
T3096	6601-6604	RB	denotes	not
T3097	6605-6609	JJ	denotes	able
T3098	6610-6612	TO	denotes	to
T3099	6613-6620	VB	denotes	restore
T3100	6621-6624	NNP	denotes	BCR
T3101	6624-6625	:	denotes	-
T3102	6626-6628	CC	denotes	or
T3103	6629-6636	VB	denotes	phorbol
T3104	6637-6650	JJ	denotes	ester-induced
T3105	6651-6656	NNP	denotes	HSP27
T3106	6657-6672	NN	denotes	phosphorylation
T3107	6673-6674	-LRB-	denotes	(
T3108	6674-6678	NNP	denotes	Fig.
T3109	6679-6681	NNP	denotes	1D
T3110	6681-6682	-RRB-	denotes	)
T3111	6682-6683	.	denotes	.
T3112	6684-6689	RB	denotes	Hence
T3113	6689-6690	,	denotes	,
T3114	6691-6695	NNP	denotes	PKD3
T3115	6696-6698	RB	denotes	as
T3116	6699-6703	RB	denotes	well
T3117	6704-6706	IN	denotes	as
T3118	6707-6711	NNP	denotes	PKD1
T3119	6712-6715	MD	denotes	can
T3120	6716-6724	VB	denotes	regulate
T3121	6725-6730	NNP	denotes	HSP27
T3122	6731-6746	NN	denotes	phosphorylation
T3123	6747-6750	CC	denotes	and
T3124	6751-6753	IN	denotes	in
T3125	6754-6758	NNP	denotes	DT40
T3126	6759-6760	NNP	denotes	B
T3127	6761-6766	NNS	denotes	cells
T3128	6767-6771	PRP	denotes	they
T3129	6772-6775	VBP	denotes	are
T3130	6776-6788	RB	denotes	functionally
T3131	6789-6798	JJ	denotes	redundant
T3132	6799-6801	IN	denotes	as
T3133	6802-6807	NNP	denotes	HSP27
T3134	6808-6815	NNS	denotes	kinases
T3135	6815-6816	.	denotes	.
T4217	0-6823	CD	denotes	Protein kinase D enzymes are dispensable for proliferation, survival and antigen receptor-regulated NFκB activity in vertebrate B-cells Abstract To investigate the importance of protein kinase D (PKD) enzymes we generated a PKD-null DT40 B-lymphocyte cell line. Previously we have shown that PKDs have an essential role in regulating class II histone deacetylases in DT40 B-cells [Matthews, S.A., Liu, P., Spitaler, M., Olson, E.N., McKinsey, T.A., Cantrell, D.A. and Scharenberg, A.M. (2006) Essential role for protein kinase D family kinases in the regulation of class II histone deacetylases in B lymphocytes. Mol. Cell Biol. 26, 1569–1577]. We now show that PKDs are also required to regulate HSP27 phosphorylation in DT40 B-cells. However, in contrast to previous observations in other cell types, PKD enzymes do not regulate basic cellular processes such as proliferation or survival responses, nor NFκB transcriptional activity downstream of the B cell antigen receptor. Thus, PKDs have a selective role in DT40 B-cell biology. 1 Introduction The protein kinase D (PKD) serine/threonine kinase family has three members: PKD1, PKD2 and PKD3. Most cell types express at least two PKD isoforms but PKD enzymes are especially highly expressed in haematopoietic cells, where they are activated in response to antigen receptors stimulation [2,3]. A conserved signalling pathway linking antigen receptors to PKDs involves the activation of PLCγ and the subsequent production of diacylglycerol (DAG) which stimulates classical and/or novel protein kinase Cs (PKC) that phosphorylate two key regulatory serine residues in the activation loop of PKD kinases [3–6]. The N-terminal regulatory region of PKD enzymes contains a DAG binding domain and direct binding of DAG also contributes to PKD1 activation [7] as well as regulating the spatial location of PKD enzymes within cells [8–12]. PKD enzymes have been proposed to regulate numerous cellular functions, including cell proliferation [13–16], anti-apoptotic signals [17,18] and thymocyte development [19]. Expression of mutant catalytically inactive and constitutively activated PKDs can also modify Golgi function, cell adhesion and cell motility (reviewed in [20]). In particular, PKDs have been widely linked to the activation of the NFκB transcription factor and in regulating cell survival during oxidative stress [17,21–23]. Another recently proposed PKD1 substrate is HSP27 [24], a small heat shock protein involved in regulating cell migration and cell survival [25]. An essential role for PKD enzymes in regulating class II histone deacetylases (HDACs), enzymes that repress MEF2-dependent gene transcription, has also been demonstrated [1,26–28]. To investigate the biological role of PKDs we have generated DT40 B cell lines that lack expression of one or more members of the PKD family [1], allowing us to investigate the function(s) of PKD isoforms following B cell antigen receptor (BCR) stimulation, as well addressing the issue of functional redundancy between the different PKD family members. Previous studies have shown that PKDs are indispensable for HDAC regulation in B cells [1]. Herein we show that PKDs are also indispensable for HSP27 phosphorylation in B cells. However, PKD-null DT40 B cells are viable and proliferate normally. Moreover, loss of the entire cellular pool of PKD does not critically affect oxidative stress responses in B cells nor do PKD kinases play an essential role in regulating NFκB transcriptional activity. Together, these findings reveal that in B lymphocytes, PKD kinases are not critical regulators of many of the cellular processes previously ascribed to them in other cellular systems. 2 Materials and methods 2.1 Cell culture, transient transfections and cell stimulation The generation, culture and activation of PKD1−/−, PKD3−/− and PKD1/3−/− knockout DT40 B cell lines have been described previously [1]. Cells were lysed and protein extracts were analysed in Western blotting experiments as previously described [1]. Chloramphenicol acetyl transferase assays have been described previously [29]. 2.2 sIgM staining DT40 B cells (2 × 106 cells per point) were resuspended in 200 μl buffer (RPMI 1640 media, 1% foetal calf serum) containing anti-chicken M1 monoclonal antibody conjugated to FITC for 20 min on ice. The cells were washed twice and fluorescent intensity was analysed by flow cytometry. All results shown are representative of at two to four independent experiments unless otherwise indicated. 3 Results 3.1 Loss of HSP27 phosphorylation in DT40 B cells lacking expression of PKD family kinases DT40 B cells express two PKD isoforms, PKD1 and PKD3, and as previously described we have recently generated DT40 B cell lines that lack expression of either PKD1 or PKD3 or both enzymes [1]. In generating the double knockout cell lines we targeted the PKD1 loci in a PKD3−/− cell line that expressed a Flag-PKD3 transgene under the control of a doxycycline-inducible promoter. Hence, in the presence of doxycycline, Flag-PKD3 expression in PKD1/3 double knockout cells is comparable to endogenous PKD3 present in wild-type DT40 cells and removal of doxycycline from the culture media for 5 days results in a completely null PKD phenotype (Fig. 1A). Previously, we have demonstrated that phosphorylation and nuclear exclusion of class II histone deacetylases (HDACs) during BCR engagement is defective in PKD1/3−/− B cells and can restored upon re-expression of a single PKD isoform [1]. The small heat shock protein HSP27 has recently been proposed as a PKD1 substrate [24] and we accordingly assessed whether PKD-null DT40 cells have defective phosphorylation of HSP27 on serine 82, the proposed PKD1 substrate sequence. We initially investigated the regulation of HSP27 phosphorylation in single knockout DT40 B cells lacking either PKD1 or PKD3. As shown in Fig. 1B, activation of the BCR or treatment with the DAG-mimetic PdBu increased the levels of HSP27 phosphorylation at S82 in wild-type DT40 B cells. BCR and phorbol ester signals were also able to increase HSP27 phosphorylation in PKD1 or PKD3 single knockout DT40 B cells (Fig. 1B). However, BCR- and phorbol ester-induced phosphorylation of HSP27 on S82 was abolished in B cells that lacked both PKD1 and PKD3 (Fig. 1C). Significantly, doxycycline-induced expression of the Flag-PKD3 transgene in the double knockout cells was sufficient to restore normal regulation of HSP27 phosphorylation (Fig. 1C). In contrast, expression of a kinase-deficient PKD3 mutant protein in the double knockout cells was not able to restore BCR- or phorbol ester-induced HSP27 phosphorylation (Fig. 1D). Hence, PKD3 as well as PKD1 can regulate HSP27 phosphorylation and in DT40 B cells they are functionally redundant as HSP27 kinases. 3.2
T4218	6823-6831	NNP	denotes	Cellular
T4219	6832-6845	NN	denotes	proliferation
T4220	6846-6849	CC	denotes	and
T4221	6850-6858	NN	denotes	survival
T4222	6859-6861	IN	denotes	in
T4223	6862-6866	NNP	denotes	DT40
T4224	6867-6868	NNP	denotes	B
T4225	6869-6874	NNS	denotes	cells
T4226	6875-6882	VBG	denotes	lacking
T4227	6883-6893	NN	denotes	expression
T4228	6894-6896	IN	denotes	of
T4229	6897-6900	NNP	denotes	PKD
T4230	6901-6907	NN	denotes	family
T4231	6908-6915	VBZ	denotes	kinases
T4232	6916-6919	NNP	denotes	PKD
T4233	6920-6927	NNS	denotes	enzymes
T4234	6928-6932	VBP	denotes	have
T4235	6933-6943	RB	denotes	previously
T4236	6944-6948	VBN	denotes	been
T4237	6949-6955	VBN	denotes	linked
T4238	6956-6958	TO	denotes	to
T4239	6959-6962	DT	denotes	the
T4240	6963-6973	NN	denotes	regulation
T4241	6974-6976	IN	denotes	of
T4242	6977-6981	NN	denotes	cell
T4243	6982-6995	NN	denotes	proliferation
T4244	6996-6999	CC	denotes	and
T4245	7000-7008	NN	denotes	survival
T4246	7009-7010	-LRB-	denotes	(
T4247	7010-7018	VBN	denotes	reviewed
T4276	7165-7167	IN	denotes	in
T4277	7168-7171	DT	denotes	the
T4278	7172-7180	NN	denotes	presence
T4279	7181-7182	-LRB-	denotes	(
T4280	7182-7188	CD	denotes	PKD1/3
T4281	7188-7189	CD	denotes	−
T4282	7189-7190	NN	denotes	/
T4283	7190-7191	NN	denotes	−
T4284	7191-7192	:	denotes	:
T4285	7193-7202	NN	denotes	Flag-PKD3
T4286	7202-7203	NN	denotes	+
T4287	7203-7205	NN	denotes	ve
T4288	7205-7206	-RRB-	denotes	)
T4289	7207-7209	CC	denotes	or
T4290	7210-7217	NN	denotes	absence
T4291	7218-7219	-LRB-	denotes	(
T4292	7219-7225	CD	denotes	PKD1/3
T4293	7225-7226	CD	denotes	−
T4294	7226-7227	NN	denotes	/
T4295	7227-7228	NN	denotes	−
T4296	7228-7229	-RRB-	denotes	)
T4297	7230-7232	IN	denotes	of
T4298	7233-7244	NN	denotes	doxycycline
T4299	7245-7248	CC	denotes	and
T4300	7249-7258	VBD	denotes	monitored
T4301	7259-7270	JJ	denotes	exponential
T4302	7271-7277	NN	denotes	growth
T4303	7277-7278	.	denotes	.
T4304	7279-7281	IN	denotes	As
T4305	7282-7287	VBN	denotes	shown
T4306	7288-7290	IN	denotes	in
T4307	7291-7295	NNP	denotes	Fig.
T4308	7296-7298	NNP	denotes	2A
T4309	7298-7299	,	denotes	,
T4310	7300-7306	NNP	denotes	PKD1/3
T4311	7306-7307	VBD	denotes	−
T4312	7307-7308	CD	denotes	/
T4313	7308-7309	NN	denotes	−
T4314	7310-7315	NNS	denotes	cells
T4315	7316-7328	VBD	denotes	proliferated
T4316	7329-7342	RB	denotes	exponentially
T4317	7343-7346	CC	denotes	and
T4318	7347-7360	NN	denotes	re-expression
T4319	7361-7363	IN	denotes	of
T4320	7364-7373	NN	denotes	Flag-PKD3
T4321	7374-7376	IN	denotes	in
T4322	7377-7382	DT	denotes	these
T4323	7383-7388	NNS	denotes	cells
T4324	7389-7392	VBD	denotes	had
T4325	7393-7395	DT	denotes	no
T4326	7396-7402	NN	denotes	impact
T4327	7403-7405	IN	denotes	on
T4328	7406-7409	DT	denotes	the
T4329	7410-7414	NN	denotes	rate
T4330	7415-7417	IN	denotes	of
T4331	7418-7431	NN	denotes	proliferation
T4332	7431-7432	.	denotes	.
T4333	7433-7444	RB	denotes	Furthermore
T4334	7444-7445	,	denotes	,
T4335	7446-7449	DT	denotes	the
T4336	7450-7459	NN	denotes	viability
T4337	7460-7462	IN	denotes	of
T4338	7463-7469	CD	denotes	PKD1/3
T4339	7469-7470	CD	denotes	−
T4340	7470-7471	NN	denotes	/
T4341	7471-7472	NN	denotes	−
T4342	7473-7474	NNP	denotes	B
T4343	7475-7480	NNS	denotes	cells
T4344	7481-7487	IN	denotes	during
T4345	7488-7495	JJ	denotes	routine
T4346	7496-7505	NN	denotes	culturing
T4347	7506-7509	VBD	denotes	was
T4348	7510-7513	RB	denotes	not
T4349	7514-7527	RB	denotes	significantly
T4350	7528-7537	JJ	denotes	different
T4351	7538-7542	IN	denotes	from
T4352	7543-7547	DT	denotes	that
T4353	7548-7550	IN	denotes	of
T4354	7551-7560	JJ	denotes	wild-type
T4355	7561-7562	NNP	denotes	B
T4356	7563-7568	NNS	denotes	cells
T4357	7569-7570	-LRB-	denotes	(
T4358	7570-7574	NNS	denotes	data
T4359	7575-7578	RB	denotes	not
T4360	7579-7584	VBN	denotes	shown
T4361	7584-7585	-RRB-	denotes	)
T4362	7585-7586	.	denotes	.
T4363	7587-7589	PRP	denotes	It
T4364	7590-7593	VBD	denotes	was
T4365	7594-7599	VBN	denotes	noted
T4366	7600-7604	IN	denotes	that
T4367	7605-7608	DT	denotes	the
T4368	7609-7619	NN	denotes	population
T4369	7620-7628	VBG	denotes	doubling
T4370	7629-7633	NN	denotes	time
T4371	7634-7636	IN	denotes	of
T4372	7637-7643	CD	denotes	PKD1/3
T4373	7643-7644	CD	denotes	−
T4374	7644-7645	NN	denotes	/
T4375	7645-7646	NN	denotes	−
T4376	7647-7652	NNS	denotes	cells
T4377	7653-7656	VBD	denotes	was
T4378	7657-7665	RB	denotes	slightly
T4379	7666-7672	JJR	denotes	slower
T4380	7673-7677	IN	denotes	than
T4381	7678-7682	DT	denotes	that
T4382	7683-7685	IN	denotes	of
T4383	7686-7690	JJ	denotes	wild
T4384	7691-7695	NN	denotes	type
T4385	7696-7700	CD	denotes	DT40
T4386	7701-7706	NNS	denotes	cells
T4387	7707-7708	-LRB-	denotes	(
T4388	7708-7712	CD	denotes	12.7
T4389	7713-7714	CD	denotes	±
T4390	7715-7718	CD	denotes	2.8
T4391	7719-7720	NN	denotes	h
T4392	7721-7727	IN	denotes	versus
T4393	7728-7732	CD	denotes	10.2
T4394	7733-7734	CD	denotes	±
T4395	7735-7738	CD	denotes	0.4
T4396	7739-7740	NN	denotes	h
T4397	7740-7741	-RRB-	denotes	)
T4398	7742-7745	CC	denotes	but
T4399	7746-7749	DT	denotes	the
T4400	7750-7757	NN	denotes	failure
T4401	7758-7760	IN	denotes	of
T4402	7761-7765	CD	denotes	PKD3
T4403	7766-7779	NN	denotes	re-expression
T4404	7780-7782	TO	denotes	to
T4405	7783-7789	VB	denotes	modify
T4406	7790-7793	DT	denotes	the
T4407	7794-7807	NN	denotes	proliferation
T4408	7808-7812	NN	denotes	rate
T4409	7813-7815	IN	denotes	of
T4410	7816-7822	CD	denotes	PKD1/3
T4411	7822-7823	CD	denotes	−
T4412	7823-7824	NN	denotes	/
T4413	7824-7825	NN	denotes	−
T4414	7826-7831	NNS	denotes	cells
T4415	7832-7840	VBZ	denotes	suggests
T4416	7841-7845	IN	denotes	that
T4417	7846-7851	DT	denotes	these
T4418	7852-7857	JJ	denotes	small
T4419	7858-7869	NNS	denotes	differences
T4420	7870-7874	VBD	denotes	were
T4421	7875-7879	RBS	denotes	most
T4422	7880-7886	JJ	denotes	likely
T4423	7887-7890	DT	denotes	the
T4424	7891-7897	NN	denotes	result
T4425	7898-7900	IN	denotes	of
T4426	7901-7907	JJ	denotes	clonal
T4427	7908-7917	NN	denotes	variation
T4428	7918-7921	CC	denotes	and
T4429	7922-7926	VBD	denotes	were
T4430	7927-7930	RB	denotes	not
T4431	7931-7937	VBN	denotes	caused
T4432	7938-7950	RB	denotes	specifically
T4433	7951-7953	IN	denotes	by
T4434	7954-7958	NN	denotes	loss
T4435	7959-7961	IN	denotes	of
T4436	7962-7965	NNP	denotes	PKD
T4437	7966-7973	VBZ	denotes	enzymes
T4438	7973-7974	.	denotes	.
T4439	7975-7979	RB	denotes	Thus
T4440	7979-7980	,	denotes	,
T4441	7981-7984	NNP	denotes	PKD
T4442	7985-7991	NN	denotes	family
T4443	7992-7999	NNS	denotes	enzymes
T4444	8000-8003	VBP	denotes	are
T4445	8004-8007	RB	denotes	not
T4446	8008-8017	JJ	denotes	essential
T4447	8018-8021	IN	denotes	for
T4448	8022-8032	VBG	denotes	regulating
T4449	8033-8038	JJ	denotes	basal
T4450	8039-8047	NN	denotes	survival
T4451	8048-8051	CC	denotes	and
T4452	8052-8065	NN	denotes	proliferation
T4453	8066-8068	IN	denotes	of
T4454	8069-8073	NNP	denotes	DT40
T4455	8074-8075	NNP	denotes	B
T4456	8076-8081	NNS	denotes	cells
T4457	8081-8082	.	denotes	.
T4458	8083-8086	NNP	denotes	PKD
T4459	8087-8094	VBZ	denotes	enzymes
T4460	8094-8095	,	denotes	,
T4461	8096-8108	RB	denotes	specifically
T4462	8109-8113	CD	denotes	PKD1
T4463	8114-8117	CC	denotes	and
T4464	8118-8122	NNP	denotes	PKD2
T4465	8122-8123	,	denotes	,
T4466	8124-8128	VBP	denotes	have
T4467	8129-8139	RB	denotes	previously
T4468	8140-8144	VBN	denotes	been
T4469	8145-8151	VBN	denotes	linked
T4470	8152-8154	TO	denotes	to
T4471	8155-8156	DT	denotes	a
T4472	8157-8167	JJ	denotes	protective
T4473	8168-8172	NN	denotes	role
T4474	8173-8180	IN	denotes	against
T4475	8181-8190	JJ	denotes	oxidative
T4476	8191-8205	JJ	denotes	stress-induced
T4477	8206-8212	NN	denotes	injury
T4478	8213-8215	IN	denotes	in
T4479	8216-8219	NNP	denotes	3T3
T4480	8220-8230	NN	denotes	fibroblast
T4481	8230-8231	,	denotes	,
T4482	8232-8236	NNP	denotes	HeLa
T4483	8237-8240	CC	denotes	and
T4484	8241-8251	JJ	denotes	epithelial
T4485	8252-8256	NN	denotes	cell
T4486	8257-8262	NNS	denotes	lines
T4487	8263-8264	NNP	denotes	[
T4488	8264-8269	CD	denotes	17,30
T4489	8270-8272	CD	denotes	32
T4490	8272-8273	NN	denotes	]
T4491	8273-8274	.	denotes	.
T4492	8275-8277	PRP	denotes	We
T4493	8278-8287	RB	denotes	therefore
T4494	8288-8297	VBD	denotes	addressed
T4495	8298-8301	DT	denotes	the
T4496	8302-8306	NN	denotes	role
T4497	8307-8309	IN	denotes	of
T4498	8310-8313	NNP	denotes	PKD
T4499	8314-8320	NN	denotes	family
T4500	8321-8328	NNS	denotes	kinases
T4501	8329-8331	IN	denotes	in
T4502	8332-8342	VBG	denotes	regulating
T4503	8343-8344	NNP	denotes	B
T4504	8345-8349	NN	denotes	cell
T4505	8350-8358	NN	denotes	survival
T4506	8359-8361	IN	denotes	in
T4507	8362-8370	NN	denotes	response
T4508	8371-8373	TO	denotes	to
T4509	8374-8383	JJ	denotes	oxidative
T4510	8384-8390	NN	denotes	stress
T4511	8391-8394	CC	denotes	and
T4512	8395-8400	JJ	denotes	other
T4513	8401-8407	NN	denotes	stress
T4514	8408-8415	NNS	denotes	stimuli
T4515	8415-8416	.	denotes	.
T4516	8417-8419	IN	denotes	As
T4517	8420-8425	VBN	denotes	shown
T4518	8426-8428	IN	denotes	in
T4519	8429-8433	NNP	denotes	Fig.
T4520	8434-8436	NNP	denotes	2B
T4521	8436-8437	,	denotes	,
T4522	8438-8442	NN	denotes	loss
T4523	8443-8445	IN	denotes	of
T4524	8446-8452	CD	denotes	PKD1/3
T4525	8453-8463	NN	denotes	expression
T4526	8464-8467	VBD	denotes	had
T4527	8468-8470	DT	denotes	no
T4528	8471-8482	JJ	denotes	significant
T4529	8483-8489	NN	denotes	impact
T4530	8490-8492	IN	denotes	on
T4531	8493-8496	DT	denotes	the
T4532	8497-8505	NN	denotes	survival
T4533	8506-8508	IN	denotes	of
T4534	8509-8513	NNP	denotes	DT40
T4535	8514-8515	NNP	denotes	B
T4536	8516-8521	NNS	denotes	cells
T4537	8522-8524	IN	denotes	in
T4538	8525-8533	NN	denotes	response
T4539	8534-8536	TO	denotes	to
T4540	8537-8550	JJ	denotes	mitochondrial
T4541	8551-8557	NN	denotes	stress
T4542	8558-8565	NNS	denotes	stimuli
T4543	8566-8567	-LRB-	denotes	(
T4544	8567-8571	CD	denotes	H2O2
T4545	8572-8574	CC	denotes	or
T4546	8575-8580	NN	denotes	serum
T4547	8581-8592	NN	denotes	deprivation
T4548	8592-8593	-RRB-	denotes	)
T4549	8593-8594	:	denotes	;
T4550	8595-8598	NNP	denotes	DNA
T4551	8599-8607	VBG	denotes	damaging
T4552	8608-8614	NNS	denotes	agents
T4553	8615-8616	-LRB-	denotes	(
T4554	8616-8625	NN	denotes	etoposide
T4555	8626-8628	CC	denotes	or
T4556	8629-8640	NN	denotes	doxorubicin
T4557	8640-8641	-RRB-	denotes	)
T4558	8641-8642	:	denotes	;
T4559	8643-8645	NNP	denotes	ER
T4560	8646-8653	NN	denotes	pathway
T4561	8654-8660	NN	denotes	stress
T4562	8661-8664	JJ	denotes	due
T4563	8665-8667	TO	denotes	to
T4564	8668-8675	NN	denotes	calcium
T4565	8676-8684	NN	denotes	overload
T4566	8685-8686	-LRB-	denotes	(
T4567	8686-8698	NN	denotes	thapsigargin
T4568	8698-8699	-RRB-	denotes	)
T4569	8700-8702	CC	denotes	or
T4570	8703-8712	VBG	denotes	following
T4571	8713-8722	JJ	denotes	prolonged
T4572	8723-8732	NN	denotes	treatment
T4573	8733-8737	IN	denotes	with
T4574	8738-8745	NN	denotes	phorbol
T4575	8746-8752	NNS	denotes	esters
T4576	8753-8755	CC	denotes	or
T4577	8756-8768	NNP	denotes	Trichostatin
T4578	8769-8770	NNP	denotes	A
T4579	8770-8771	,	denotes	,
T4580	8772-8774	DT	denotes	an
T4581	8775-8784	NN	denotes	inhibitor
T4582	8785-8787	IN	denotes	of
T4583	8788-8793	NN	denotes	class
T4584	8794-8798	NNP	denotes	I/II
T4585	8799-8804	NNP	denotes	HDACs
T4586	8804-8805	.	denotes	.
T4587	8806-8810	RB	denotes	Thus
T4588	8810-8811	,	denotes	,
T4589	8812-8815	NNP	denotes	PKD
T4590	8816-8823	NNS	denotes	kinases
T4591	8824-8826	VBP	denotes	do
T4592	8827-8830	RB	denotes	not
T4593	8831-8835	VB	denotes	play
T4594	8836-8838	DT	denotes	an
T4595	8839-8848	JJ	denotes	essential
T4596	8849-8853	NN	denotes	role
T4597	8854-8856	IN	denotes	in
T4598	8857-8867	VBG	denotes	regulating
T4599	8868-8869	NNP	denotes	B
T4600	8870-8874	NN	denotes	cell
T4601	8875-8883	NN	denotes	survival
T4602	8884-8886	IN	denotes	in
T4603	8887-8895	NN	denotes	response
T4604	8896-8898	TO	denotes	to
T4605	8899-8900	DT	denotes	a
T4606	8901-8906	NN	denotes	range
T4607	8907-8909	IN	denotes	of
T4608	8910-8919	JJ	denotes	different
T4609	8920-8926	NN	denotes	stress
T4610	8927-8934	NNS	denotes	stimuli
T4611	8934-8935	.	denotes	.
T5768	0-8942	CD	denotes	Protein kinase D enzymes are dispensable for proliferation, survival and antigen receptor-regulated NFκB activity in vertebrate B-cells Abstract To investigate the importance of protein kinase D (PKD) enzymes we generated a PKD-null DT40 B-lymphocyte cell line. Previously we have shown that PKDs have an essential role in regulating class II histone deacetylases in DT40 B-cells [Matthews, S.A., Liu, P., Spitaler, M., Olson, E.N., McKinsey, T.A., Cantrell, D.A. and Scharenberg, A.M. (2006) Essential role for protein kinase D family kinases in the regulation of class II histone deacetylases in B lymphocytes. Mol. Cell Biol. 26, 1569–1577]. We now show that PKDs are also required to regulate HSP27 phosphorylation in DT40 B-cells. However, in contrast to previous observations in other cell types, PKD enzymes do not regulate basic cellular processes such as proliferation or survival responses, nor NFκB transcriptional activity downstream of the B cell antigen receptor. Thus, PKDs have a selective role in DT40 B-cell biology. 1 Introduction The protein kinase D (PKD) serine/threonine kinase family has three members: PKD1, PKD2 and PKD3. Most cell types express at least two PKD isoforms but PKD enzymes are especially highly expressed in haematopoietic cells, where they are activated in response to antigen receptors stimulation [2,3]. A conserved signalling pathway linking antigen receptors to PKDs involves the activation of PLCγ and the subsequent production of diacylglycerol (DAG) which stimulates classical and/or novel protein kinase Cs (PKC) that phosphorylate two key regulatory serine residues in the activation loop of PKD kinases [3–6]. The N-terminal regulatory region of PKD enzymes contains a DAG binding domain and direct binding of DAG also contributes to PKD1 activation [7] as well as regulating the spatial location of PKD enzymes within cells [8–12]. PKD enzymes have been proposed to regulate numerous cellular functions, including cell proliferation [13–16], anti-apoptotic signals [17,18] and thymocyte development [19]. Expression of mutant catalytically inactive and constitutively activated PKDs can also modify Golgi function, cell adhesion and cell motility (reviewed in [20]). In particular, PKDs have been widely linked to the activation of the NFκB transcription factor and in regulating cell survival during oxidative stress [17,21–23]. Another recently proposed PKD1 substrate is HSP27 [24], a small heat shock protein involved in regulating cell migration and cell survival [25]. An essential role for PKD enzymes in regulating class II histone deacetylases (HDACs), enzymes that repress MEF2-dependent gene transcription, has also been demonstrated [1,26–28]. To investigate the biological role of PKDs we have generated DT40 B cell lines that lack expression of one or more members of the PKD family [1], allowing us to investigate the function(s) of PKD isoforms following B cell antigen receptor (BCR) stimulation, as well addressing the issue of functional redundancy between the different PKD family members. Previous studies have shown that PKDs are indispensable for HDAC regulation in B cells [1]. Herein we show that PKDs are also indispensable for HSP27 phosphorylation in B cells. However, PKD-null DT40 B cells are viable and proliferate normally. Moreover, loss of the entire cellular pool of PKD does not critically affect oxidative stress responses in B cells nor do PKD kinases play an essential role in regulating NFκB transcriptional activity. Together, these findings reveal that in B lymphocytes, PKD kinases are not critical regulators of many of the cellular processes previously ascribed to them in other cellular systems. 2 Materials and methods 2.1 Cell culture, transient transfections and cell stimulation The generation, culture and activation of PKD1−/−, PKD3−/− and PKD1/3−/− knockout DT40 B cell lines have been described previously [1]. Cells were lysed and protein extracts were analysed in Western blotting experiments as previously described [1]. Chloramphenicol acetyl transferase assays have been described previously [29]. 2.2 sIgM staining DT40 B cells (2 × 106 cells per point) were resuspended in 200 μl buffer (RPMI 1640 media, 1% foetal calf serum) containing anti-chicken M1 monoclonal antibody conjugated to FITC for 20 min on ice. The cells were washed twice and fluorescent intensity was analysed by flow cytometry. All results shown are representative of at two to four independent experiments unless otherwise indicated. 3 Results 3.1 Loss of HSP27 phosphorylation in DT40 B cells lacking expression of PKD family kinases DT40 B cells express two PKD isoforms, PKD1 and PKD3, and as previously described we have recently generated DT40 B cell lines that lack expression of either PKD1 or PKD3 or both enzymes [1]. In generating the double knockout cell lines we targeted the PKD1 loci in a PKD3−/− cell line that expressed a Flag-PKD3 transgene under the control of a doxycycline-inducible promoter. Hence, in the presence of doxycycline, Flag-PKD3 expression in PKD1/3 double knockout cells is comparable to endogenous PKD3 present in wild-type DT40 cells and removal of doxycycline from the culture media for 5 days results in a completely null PKD phenotype (Fig. 1A). Previously, we have demonstrated that phosphorylation and nuclear exclusion of class II histone deacetylases (HDACs) during BCR engagement is defective in PKD1/3−/− B cells and can restored upon re-expression of a single PKD isoform [1]. The small heat shock protein HSP27 has recently been proposed as a PKD1 substrate [24] and we accordingly assessed whether PKD-null DT40 cells have defective phosphorylation of HSP27 on serine 82, the proposed PKD1 substrate sequence. We initially investigated the regulation of HSP27 phosphorylation in single knockout DT40 B cells lacking either PKD1 or PKD3. As shown in Fig. 1B, activation of the BCR or treatment with the DAG-mimetic PdBu increased the levels of HSP27 phosphorylation at S82 in wild-type DT40 B cells. BCR and phorbol ester signals were also able to increase HSP27 phosphorylation in PKD1 or PKD3 single knockout DT40 B cells (Fig. 1B). However, BCR- and phorbol ester-induced phosphorylation of HSP27 on S82 was abolished in B cells that lacked both PKD1 and PKD3 (Fig. 1C). Significantly, doxycycline-induced expression of the Flag-PKD3 transgene in the double knockout cells was sufficient to restore normal regulation of HSP27 phosphorylation (Fig. 1C). In contrast, expression of a kinase-deficient PKD3 mutant protein in the double knockout cells was not able to restore BCR- or phorbol ester-induced HSP27 phosphorylation (Fig. 1D). Hence, PKD3 as well as PKD1 can regulate HSP27 phosphorylation and in DT40 B cells they are functionally redundant as HSP27 kinases. 3.2 Cellular proliferation and survival in DT40 B cells lacking expression of PKD family kinases PKD enzymes have previously been linked to the regulation of cell proliferation and survival (reviewed in [20]). To investigate the effect that loss of PKD kinases had on B cell survival and/or proliferation we cultured wild-type and PKD-null cells in the presence (PKD1/3−/−: Flag-PKD3+ve) or absence (PKD1/3−/−) of doxycycline and monitored exponential growth. As shown in Fig. 2A, PKD1/3−/− cells proliferated exponentially and re-expression of Flag-PKD3 in these cells had no impact on the rate of proliferation. Furthermore, the viability of PKD1/3−/− B cells during routine culturing was not significantly different from that of wild-type B cells (data not shown). It was noted that the population doubling time of PKD1/3−/− cells was slightly slower than that of wild type DT40 cells (12.7 ± 2.8 h versus 10.2 ± 0.4 h) but the failure of PKD3 re-expression to modify the proliferation rate of PKD1/3−/− cells suggests that these small differences were most likely the result of clonal variation and were not caused specifically by loss of PKD enzymes. Thus, PKD family enzymes are not essential for regulating basal survival and proliferation of DT40 B cells. PKD enzymes, specifically PKD1 and PKD2, have previously been linked to a protective role against oxidative stress-induced injury in 3T3 fibroblast, HeLa and epithelial cell lines [17,30–32]. We therefore addressed the role of PKD family kinases in regulating B cell survival in response to oxidative stress and other stress stimuli. As shown in Fig. 2B, loss of PKD1/3 expression had no significant impact on the survival of DT40 B cells in response to mitochondrial stress stimuli (H2O2 or serum deprivation); DNA damaging agents (etoposide or doxorubicin); ER pathway stress due to calcium overload (thapsigargin) or following prolonged treatment with phorbol esters or Trichostatin A, an inhibitor of class I/II HDACs. Thus, PKD kinases do not play an essential role in regulating B cell survival in response to a range of different stress stimuli. 3.3
T4248	7019-7021	IN	denotes	in
T4249	7022-7023	NNP	denotes	[
T4250	7023-7025	CD	denotes	20
T4251	7025-7026	NNP	denotes	]
T4252	7026-7027	-RRB-	denotes	)
T4253	7027-7028	.	denotes	.
T4254	7029-7031	TO	denotes	To
T4255	7032-7043	VB	denotes	investigate
T4256	7044-7047	DT	denotes	the
T4257	7048-7054	NN	denotes	effect
T4258	7055-7059	WDT	denotes	that
T4259	7060-7064	NN	denotes	loss
T4260	7065-7067	IN	denotes	of
T4261	7068-7071	NNP	denotes	PKD
T4262	7072-7079	NNS	denotes	kinases
T4263	7080-7083	VBD	denotes	had
T4264	7084-7086	IN	denotes	on
T4265	7087-7088	NNP	denotes	B
T4266	7089-7093	NN	denotes	cell
T4267	7094-7102	NN	denotes	survival
T4268	7103-7109	CC	denotes	and/or
T4269	7110-7123	NN	denotes	proliferation
T4270	7124-7126	PRP	denotes	we
T4271	7127-7135	VBD	denotes	cultured
T4272	7136-7145	JJ	denotes	wild-type
T4273	7146-7149	CC	denotes	and
T4274	7150-7158	JJ	denotes	PKD-null
T4275	7159-7164	NNS	denotes	cells
R204	T241	T231	dobj	enzymes,investigate
R205	T242	T243	nsubj	we,generated
R206	T243	T241	relcl	generated,enzymes
R207	T244	T249	det	a,line
R208	T245	T249	compound	PKD-null,line
R209	T246	T247	compound	DT40,B-lymphocyte
R210	T247	T249	compound	B-lymphocyte,line
R211	T248	T249	compound	cell,line
R212	T249	T243	dobj	line,generated
R213	T250	T216	punct	.,are
R214	T251	T254	advmod	Previously,shown
R215	T252	T254	nsubj	we,shown
R216	T253	T254	aux	have,shown
R217	T254	T254	ROOT	shown,shown
R218	T255	T257	mark	that,have
R219	T256	T257	nsubj	PKDs,have
R220	T257	T254	ccomp	have,shown
R221	T258	T260	det	an,role
R222	T259	T260	amod	essential,role
R223	T260	T257	dobj	role,have
R224	T261	T260	prep	in,role
R225	T262	T261	pcomp	regulating,in
R226	T263	T266	compound	class,deacetylases
R227	T264	T266	compound	II,deacetylases
R228	T265	T266	compound	histone,deacetylases
R229	T266	T262	dobj	deacetylases,regulating
R230	T267	T262	prep	in,regulating
R231	T268	T271	compound	DT40,Matthews
R232	T269	T271	compound	B-cells,Matthews
R233	T270	T271	compound	[,Matthews
R234	T271	T267	pobj	Matthews,in
R235	T272	T271	punct	",",Matthews
R236	T273	T271	conj	S.A.,Matthews
R237	T274	T271	punct	",",Matthews
R238	T275	T271	conj	Liu,Matthews
R239	T276	T275	punct	",",Liu
R240	T277	T275	conj	P.,Liu
R241	T278	T277	punct	",",P.
R242	T279	T277	conj	Spitaler,P.
R243	T280	T279	punct	",",Spitaler
R244	T281	T279	conj	M.,Spitaler
R245	T282	T281	punct	",",M.
R246	T283	T281	conj	Olson,M.
R247	T284	T283	punct	",",Olson
R248	T285	T283	conj	E.N.,Olson
R249	T286	T285	punct	",",E.N.
R250	T287	T285	conj	McKinsey,E.N.
R251	T288	T287	punct	",",McKinsey
R252	T289	T287	conj	T.A.,McKinsey
R253	T290	T287	punct	",",McKinsey
R254	T291	T287	conj	Cantrell,McKinsey
R255	T292	T291	punct	",",Cantrell
R256	T293	T291	conj	D.A.,Cantrell
R257	T294	T293	cc	and,D.A.
R258	T295	T293	conj	Scharenberg,D.A.
R259	T296	T295	punct	",",Scharenberg
R260	T297	T295	conj	A.M.,Scharenberg
R261	T298	T297	punct	(,A.M.
R262	T299	T297	appos	2006,A.M.
R263	T300	T297	punct	),A.M.
R264	T301	T302	amod	Essential,role
R265	T302	T260	appos	role,role
R266	T303	T302	prep	for,role
R267	T304	T303	pobj	protein,for
R268	T305	T308	compound	kinase,kinases
R269	T306	T307	compound	D,family
R270	T307	T308	compound	family,kinases
R271	T308	T302	conj	kinases,role
R272	T309	T308	prep	in,kinases
R273	T310	T311	det	the,regulation
R274	T311	T309	pobj	regulation,in
R275	T312	T311	prep	of,regulation
R276	T313	T316	compound	class,deacetylases
R277	T314	T316	compound	II,deacetylases
R278	T315	T316	compound	histone,deacetylases
R279	T316	T312	pobj	deacetylases,of
R280	T317	T316	prep	in,deacetylases
R281	T318	T319	compound	B,lymphocytes
R282	T319	T317	pobj	lymphocytes,in
R283	T320	T254	punct	.,shown
R284	T321	T321	ROOT	Mol,Mol
R285	T322	T321	punct	.,Mol
R286	T323	T324	compound	Cell,Biol
R287	T324	T324	ROOT	Biol,Biol
R288	T325	T326	punct	.,26
R289	T326	T324	nummod	26,Biol
R290	T327	T324	punct	",",Biol
R291	T328	T324	appos	1569,Biol
R292	T329	T324	appos	1577,Biol
R293	T330	T330	ROOT	],]
R294	T331	T324	punct	.,Biol
R295	T332	T334	nsubj	We,show
R296	T333	T334	advmod	now,show
R297	T334	T334	ROOT	show,show
R298	T335	T339	mark	that,required
R299	T336	T339	nsubjpass	PKDs,required
R300	T337	T339	auxpass	are,required
R301	T338	T339	advmod	also,required
R302	T339	T334	ccomp	required,show
R303	T340	T341	aux	to,regulate
R304	T341	T339	xcomp	regulate,required
R305	T342	T343	compound	HSP27,phosphorylation
R306	T343	T341	dobj	phosphorylation,regulate
R307	T344	T343	prep	in,phosphorylation
R308	T345	T346	compound	DT40,B-cells
R309	T346	T344	pobj	B-cells,in
R310	T347	T334	punct	.,show
R311	T348	T364	advmod	However,regulate
R312	T349	T364	punct	",",regulate
R313	T350	T364	prep	in,regulate
R314	T351	T350	pobj	contrast,in
R315	T352	T351	prep	to,contrast
R316	T353	T354	amod	previous,observations
R317	T354	T352	pobj	observations,to
R318	T355	T354	prep	in,observations
R319	T356	T358	amod	other,types
R320	T357	T358	compound	cell,types
R321	T358	T355	pobj	types,in
R322	T359	T364	punct	",",regulate
R323	T360	T364	nsubj	PKD,regulate
R324	T361	T364	nsubj	enzymes,regulate
R325	T362	T364	aux	do,regulate
R326	T363	T364	neg	not,regulate
R327	T364	T364	ROOT	regulate,regulate
R328	T365	T367	amod	basic,processes
R329	T366	T367	amod	cellular,processes
R330	T367	T364	dobj	processes,regulate
R331	T368	T369	amod	such,as
R332	T369	T367	prep	as,processes
R333	T370	T369	pobj	proliferation,as
R334	T371	T370	cc	or,proliferation
R335	T372	T370	conj	survival,proliferation
R336	T373	T370	conj	responses,proliferation
R337	T374	T373	punct	",",responses
R338	T375	T364	cc	nor,regulate
R339	T376	T378	nmod	NFκB,activity
R340	T377	T378	amod	transcriptional,activity
R341	T378	T364	conj	activity,regulate
R342	T379	T378	amod	downstream,activity
R343	T380	T379	prep	of,downstream
R344	T381	T385	det	the,receptor
R345	T382	T384	compound	B,antigen
R346	T383	T384	compound	cell,antigen
R347	T384	T385	compound	antigen,receptor
R348	T385	T380	pobj	receptor,of
R349	T386	T364	punct	.,regulate
R350	T387	T390	advmod	Thus,have
R351	T388	T390	punct	",",have
R352	T389	T390	nsubj	PKDs,have
R353	T390	T390	ROOT	have,have
R354	T391	T393	det	a,role
R355	T392	T393	amod	selective,role
R356	T393	T390	dobj	role,have
R357	T394	T393	prep	in,role
R358	T395	T396	compound	DT40,B-cell
R359	T396	T397	compound	B-cell,biology
R360	T397	T394	pobj	biology,in
R361	T398	T390	punct	.,have
R894	T1008	T1011	amod	"1569–1577]. We now show that PKDs are also required to regulate HSP27 phosphorylation in DT40 B-cells. However, in contrast to previous observations in other cell types, PKD enzymes do not regulate basic cellular processes such as proliferation or survival responses, nor NFκB transcriptional activity downstream of the B cell antigen receptor. Thus, PKDs have a selective role in DT40 B-cell biology. 1 Introduction The",D
R895	T1009	T1011	nmod	protein,D
R896	T1010	T1011	compound	kinase,D
R897	T1011	T1011	ROOT	D,D
R898	T1012	T1011	punct	(,D
R899	T1013	T1011	appos	PKD,D
R900	T1014	T1011	punct	),D
R901	T1015	T1017	amod	serine/threonine,family
R902	T1016	T1017	compound	kinase,family
R903	T1017	T1018	nsubj	family,has
R904	T1018	T1018	ROOT	has,has
R905	T1019	T1020	nummod	three,members
R906	T1020	T1018	dobj	members,has
R907	T1021	T1018	punct	:,has
R908	T1022	T1022	ROOT	PKD1,PKD1
R909	T1023	T1022	punct	",",PKD1
R910	T1024	T1022	conj	PKD2,PKD1
R911	T1025	T1024	cc	and,PKD2
R912	T1026	T1024	conj	PKD3,PKD2
R913	T1027	T1018	punct	.,has
R914	T1028	T1030	amod	Most,types
R915	T1029	T1030	compound	cell,types
R916	T1030	T1031	nsubj	types,express
R917	T1031	T1031	ROOT	express,express
R918	T1032	T1033	advmod	at,least
R919	T1033	T1034	advmod	least,two
R920	T1034	T1036	nummod	two,isoforms
R921	T1035	T1036	compound	PKD,isoforms
R922	T1036	T1031	dobj	isoforms,express
R923	T1037	T1031	cc	but,express
R924	T1038	T1039	compound	PKD,enzymes
R925	T1039	T1040	nsubj	enzymes,are
R926	T1040	T1043	auxpass	are,expressed
R927	T1041	T1042	advmod	especially,highly
R928	T1042	T1043	advmod	highly,expressed
R929	T1043	T1031	conj	expressed,express
R930	T1044	T1043	prep	in,expressed
R931	T1045	T1046	amod	haematopoietic,cells
R932	T1046	T1044	pobj	cells,in
R933	T1047	T1043	punct	",",expressed
R934	T1048	T1051	advmod	where,activated
R935	T1049	T1051	nsubjpass	they,activated
R936	T1050	T1051	auxpass	are,activated
R937	T1051	T1043	advcl	activated,expressed
R938	T1052	T1051	prep	in,activated
R939	T1053	T1052	pobj	response,in
R940	T1054	T1053	prep	to,response
R941	T1055	T1056	compound	antigen,receptors
R942	T1056	T1057	compound	receptors,stimulation
R943	T1057	T1054	pobj	stimulation,to
R944	T1058	T1060	compound	[,]
R945	T1059	T1060	compound	"2,3",]
R946	T1060	T1057	appos	],stimulation
R947	T1061	T1043	punct	.,expressed
R948	T1062	T1065	det	A,pathway
R949	T1063	T1065	amod	conserved,pathway
R950	T1064	T1065	amod	signalling,pathway
R951	T1065	T1071	nsubj	pathway,involves
R952	T1066	T1065	acl	linking,pathway
R953	T1067	T1068	compound	antigen,receptors
R954	T1068	T1066	dobj	receptors,linking
R955	T1069	T1066	prep	to,linking
R956	T1070	T1069	pobj	PKDs,to
R957	T1071	T1071	ROOT	involves,involves
R958	T1072	T1073	det	the,activation
R959	T1073	T1071	dobj	activation,involves
R960	T1074	T1073	prep	of,activation
R961	T1075	T1074	pobj	PLCγ,of
R962	T1076	T1073	cc	and,activation
R963	T1077	T1079	det	the,production
R964	T1078	T1079	amod	subsequent,production
R965	T1079	T1073	conj	production,activation
R966	T1080	T1079	prep	of,production
R967	T1081	T1080	pobj	diacylglycerol,of
R968	T1082	T1083	punct	(,DAG
R969	T1083	T1081	appos	DAG,diacylglycerol
R970	T1084	T1081	punct	),diacylglycerol
R971	T1085	T1086	nsubj	which,stimulates
R972	T1086	T1079	relcl	stimulates,production
R973	T1087	T1090	amod	classical,protein
R974	T1088	T1087	cc	and/or,classical
R975	T1089	T1087	conj	novel,classical
R976	T1090	T1092	compound	protein,Cs
R977	T1091	T1092	compound	kinase,Cs
R978	T1092	T1086	dobj	Cs,stimulates
R979	T1093	T1092	punct	(,Cs
R980	T1094	T1092	appos	PKC,Cs
R981	T1095	T1092	punct	),Cs
R982	T1096	T1109	nsubj	that,kinases
R983	T1097	T1096	aux	phosphorylate,that
R984	T1098	T1102	nummod	two,residues
R985	T1099	T1102	amod	key,residues
R986	T1100	T1102	amod	regulatory,residues
R987	T1101	T1102	compound	serine,residues
R988	T1102	T1097	dobj	residues,phosphorylate
R989	T1103	T1102	prep	in,residues
R990	T1104	T1106	det	the,loop
R991	T1105	T1106	compound	activation,loop
R992	T1106	T1103	pobj	loop,in
R993	T1107	T1106	prep	of,loop
R994	T1108	T1107	pobj	PKD,of
R995	T1109	T1079	relcl	kinases,production
R996	T1110	T1109	dobj	[,kinases
R997	T1111	T1110	nummod	3,[
R998	T1112	T1113	nummod	6,]
R999	T1113	T1079	appos	],production
R1000	T1114	T1071	punct	.,involves
R1001	T1115	T1118	det	The,region
R1002	T1116	T1118	amod	N-terminal,region
R1003	T1117	T1118	amod	regulatory,region
R1004	T1118	T1121	nsubj	region,enzymes
R1005	T1119	T1118	prep	of,region
R1006	T1120	T1119	pobj	PKD,of
R1007	T1121	T1121	ROOT	enzymes,enzymes
R1008	T1122	T1121	conj	contains,enzymes
R1009	T1123	T1124	det	a,DAG
R1010	T1124	T1126	nmod	DAG,domain
R1011	T1125	T1126	amod	binding,domain
R1012	T1126	T1122	dobj	domain,contains
R1013	T1127	T1126	cc	and,domain
R1014	T1128	T1129	amod	direct,binding
R1015	T1129	T1126	conj	binding,domain
R1016	T1130	T1129	prep	of,binding
R1017	T1131	T1130	pobj	DAG,of
R1018	T1132	T1133	advmod	also,contributes
R1019	T1133	T1121	conj	contributes,enzymes
R1020	T1134	T1133	prep	to,contributes
R1021	T1135	T1139	nummod	PKD1,]
R1022	T1136	T1137	compound	activation,[
R1023	T1137	T1139	nmod	[,]
R1024	T1138	T1137	nummod	7,[
R1025	T1139	T1134	pobj	],to
R1026	T1140	T1142	advmod	as,as
R1027	T1141	T1142	advmod	well,as
R1028	T1142	T1133	prep	as,contributes
R1029	T1143	T1142	pcomp	regulating,as
R1030	T1144	T1146	det	the,location
R1031	T1145	T1146	amod	spatial,location
R1032	T1146	T1143	dobj	location,regulating
R1033	T1147	T1146	prep	of,location
R1034	T1148	T1149	compound	PKD,enzymes
R1035	T1149	T1147	pobj	enzymes,of
R1036	T1150	T1143	prep	within,regulating
R1037	T1151	T1150	pobj	cells,within
R1038	T1152	T1153	nmod	[,8
R1039	T1153	T1143	npadvmod	8,regulating
R1040	T1154	T1155	nummod	12,]
R1041	T1155	T1153	appos	],8
R1042	T1156	T1121	punct	.,enzymes
R1043	T1157	T1158	compound	PKD,enzymes
R1044	T1158	T1161	nsubjpass	enzymes,proposed
R1045	T1159	T1161	aux	have,proposed
R1062	T1176	T1177	amod	anti-apoptotic,signals
R1063	T1177	T1171	appos	signals,[
R1064	T1178	T1180	compound	[,]
R1065	T1179	T1180	compound	"17,18",]
R1066	T1180	T1177	dobj	],signals
R1067	T1181	T1180	cc	and,]
R1068	T1182	T1183	amod	thymocyte,development
R1069	T1183	T1186	nmod	development,]
R1070	T1184	T1186	nmod	[,]
R1071	T1185	T1186	nummod	19,]
R1072	T1186	T1180	conj	],]
R1073	T1187	T1177	punct	.,signals
R1074	T1188	T1199	nsubj	Expression,modify
R1075	T1189	T1188	prep	of,Expression
R1076	T1190	T1192	amod	mutant,inactive
R1077	T1191	T1192	advmod	catalytically,inactive
R1078	T1192	T1196	amod	inactive,PKDs
R1079	T1193	T1192	cc	and,inactive
R1080	T1194	T1195	advmod	constitutively,activated
R1081	T1195	T1192	conj	activated,inactive
R1082	T1196	T1188	appos	PKDs,Expression
R1083	T1197	T1199	aux	can,modify
R1084	T1198	T1199	advmod	also,modify
R1085	T1199	T1199	ROOT	modify,modify
R1086	T1200	T1201	compound	Golgi,function
R1087	T1201	T1199	dobj	function,modify
R1088	T1202	T1201	punct	",",function
R1089	T1203	T1204	compound	cell,adhesion
R1090	T1204	T1201	conj	adhesion,function
R1091	T1205	T1204	cc	and,adhesion
R1092	T1206	T1207	compound	cell,motility
R1093	T1207	T1204	conj	motility,adhesion
R1094	T1208	T1209	punct	(,reviewed
R1095	T1209	T1199	advcl	reviewed,modify
R1096	T1210	T1209	prep	in,reviewed
R1097	T1211	T1210	pobj	[,in
R1098	T1212	T1213	nummod	20,]
R1099	T1213	T1211	appos	],[
R1100	T1214	T1211	punct	),[
R1101	T1215	T1199	punct	.,modify
R1102	T1216	T1223	prep	In,linked
R1103	T1217	T1216	amod	particular,In
R1104	T1218	T1223	punct	",",linked
R1105	T1219	T1223	nsubjpass	PKDs,linked
R1106	T1220	T1223	aux	have,linked
R1107	T1221	T1223	auxpass	been,linked
R1108	T1222	T1223	advmod	widely,linked
R1109	T1223	T1223	ROOT	linked,linked
R1110	T1224	T1223	prep	to,linked
R1111	T1225	T1226	det	the,activation
R1112	T1226	T1224	pobj	activation,to
R1113	T1227	T1226	prep	of,activation
R1114	T1228	T1231	det	the,factor
R1115	T1229	T1231	compound	NFκB,factor
R1116	T1230	T1231	compound	transcription,factor
R1117	T1231	T1227	pobj	factor,of
R1118	T1232	T1224	cc	and,to
R1119	T1233	T1223	prep	in,linked
R1120	T1234	T1233	pcomp	regulating,in
R1121	T1235	T1236	compound	cell,survival
R1122	T1236	T1234	dobj	survival,regulating
R1123	T1237	T1234	prep	during,regulating
R1124	T1238	T1239	amod	oxidative,stress
R1125	T1239	T1241	compound	stress,"17,21"
R1126	T1240	T1241	compound	[,"17,21"
R1127	T1241	T1237	pobj	"17,21",during
R1175	T1289	T1289	ROOT	enzymes,enzymes
R1176	T1290	T1291	mark	that,repress
R1196	T1310	T1309	prep	of,role
R1197	T1311	T1310	pobj	PKDs,of
R1198	T1312	T1314	nsubj	we,generated
R1199	T1313	T1314	aux	have,generated
R1200	T1314	T1314	ROOT	generated,generated
R1201	T1315	T1318	compound	DT40,lines
R1202	T1316	T1318	compound	B,lines
R1203	T1317	T1318	compound	cell,lines
R1204	T1318	T1314	dobj	lines,generated
R1205	T1319	T1320	nsubj	that,lack
R1206	T1320	T1318	relcl	lack,lines
R1207	T1321	T1320	dobj	expression,lack
R1208	T1322	T1321	prep	of,expression
R1209	T1323	T1326	nummod	one,members
R1210	T1324	T1323	cc	or,one
R1211	T1325	T1323	conj	more,one
R1212	T1326	T1322	pobj	members,of
R1213	T1327	T1326	prep	of,members
R1214	T1328	T1330	det	the,family
R1215	T1329	T1330	compound	PKD,family
R1216	T1330	T1327	pobj	family,of
R1217	T1331	T1333	nmod	[,]
R1218	T1332	T1333	nummod	1,]
R1219	T1333	T1326	appos	],members
R1220	T1334	T1314	punct	",",generated
R1221	T1335	T1314	advcl	allowing,generated
R1222	T1336	T1338	nsubj	us,investigate
R1223	T1337	T1338	aux	to,investigate
R1224	T1338	T1335	ccomp	investigate,allowing
R1225	T1339	T1340	det	the,function
R1226	T1340	T1338	dobj	function,investigate
R1227	T1341	T1340	punct	(,function
R1228	T1342	T1340	appos	s,function
R1229	T1343	T1340	punct	),function
R1230	T1344	T1340	prep	of,function
R1231	T1345	T1344	pobj	PKD,of
R1232	T1346	T1344	pobj	isoforms,of
R1233	T1347	T1346	acl	following,isoforms
R1234	T1348	T1350	compound	B,antigen
R1235	T1349	T1350	compound	cell,antigen
R1236	T1350	T1351	compound	antigen,receptor
R1237	T1351	T1347	dobj	receptor,following
R1238	T1352	T1353	punct	(,BCR
R1239	T1353	T1351	appos	BCR,receptor
R1240	T1354	T1353	punct	),BCR
R1241	T1355	T1351	conj	stimulation,receptor
R1242	T1356	T1347	punct	",",following
R1243	T1357	T1359	advmod	as,addressing
R1244	T1358	T1359	advmod	well,addressing
R1245	T1359	T1338	advcl	addressing,investigate
R1246	T1360	T1361	det	the,issue
R1247	T1361	T1359	dobj	issue,addressing
R1248	T1362	T1361	prep	of,issue
R1249	T1363	T1364	amod	functional,redundancy
R1250	T1364	T1362	pobj	redundancy,of
R1251	T1365	T1364	prep	between,redundancy
R1252	T1366	T1370	det	the,members
R1253	T1367	T1370	amod	different,members
R1254	T1368	T1370	compound	PKD,members
R1255	T1369	T1370	compound	family,members
R1256	T1370	T1365	pobj	members,between
R1257	T1371	T1314	punct	.,generated
R1258	T1372	T1373	amod	Previous,studies
R1259	T1373	T1375	nsubj	studies,shown
R1261	T1374	T1375	aux	have,shown
R1264	T1375	T1375	ROOT	shown,shown
R1267	T1376	T1378	mark	that,are
R1275	T1377	T1378	nsubj	PKDs,are
R1279	T1378	T1375	ccomp	are,shown
R1284	T1379	T1378	acomp	indispensable,are
R1288	T1380	T1378	prep	for,are
R1292	T1381	T1382	compound	HDAC,regulation
R1296	T1382	T1380	pobj	regulation,for
R1298	T1383	T1382	prep	in,regulation
R1299	T1477	T1474	prep	in,ascribed
R1300	T1478	T1480	amod	other,systems
R1301	T1479	T1480	amod	cellular,systems
R1302	T1480	T1477	pobj	systems,in
R1303	T1384	T1385	compound	B,cells
R1304	T1481	T1455	punct	.,reveal
R1305	T1385	T1383	pobj	cells,in
R1306	T1386	T1388	nmod	[,]
R1307	T1387	T1388	nummod	1,]
R1308	T1388	T1382	appos	],regulation
R1309	T1389	T1375	punct	.,shown
R1310	T1390	T1392	npadvmod	Herein,show
R1311	T1391	T1392	nsubj	we,show
R1312	T1392	T1392	ROOT	show,show
R1313	T1393	T1395	mark	that,are
R1314	T1394	T1395	nsubj	PKDs,are
R1315	T1395	T1392	ccomp	are,show
R1316	T1396	T1395	advmod	also,are
R1317	T1397	T1395	acomp	indispensable,are
R1318	T1398	T1395	prep	for,are
R1319	T1399	T1400	compound	HSP27,phosphorylation
R1320	T1400	T1398	pobj	phosphorylation,for
R1321	T1401	T1400	prep	in,phosphorylation
R1322	T1402	T1403	compound	B,cells
R1323	T1403	T1401	pobj	cells,in
R1324	T1404	T1392	punct	.,show
R1325	T1405	T1411	advmod	However,are
R1326	T1406	T1411	punct	",",are
R1327	T1407	T1409	compound	PKD-null,B
R1328	T1408	T1409	compound	DT40,B
R1329	T1409	T1410	compound	B,cells
R1331	T1410	T1411	nsubj	cells,are
R1335	T1411	T1411	ROOT	are,are
R1336	T1412	T1411	acomp	viable,are
R1338	T1413	T1411	cc	and,are
R1340	T1414	T1411	conj	proliferate,are
R1341	T1415	T1414	advmod	normally,proliferate
R1342	T1416	T1411	punct	.,are
R1343	T1417	T1430	advmod	Moreover,affect
R1344	T1418	T1430	punct	",",affect
R1345	T1419	T1430	nsubj	loss,affect
R1347	T1420	T1419	prep	of,loss
R1351	T1421	T1424	det	the,pool
R1352	T1422	T1424	amod	entire,pool
R1355	T1423	T1424	amod	cellular,pool
R1356	T1424	T1420	pobj	pool,of
R1357	T1425	T1424	prep	of,pool
R1358	T1426	T1425	pobj	PKD,of
R1359	T1427	T1430	aux	does,affect
R1360	T1428	T1430	neg	not,affect
R1361	T1429	T1430	advmod	critically,affect
R1362	T1430	T1430	ROOT	affect,affect
R1365	T1431	T1433	amod	oxidative,responses
R1367	T1432	T1433	compound	stress,responses
R1369	T1433	T1430	dobj	responses,affect
R1371	T1434	T1433	prep	in,responses
R1373	T1435	T1436	compound	B,cells
R1374	T1436	T1434	pobj	cells,in
R1376	T1437	T1430	cc	nor,affect
R1378	T1438	T1441	aux	do,play
R1379	T1439	T1440	compound	PKD,kinases
R1380	T1440	T1441	nsubj	kinases,play
R1381	T1441	T1430	conj	play,affect
R1382	T1442	T1444	det	an,role
R1383	T1443	T1444	amod	essential,role
R1384	T1444	T1441	dobj	role,play
R1385	T1445	T1444	prep	in,role
R1387	T1446	T1445	pcomp	regulating,in
R1391	T1447	T1449	nmod	NFκB,activity
R1519	T1759	T1761	nummod	−,−
R1520	T1760	T1761	compound	/,−
R1521	T1761	T1757	pobj	−,of
R1522	T1762	T1765	punct	",",/
R1523	T1763	T1764	compound	PKD3,−
R1524	T1764	T1765	nsubj	−,/
R1525	T1765	T1765	ROOT	/,/
R1526	T1766	T1776	nummod	−,lines
R1527	T1767	T1766	cc	and,−
R1528	T1768	T1776	nummod	PKD1/3,lines
R1529	T1769	T1776	nummod	−,lines
R1530	T1770	T1772	nmod	/,knockout
R1531	T1771	T1772	compound	−,knockout
R1532	T1772	T1776	compound	knockout,lines
R1533	T1773	T1774	compound	DT40,B
R1534	T1774	T1775	compound	B,cell
R1535	T1775	T1776	compound	cell,lines
R1536	T1776	T1779	nsubjpass	lines,described
R1537	T1777	T1779	aux	have,described
R1538	T1778	T1779	auxpass	been,described
R1539	T1779	T1765	ccomp	described,/
R1540	T1780	T1783	advmod	previously,]
R1541	T1781	T1783	nmod	[,]
R1542	T1782	T1781	nummod	1,[
R1543	T1783	T1779	dobj	],described
R1544	T1784	T1765	punct	.,/
R1545	T1785	T1787	nsubjpass	Cells,lysed
R1546	T1786	T1787	auxpass	were,lysed
R1547	T1787	T1787	ROOT	lysed,lysed
R1548	T1788	T1787	cc	and,lysed
R1549	T1789	T1790	compound	protein,extracts
R1550	T1790	T1792	nsubjpass	extracts,analysed
R1551	T1791	T1792	auxpass	were,analysed
R1552	T1792	T1787	conj	analysed,lysed
R1553	T1793	T1792	prep	in,analysed
R1554	T1794	T1796	amod	Western,experiments
R1555	T1795	T1796	amod	blotting,experiments
R1556	T1796	T1793	pobj	experiments,in
R1557	T1797	T1799	mark	as,described
R1558	T1798	T1799	advmod	previously,described
R1559	T1799	T1792	advcl	described,analysed
R1560	T1800	T1802	nmod	[,]
R1561	T1801	T1802	nummod	1,]
R1562	T1802	T1799	dobj	],described
R1563	T1803	T1792	punct	.,analysed
R1564	T1804	T1807	nmod	Chloramphenicol,assays
R1565	T1805	T1807	nmod	acetyl,assays
R1566	T1806	T1807	compound	transferase,assays
R1567	T1807	T1810	nsubjpass	assays,described
R1568	T1808	T1810	aux	have,described
R1569	T1809	T1810	auxpass	been,described
R1570	T1810	T1810	ROOT	described,described
R1571	T1811	T1814	advmod	previously,]
R1572	T1812	T1814	nmod	[,]
R1573	T1813	T1814	nummod	29,]
R1574	T1814	T1810	dobj	],described
R1575	T1815	T1810	punct	.,described
R1663	T1939	T1940	nummod	"Protein kinase D enzymes are dispensable for proliferation, survival and antigen receptor-regulated NFκB activity in vertebrate B-cells Abstract To investigate the importance of protein kinase D (PKD) enzymes we generated a PKD-null DT40 B-lymphocyte cell line. Previously we have shown that PKDs have an essential role in regulating class II histone deacetylases in DT40 B-cells [Matthews, S.A., Liu, P., Spitaler, M., Olson, E.N., McKinsey, T.A., Cantrell, D.A. and Scharenberg, A.M. (2006) Essential role for protein kinase D family kinases in the regulation of class II histone deacetylases in B lymphocytes. Mol. Cell Biol. 26, 1569–1577]. We now show that PKDs are also required to regulate HSP27 phosphorylation in DT40 B-cells. However, in contrast to previous observations in other cell types, PKD enzymes do not regulate basic cellular processes such as proliferation or survival responses, nor NFκB transcriptional activity downstream of the B cell antigen receptor. Thus, PKDs have a selective role in DT40 B-cell biology. 1 Introduction The protein kinase D (PKD) serine/threonine kinase family has three members: PKD1, PKD2 and PKD3. Most cell types express at least two PKD isoforms but PKD enzymes are especially highly expressed in haematopoietic cells, where they are activated in response to antigen receptors stimulation [2,3]. A conserved signalling pathway linking antigen receptors to PKDs involves the activation of PLCγ and the subsequent production of diacylglycerol (DAG) which stimulates classical and/or novel protein kinase Cs (PKC) that phosphorylate two key regulatory serine residues in the activation loop of PKD kinases [3–6]. The N-terminal regulatory region of PKD enzymes contains a DAG binding domain and direct binding of DAG also contributes to PKD1 activation [7] as well as regulating the spatial location of PKD enzymes within cells [8–12]. PKD enzymes have been proposed to regulate numerous cellular functions, including cell proliferation [13–16], anti-apoptotic signals [17,18] and thymocyte development [19]. Expression of mutant catalytically inactive and constitutively activated PKDs can also modify Golgi function, cell adhesion and cell motility (reviewed in [20]). In particular, PKDs have been widely linked to the activation of the NFκB transcription factor and in regulating cell survival during oxidative stress [17,21–23]. Another recently proposed PKD1 substrate is HSP27 [24], a small heat shock protein involved in regulating cell migration and cell survival [25]. An essential role for PKD enzymes in regulating class II histone deacetylases (HDACs), enzymes that repress MEF2-dependent gene transcription, has also been demonstrated [1,26–28]. To investigate the biological role of PKDs we have generated DT40 B cell lines that lack expression of one or more members of the PKD family [1], allowing us to investigate the function(s) of PKD isoforms following B cell antigen receptor (BCR) stimulation, as well addressing the issue of functional redundancy between the different PKD family members. Previous studies have shown that PKDs are indispensable for HDAC regulation in B cells [1]. Herein we show that PKDs are also indispensable for HSP27 phosphorylation in B cells. However, PKD-null DT40 B cells are viable and proliferate normally. Moreover, loss of the entire cellular pool of PKD does not critically affect oxidative stress responses in B cells nor do PKD kinases play an essential role in regulating NFκB transcriptional activity. Together, these findings reveal that in B lymphocytes, PKD kinases are not critical regulators of many of the cellular processes previously ascribed to them in other cellular systems. 2 Materials and methods 2.1 Cell culture, transient transfections and cell stimulation The generation, culture and activation of PKD1−/−, PKD3−/− and PKD1/3−/− knockout DT40 B cell lines have been described previously [1]. Cells were lysed and protein extracts were analysed in Western blotting experiments as previously described [1]. Chloramphenicol acetyl transferase assays have been described previously [29]. 2.2 ",sIgM
R1664	T1940	T1944	amod	sIgM,cells
R1665	T1941	T1944	amod	staining,cells
R1666	T1942	T1943	compound	DT40,B
R1667	T1943	T1944	compound	B,cells
R1668	T1944	T1954	nsubjpass	cells,resuspended
R1669	T1945	T1944	punct	(,cells
R1670	T1946	T1949	nummod	2,cells
R1671	T1947	T1949	nummod	×,cells
R1677	T1953	T1954	auxpass	were,resuspended
R1678	T1954	T1954	ROOT	resuspended,resuspended
R1679	T1955	T1954	prep	in,resuspended
R1680	T1956	T1958	nummod	200,buffer
R1681	T1957	T1958	compound	μl,buffer
R1682	T1958	T1955	pobj	buffer,in
R1683	T1959	T1958	punct	(,buffer
R1684	T1960	T1962	compound	RPMI,media
R1685	T1961	T1962	compound	1640,media
R1686	T1962	T1958	appos	media,buffer
R1687	T1963	T1962	punct	",",media
R1688	T1964	T1965	nummod	1,%
R1689	T1965	T1968	nmod	%,serum
R1690	T1966	T1968	amod	foetal,serum
R1691	T1967	T1968	compound	calf,serum
R1692	T1968	T1962	appos	serum,media
R1693	T1969	T1962	punct	),media
R1694	T1970	T1958	acl	containing,buffer
R1695	T1971	T1975	amod	anti-chicken,conjugated
R1696	T1972	T1974	nummod	M1,antibody
R1697	T1973	T1974	amod	monoclonal,antibody
R1698	T1974	T1975	compound	antibody,conjugated
R1699	T1975	T1954	conj	conjugated,resuspended
R1700	T1976	T1975	prep	to,conjugated
R1701	T1977	T1976	pobj	FITC,to
R1702	T1978	T1975	prep	for,conjugated
R1703	T1979	T1980	nummod	20,min
R1704	T1980	T1978	pobj	min,for
R1705	T1981	T1980	prep	on,min
R1706	T1982	T1981	pobj	ice,on
R1707	T1983	T1954	punct	.,resuspended
R1708	T1984	T1985	det	The,cells
R1709	T1985	T1987	nsubjpass	cells,washed
R1710	T1986	T1987	auxpass	were,washed
R1711	T1987	T1987	ROOT	washed,washed
R1712	T1988	T1987	advmod	twice,washed
R1713	T1989	T1988	cc	and,twice
R1714	T1990	T1991	amod	fluorescent,intensity
R1715	T1991	T1993	nsubjpass	intensity,analysed
R1716	T1992	T1993	auxpass	was,analysed
R1717	T1993	T1987	conj	analysed,washed
R1718	T1994	T1993	agent	by,analysed
R1719	T1995	T1996	compound	flow,cytometry
R1720	T1996	T1994	pobj	cytometry,by
R1721	T1997	T1993	punct	.,analysed
R1722	T1998	T1999	det	All,results
R1723	T1999	T2001	nsubj	results,are
R1724	T2000	T1999	acl	shown,results
R1725	T2001	T2001	ROOT	are,are
R1726	T2002	T2001	attr	representative,are
R1727	T2003	T2002	prep	of,representative
R1728	T2004	T2001	prep	at,are
R1729	T2005	T2007	quantmod	two,four
R1730	T2006	T2007	quantmod	to,four
R1731	T2007	T2009	nummod	four,experiments
R1732	T2008	T2009	amod	independent,experiments
R1733	T2009	T2004	pobj	experiments,at
R1734	T2010	T2012	mark	unless,indicated
R1735	T2011	T2012	advmod	otherwise,indicated
R1736	T2012	T2001	advcl	indicated,are
R1737	T2013	T2001	punct	.,are
R2279	T2730	T2731	nummod	"Protein kinase D enzymes are dispensable for proliferation, survival and antigen receptor-regulated NFκB activity in vertebrate B-cells Abstract To investigate the importance of protein kinase D (PKD) enzymes we generated a PKD-null DT40 B-lymphocyte cell line. Previously we have shown that PKDs have an essential role in regulating class II histone deacetylases in DT40 B-cells [Matthews, S.A., Liu, P., Spitaler, M., Olson, E.N., McKinsey, T.A., Cantrell, D.A. and Scharenberg, A.M. (2006) Essential role for protein kinase D family kinases in the regulation of class II histone deacetylases in B lymphocytes. Mol. Cell Biol. 26, 1569–1577]. We now show that PKDs are also required to regulate HSP27 phosphorylation in DT40 B-cells. However, in contrast to previous observations in other cell types, PKD enzymes do not regulate basic cellular processes such as proliferation or survival responses, nor NFκB transcriptional activity downstream of the B cell antigen receptor. Thus, PKDs have a selective role in DT40 B-cell biology. 1 Introduction The protein kinase D (PKD) serine/threonine kinase family has three members: PKD1, PKD2 and PKD3. Most cell types express at least two PKD isoforms but PKD enzymes are especially highly expressed in haematopoietic cells, where they are activated in response to antigen receptors stimulation [2,3]. A conserved signalling pathway linking antigen receptors to PKDs involves the activation of PLCγ and the subsequent production of diacylglycerol (DAG) which stimulates classical and/or novel protein kinase Cs (PKC) that phosphorylate two key regulatory serine residues in the activation loop of PKD kinases [3–6]. The N-terminal regulatory region of PKD enzymes contains a DAG binding domain and direct binding of DAG also contributes to PKD1 activation [7] as well as regulating the spatial location of PKD enzymes within cells [8–12]. PKD enzymes have been proposed to regulate numerous cellular functions, including cell proliferation [13–16], anti-apoptotic signals [17,18] and thymocyte development [19]. Expression of mutant catalytically inactive and constitutively activated PKDs can also modify Golgi function, cell adhesion and cell motility (reviewed in [20]). In particular, PKDs have been widely linked to the activation of the NFκB transcription factor and in regulating cell survival during oxidative stress [17,21–23]. Another recently proposed PKD1 substrate is HSP27 [24], a small heat shock protein involved in regulating cell migration and cell survival [25]. An essential role for PKD enzymes in regulating class II histone deacetylases (HDACs), enzymes that repress MEF2-dependent gene transcription, has also been demonstrated [1,26–28]. To investigate the biological role of PKDs we have generated DT40 B cell lines that lack expression of one or more members of the PKD family [1], allowing us to investigate the function(s) of PKD isoforms following B cell antigen receptor (BCR) stimulation, as well addressing the issue of functional redundancy between the different PKD family members. Previous studies have shown that PKDs are indispensable for HDAC regulation in B cells [1]. Herein we show that PKDs are also indispensable for HSP27 phosphorylation in B cells. However, PKD-null DT40 B cells are viable and proliferate normally. Moreover, loss of the entire cellular pool of PKD does not critically affect oxidative stress responses in B cells nor do PKD kinases play an essential role in regulating NFκB transcriptional activity. Together, these findings reveal that in B lymphocytes, PKD kinases are not critical regulators of many of the cellular processes previously ascribed to them in other cellular systems. 2 Materials and methods 2.1 Cell culture, transient transfections and cell stimulation The generation, culture and activation of PKD1−/−, PKD3−/− and PKD1/3−/− knockout DT40 B cell lines have been described previously [1]. Cells were lysed and protein extracts were analysed in Western blotting experiments as previously described [1]. Chloramphenicol acetyl transferase assays have been described previously [29]. 2.2 sIgM staining DT40 B cells (2 × 106 cells per point) were resuspended in 200 μl buffer (RPMI 1640 media, 1% foetal calf serum) containing anti-chicken M1 monoclonal antibody conjugated to FITC for 20 min on ice. The cells were washed twice and fluorescent intensity was analysed by flow cytometry. All results shown are representative of at two to four independent experiments unless otherwise indicated. 3 Results 3.1 ",Loss
R2280	T2731	T2744	nsubj	Loss,kinases
R2281	T2732	T2731	prep	of,Loss
R2282	T2733	T2734	compound	HSP27,phosphorylation
R2283	T2734	T2732	pobj	phosphorylation,of
R2284	T2735	T2731	prep	in,Loss
R2285	T2736	T2737	compound	DT40,B
R2286	T2737	T2738	compound	B,cells
R2287	T2738	T2735	pobj	cells,in
R2288	T2739	T2744	advcl	lacking,kinases
R2289	T2740	T2739	dobj	expression,lacking
R2290	T2741	T2740	prep	of,expression
R2291	T2742	T2743	compound	PKD,family
R2292	T2743	T2741	pobj	family,of
R2293	T2744	T2744	ROOT	kinases,kinases
R2294	T2745	T2746	compound	DT40,B
R2295	T2746	T2747	compound	B,cells
R2296	T2747	T2748	nsubj	cells,express
R2297	T2748	T2744	ccomp	express,kinases
R2298	T2749	T2751	nummod	two,isoforms
R2299	T2750	T2751	compound	PKD,isoforms
R2300	T2751	T2748	dobj	isoforms,express
R2301	T2752	T2751	punct	",",isoforms
R2302	T2753	T2751	conj	PKD1,isoforms
R2303	T2754	T2753	cc	and,PKD1
R2304	T2755	T2753	conj	PKD3,PKD1
R2305	T2756	T2755	punct	",",PKD3
R2306	T2757	T2748	cc	and,express
R2307	T2758	T2760	mark	as,described
R2308	T2759	T2760	advmod	previously,described
R2309	T2760	T2764	advcl	described,generated
R2310	T2761	T2764	nsubj	we,generated
R2311	T2762	T2764	aux	have,generated
R2312	T2763	T2764	advmod	recently,generated
R2313	T2764	T2748	conj	generated,express
R2314	T2765	T2768	compound	DT40,lines
R2315	T2766	T2768	compound	B,lines
R2316	T2767	T2768	compound	cell,lines
R2317	T2768	T2764	dobj	lines,generated
R2318	T2769	T2770	nsubj	that,lack
R2319	T2770	T2768	relcl	lack,lines
R2320	T2771	T2770	dobj	expression,lack
R2321	T2772	T2771	prep	of,expression
R2322	T2773	T2774	det	either,PKD1
R2323	T2774	T2772	pobj	PKD1,of
R2324	T2775	T2774	cc	or,PKD1
R2325	T2776	T2774	conj	PKD3,PKD1
R2326	T2777	T2776	cc	or,PKD3
R2327	T2778	T2779	det	both,enzymes
R2328	T2779	T2772	pobj	enzymes,of
R2329	T2780	T2782	nmod	[,]
R2330	T2781	T2782	nummod	1,]
R2331	T2782	T2764	npadvmod	],generated
R2332	T2783	T2744	punct	.,kinases
R2333	T2784	T2800	prep	In,/
R2334	T2785	T2784	pcomp	generating,In
R2335	T2786	T2790	det	the,lines
R2336	T2787	T2789	amod	double,cell
R2337	T2788	T2789	compound	knockout,cell
R2338	T2789	T2790	compound	cell,lines
R2339	T2790	T2785	dobj	lines,generating
R2340	T2791	T2792	nsubj	we,targeted
R2341	T2792	T2790	relcl	targeted,lines
R2342	T2793	T2795	det	the,loci
R2343	T2794	T2795	compound	PKD1,loci
R2344	T2795	T2792	dobj	loci,targeted
R2345	T2796	T2792	prep	in,targeted
R2346	T2797	T2799	det	a,−
R2347	T2798	T2799	compound	PKD3,−
R2348	T2799	T2796	pobj	−,in
R2349	T2800	T2800	ROOT	/,/
R2350	T2801	T2803	nummod	−,line
R2351	T2802	T2803	compound	cell,line
R2352	T2803	T2800	dobj	line,/
R2353	T2804	T2805	nsubj	that,expressed
R2354	T2805	T2803	relcl	expressed,line
R2355	T2806	T2808	det	a,transgene
R2356	T2807	T2808	amod	Flag-PKD3,transgene
R2357	T2808	T2805	dobj	transgene,expressed
R2358	T2809	T2808	prep	under,transgene
R2359	T2810	T2811	det	the,control
R2360	T2811	T2809	pobj	control,under
R2361	T2812	T2811	prep	of,control
R2362	T2813	T2815	det	a,promoter
R2363	T2814	T2815	amod	doxycycline-inducible,promoter
R2364	T2815	T2812	pobj	promoter,of
R2365	T2816	T2800	punct	.,/
R2366	T2817	T2832	advmod	Hence,is
R2367	T2818	T2832	punct	",",is
R2368	T2819	T2826	nmod	in,expression
R2369	T2820	T2821	det	the,presence
R2370	T2821	T2819	pobj	presence,in
R2371	T2822	T2821	prep	of,presence
R2372	T2823	T2822	pobj	doxycycline,of
R2373	T2824	T2821	punct	",",presence
R2374	T2825	T2826	compound	Flag-PKD3,expression
R2375	T2826	T2832	nsubj	expression,is
R2376	T2827	T2826	prep	in,expression
R2377	T2828	T2831	nummod	PKD1/3,cells
R2378	T2829	T2831	amod	double,cells
R2379	T2830	T2831	compound	knockout,cells
R2380	T2831	T2827	pobj	cells,in
R2381	T2832	T2832	ROOT	is,is
R2382	T2833	T2832	acomp	comparable,is
R2383	T2834	T2833	prep	to,comparable
R2384	T2835	T2837	amod	endogenous,present
R2385	T2836	T2837	amod	PKD3,present
R2386	T2837	T2834	pobj	present,to
R2387	T2838	T2837	prep	in,present
R2388	T2839	T2841	amod	wild-type,cells
R2389	T2840	T2841	compound	DT40,cells
R2390	T2841	T2838	pobj	cells,in
R2391	T2842	T2837	cc	and,present
R2392	T2843	T2837	conj	removal,present
R2393	T2844	T2843	prep	of,removal
R2394	T2845	T2844	pobj	doxycycline,of
R2395	T2846	T2843	prep	from,removal
R2396	T2847	T2849	det	the,media
R2397	T2848	T2849	compound	culture,media
R2398	T2849	T2846	pobj	media,from
R2399	T2850	T2832	prep	for,is
R2400	T2851	T2852	nummod	5,days
R2401	T2852	T2853	compound	days,results
R2402	T2853	T2850	pobj	results,for
R2403	T2854	T2853	prep	in,results
R2404	T2855	T2859	det	a,phenotype
R2405	T2856	T2857	advmod	completely,null
R2406	T2857	T2859	amod	null,phenotype
R2407	T2858	T2859	compound	PKD,phenotype
R2408	T2859	T2854	pobj	phenotype,in
R2409	T2860	T2862	punct	(,1A
R2410	T2861	T2862	compound	Fig.,1A
R2411	T2862	T2859	appos	1A,phenotype
R2412	T2863	T2862	punct	),1A
R2413	T2864	T2832	punct	.,is
R2414	T2865	T2869	advmod	Previously,demonstrated
R2415	T2866	T2869	punct	",",demonstrated
R2416	T2867	T2869	nsubj	we,demonstrated
R2417	T2868	T2869	aux	have,demonstrated
R2418	T2869	T2869	ROOT	demonstrated,demonstrated
R2419	T2870	T2886	mark	that,is
R2420	T2871	T2886	nsubj	phosphorylation,is
R2421	T2872	T2871	cc	and,phosphorylation
R2422	T2873	T2874	amod	nuclear,exclusion
R2423	T2874	T2871	conj	exclusion,phosphorylation
R2424	T2875	T2874	prep	of,exclusion
R2425	T2876	T2879	nmod	class,deacetylases
R2426	T2877	T2879	nmod	II,deacetylases
R2427	T2878	T2879	compound	histone,deacetylases
R2428	T2879	T2875	pobj	deacetylases,of
R2429	T2880	T2879	punct	(,deacetylases
R2430	T2881	T2879	appos	HDACs,deacetylases
R2431	T2882	T2879	punct	),deacetylases
R2432	T2883	T2871	prep	during,phosphorylation
R2433	T2884	T2885	compound	BCR,engagement
R2434	T2885	T2883	pobj	engagement,during
R2435	T2886	T2869	ccomp	is,demonstrated
R2436	T2887	T2886	acomp	defective,is
R2437	T2888	T2886	prep	in,is
R2438	T2889	T2894	nummod	PKD1/3,cells
R2439	T2890	T2894	nummod	−,cells
R2440	T2891	T2894	nmod	/,cells
R2441	T2892	T2894	compound	−,cells
R2442	T2893	T2894	compound	B,cells
R2443	T2894	T2888	pobj	cells,in
R2444	T2895	T2886	cc	and,is
R2445	T2896	T2897	aux	can,restored
R2446	T2897	T2886	conj	restored,is
R2451	T2902	T2904	amod	single,isoform
R2488	T2939	T2936	prep	on,phosphorylation
R2489	T2940	T2939	pobj	serine,on
R2490	T2941	T2940	nummod	82,serine
R2491	T2942	T2936	punct	",",phosphorylation
R2492	T2943	T2947	det	the,sequence
R2493	T2944	T2947	amod	proposed,sequence
R2494	T2945	T2946	compound	PKD1,substrate
R2495	T2946	T2947	compound	substrate,sequence
R2496	T2947	T2936	appos	sequence,phosphorylation
R2497	T2948	T2929	punct	.,assessed
R2498	T2949	T2951	nsubj	We,investigated
R2499	T2950	T2951	advmod	initially,investigated
R2500	T2951	T2951	ROOT	investigated,investigated
R2501	T2952	T2953	det	the,regulation
R2502	T2953	T2951	dobj	regulation,investigated
R2503	T2954	T2953	prep	of,regulation
R2504	T2955	T2956	compound	HSP27,phosphorylation
R2505	T2956	T2954	pobj	phosphorylation,of
R2506	T2957	T2956	prep	in,phosphorylation
R2507	T2958	T2959	amod	single,knockout
R2508	T2959	T2962	compound	knockout,cells
R2509	T2960	T2961	compound	DT40,B
R2510	T2961	T2962	compound	B,cells
R2511	T2962	T2957	pobj	cells,in
R2512	T2963	T2951	advcl	lacking,investigated
R2513	T2964	T2965	preconj	either,PKD1
R2514	T2965	T2963	dobj	PKD1,lacking
R2515	T2966	T2965	cc	or,PKD1
R2516	T2967	T2965	conj	PKD3,PKD1
R2517	T2968	T2951	punct	.,investigated
R2518	T2969	T2970	mark	As,shown
R2519	T2970	T2985	advcl	shown,increased
R2520	T2971	T2970	prep	in,shown
R2521	T2972	T2973	compound	Fig.,1B
R2522	T2973	T2971	pobj	1B,in
R2523	T2974	T2973	punct	",",1B
R2524	T2975	T2973	appos	activation,1B
R2525	T2976	T2975	prep	of,activation
R2526	T2977	T2978	det	the,BCR
R2527	T2978	T2976	pobj	BCR,of
R2528	T2979	T2975	cc	or,activation
R2529	T2980	T2975	conj	treatment,activation
R2530	T2981	T2975	prep	with,activation
R2531	T2982	T2984	det	the,PdBu
R2532	T2983	T2984	compound	DAG-mimetic,PdBu
R2533	T2984	T2981	pobj	PdBu,with
R2534	T2985	T2985	ROOT	increased,increased
R2535	T2986	T2987	det	the,levels
R2536	T2987	T2985	dobj	levels,increased
R2537	T2988	T2987	prep	of,levels
R2538	T2989	T2990	compound	HSP27,phosphorylation
R2539	T2990	T2988	pobj	phosphorylation,of
R2540	T2991	T2987	prep	at,levels
R2541	T2992	T2991	pobj	S82,at
R2542	T2993	T2992	prep	in,S82
R2543	T2994	T2997	amod	wild-type,cells
R2544	T2995	T2996	compound	DT40,B
R2545	T2996	T2997	compound	B,cells
R2546	T2997	T2993	pobj	cells,in
R2547	T2998	T2985	punct	.,increased
R2548	T2999	T3004	nsubj	BCR,were
R2549	T3000	T2999	cc	and,BCR
R2550	T3001	T3003	amod	phorbol,signals
R2551	T3002	T3003	compound	ester,signals
R2552	T3003	T2999	conj	signals,BCR
R2553	T3004	T3004	ROOT	were,were
R2554	T3005	T3004	advmod	also,were
R2555	T3006	T3004	acomp	able,were
R2556	T3007	T3008	aux	to,increase
R2557	T3008	T3006	xcomp	increase,able
R2558	T3009	T3010	compound	HSP27,phosphorylation
R2559	T3010	T3008	dobj	phosphorylation,increase
R2560	T3011	T3008	prep	in,increase
R2561	T3012	T3016	nummod	PKD1,knockout
R2562	T3013	T3012	cc	or,PKD1
R2563	T3014	T3012	conj	PKD3,PKD1
R2564	T3015	T3016	amod	single,knockout
R2565	T3016	T3019	compound	knockout,cells
R2566	T3017	T3018	compound	DT40,B
R2567	T3018	T3019	compound	B,cells
R2568	T3019	T3011	pobj	cells,in
R2569	T3020	T3004	punct	(,were
R2570	T3021	T3022	compound	Fig.,1B
R2571	T3022	T3004	npadvmod	1B,were
R2572	T3023	T3004	punct	),were
R2573	T3024	T3004	punct	.,were
R2574	T3025	T3038	advmod	However,abolished
R2575	T3026	T3038	punct	",",abolished
R2576	T3027	T3038	nsubjpass	BCR,abolished
R2577	T3028	T3027	punct	-,BCR
R2578	T3029	T3027	cc	and,BCR
R2579	T3030	T3032	nmod	phorbol,phosphorylation
R2580	T3031	T3032	amod	ester-induced,phosphorylation
R2581	T3032	T3027	conj	phosphorylation,BCR
R2582	T3033	T3032	prep	of,phosphorylation
R2583	T3034	T3033	pobj	HSP27,of
R2584	T3035	T3032	prep	on,phosphorylation
R2585	T3036	T3035	pobj	S82,on
R2586	T3037	T3038	auxpass	was,abolished
R2587	T3038	T3038	ROOT	abolished,abolished
R2588	T3039	T3038	prep	in,abolished
R2589	T3040	T3041	compound	B,cells
R2590	T3041	T3039	pobj	cells,in
R2591	T3042	T3043	nsubj	that,lacked
R2592	T3043	T3041	relcl	lacked,cells
R2593	T3044	T3045	preconj	both,PKD1
R2594	T3045	T3043	dobj	PKD1,lacked
R2595	T3046	T3045	cc	and,PKD1
R2596	T3047	T3045	conj	PKD3,PKD1
R2597	T3048	T3047	punct	(,PKD3
R2598	T3049	T3050	compound	Fig.,1C
R2599	T3050	T3047	appos	1C,PKD3
R2600	T3051	T3047	punct	),PKD3
R2601	T3052	T3038	punct	.,abolished
R2602	T3053	T3066	advmod	Significantly,was
R2603	T3054	T3056	punct	",",expression
R2604	T3055	T3056	amod	doxycycline-induced,expression
R2605	T3056	T3066	nsubj	expression,was
R2606	T3057	T3056	prep	of,expression
R2607	T3058	T3060	det	the,transgene
R2608	T3059	T3060	compound	Flag-PKD3,transgene
R2609	T3060	T3057	pobj	transgene,of
R2610	T3061	T3060	prep	in,transgene
R2611	T3062	T3065	det	the,cells
R2612	T3063	T3065	amod	double,cells
R2613	T3064	T3065	compound	knockout,cells
R2614	T3065	T3061	pobj	cells,in
R2615	T3066	T3066	ROOT	was,was
R2616	T3067	T3066	acomp	sufficient,was
R2617	T3068	T3069	aux	to,restore
R2618	T3069	T3067	xcomp	restore,sufficient
R2619	T3070	T3071	amod	normal,regulation
R2620	T3071	T3069	dobj	regulation,restore
R2621	T3072	T3071	prep	of,regulation
R2622	T3073	T3074	compound	HSP27,phosphorylation
R2623	T3074	T3072	pobj	phosphorylation,of
R2624	T3075	T3077	punct	(,1C
R2625	T3076	T3077	compound	Fig.,1C
R2626	T3077	T3074	appos	1C,phosphorylation
R2627	T3078	T3077	punct	),1C
R2628	T3079	T3066	punct	.,was
R2629	T3080	T3095	prep	In,was
R2630	T3081	T3080	pobj	contrast,In
R2631	T3082	T3095	punct	",",was
R2632	T3083	T3095	nsubj	expression,was
R2633	T3084	T3083	prep	of,expression
R2634	T3085	T3089	det	a,protein
R2635	T3086	T3089	amod	kinase-deficient,protein
R2636	T3087	T3089	amod	PKD3,protein
R2637	T3088	T3089	amod	mutant,protein
R2638	T3089	T3084	pobj	protein,of
R2639	T3090	T3089	prep	in,protein
R2640	T3091	T3094	det	the,cells
R2641	T3092	T3094	amod	double,cells
R2642	T3093	T3094	compound	knockout,cells
R2643	T3094	T3090	pobj	cells,in
R2644	T3095	T3095	ROOT	was,was
R2645	T3096	T3095	neg	not,was
R2646	T3097	T3095	acomp	able,was
R2647	T3098	T3099	aux	to,restore
R2648	T3099	T3097	xcomp	restore,able
R2649	T3100	T3099	dobj	BCR,restore
R2650	T3101	T3100	punct	-,BCR
R2651	T3102	T3099	cc	or,restore
R2652	T3103	T3099	conj	phorbol,restore
R2653	T3104	T3106	amod	ester-induced,phosphorylation
R2654	T3105	T3106	compound	HSP27,phosphorylation
R2655	T3106	T3103	dobj	phosphorylation,phorbol
R2656	T3107	T3106	punct	(,phosphorylation
R2657	T3108	T3109	compound	Fig.,1D
R2658	T3109	T3106	appos	1D,phosphorylation
R2659	T3110	T3106	punct	),phosphorylation
R2660	T3111	T3095	punct	.,was
R2661	T3112	T3120	advmod	Hence,regulate
R2662	T3113	T3120	punct	",",regulate
R2663	T3114	T3120	nsubj	PKD3,regulate
R2664	T3115	T3117	advmod	as,as
R2665	T3116	T3117	advmod	well,as
R2666	T3117	T3114	cc	as,PKD3
R2667	T3118	T3114	conj	PKD1,PKD3
R2668	T3119	T3120	aux	can,regulate
R2669	T3120	T3120	ROOT	regulate,regulate
R2670	T3121	T3122	compound	HSP27,phosphorylation
R2671	T3122	T3120	dobj	phosphorylation,regulate
R2672	T3123	T3120	cc	and,regulate
R2673	T3124	T3120	prep	in,regulate
R2674	T3125	T3126	compound	DT40,B
R2675	T3126	T3127	compound	B,cells
R2676	T3127	T3124	pobj	cells,in
R2677	T3128	T3129	nsubj	they,are
R2678	T3129	T3120	conj	are,regulate
R2679	T3130	T3131	advmod	functionally,redundant
R2680	T3131	T3129	acomp	redundant,are
R2681	T3132	T3131	prep	as,redundant
R2682	T3133	T3134	compound	HSP27,kinases
R2683	T3134	T3132	pobj	kinases,as
R2684	T3135	T3129	punct	.,are
R3416	T4217	T4219	nummod	"Protein kinase D enzymes are dispensable for proliferation, survival and antigen receptor-regulated NFκB activity in vertebrate B-cells Abstract To investigate the importance of protein kinase D (PKD) enzymes we generated a PKD-null DT40 B-lymphocyte cell line. Previously we have shown that PKDs have an essential role in regulating class II histone deacetylases in DT40 B-cells [Matthews, S.A., Liu, P., Spitaler, M., Olson, E.N., McKinsey, T.A., Cantrell, D.A. and Scharenberg, A.M. (2006) Essential role for protein kinase D family kinases in the regulation of class II histone deacetylases in B lymphocytes. Mol. Cell Biol. 26, 1569–1577]. We now show that PKDs are also required to regulate HSP27 phosphorylation in DT40 B-cells. However, in contrast to previous observations in other cell types, PKD enzymes do not regulate basic cellular processes such as proliferation or survival responses, nor NFκB transcriptional activity downstream of the B cell antigen receptor. Thus, PKDs have a selective role in DT40 B-cell biology. 1 Introduction The protein kinase D (PKD) serine/threonine kinase family has three members: PKD1, PKD2 and PKD3. Most cell types express at least two PKD isoforms but PKD enzymes are especially highly expressed in haematopoietic cells, where they are activated in response to antigen receptors stimulation [2,3]. A conserved signalling pathway linking antigen receptors to PKDs involves the activation of PLCγ and the subsequent production of diacylglycerol (DAG) which stimulates classical and/or novel protein kinase Cs (PKC) that phosphorylate two key regulatory serine residues in the activation loop of PKD kinases [3–6]. The N-terminal regulatory region of PKD enzymes contains a DAG binding domain and direct binding of DAG also contributes to PKD1 activation [7] as well as regulating the spatial location of PKD enzymes within cells [8–12]. PKD enzymes have been proposed to regulate numerous cellular functions, including cell proliferation [13–16], anti-apoptotic signals [17,18] and thymocyte development [19]. Expression of mutant catalytically inactive and constitutively activated PKDs can also modify Golgi function, cell adhesion and cell motility (reviewed in [20]). In particular, PKDs have been widely linked to the activation of the NFκB transcription factor and in regulating cell survival during oxidative stress [17,21–23]. Another recently proposed PKD1 substrate is HSP27 [24], a small heat shock protein involved in regulating cell migration and cell survival [25]. An essential role for PKD enzymes in regulating class II histone deacetylases (HDACs), enzymes that repress MEF2-dependent gene transcription, has also been demonstrated [1,26–28]. To investigate the biological role of PKDs we have generated DT40 B cell lines that lack expression of one or more members of the PKD family [1], allowing us to investigate the function(s) of PKD isoforms following B cell antigen receptor (BCR) stimulation, as well addressing the issue of functional redundancy between the different PKD family members. Previous studies have shown that PKDs are indispensable for HDAC regulation in B cells [1]. Herein we show that PKDs are also indispensable for HSP27 phosphorylation in B cells. However, PKD-null DT40 B cells are viable and proliferate normally. Moreover, loss of the entire cellular pool of PKD does not critically affect oxidative stress responses in B cells nor do PKD kinases play an essential role in regulating NFκB transcriptional activity. Together, these findings reveal that in B lymphocytes, PKD kinases are not critical regulators of many of the cellular processes previously ascribed to them in other cellular systems. 2 Materials and methods 2.1 Cell culture, transient transfections and cell stimulation The generation, culture and activation of PKD1−/−, PKD3−/− and PKD1/3−/− knockout DT40 B cell lines have been described previously [1]. Cells were lysed and protein extracts were analysed in Western blotting experiments as previously described [1]. Chloramphenicol acetyl transferase assays have been described previously [29]. 2.2 sIgM staining DT40 B cells (2 × 106 cells per point) were resuspended in 200 μl buffer (RPMI 1640 media, 1% foetal calf serum) containing anti-chicken M1 monoclonal antibody conjugated to FITC for 20 min on ice. The cells were washed twice and fluorescent intensity was analysed by flow cytometry. All results shown are representative of at two to four independent experiments unless otherwise indicated. 3 Results 3.1 Loss of HSP27 phosphorylation in DT40 B cells lacking expression of PKD family kinases DT40 B cells express two PKD isoforms, PKD1 and PKD3, and as previously described we have recently generated DT40 B cell lines that lack expression of either PKD1 or PKD3 or both enzymes [1]. In generating the double knockout cell lines we targeted the PKD1 loci in a PKD3−/− cell line that expressed a Flag-PKD3 transgene under the control of a doxycycline-inducible promoter. Hence, in the presence of doxycycline, Flag-PKD3 expression in PKD1/3 double knockout cells is comparable to endogenous PKD3 present in wild-type DT40 cells and removal of doxycycline from the culture media for 5 days results in a completely null PKD phenotype (Fig. 1A). Previously, we have demonstrated that phosphorylation and nuclear exclusion of class II histone deacetylases (HDACs) during BCR engagement is defective in PKD1/3−/− B cells and can restored upon re-expression of a single PKD isoform [1]. The small heat shock protein HSP27 has recently been proposed as a PKD1 substrate [24] and we accordingly assessed whether PKD-null DT40 cells have defective phosphorylation of HSP27 on serine 82, the proposed PKD1 substrate sequence. We initially investigated the regulation of HSP27 phosphorylation in single knockout DT40 B cells lacking either PKD1 or PKD3. As shown in Fig. 1B, activation of the BCR or treatment with the DAG-mimetic PdBu increased the levels of HSP27 phosphorylation at S82 in wild-type DT40 B cells. BCR and phorbol ester signals were also able to increase HSP27 phosphorylation in PKD1 or PKD3 single knockout DT40 B cells (Fig. 1B). However, BCR- and phorbol ester-induced phosphorylation of HSP27 on S82 was abolished in B cells that lacked both PKD1 and PKD3 (Fig. 1C). Significantly, doxycycline-induced expression of the Flag-PKD3 transgene in the double knockout cells was sufficient to restore normal regulation of HSP27 phosphorylation (Fig. 1C). In contrast, expression of a kinase-deficient PKD3 mutant protein in the double knockout cells was not able to restore BCR- or phorbol ester-induced HSP27 phosphorylation (Fig. 1D). Hence, PKD3 as well as PKD1 can regulate HSP27 phosphorylation and in DT40 B cells they are functionally redundant as HSP27 kinases. 3.2 ",proliferation
R3417	T4218	T4219	compound	Cellular,proliferation
R3418	T4219	T4231	nsubj	proliferation,kinases
R3419	T4220	T4219	cc	and,proliferation
R3420	T4221	T4219	conj	survival,proliferation
R3421	T4222	T4219	prep	in,proliferation
R3422	T4223	T4224	compound	DT40,B
R3423	T4224	T4225	compound	B,cells
R3424	T4225	T4222	pobj	cells,in
R3425	T4226	T4231	advcl	lacking,kinases
R3426	T4227	T4226	dobj	expression,lacking
R3427	T4228	T4227	prep	of,expression
R3428	T4229	T4230	compound	PKD,family
R3429	T4230	T4228	pobj	family,of
R3430	T4231	T4231	ROOT	kinases,kinases
R3431	T4232	T4233	compound	PKD,enzymes
R3432	T4233	T4237	nsubjpass	enzymes,linked
R3433	T4234	T4237	aux	have,linked
R3434	T4235	T4237	advmod	previously,linked
R3435	T4236	T4237	auxpass	been,linked
R3436	T4237	T4231	ccomp	linked,kinases
R3437	T4238	T4237	prep	to,linked
R3438	T4239	T4240	det	the,regulation
R3439	T4240	T4238	pobj	regulation,to
R3440	T4241	T4240	prep	of,regulation
R3441	T4242	T4243	compound	cell,proliferation
R3442	T4243	T4241	pobj	proliferation,of
R3443	T4244	T4243	cc	and,proliferation
R3444	T4245	T4243	conj	survival,proliferation
R3445	T4246	T4243	punct	(,proliferation
R3446	T4247	T4240	acl	reviewed,regulation
R3447	T4248	T4247	prep	in,reviewed
R3448	T4249	T4248	pobj	[,in
R3449	T4250	T4251	nummod	20,]
R3450	T4251	T4249	appos	],[
R3451	T4252	T4249	punct	),[
R3452	T4253	T4231	punct	.,kinases
R3453	T4254	T4255	aux	To,investigate
R3454	T4255	T4271	advcl	investigate,cultured
R3455	T4256	T4257	det	the,effect
R3456	T4257	T4255	dobj	effect,investigate
R3457	T4258	T4263	dobj	that,had
R3458	T4259	T4263	nsubj	loss,had
R3459	T4260	T4259	prep	of,loss
R3460	T4261	T4262	compound	PKD,kinases
R3461	T4262	T4260	pobj	kinases,of
R3462	T4263	T4257	relcl	had,effect
R3463	T4264	T4263	prep	on,had
R3464	T4265	T4267	compound	B,survival
R3465	T4266	T4267	compound	cell,survival
R3466	T4267	T4264	pobj	survival,on
R3467	T4268	T4263	cc	and/or,had
R3468	T4269	T4271	dobj	proliferation,cultured
R3469	T4270	T4271	nsubj	we,cultured
R3470	T4271	T4271	ROOT	cultured,cultured
R3471	T4272	T4275	amod	wild-type,cells
R3472	T4273	T4272	cc	and,wild-type
R3473	T4274	T4272	conj	PKD-null,wild-type
R3474	T4275	T4271	dobj	cells,cultured
R3475	T4276	T4271	prep	in,cultured
R3476	T4277	T4278	det	the,presence
R3477	T4278	T4276	pobj	presence,in
R3478	T4279	T4278	punct	(,presence
R3479	T4280	T4300	nsubj	PKD1/3,monitored
R3480	T4281	T4283	nummod	−,−
R3481	T4282	T4283	compound	/,−
R3482	T4283	T4300	dep	−,monitored
R3483	T4284	T4283	punct	:,−
R3484	T4285	T4287	nmod	Flag-PKD3,ve
R3485	T4286	T4287	compound	+,ve
R3486	T4287	T4283	appos	ve,−
R3487	T4288	T4287	punct	),ve
R3488	T4289	T4287	cc	or,ve
R3489	T4290	T4287	conj	absence,ve
R3490	T4291	T4290	punct	(,absence
R3491	T4292	T4295	nummod	PKD1/3,−
R3492	T4293	T4295	nummod	−,−
R3493	T4294	T4295	compound	/,−
R3494	T4295	T4290	appos	−,absence
R3495	T4296	T4295	punct	),−
R3496	T4297	T4290	prep	of,absence
R3497	T4298	T4297	pobj	doxycycline,of
R3498	T4299	T4290	cc	and,absence
R3499	T4300	T4271	advcl	monitored,cultured
R3500	T4301	T4302	amod	exponential,growth
R3501	T4302	T4300	dobj	growth,monitored
R3502	T4303	T4271	punct	.,cultured
R3503	T4304	T4305	mark	As,shown
R3504	T4305	T4311	advcl	shown,−
R3505	T4306	T4305	prep	in,shown
R3506	T4307	T4308	compound	Fig.,2A
R3507	T4308	T4306	pobj	2A,in
R3508	T4309	T4311	punct	",",−
R3509	T4310	T4311	nsubj	PKD1/3,−
R3510	T4311	T4311	ROOT	−,−
R3511	T4312	T4314	nummod	/,cells
R3512	T4313	T4314	compound	−,cells
R3513	T4314	T4315	nsubj	cells,proliferated
R3514	T4315	T4311	ccomp	proliferated,−
R3515	T4316	T4315	advmod	exponentially,proliferated
R3516	T4317	T4315	cc	and,proliferated
R3517	T4318	T4324	nsubj	re-expression,had
R3518	T4319	T4318	prep	of,re-expression
R3519	T4320	T4319	pobj	Flag-PKD3,of
R3520	T4321	T4318	prep	in,re-expression
R3521	T4322	T4323	det	these,cells
R3522	T4323	T4321	pobj	cells,in
R3523	T4324	T4315	conj	had,proliferated
R3524	T4325	T4326	det	no,impact
R3525	T4326	T4324	dobj	impact,had
R3526	T4327	T4326	prep	on,impact
R3527	T4328	T4329	det	the,rate
R3528	T4329	T4327	pobj	rate,on
R3529	T4330	T4329	prep	of,rate
R3530	T4331	T4330	pobj	proliferation,of
R3531	T4332	T4311	punct	.,−
R3532	T4333	T4347	advmod	Furthermore,was
R3533	T4334	T4347	punct	",",was
R3534	T4335	T4336	det	the,viability
R3535	T4336	T4347	nsubj	viability,was
R3536	T4337	T4336	prep	of,viability
R3537	T4338	T4343	nummod	PKD1/3,cells
R3538	T4339	T4343	nummod	−,cells
R3539	T4340	T4343	compound	/,cells
R3540	T4341	T4343	compound	−,cells
R3541	T4342	T4343	compound	B,cells
R3542	T4343	T4337	pobj	cells,of
R3543	T4344	T4336	prep	during,viability
R3544	T4345	T4346	amod	routine,culturing
R3545	T4346	T4344	pobj	culturing,during
R3546	T4347	T4347	ROOT	was,was
R3547	T4348	T4347	neg	not,was
R3548	T4349	T4350	advmod	significantly,different
R3549	T4350	T4347	acomp	different,was
R3550	T4351	T4350	prep	from,different
R3551	T4352	T4351	pobj	that,from
R3552	T4353	T4352	prep	of,that
R3553	T4354	T4356	amod	wild-type,cells
R3554	T4355	T4356	compound	B,cells
R3555	T4356	T4353	pobj	cells,of
R3556	T4357	T4360	punct	(,shown
R3557	T4358	T4360	nsubjpass	data,shown
R3558	T4359	T4360	neg	not,shown
R3559	T4360	T4347	conj	shown,was
R3560	T4361	T4360	punct	),shown
R3561	T4362	T4347	punct	.,was
R3562	T4363	T4365	nsubjpass	It,noted
R3563	T4364	T4365	auxpass	was,noted
R3564	T4365	T4365	ROOT	noted,noted
R3565	T4366	T4377	mark	that,was
R3566	T4367	T4370	det	the,time
R3567	T4368	T4369	compound	population,doubling
R3568	T4369	T4370	amod	doubling,time
R3569	T4370	T4377	nsubj	time,was
R3570	T4371	T4370	prep	of,time
R3571	T4372	T4376	nummod	PKD1/3,cells
R3572	T4373	T4376	nummod	−,cells
R3573	T4374	T4376	compound	/,cells
R3574	T4375	T4376	compound	−,cells
R3575	T4376	T4371	pobj	cells,of
R3576	T4377	T4365	ccomp	was,noted
R3577	T4378	T4379	advmod	slightly,slower
R3578	T4379	T4377	acomp	slower,was
R3579	T4380	T4379	prep	than,slower
R3580	T4381	T4380	pobj	that,than
R3581	T4382	T4381	prep	of,that
R3582	T4383	T4384	amod	wild,type
R3583	T4384	T4382	pobj	type,of
R3584	T4385	T4386	nummod	DT40,cells
R3585	T4386	T4384	appos	cells,type
R3586	T4387	T4386	punct	(,cells
R3587	T4388	T4391	nummod	12.7,h
R3588	T4389	T4391	nmod	±,h
R3589	T4390	T4391	nummod	2.8,h
R3590	T4391	T4386	appos	h,cells
R3591	T4392	T4391	prep	versus,h
R3592	T4393	T4396	nummod	10.2,h
R3593	T4394	T4396	nmod	±,h
R3594	T4395	T4396	nummod	0.4,h
R3595	T4396	T4392	pobj	h,versus
R3596	T4397	T4386	punct	),cells
R3597	T4398	T4365	cc	but,noted
R3598	T4399	T4400	det	the,failure
R3599	T4400	T4415	nsubj	failure,suggests
R3600	T4401	T4400	prep	of,failure
R3601	T4402	T4403	nummod	PKD3,re-expression
R3602	T4403	T4401	pobj	re-expression,of
R3603	T4404	T4405	aux	to,modify
R3604	T4405	T4400	acl	modify,failure
R3605	T4406	T4408	det	the,rate
R3606	T4407	T4408	compound	proliferation,rate
R3607	T4408	T4405	dobj	rate,modify
R3608	T4409	T4408	prep	of,rate
R3609	T4410	T4414	nummod	PKD1/3,cells
R3610	T4411	T4414	nummod	−,cells
R3611	T4412	T4414	compound	/,cells
R3612	T4413	T4414	compound	−,cells
R3613	T4414	T4409	pobj	cells,of
R3614	T4415	T4365	conj	suggests,noted
R3615	T4416	T4420	mark	that,were
R3616	T4417	T4419	det	these,differences
R3617	T4418	T4419	amod	small,differences
R3618	T4419	T4420	nsubj	differences,were
R3619	T4420	T4415	ccomp	were,suggests
R3620	T4421	T4422	advmod	most,likely
R3621	T4422	T4420	acomp	likely,were
R3622	T4423	T4424	det	the,result
R3623	T4424	T4420	attr	result,were
R3624	T4425	T4424	prep	of,result
R3625	T4426	T4427	amod	clonal,variation
R3626	T4427	T4425	pobj	variation,of
R3627	T4428	T4420	cc	and,were
R3628	T4429	T4431	auxpass	were,caused
R3629	T4430	T4431	neg	not,caused
R3630	T4431	T4420	conj	caused,were
R3631	T4432	T4431	advmod	specifically,caused
R3632	T4433	T4431	agent	by,caused
R3633	T4434	T4433	pobj	loss,by
R3634	T4435	T4434	prep	of,loss
R3635	T4436	T4437	compound	PKD,enzymes
R3636	T4437	T4435	pobj	enzymes,of
R3637	T4438	T4415	punct	.,suggests
R3638	T4439	T4444	advmod	Thus,are
R3639	T4440	T4444	punct	",",are
R3640	T4441	T4443	compound	PKD,enzymes
R3641	T4442	T4443	compound	family,enzymes
R3642	T4443	T4444	nsubj	enzymes,are
R3643	T4444	T4444	ROOT	are,are
R3644	T4445	T4444	neg	not,are
R3645	T4446	T4444	acomp	essential,are
R3646	T4447	T4446	prep	for,essential
R3647	T4448	T4447	pcomp	regulating,for
R3648	T4449	T4450	amod	basal,survival
R3649	T4450	T4448	dobj	survival,regulating
R3650	T4451	T4450	cc	and,survival
R3658	T4459	T4469	nsubj	enzymes,linked
R3659	T4460	T4459	punct	",",enzymes
R3660	T4461	T4462	advmod	specifically,PKD1
R3661	T4462	T4459	appos	PKD1,enzymes
R3662	T4463	T4462	cc	and,PKD1
R3663	T4464	T4462	conj	PKD2,PKD1
R3664	T4465	T4469	punct	",",linked
R3665	T4466	T4469	aux	have,linked
R3666	T4467	T4469	advmod	previously,linked
R3667	T4468	T4469	auxpass	been,linked
R3668	T4469	T4469	ROOT	linked,linked
R3669	T4470	T4469	prep	to,linked
R3670	T4471	T4473	det	a,role
R3671	T4472	T4473	amod	protective,role
R3672	T4473	T4470	pobj	role,to
R3673	T4474	T4473	prep	against,role
R3674	T4475	T4477	amod	oxidative,injury
R3675	T4476	T4477	amod	stress-induced,injury
R3676	T4477	T4486	nmod	injury,lines
R3677	T4478	T4477	prep	in,injury
R3678	T4479	T4480	compound	3T3,fibroblast
R3679	T4480	T4478	pobj	fibroblast,in
R3680	T4481	T4480	punct	",",fibroblast
R3681	T4482	T4480	conj	HeLa,fibroblast
R3682	T4483	T4482	cc	and,HeLa
R3683	T4484	T4486	amod	epithelial,lines
R3684	T4485	T4486	compound	cell,lines
R3685	T4486	T4474	pobj	lines,against
R3686	T4487	T4488	compound	[,"17,30"
R3687	T4488	T4486	appos	"17,30",lines
R3688	T4489	T4490	nummod	32,]
R3689	T4490	T4486	appos	],lines
R3690	T4491	T4469	punct	.,linked
R3691	T4492	T4494	nsubj	We,addressed
R3692	T4493	T4494	advmod	therefore,addressed
R3693	T4494	T4494	ROOT	addressed,addressed
R3694	T4495	T4496	det	the,role
R3695	T4496	T4494	dobj	role,addressed
R3696	T4497	T4496	prep	of,role
R3697	T4498	T4499	compound	PKD,family
R3698	T4499	T4500	compound	family,kinases
R3699	T4500	T4497	pobj	kinases,of
R3700	T4501	T4496	prep	in,role
R3701	T4502	T4501	pcomp	regulating,in
R3702	T4503	T4504	compound	B,cell
R3703	T4504	T4505	compound	cell,survival
R3704	T4505	T4502	dobj	survival,regulating
R3705	T4506	T4502	prep	in,regulating
R3706	T4507	T4506	pobj	response,in
R3707	T4508	T4507	prep	to,response
R3708	T4509	T4510	amod	oxidative,stress
R3709	T4510	T4508	pobj	stress,to
R3710	T4511	T4510	cc	and,stress
R3711	T4512	T4514	amod	other,stimuli
R3712	T4513	T4514	compound	stress,stimuli
R3713	T4514	T4510	conj	stimuli,stress
R3714	T4515	T4494	punct	.,addressed
R3715	T4516	T4517	mark	As,shown
R3716	T4517	T4526	advcl	shown,had
R3717	T4518	T4517	prep	in,shown
R3718	T4519	T4520	compound	Fig.,2B
R3719	T4520	T4518	pobj	2B,in
R3720	T4521	T4520	punct	",",2B
R3721	T4522	T4520	appos	loss,2B
R3722	T4523	T4522	prep	of,loss
R3723	T4524	T4525	nummod	PKD1/3,expression
R3724	T4525	T4523	pobj	expression,of
R3725	T4526	T4526	ROOT	had,had
R3726	T4527	T4529	det	no,impact
R3727	T4528	T4529	amod	significant,impact
R3728	T4529	T4526	dobj	impact,had
R3729	T4530	T4529	prep	on,impact
R3730	T4531	T4532	det	the,survival
R3731	T4532	T4530	pobj	survival,on
R3732	T4533	T4532	prep	of,survival
R3733	T4534	T4535	compound	DT40,B
R3734	T4535	T4536	compound	B,cells
R3735	T4536	T4533	pobj	cells,of
R3736	T4537	T4526	prep	in,had
R3737	T4538	T4537	pobj	response,in
R3738	T4539	T4538	prep	to,response
R3739	T4540	T4542	amod	mitochondrial,stimuli
R3740	T4541	T4542	compound	stress,stimuli
R3741	T4542	T4539	pobj	stimuli,to
R3742	T4543	T4542	punct	(,stimuli
R3743	T4544	T4542	appos	H2O2,stimuli
R3744	T4545	T4544	cc	or,H2O2
R3745	T4546	T4547	compound	serum,deprivation
R3746	T4547	T4544	conj	deprivation,H2O2
R3747	T4548	T4542	punct	),stimuli
R3748	T4549	T4542	punct	;,stimuli
R3749	T4550	T4552	amod	DNA,agents
R3750	T4551	T4552	amod	damaging,agents
R3751	T4552	T4542	conj	agents,stimuli
R3752	T4553	T4552	punct	(,agents
R3753	T4554	T4552	appos	etoposide,agents
R3754	T4555	T4554	cc	or,etoposide
R3755	T4556	T4554	conj	doxorubicin,etoposide
R3756	T4557	T4552	punct	),agents
R3757	T4558	T4526	punct	;,had
R3758	T4559	T4561	compound	ER,stress
R3759	T4560	T4561	compound	pathway,stress
R3760	T4561	T4570	nsubj	stress,following
R3761	T4562	T4561	amod	due,stress
R3762	T4563	T4562	pcomp	to,due
R3763	T4564	T4565	compound	calcium,overload
R3764	T4565	T4562	pobj	overload,due
R3765	T4566	T4567	punct	(,thapsigargin
R3766	T4567	T4565	appos	thapsigargin,overload
R3767	T4568	T4567	punct	),thapsigargin
R3768	T4569	T4565	cc	or,overload
R3769	T4570	T4526	advcl	following,had
R3770	T4571	T4572	amod	prolonged,treatment
R3771	T4572	T4570	dobj	treatment,following
R3772	T4573	T4572	prep	with,treatment
R3773	T4574	T4575	compound	phorbol,esters
R3774	T4575	T4573	pobj	esters,with
R3775	T4576	T4575	cc	or,esters
R3776	T4577	T4578	compound	Trichostatin,A
R3777	T4578	T4575	conj	A,esters
R3778	T4579	T4578	punct	",",A
R3779	T4580	T4581	det	an,inhibitor
R3780	T4581	T4578	appos	inhibitor,A
R3781	T4582	T4581	prep	of,inhibitor
R3782	T4583	T4585	compound	class,HDACs
R3783	T4584	T4585	compound	I/II,HDACs
R3784	T4585	T4582	pobj	HDACs,of
R3785	T4586	T4526	punct	.,had
R3786	T4587	T4593	advmod	Thus,play
R3787	T4588	T4593	punct	",",play
R3788	T4589	T4590	compound	PKD,kinases
R3789	T4590	T4593	nsubj	kinases,play
R3790	T4591	T4593	aux	do,play
R3791	T4592	T4593	neg	not,play
R3792	T4593	T4593	ROOT	play,play
R3793	T4594	T4596	det	an,role
R3794	T4595	T4596	amod	essential,role
R3795	T4596	T4593	dobj	role,play
R3796	T4597	T4593	prep	in,play
R3797	T4598	T4597	pcomp	regulating,in
R3798	T4599	T4600	compound	B,cell
R3799	T4600	T4601	compound	cell,survival
R3800	T4601	T4598	dobj	survival,regulating
R3801	T4602	T4598	prep	in,regulating
R3802	T4603	T4602	pobj	response,in
R3803	T4604	T4603	prep	to,response
R3804	T4605	T4606	det	a,range
R3805	T4606	T4604	pobj	range,to
R3806	T4607	T4606	prep	of,range
R3807	T4608	T4610	amod	different,stimuli
R3808	T4609	T4610	compound	stress,stimuli
R3809	T4610	T4607	pobj	stimuli,of
R3810	T4611	T4593	punct	.,play
R1050	T1164	T1166	amod	numerous,functions
R1051	T1165	T1166	amod	cellular,functions
R1052	T1166	T1163	dobj	functions,regulate
R1053	T1167	T1166	punct	",",functions
R1054	T1168	T1166	prep	including,functions
R1055	T1169	T1170	compound	cell,proliferation
R1056	T1170	T1168	pobj	proliferation,including
R1057	T1171	T1170	dobj	[,proliferation
R1058	T1172	T1171	nummod	13,[
R1059	T1173	T1174	nummod	16,]
R1060	T1174	T1171	appos	],[
R1061	T1175	T1177	punct	",",signals
R1131	T1245	T1249	det	Another,substrate
R1132	T1246	T1247	advmod	recently,proposed
R1133	T1247	T1249	amod	proposed,substrate
R1134	T1248	T1249	compound	PKD1,substrate
R1135	T1249	T1250	nsubj	substrate,is
R1136	T1250	T1250	ROOT	is,is
R1137	T1251	T1252	compound	HSP27,[
R1138	T1252	T1250	attr	[,is
R1139	T1253	T1254	nummod	24,]
R1140	T1254	T1252	appos	],[
R1141	T1255	T1254	punct	",",]
R1142	T1256	T1260	det	a,protein
R1143	T1257	T1260	amod	small,protein
R1144	T1258	T1259	compound	heat,shock
R1145	T1259	T1260	compound	shock,protein
R1146	T1260	T1254	appos	protein,]
R1147	T1261	T1260	amod	involved,protein
R1148	T1262	T1261	prep	in,involved
R1149	T1263	T1262	pcomp	regulating,in
R1150	T1264	T1265	compound	cell,migration
R1151	T1265	T1263	dobj	migration,regulating
R1152	T1266	T1265	cc	and,migration
R1153	T1267	T1268	compound	cell,survival
R1154	T1268	T1265	conj	survival,migration
R1155	T1269	T1271	nmod	[,]
R1156	T1270	T1271	nummod	25,]
R1157	T1271	T1265	appos	],migration
R1158	T1272	T1250	punct	.,is
R1159	T1273	T1275	det	An,role
R1160	T1274	T1275	amod	essential,role
R1161	T1275	T1289	nsubj	role,enzymes
R1162	T1276	T1275	prep	for,role
R1163	T1277	T1278	compound	PKD,enzymes
R1164	T1278	T1276	pobj	enzymes,for
R1165	T1279	T1278	prep	in,enzymes
R1166	T1280	T1279	pcomp	regulating,in
R1167	T1281	T1284	nmod	class,deacetylases
R1168	T1282	T1281	nummod	II,class
R1169	T1283	T1284	compound	histone,deacetylases
R1170	T1284	T1280	dobj	deacetylases,regulating
R1171	T1285	T1284	punct	(,deacetylases
R1172	T1286	T1284	appos	HDACs,deacetylases
R1173	T1287	T1284	punct	),deacetylases
R1174	T1288	T1278	punct	",",enzymes
R1177	T1291	T1289	ccomp	repress,enzymes
R1178	T1292	T1294	amod	MEF2-dependent,transcription
R1179	T1293	T1294	compound	gene,transcription
R1180	T1294	T1291	dobj	transcription,repress
R1181	T1295	T1299	punct	",",demonstrated
R1182	T1296	T1299	aux	has,demonstrated
R1183	T1297	T1299	advmod	also,demonstrated
R1184	T1298	T1299	auxpass	been,demonstrated
R1185	T1299	T1289	conj	demonstrated,enzymes
R1186	T1300	T1301	compound	[,"1,26"
R1187	T1301	T1299	dobj	"1,26",demonstrated
R1188	T1302	T1303	nummod	28,]
R1189	T1303	T1299	npadvmod	],demonstrated
R1190	T1304	T1299	punct	.,demonstrated
R1191	T1305	T1306	aux	To,investigate
R1192	T1306	T1314	advcl	investigate,generated
R1193	T1307	T1309	det	the,role
R1194	T1308	T1309	amod	biological,role
R1195	T1309	T1306	dobj	role,investigate
R1260	T1448	T1449	amod	transcriptional,activity
R1262	T1449	T1446	dobj	activity,regulating
R1263	T1450	T1441	punct	.,play
R1265	T1451	T1455	advmod	Together,reveal
R1266	T1452	T1455	punct	",",reveal
R1268	T1453	T1454	det	these,findings
R1269	T1454	T1455	nsubj	findings,reveal
R1270	T1455	T1455	ROOT	reveal,reveal
R1271	T1456	T1463	mark	that,are
R1272	T1457	T1463	prep	in,are
R1273	T1458	T1459	compound	B,lymphocytes
R1274	T1459	T1457	pobj	lymphocytes,in
R1276	T1460	T1463	punct	",",are
R1277	T1461	T1462	compound	PKD,kinases
R1278	T1462	T1463	nsubj	kinases,are
R1280	T1463	T1455	ccomp	are,reveal
R1281	T1464	T1463	neg	not,are
R1282	T1465	T1466	amod	critical,regulators
R1283	T1466	T1463	attr	regulators,are
R1285	T1467	T1466	prep	of,regulators
R1286	T1468	T1467	pobj	many,of
R1287	T1469	T1468	prep	of,many
R1289	T1470	T1472	det	the,processes
R1290	T1471	T1472	amod	cellular,processes
R1291	T1472	T1469	pobj	processes,of
R1293	T1473	T1474	advmod	previously,ascribed
R1294	T1474	T1463	conj	ascribed,are
R1295	T1475	T1474	prep	to,ascribed
R1297	T1476	T1475	pobj	them,to
R1502	T1742	T1744	nummod	"Protein kinase D enzymes are dispensable for proliferation, survival and antigen receptor-regulated NFκB activity in vertebrate B-cells Abstract To investigate the importance of protein kinase D (PKD) enzymes we generated a PKD-null DT40 B-lymphocyte cell line. Previously we have shown that PKDs have an essential role in regulating class II histone deacetylases in DT40 B-cells [Matthews, S.A., Liu, P., Spitaler, M., Olson, E.N., McKinsey, T.A., Cantrell, D.A. and Scharenberg, A.M. (2006) Essential role for protein kinase D family kinases in the regulation of class II histone deacetylases in B lymphocytes. Mol. Cell Biol. 26, 1569–1577]. We now show that PKDs are also required to regulate HSP27 phosphorylation in DT40 B-cells. However, in contrast to previous observations in other cell types, PKD enzymes do not regulate basic cellular processes such as proliferation or survival responses, nor NFκB transcriptional activity downstream of the B cell antigen receptor. Thus, PKDs have a selective role in DT40 B-cell biology. 1 Introduction The protein kinase D (PKD) serine/threonine kinase family has three members: PKD1, PKD2 and PKD3. Most cell types express at least two PKD isoforms but PKD enzymes are especially highly expressed in haematopoietic cells, where they are activated in response to antigen receptors stimulation [2,3]. A conserved signalling pathway linking antigen receptors to PKDs involves the activation of PLCγ and the subsequent production of diacylglycerol (DAG) which stimulates classical and/or novel protein kinase Cs (PKC) that phosphorylate two key regulatory serine residues in the activation loop of PKD kinases [3–6]. The N-terminal regulatory region of PKD enzymes contains a DAG binding domain and direct binding of DAG also contributes to PKD1 activation [7] as well as regulating the spatial location of PKD enzymes within cells [8–12]. PKD enzymes have been proposed to regulate numerous cellular functions, including cell proliferation [13–16], anti-apoptotic signals [17,18] and thymocyte development [19]. Expression of mutant catalytically inactive and constitutively activated PKDs can also modify Golgi function, cell adhesion and cell motility (reviewed in [20]). In particular, PKDs have been widely linked to the activation of the NFκB transcription factor and in regulating cell survival during oxidative stress [17,21–23]. Another recently proposed PKD1 substrate is HSP27 [24], a small heat shock protein involved in regulating cell migration and cell survival [25]. An essential role for PKD enzymes in regulating class II histone deacetylases (HDACs), enzymes that repress MEF2-dependent gene transcription, has also been demonstrated [1,26–28]. To investigate the biological role of PKDs we have generated DT40 B cell lines that lack expression of one or more members of the PKD family [1], allowing us to investigate the function(s) of PKD isoforms following B cell antigen receptor (BCR) stimulation, as well addressing the issue of functional redundancy between the different PKD family members. Previous studies have shown that PKDs are indispensable for HDAC regulation in B cells [1]. Herein we show that PKDs are also indispensable for HSP27 phosphorylation in B cells. However, PKD-null DT40 B cells are viable and proliferate normally. Moreover, loss of the entire cellular pool of PKD does not critically affect oxidative stress responses in B cells nor do PKD kinases play an essential role in regulating NFκB transcriptional activity. Together, these findings reveal that in B lymphocytes, PKD kinases are not critical regulators of many of the cellular processes previously ascribed to them in other cellular systems. 2 Materials and methods 2.1 ",culture
R1503	T1743	T1744	compound	Cell,culture
R1504	T1744	T1744	ROOT	culture,culture
R1505	T1745	T1744	punct	",",culture
R1506	T1746	T1747	compound	transient,transfections
R1507	T1747	T1744	appos	transfections,culture
R1508	T1748	T1747	cc	and,transfections
R1509	T1749	T1750	compound	cell,stimulation
R1510	T1750	T1747	conj	stimulation,transfections
R1511	T1751	T1752	det	The,generation
R1512	T1752	T1765	nsubj	generation,/
R1513	T1753	T1752	punct	",",generation
R1514	T1754	T1752	conj	culture,generation
R1515	T1755	T1754	cc	and,culture
R1046	T1160	T1161	auxpass	been,proposed
R1047	T1161	T1161	ROOT	proposed,proposed
R1048	T1162	T1163	aux	to,regulate
R1049	T1163	T1161	xcomp	regulate,proposed
R1128	T1242	T1243	nummod	23,]
R1129	T1243	T1243	ROOT	],]
R1130	T1244	T1223	punct	.,linked
R1516	T1756	T1754	conj	activation,culture
R1517	T1757	T1752	prep	of,generation
R1518	T1758	T1761	nummod	PKD1,−
R1672	T1948	T1949	nummod	106,cells
R1673	T1949	T1944	appos	cells,cells
R1674	T1950	T1949	prep	per,cells
R1675	T1951	T1950	pobj	point,per
R1676	T1952	T1944	punct	),cells
R2447	T2898	T2897	prep	upon,restored
R2448	T2899	T2898	pobj	re-expression,upon
R2449	T2900	T2899	prep	of,re-expression
R2450	T2901	T2904	det	a,isoform
R2452	T2903	T2904	compound	PKD,isoform
R2453	T2904	T2900	pobj	isoform,of
R2454	T2905	T2907	nmod	[,]
R2455	T2906	T2907	nummod	1,]
R2456	T2907	T2869	dobj	],demonstrated
R2457	T2908	T2869	punct	.,demonstrated
R2458	T2909	T2913	det	The,protein
R2459	T2910	T2913	amod	small,protein
R2460	T2911	T2912	compound	heat,shock
R2461	T2912	T2913	compound	shock,protein
R2462	T2913	T2918	nsubj	protein,proposed
R2463	T2914	T2913	appos	HSP27,protein
R2464	T2915	T2918	aux	has,proposed
R2465	T2916	T2918	advmod	recently,proposed
R2466	T2917	T2918	auxpass	been,proposed
R2467	T2918	T2918	ROOT	proposed,proposed
R2468	T2919	T2918	prep	as,proposed
R2469	T2920	T2922	det	a,substrate
R2470	T2921	T2922	compound	PKD1,substrate
R2471	T2922	T2919	pobj	substrate,as
R2472	T2923	T2925	nmod	[,]
R2473	T2924	T2925	nummod	24,]
R2474	T2925	T2922	dep	],substrate
R2475	T2926	T2922	cc	and,substrate
R2476	T2927	T2929	nsubj	we,assessed
R2477	T2928	T2929	advmod	accordingly,assessed
R2478	T2929	T2922	conj	assessed,substrate
R2479	T2930	T2934	mark	whether,have
R2480	T2931	T2932	compound	PKD-null,DT40
R2481	T2932	T2933	compound	DT40,cells
R2482	T2933	T2934	nsubj	cells,have
R2483	T2934	T2929	ccomp	have,assessed
R2484	T2935	T2936	amod	defective,phosphorylation
R2485	T2936	T2934	dobj	phosphorylation,have
R2486	T2937	T2936	prep	of,phosphorylation
R2487	T2938	T2937	pobj	HSP27,of
R3651	T4452	T4450	conj	proliferation,survival
R3652	T4453	T4452	prep	of,proliferation
R3653	T4454	T4455	compound	DT40,B
R3654	T4455	T4456	compound	B,cells
R3655	T4456	T4453	pobj	cells,of
R3656	T4457	T4444	punct	.,are
R3657	T4458	T4459	compound	PKD,enzymes
R175	T212	T215	compound	Protein,enzymes
R176	T213	T215	compound	kinase,enzymes
R177	T214	T215	compound	D,enzymes
R178	T215	T216	nsubj	enzymes,are
R179	T216	T216	ROOT	are,are
R180	T217	T216	acomp	dispensable,are
R181	T218	T217	prep	for,dispensable
R182	T219	T226	nmod	proliferation,activity
R183	T220	T219	punct	",",proliferation
R184	T221	T219	conj	survival,proliferation
R185	T222	T221	cc	and,survival
R186	T223	T221	conj	antigen,survival
R187	T224	T226	amod	receptor-regulated,activity
R188	T225	T226	compound	NFκB,activity
R189	T226	T218	pobj	activity,for
R190	T227	T226	prep	in,activity
R191	T228	T229	amod	vertebrate,B-cells
R192	T229	T227	pobj	B-cells,in
R193	T230	T231	aux	To,investigate
R194	T231	T216	advcl	investigate,are
R195	T232	T233	det	the,importance
R196	T233	T231	dobj	importance,investigate
R197	T234	T233	prep	of,importance
R198	T235	T234	pobj	protein,of
R199	T236	T237	compound	kinase,D
R200	T237	T235	appos	D,protein
R201	T238	T237	punct	(,D
R202	T239	T237	appos	PKD,D
R203	T240	T237	punct	),D

UBERON-AE

Id	Subject	Object	Predicate	Lexical cue
T9	117-127	http://purl.obolibrary.org/obo/UBERON_3010224	denotes	vertebrate
T1863	4244-4249	http://purl.obolibrary.org/obo/UBERON_0001977	denotes	serum
T3782	8575-8580	http://purl.obolibrary.org/obo/UBERON_0001977	denotes	serum

GO-BP

Id	Subject	Object	Predicate	Lexical cue
T504	1364-1374	http://purl.obolibrary.org/obo/GO_0023052	denotes	signalling
T505	2014-2021	http://purl.obolibrary.org/obo/GO_0023052	denotes	signals
T506	1364-1382	http://purl.obolibrary.org/obo/GO_0007165	denotes	signalling pathway
T507	1562-1565	http://purl.obolibrary.org/obo/GO_0004697	denotes	PKC
T508	1941-1949	http://purl.obolibrary.org/obo/GO_0007349	denotes	cellular
T509	3342-3350	http://purl.obolibrary.org/obo/GO_0007349	denotes	cellular
T510	3625-3633	http://purl.obolibrary.org/obo/GO_0007349	denotes	cellular
T511	3681-3689	http://purl.obolibrary.org/obo/GO_0007349	denotes	cellular
T512	1971-1989	http://purl.obolibrary.org/obo/GO_0008283	denotes	cell proliferation
T513	2044-2055	http://purl.obolibrary.org/obo/GO_0032502	denotes	development
T514	2172-2185	http://purl.obolibrary.org/obo/GO_0007155	denotes	cell adhesion
T515	2190-2203	http://purl.obolibrary.org/obo/GO_0048870	denotes	cell motility
T516	2298-2311	http://purl.obolibrary.org/obo/GO_0006351	denotes	transcription
T517	2660-2673	http://purl.obolibrary.org/obo/GO_0006351	denotes	transcription
T518	3489-3504	http://purl.obolibrary.org/obo/GO_0006351	denotes	transcriptional
T519	2451-2469	http://purl.obolibrary.org/obo/GO_0006986	denotes	heat shock protein
T520	2451-2469	http://purl.obolibrary.org/obo/GO_0034620	denotes	heat shock protein
T521	2451-2469	http://purl.obolibrary.org/obo/GO_0042026	denotes	heat shock protein
T522	2493-2507	http://purl.obolibrary.org/obo/GO_0016477	denotes	cell migration
T523	3132-3142	http://purl.obolibrary.org/obo/GO_0065007	denotes	regulation
T524	3217-3232	http://purl.obolibrary.org/obo/GO_0016310	denotes	phosphorylation
T525	3390-3416	http://purl.obolibrary.org/obo/GO_1902883	denotes	oxidative stress responses
T526	3390-3416	http://purl.obolibrary.org/obo/GO_0006979	denotes	oxidative stress responses
T527	3390-3416	http://purl.obolibrary.org/obo/GO_0097468	denotes	oxidative stress responses
T528	3390-3416	http://purl.obolibrary.org/obo/GO_1902884	denotes	oxidative stress responses
T529	3390-3416	http://purl.obolibrary.org/obo/GO_0034599	denotes	oxidative stress responses
T530	3400-3416	http://purl.obolibrary.org/obo/GO_0006950	denotes	stress responses
T531	3625-3643	http://purl.obolibrary.org/obo/GO_0009987	denotes	cellular processes
T2272	4561-4576	http://purl.obolibrary.org/obo/GO_0016310	denotes	phosphorylation
T2273	5322-5337	http://purl.obolibrary.org/obo/GO_0016310	denotes	phosphorylation
T2274	5680-5695	http://purl.obolibrary.org/obo/GO_0016310	denotes	phosphorylation
T2275	5807-5822	http://purl.obolibrary.org/obo/GO_0016310	denotes	phosphorylation
T2276	5996-6011	http://purl.obolibrary.org/obo/GO_0016310	denotes	phosphorylation
T2277	6109-6124	http://purl.obolibrary.org/obo/GO_0016310	denotes	phosphorylation
T2278	6221-6236	http://purl.obolibrary.org/obo/GO_0016310	denotes	phosphorylation
T2279	6475-6490	http://purl.obolibrary.org/obo/GO_0016310	denotes	phosphorylation
T2280	6657-6672	http://purl.obolibrary.org/obo/GO_0016310	denotes	phosphorylation
T2281	6731-6746	http://purl.obolibrary.org/obo/GO_0016310	denotes	phosphorylation
T2282	5532-5550	http://purl.obolibrary.org/obo/GO_0006986	denotes	heat shock protein
T2283	5532-5550	http://purl.obolibrary.org/obo/GO_0034620	denotes	heat shock protein
T3833	8362-8390	http://purl.obolibrary.org/obo/GO_1902884	denotes	response to oxidative stress
T3834	8362-8390	http://purl.obolibrary.org/obo/GO_0034599	denotes	response to oxidative stress
T3835	8525-8557	http://purl.obolibrary.org/obo/GO_1990046	denotes	response to mitochondrial stress
T21	553-563	http://purl.obolibrary.org/obo/GO_0065007	denotes	regulation
T22	705-720	http://purl.obolibrary.org/obo/GO_0016310	denotes	phosphorylation
T23	839-847	http://purl.obolibrary.org/obo/GO_0007349	denotes	cellular
T24	839-857	http://purl.obolibrary.org/obo/GO_0009987	denotes	cellular processes
T25	912-927	http://purl.obolibrary.org/obo/GO_0006351	denotes	transcriptional
T3820	6963-6973	http://purl.obolibrary.org/obo/GO_0065007	denotes	regulation
T3821	6963-6995	http://purl.obolibrary.org/obo/GO_0030888	denotes	regulation of cell proliferation
T3822	6963-6995	http://purl.obolibrary.org/obo/GO_0051140	denotes	regulation of cell proliferation
T3823	6963-6995	http://purl.obolibrary.org/obo/GO_0060251	denotes	regulation of cell proliferation
T3824	6963-6995	http://purl.obolibrary.org/obo/GO_0070666	denotes	regulation of cell proliferation
T3825	6963-6995	http://purl.obolibrary.org/obo/GO_1904073	denotes	regulation of cell proliferation
T3826	6963-6995	http://purl.obolibrary.org/obo/GO_1904697	denotes	regulation of cell proliferation
T3827	6963-6995	http://purl.obolibrary.org/obo/GO_1904441	denotes	regulation of cell proliferation
T3828	6977-6995	http://purl.obolibrary.org/obo/GO_0008283	denotes	cell proliferation
T3829	7271-7277	http://purl.obolibrary.org/obo/GO_0040007	denotes	growth
T3830	8362-8390	http://purl.obolibrary.org/obo/GO_1902883	denotes	response to oxidative stress
T3831	8362-8390	http://purl.obolibrary.org/obo/GO_0006979	denotes	response to oxidative stress
T3832	8362-8390	http://purl.obolibrary.org/obo/GO_0097468	denotes	response to oxidative stress
T2284	5532-5550	http://purl.obolibrary.org/obo/GO_0042026	denotes	heat shock protein
T2285	5787-5797	http://purl.obolibrary.org/obo/GO_0065007	denotes	regulation
T2286	6455-6465	http://purl.obolibrary.org/obo/GO_0065007	denotes	regulation
T2287	6068-6075	http://purl.obolibrary.org/obo/GO_0023052	denotes	signals

GO-MF

Id	Subject	Object	Predicate	Lexical cue
T1483	1562-1565	http://purl.obolibrary.org/obo/GO_0004697	denotes	PKC
T1484	1729-1736	http://purl.obolibrary.org/obo/GO_0005488	denotes	binding
T1485	1755-1762	http://purl.obolibrary.org/obo/GO_0005488	denotes	binding
T2014	4289-4297	http://purl.obolibrary.org/obo/GO_0003823	denotes	antibody

GO-CC

Id	Subject	Object	Predicate	Lexical cue
T1499	2156-2161	http://purl.obolibrary.org/obo/GO_0005794	denotes	Golgi
T1500	3625-3643	http://purl.obolibrary.org/obo/GO_0042995	denotes	cellular processes
T1816	3773-3777	http://purl.obolibrary.org/obo/GO_0005623	denotes	cell
T1817	3879-3883	http://purl.obolibrary.org/obo/GO_0005623	denotes	cell
T2015	4145-4150	http://purl.obolibrary.org/obo/GO_0005623	denotes	cells
T2016	4160-4165	http://purl.obolibrary.org/obo/GO_0005623	denotes	cells
T2017	4340-4345	http://purl.obolibrary.org/obo/GO_0005623	denotes	cells
T2018	4289-4297	http://purl.obolibrary.org/obo/GO_0019815	denotes	antibody
T2019	4289-4297	http://purl.obolibrary.org/obo/GO_0042571	denotes	antibody
T4613	6869-6874	http://purl.obolibrary.org/obo/GO_0005623	denotes	cells
T4614	7159-7164	http://purl.obolibrary.org/obo/GO_0005623	denotes	cells
T4615	7310-7315	http://purl.obolibrary.org/obo/GO_0005623	denotes	cells
T4616	7383-7388	http://purl.obolibrary.org/obo/GO_0005623	denotes	cells
T4617	7475-7480	http://purl.obolibrary.org/obo/GO_0005623	denotes	cells
T4618	7563-7568	http://purl.obolibrary.org/obo/GO_0005623	denotes	cells
T4619	7647-7652	http://purl.obolibrary.org/obo/GO_0005623	denotes	cells
T4620	7701-7706	http://purl.obolibrary.org/obo/GO_0005623	denotes	cells
T4621	7826-7831	http://purl.obolibrary.org/obo/GO_0005623	denotes	cells
T4622	8076-8081	http://purl.obolibrary.org/obo/GO_0005623	denotes	cells
T4623	8516-8521	http://purl.obolibrary.org/obo/GO_0005623	denotes	cells
T4624	8870-8874	http://purl.obolibrary.org/obo/GO_0005623	denotes	cell
T4625	8643-8645	http://purl.obolibrary.org/obo/GO_0005783	denotes	ER
T1486	1157-1161	http://purl.obolibrary.org/obo/GO_0005623	denotes	cell
T1487	1971-1975	http://purl.obolibrary.org/obo/GO_0005623	denotes	cell
T1488	2172-2176	http://purl.obolibrary.org/obo/GO_0005623	denotes	cell
T1489	2190-2194	http://purl.obolibrary.org/obo/GO_0005623	denotes	cell
T1490	2337-2341	http://purl.obolibrary.org/obo/GO_0005623	denotes	cell
T1491	2493-2497	http://purl.obolibrary.org/obo/GO_0005623	denotes	cell
T1492	2512-2516	http://purl.obolibrary.org/obo/GO_0005623	denotes	cell
T1493	2781-2785	http://purl.obolibrary.org/obo/GO_0005623	denotes	cell
T1494	2930-2934	http://purl.obolibrary.org/obo/GO_0005623	denotes	cell
T3137	4587-4592	http://purl.obolibrary.org/obo/GO_0005623	denotes	cells
T3138	4641-4646	http://purl.obolibrary.org/obo/GO_0005623	denotes	cells
T3139	5098-5103	http://purl.obolibrary.org/obo/GO_0005623	denotes	cells
T3140	5163-5168	http://purl.obolibrary.org/obo/GO_0005623	denotes	cells
T3141	5451-5456	http://purl.obolibrary.org/obo/GO_0005623	denotes	cells
T3142	5659-5664	http://purl.obolibrary.org/obo/GO_0005623	denotes	cells
T3143	5849-5854	http://purl.obolibrary.org/obo/GO_0005623	denotes	cells
T3144	6039-6044	http://purl.obolibrary.org/obo/GO_0005623	denotes	cells
T3145	6164-6169	http://purl.obolibrary.org/obo/GO_0005623	denotes	cells
T3146	6272-6277	http://purl.obolibrary.org/obo/GO_0005623	denotes	cells
T3147	6416-6421	http://purl.obolibrary.org/obo/GO_0005623	denotes	cells
T3148	6591-6596	http://purl.obolibrary.org/obo/GO_0005623	denotes	cells
T3149	6761-6766	http://purl.obolibrary.org/obo/GO_0005623	denotes	cells
T399	130-135	http://purl.obolibrary.org/obo/GO_0005623	denotes	cells
T400	376-381	http://purl.obolibrary.org/obo/GO_0005623	denotes	cells
T401	731-736	http://purl.obolibrary.org/obo/GO_0005623	denotes	cells
T402	793-797	http://purl.obolibrary.org/obo/GO_0005623	denotes	cell
T403	957-961	http://purl.obolibrary.org/obo/GO_0005623	denotes	cell
T404	1023-1027	http://purl.obolibrary.org/obo/GO_0005623	denotes	cell
T405	839-857	http://purl.obolibrary.org/obo/GO_0042995	denotes	cellular processes
T1495	3148-3153	http://purl.obolibrary.org/obo/GO_0005623	denotes	cells
T1496	3238-3243	http://purl.obolibrary.org/obo/GO_0005623	denotes	cells
T1497	3270-3275	http://purl.obolibrary.org/obo/GO_0005623	denotes	cells
T1498	3422-3427	http://purl.obolibrary.org/obo/GO_0005623	denotes	cells

sentences

Id	Subject	Object	Predicate	Lexical cue
T17	615-646	Sentence	denotes	Mol. Cell Biol. 26, 1569–1577].
T489	635-1151	Sentence	denotes	1569–1577]. We now show that PKDs are also required to regulate HSP27 phosphorylation in DT40 B-cells. However, in contrast to previous observations in other cell types, PKD enzymes do not regulate basic cellular processes such as proliferation or survival responses, nor NFκB transcriptional activity downstream of the B cell antigen receptor. Thus, PKDs have a selective role in DT40 B-cell biology. 1 Introduction The protein kinase D (PKD) serine/threonine kinase family has three members: PKD1, PKD2 and PKD3.
T490	1152-1351	Sentence	denotes	Most cell types express at least two PKD isoforms but PKD enzymes are especially highly expressed in haematopoietic cells, where they are activated in response to antigen receptors stimulation [2,3].
T491	1352-1665	Sentence	denotes	A conserved signalling pathway linking antigen receptors to PKDs involves the activation of PLCγ and the subsequent production of diacylglycerol (DAG) which stimulates classical and/or novel protein kinase Cs (PKC) that phosphorylate two key regulatory serine residues in the activation loop of PKD kinases [3–6].
T492	1666-1888	Sentence	denotes	The N-terminal regulatory region of PKD enzymes contains a DAG binding domain and direct binding of DAG also contributes to PKD1 activation [7] as well as regulating the spatial location of PKD enzymes within cells [8–12].
T493	1889-2061	Sentence	denotes	PKD enzymes have been proposed to regulate numerous cellular functions, including cell proliferation [13–16], anti-apoptotic signals [17,18] and thymocyte development [19].
T494	2062-2223	Sentence	denotes	Expression of mutant catalytically inactive and constitutively activated PKDs can also modify Golgi function, cell adhesion and cell motility (reviewed in [20]).
T495	2224-2386	Sentence	denotes	In particular, PKDs have been widely linked to the activation of the NFκB transcription factor and in regulating cell survival during oxidative stress [17,21–23].
T496	2387-2531	Sentence	denotes	Another recently proposed PKD1 substrate is HSP27 [24], a small heat shock protein involved in regulating cell migration and cell survival [25].
T497	2532-2712	Sentence	denotes	An essential role for PKD enzymes in regulating class II histone deacetylases (HDACs), enzymes that repress MEF2-dependent gene transcription, has also been demonstrated [1,26–28].
T498	2713-3066	Sentence	denotes	To investigate the biological role of PKDs we have generated DT40 B cell lines that lack expression of one or more members of the PKD family [1], allowing us to investigate the function(s) of PKD isoforms following B cell antigen receptor (BCR) stimulation, as well addressing the issue of functional redundancy between the different PKD family members.
T499	3067-3158	Sentence	denotes	Previous studies have shown that PKDs are indispensable for HDAC regulation in B cells [1].
T500	3159-3244	Sentence	denotes	Herein we show that PKDs are also indispensable for HSP27 phosphorylation in B cells.
T501	3245-3312	Sentence	denotes	However, PKD-null DT40 B cells are viable and proliferate normally.
T502	3313-3514	Sentence	denotes	Moreover, loss of the entire cellular pool of PKD does not critically affect oxidative stress responses in B cells nor do PKD kinases play an essential role in regulating NFκB transcriptional activity.
T503	3515-3698	Sentence	denotes	Together, these findings reveal that in B lymphocytes, PKD kinases are not critical regulators of many of the cellular processes previously ascribed to them in other cellular systems.
T1670	0-3789	Sentence	denotes	Protein kinase D enzymes are dispensable for proliferation, survival and antigen receptor-regulated NFκB activity in vertebrate B-cells Abstract To investigate the importance of protein kinase D (PKD) enzymes we generated a PKD-null DT40 B-lymphocyte cell line. Previously we have shown that PKDs have an essential role in regulating class II histone deacetylases in DT40 B-cells [Matthews, S.A., Liu, P., Spitaler, M., Olson, E.N., McKinsey, T.A., Cantrell, D.A. and Scharenberg, A.M. (2006) Essential role for protein kinase D family kinases in the regulation of class II histone deacetylases in B lymphocytes. Mol. Cell Biol. 26, 1569–1577]. We now show that PKDs are also required to regulate HSP27 phosphorylation in DT40 B-cells. However, in contrast to previous observations in other cell types, PKD enzymes do not regulate basic cellular processes such as proliferation or survival responses, nor NFκB transcriptional activity downstream of the B cell antigen receptor. Thus, PKDs have a selective role in DT40 B-cell biology. 1 Introduction The protein kinase D (PKD) serine/threonine kinase family has three members: PKD1, PKD2 and PKD3. Most cell types express at least two PKD isoforms but PKD enzymes are especially highly expressed in haematopoietic cells, where they are activated in response to antigen receptors stimulation [2,3]. A conserved signalling pathway linking antigen receptors to PKDs involves the activation of PLCγ and the subsequent production of diacylglycerol (DAG) which stimulates classical and/or novel protein kinase Cs (PKC) that phosphorylate two key regulatory serine residues in the activation loop of PKD kinases [3–6]. The N-terminal regulatory region of PKD enzymes contains a DAG binding domain and direct binding of DAG also contributes to PKD1 activation [7] as well as regulating the spatial location of PKD enzymes within cells [8–12]. PKD enzymes have been proposed to regulate numerous cellular functions, including cell proliferation [13–16], anti-apoptotic signals [17,18] and thymocyte development [19]. Expression of mutant catalytically inactive and constitutively activated PKDs can also modify Golgi function, cell adhesion and cell motility (reviewed in [20]). In particular, PKDs have been widely linked to the activation of the NFκB transcription factor and in regulating cell survival during oxidative stress [17,21–23]. Another recently proposed PKD1 substrate is HSP27 [24], a small heat shock protein involved in regulating cell migration and cell survival [25]. An essential role for PKD enzymes in regulating class II histone deacetylases (HDACs), enzymes that repress MEF2-dependent gene transcription, has also been demonstrated [1,26–28]. To investigate the biological role of PKDs we have generated DT40 B cell lines that lack expression of one or more members of the PKD family [1], allowing us to investigate the function(s) of PKD isoforms following B cell antigen receptor (BCR) stimulation, as well addressing the issue of functional redundancy between the different PKD family members. Previous studies have shown that PKDs are indispensable for HDAC regulation in B cells [1]. Herein we show that PKDs are also indispensable for HSP27 phosphorylation in B cells. However, PKD-null DT40 B cells are viable and proliferate normally. Moreover, loss of the entire cellular pool of PKD does not critically affect oxidative stress responses in B cells nor do PKD kinases play an essential role in regulating NFκB transcriptional activity. Together, these findings reveal that in B lymphocytes, PKD kinases are not critical regulators of many of the cellular processes previously ascribed to them in other cellular systems. 2 Materials and methods 2.1 Cell culture, transient transfections and cell stimulation
T1671	3790-3925	Sentence	denotes	The generation, culture and activation of PKD1−/−, PKD3−/− and PKD1/3−/− knockout DT40 B cell lines have been described previously [1].
T1672	3926-4038	Sentence	denotes	Cells were lysed and protein extracts were analysed in Western blotting experiments as previously described [1].
T1673	4039-4117	Sentence	denotes	Chloramphenicol acetyl transferase assays have been described previously [29].
T1864	0-4137	Sentence	denotes	Protein kinase D enzymes are dispensable for proliferation, survival and antigen receptor-regulated NFκB activity in vertebrate B-cells Abstract To investigate the importance of protein kinase D (PKD) enzymes we generated a PKD-null DT40 B-lymphocyte cell line. Previously we have shown that PKDs have an essential role in regulating class II histone deacetylases in DT40 B-cells [Matthews, S.A., Liu, P., Spitaler, M., Olson, E.N., McKinsey, T.A., Cantrell, D.A. and Scharenberg, A.M. (2006) Essential role for protein kinase D family kinases in the regulation of class II histone deacetylases in B lymphocytes. Mol. Cell Biol. 26, 1569–1577]. We now show that PKDs are also required to regulate HSP27 phosphorylation in DT40 B-cells. However, in contrast to previous observations in other cell types, PKD enzymes do not regulate basic cellular processes such as proliferation or survival responses, nor NFκB transcriptional activity downstream of the B cell antigen receptor. Thus, PKDs have a selective role in DT40 B-cell biology. 1 Introduction The protein kinase D (PKD) serine/threonine kinase family has three members: PKD1, PKD2 and PKD3. Most cell types express at least two PKD isoforms but PKD enzymes are especially highly expressed in haematopoietic cells, where they are activated in response to antigen receptors stimulation [2,3]. A conserved signalling pathway linking antigen receptors to PKDs involves the activation of PLCγ and the subsequent production of diacylglycerol (DAG) which stimulates classical and/or novel protein kinase Cs (PKC) that phosphorylate two key regulatory serine residues in the activation loop of PKD kinases [3–6]. The N-terminal regulatory region of PKD enzymes contains a DAG binding domain and direct binding of DAG also contributes to PKD1 activation [7] as well as regulating the spatial location of PKD enzymes within cells [8–12]. PKD enzymes have been proposed to regulate numerous cellular functions, including cell proliferation [13–16], anti-apoptotic signals [17,18] and thymocyte development [19]. Expression of mutant catalytically inactive and constitutively activated PKDs can also modify Golgi function, cell adhesion and cell motility (reviewed in [20]). In particular, PKDs have been widely linked to the activation of the NFκB transcription factor and in regulating cell survival during oxidative stress [17,21–23]. Another recently proposed PKD1 substrate is HSP27 [24], a small heat shock protein involved in regulating cell migration and cell survival [25]. An essential role for PKD enzymes in regulating class II histone deacetylases (HDACs), enzymes that repress MEF2-dependent gene transcription, has also been demonstrated [1,26–28]. To investigate the biological role of PKDs we have generated DT40 B cell lines that lack expression of one or more members of the PKD family [1], allowing us to investigate the function(s) of PKD isoforms following B cell antigen receptor (BCR) stimulation, as well addressing the issue of functional redundancy between the different PKD family members. Previous studies have shown that PKDs are indispensable for HDAC regulation in B cells [1]. Herein we show that PKDs are also indispensable for HSP27 phosphorylation in B cells. However, PKD-null DT40 B cells are viable and proliferate normally. Moreover, loss of the entire cellular pool of PKD does not critically affect oxidative stress responses in B cells nor do PKD kinases play an essential role in regulating NFκB transcriptional activity. Together, these findings reveal that in B lymphocytes, PKD kinases are not critical regulators of many of the cellular processes previously ascribed to them in other cellular systems. 2 Materials and methods 2.1 Cell culture, transient transfections and cell stimulation The generation, culture and activation of PKD1−/−, PKD3−/− and PKD1/3−/− knockout DT40 B cell lines have been described previously [1]. Cells were lysed and protein extracts were analysed in Western blotting experiments as previously described [1]. Chloramphenicol acetyl transferase assays have been described previously [29]. 2.2 sIgM staining
T1865	4138-4335	Sentence	denotes	DT40 B cells (2 × 106 cells per point) were resuspended in 200 μl buffer (RPMI 1640 media, 1% foetal calf serum) containing anti-chicken M1 monoclonal antibody conjugated to FITC for 20 min on ice.
T1866	4336-4421	Sentence	denotes	The cells were washed twice and fluorescent intensity was analysed by flow cytometry.
T1867	4422-4528	Sentence	denotes	All results shown are representative of at two to four independent experiments unless otherwise indicated.
T2259	0-4633	Sentence	denotes	Protein kinase D enzymes are dispensable for proliferation, survival and antigen receptor-regulated NFκB activity in vertebrate B-cells Abstract To investigate the importance of protein kinase D (PKD) enzymes we generated a PKD-null DT40 B-lymphocyte cell line. Previously we have shown that PKDs have an essential role in regulating class II histone deacetylases in DT40 B-cells [Matthews, S.A., Liu, P., Spitaler, M., Olson, E.N., McKinsey, T.A., Cantrell, D.A. and Scharenberg, A.M. (2006) Essential role for protein kinase D family kinases in the regulation of class II histone deacetylases in B lymphocytes. Mol. Cell Biol. 26, 1569–1577]. We now show that PKDs are also required to regulate HSP27 phosphorylation in DT40 B-cells. However, in contrast to previous observations in other cell types, PKD enzymes do not regulate basic cellular processes such as proliferation or survival responses, nor NFκB transcriptional activity downstream of the B cell antigen receptor. Thus, PKDs have a selective role in DT40 B-cell biology. 1 Introduction The protein kinase D (PKD) serine/threonine kinase family has three members: PKD1, PKD2 and PKD3. Most cell types express at least two PKD isoforms but PKD enzymes are especially highly expressed in haematopoietic cells, where they are activated in response to antigen receptors stimulation [2,3]. A conserved signalling pathway linking antigen receptors to PKDs involves the activation of PLCγ and the subsequent production of diacylglycerol (DAG) which stimulates classical and/or novel protein kinase Cs (PKC) that phosphorylate two key regulatory serine residues in the activation loop of PKD kinases [3–6]. The N-terminal regulatory region of PKD enzymes contains a DAG binding domain and direct binding of DAG also contributes to PKD1 activation [7] as well as regulating the spatial location of PKD enzymes within cells [8–12]. PKD enzymes have been proposed to regulate numerous cellular functions, including cell proliferation [13–16], anti-apoptotic signals [17,18] and thymocyte development [19]. Expression of mutant catalytically inactive and constitutively activated PKDs can also modify Golgi function, cell adhesion and cell motility (reviewed in [20]). In particular, PKDs have been widely linked to the activation of the NFκB transcription factor and in regulating cell survival during oxidative stress [17,21–23]. Another recently proposed PKD1 substrate is HSP27 [24], a small heat shock protein involved in regulating cell migration and cell survival [25]. An essential role for PKD enzymes in regulating class II histone deacetylases (HDACs), enzymes that repress MEF2-dependent gene transcription, has also been demonstrated [1,26–28]. To investigate the biological role of PKDs we have generated DT40 B cell lines that lack expression of one or more members of the PKD family [1], allowing us to investigate the function(s) of PKD isoforms following B cell antigen receptor (BCR) stimulation, as well addressing the issue of functional redundancy between the different PKD family members. Previous studies have shown that PKDs are indispensable for HDAC regulation in B cells [1]. Herein we show that PKDs are also indispensable for HSP27 phosphorylation in B cells. However, PKD-null DT40 B cells are viable and proliferate normally. Moreover, loss of the entire cellular pool of PKD does not critically affect oxidative stress responses in B cells nor do PKD kinases play an essential role in regulating NFκB transcriptional activity. Together, these findings reveal that in B lymphocytes, PKD kinases are not critical regulators of many of the cellular processes previously ascribed to them in other cellular systems. 2 Materials and methods 2.1 Cell culture, transient transfections and cell stimulation The generation, culture and activation of PKD1−/−, PKD3−/− and PKD1/3−/− knockout DT40 B cell lines have been described previously [1]. Cells were lysed and protein extracts were analysed in Western blotting experiments as previously described [1]. Chloramphenicol acetyl transferase assays have been described previously [29]. 2.2 sIgM staining DT40 B cells (2 × 106 cells per point) were resuspended in 200 μl buffer (RPMI 1640 media, 1% foetal calf serum) containing anti-chicken M1 monoclonal antibody conjugated to FITC for 20 min on ice. The cells were washed twice and fluorescent intensity was analysed by flow cytometry. All results shown are representative of at two to four independent experiments unless otherwise indicated. 3 Results 3.1 Loss of HSP27 phosphorylation in DT40 B cells lacking expression of PKD family kinases
T2260	4634-4825	Sentence	denotes	DT40 B cells express two PKD isoforms, PKD1 and PKD3, and as previously described we have recently generated DT40 B cell lines that lack expression of either PKD1 or PKD3 or both enzymes [1].
T2261	4826-5011	Sentence	denotes	In generating the double knockout cell lines we targeted the PKD1 loci in a PKD3−/− cell line that expressed a Flag-PKD3 transgene under the control of a doxycycline-inducible promoter.
T2262	5012-5283	Sentence	denotes	Hence, in the presence of doxycycline, Flag-PKD3 expression in PKD1/3 double knockout cells is comparable to endogenous PKD3 present in wild-type DT40 cells and removal of doxycycline from the culture media for 5 days results in a completely null PKD phenotype (Fig. 1A).
T2263	5284-5521	Sentence	denotes	Previously, we have demonstrated that phosphorylation and nuclear exclusion of class II histone deacetylases (HDACs) during BCR engagement is defective in PKD1/3−/− B cells and can restored upon re-expression of a single PKD isoform [1].
T2264	5522-5756	Sentence	denotes	The small heat shock protein HSP27 has recently been proposed as a PKD1 substrate [24] and we accordingly assessed whether PKD-null DT40 cells have defective phosphorylation of HSP27 on serine 82, the proposed PKD1 substrate sequence.
T2265	5757-5883	Sentence	denotes	We initially investigated the regulation of HSP27 phosphorylation in single knockout DT40 B cells lacking either PKD1 or PKD3.
T2266	5884-6045	Sentence	denotes	As shown in Fig. 1B, activation of the BCR or treatment with the DAG-mimetic PdBu increased the levels of HSP27 phosphorylation at S82 in wild-type DT40 B cells.
T2267	6046-6180	Sentence	denotes	BCR and phorbol ester signals were also able to increase HSP27 phosphorylation in PKD1 or PKD3 single knockout DT40 B cells (Fig. 1B).
T2268	6181-6319	Sentence	denotes	However, BCR- and phorbol ester-induced phosphorylation of HSP27 on S82 was abolished in B cells that lacked both PKD1 and PKD3 (Fig. 1C).
T2269	6320-6501	Sentence	denotes	Significantly, doxycycline-induced expression of the Flag-PKD3 transgene in the double knockout cells was sufficient to restore normal regulation of HSP27 phosphorylation (Fig. 1C).
T2270	6502-6683	Sentence	denotes	In contrast, expression of a kinase-deficient PKD3 mutant protein in the double knockout cells was not able to restore BCR- or phorbol ester-induced HSP27 phosphorylation (Fig. 1D).
T2271	6684-6816	Sentence	denotes	Hence, PKD3 as well as PKD1 can regulate HSP27 phosphorylation and in DT40 B cells they are functionally redundant as HSP27 kinases.
T3808	0-6915	Sentence	denotes	Protein kinase D enzymes are dispensable for proliferation, survival and antigen receptor-regulated NFκB activity in vertebrate B-cells Abstract To investigate the importance of protein kinase D (PKD) enzymes we generated a PKD-null DT40 B-lymphocyte cell line. Previously we have shown that PKDs have an essential role in regulating class II histone deacetylases in DT40 B-cells [Matthews, S.A., Liu, P., Spitaler, M., Olson, E.N., McKinsey, T.A., Cantrell, D.A. and Scharenberg, A.M. (2006) Essential role for protein kinase D family kinases in the regulation of class II histone deacetylases in B lymphocytes. Mol. Cell Biol. 26, 1569–1577]. We now show that PKDs are also required to regulate HSP27 phosphorylation in DT40 B-cells. However, in contrast to previous observations in other cell types, PKD enzymes do not regulate basic cellular processes such as proliferation or survival responses, nor NFκB transcriptional activity downstream of the B cell antigen receptor. Thus, PKDs have a selective role in DT40 B-cell biology. 1 Introduction The protein kinase D (PKD) serine/threonine kinase family has three members: PKD1, PKD2 and PKD3. Most cell types express at least two PKD isoforms but PKD enzymes are especially highly expressed in haematopoietic cells, where they are activated in response to antigen receptors stimulation [2,3]. A conserved signalling pathway linking antigen receptors to PKDs involves the activation of PLCγ and the subsequent production of diacylglycerol (DAG) which stimulates classical and/or novel protein kinase Cs (PKC) that phosphorylate two key regulatory serine residues in the activation loop of PKD kinases [3–6]. The N-terminal regulatory region of PKD enzymes contains a DAG binding domain and direct binding of DAG also contributes to PKD1 activation [7] as well as regulating the spatial location of PKD enzymes within cells [8–12]. PKD enzymes have been proposed to regulate numerous cellular functions, including cell proliferation [13–16], anti-apoptotic signals [17,18] and thymocyte development [19]. Expression of mutant catalytically inactive and constitutively activated PKDs can also modify Golgi function, cell adhesion and cell motility (reviewed in [20]). In particular, PKDs have been widely linked to the activation of the NFκB transcription factor and in regulating cell survival during oxidative stress [17,21–23]. Another recently proposed PKD1 substrate is HSP27 [24], a small heat shock protein involved in regulating cell migration and cell survival [25]. An essential role for PKD enzymes in regulating class II histone deacetylases (HDACs), enzymes that repress MEF2-dependent gene transcription, has also been demonstrated [1,26–28]. To investigate the biological role of PKDs we have generated DT40 B cell lines that lack expression of one or more members of the PKD family [1], allowing us to investigate the function(s) of PKD isoforms following B cell antigen receptor (BCR) stimulation, as well addressing the issue of functional redundancy between the different PKD family members. Previous studies have shown that PKDs are indispensable for HDAC regulation in B cells [1]. Herein we show that PKDs are also indispensable for HSP27 phosphorylation in B cells. However, PKD-null DT40 B cells are viable and proliferate normally. Moreover, loss of the entire cellular pool of PKD does not critically affect oxidative stress responses in B cells nor do PKD kinases play an essential role in regulating NFκB transcriptional activity. Together, these findings reveal that in B lymphocytes, PKD kinases are not critical regulators of many of the cellular processes previously ascribed to them in other cellular systems. 2 Materials and methods 2.1 Cell culture, transient transfections and cell stimulation The generation, culture and activation of PKD1−/−, PKD3−/− and PKD1/3−/− knockout DT40 B cell lines have been described previously [1]. Cells were lysed and protein extracts were analysed in Western blotting experiments as previously described [1]. Chloramphenicol acetyl transferase assays have been described previously [29]. 2.2 sIgM staining DT40 B cells (2 × 106 cells per point) were resuspended in 200 μl buffer (RPMI 1640 media, 1% foetal calf serum) containing anti-chicken M1 monoclonal antibody conjugated to FITC for 20 min on ice. The cells were washed twice and fluorescent intensity was analysed by flow cytometry. All results shown are representative of at two to four independent experiments unless otherwise indicated. 3 Results 3.1 Loss of HSP27 phosphorylation in DT40 B cells lacking expression of PKD family kinases DT40 B cells express two PKD isoforms, PKD1 and PKD3, and as previously described we have recently generated DT40 B cell lines that lack expression of either PKD1 or PKD3 or both enzymes [1]. In generating the double knockout cell lines we targeted the PKD1 loci in a PKD3−/− cell line that expressed a Flag-PKD3 transgene under the control of a doxycycline-inducible promoter. Hence, in the presence of doxycycline, Flag-PKD3 expression in PKD1/3 double knockout cells is comparable to endogenous PKD3 present in wild-type DT40 cells and removal of doxycycline from the culture media for 5 days results in a completely null PKD phenotype (Fig. 1A). Previously, we have demonstrated that phosphorylation and nuclear exclusion of class II histone deacetylases (HDACs) during BCR engagement is defective in PKD1/3−/− B cells and can restored upon re-expression of a single PKD isoform [1]. The small heat shock protein HSP27 has recently been proposed as a PKD1 substrate [24] and we accordingly assessed whether PKD-null DT40 cells have defective phosphorylation of HSP27 on serine 82, the proposed PKD1 substrate sequence. We initially investigated the regulation of HSP27 phosphorylation in single knockout DT40 B cells lacking either PKD1 or PKD3. As shown in Fig. 1B, activation of the BCR or treatment with the DAG-mimetic PdBu increased the levels of HSP27 phosphorylation at S82 in wild-type DT40 B cells. BCR and phorbol ester signals were also able to increase HSP27 phosphorylation in PKD1 or PKD3 single knockout DT40 B cells (Fig. 1B). However, BCR- and phorbol ester-induced phosphorylation of HSP27 on S82 was abolished in B cells that lacked both PKD1 and PKD3 (Fig. 1C). Significantly, doxycycline-induced expression of the Flag-PKD3 transgene in the double knockout cells was sufficient to restore normal regulation of HSP27 phosphorylation (Fig. 1C). In contrast, expression of a kinase-deficient PKD3 mutant protein in the double knockout cells was not able to restore BCR- or phorbol ester-induced HSP27 phosphorylation (Fig. 1D). Hence, PKD3 as well as PKD1 can regulate HSP27 phosphorylation and in DT40 B cells they are functionally redundant as HSP27 kinases. 3.2 Cellular proliferation and survival in DT40 B cells lacking expression of PKD family kinases
T3809	6916-7028	Sentence	denotes	PKD enzymes have previously been linked to the regulation of cell proliferation and survival (reviewed in [20]).
T3810	7029-7192	Sentence	denotes	To investigate the effect that loss of PKD kinases had on B cell survival and/or proliferation we cultured wild-type and PKD-null cells in the presence (PKD1/3−/−:
T3811	7193-7278	Sentence	denotes	Flag-PKD3+ve) or absence (PKD1/3−/−) of doxycycline and monitored exponential growth.
T3812	7279-7432	Sentence	denotes	As shown in Fig. 2A, PKD1/3−/− cells proliferated exponentially and re-expression of Flag-PKD3 in these cells had no impact on the rate of proliferation.
T3813	7433-7586	Sentence	denotes	Furthermore, the viability of PKD1/3−/− B cells during routine culturing was not significantly different from that of wild-type B cells (data not shown).
T3814	7587-7974	Sentence	denotes	It was noted that the population doubling time of PKD1/3−/− cells was slightly slower than that of wild type DT40 cells (12.7 ± 2.8 h versus 10.2 ± 0.4 h) but the failure of PKD3 re-expression to modify the proliferation rate of PKD1/3−/− cells suggests that these small differences were most likely the result of clonal variation and were not caused specifically by loss of PKD enzymes.
T3815	7975-8082	Sentence	denotes	Thus, PKD family enzymes are not essential for regulating basal survival and proliferation of DT40 B cells.
T3816	8083-8274	Sentence	denotes	PKD enzymes, specifically PKD1 and PKD2, have previously been linked to a protective role against oxidative stress-induced injury in 3T3 fibroblast, HeLa and epithelial cell lines [17,30–32].
T3817	8275-8416	Sentence	denotes	We therefore addressed the role of PKD family kinases in regulating B cell survival in response to oxidative stress and other stress stimuli.
T3818	8417-8805	Sentence	denotes	As shown in Fig. 2B, loss of PKD1/3 expression had no significant impact on the survival of DT40 B cells in response to mitochondrial stress stimuli (H2O2 or serum deprivation); DNA damaging agents (etoposide or doxorubicin); ER pathway stress due to calcium overload (thapsigargin) or following prolonged treatment with phorbol esters or Trichostatin A, an inhibitor of class I/II HDACs.
T3819	8806-8935	Sentence	denotes	Thus, PKD kinases do not play an essential role in regulating B cell survival in response to a range of different stress stimuli.
T14	0-135	Sentence	denotes	Protein kinase D enzymes are dispensable for proliferation, survival and antigen receptor-regulated NFκB activity in vertebrate B-cells
T15	147-263	Sentence	denotes	To investigate the importance of protein kinase D (PKD) enzymes we generated a PKD-null DT40 B-lymphocyte cell line.
T16	264-614	Sentence	denotes	Previously we have shown that PKDs have an essential role in regulating class II histone deacetylases in DT40 B-cells [Matthews, S.A., Liu, P., Spitaler, M., Olson, E.N., McKinsey, T.A., Cantrell, D.A. and Scharenberg, A.M. (2006) Essential role for protein kinase D family kinases in the regulation of class II histone deacetylases in B lymphocytes.
T18	647-737	Sentence	denotes	We now show that PKDs are also required to regulate HSP27 phosphorylation in DT40 B-cells.
T19	738-979	Sentence	denotes	However, in contrast to previous observations in other cell types, PKD enzymes do not regulate basic cellular processes such as proliferation or survival responses, nor NFκB transcriptional activity downstream of the B cell antigen receptor.
T20	980-1036	Sentence	denotes	Thus, PKDs have a selective role in DT40 B-cell biology.
T1	0-135	Sentence	denotes	Protein kinase D enzymes are dispensable for proliferation, survival and antigen receptor-regulated NFκB activity in vertebrate B-cells
T2	138-146	Sentence	denotes	Abstract
T3	147-263	Sentence	denotes	To investigate the importance of protein kinase D (PKD) enzymes we generated a PKD-null DT40 B-lymphocyte cell line.
T4	264-614	Sentence	denotes	Previously we have shown that PKDs have an essential role in regulating class II histone deacetylases in DT40 B-cells [Matthews, S.A., Liu, P., Spitaler, M., Olson, E.N., McKinsey, T.A., Cantrell, D.A. and Scharenberg, A.M. (2006) Essential role for protein kinase D family kinases in the regulation of class II histone deacetylases in B lymphocytes.
T5	615-619	Sentence	denotes	Mol.
T6	620-630	Sentence	denotes	Cell Biol.
T7	631-646	Sentence	denotes	26, 1569–1577].
T8	647-737	Sentence	denotes	We now show that PKDs are also required to regulate HSP27 phosphorylation in DT40 B-cells.
T9	738-979	Sentence	denotes	However, in contrast to previous observations in other cell types, PKD enzymes do not regulate basic cellular processes such as proliferation or survival responses, nor NFκB transcriptional activity downstream of the B cell antigen receptor.
T10	980-1036	Sentence	denotes	Thus, PKDs have a selective role in DT40 B-cell biology.
T11	1038-1053	Sentence	denotes	1 Introduction
T12	1054-1151	Sentence	denotes	The protein kinase D (PKD) serine/threonine kinase family has three members: PKD1, PKD2 and PKD3.
T13	1152-1351	Sentence	denotes	Most cell types express at least two PKD isoforms but PKD enzymes are especially highly expressed in haematopoietic cells, where they are activated in response to antigen receptors stimulation [2,3].
T14	1352-1665	Sentence	denotes	A conserved signalling pathway linking antigen receptors to PKDs involves the activation of PLCγ and the subsequent production of diacylglycerol (DAG) which stimulates classical and/or novel protein kinase Cs (PKC) that phosphorylate two key regulatory serine residues in the activation loop of PKD kinases [3–6].
T15	1666-1888	Sentence	denotes	The N-terminal regulatory region of PKD enzymes contains a DAG binding domain and direct binding of DAG also contributes to PKD1 activation [7] as well as regulating the spatial location of PKD enzymes within cells [8–12].
T16	1889-2061	Sentence	denotes	PKD enzymes have been proposed to regulate numerous cellular functions, including cell proliferation [13–16], anti-apoptotic signals [17,18] and thymocyte development [19].
T17	2062-2223	Sentence	denotes	Expression of mutant catalytically inactive and constitutively activated PKDs can also modify Golgi function, cell adhesion and cell motility (reviewed in [20]).
T18	2224-2386	Sentence	denotes	In particular, PKDs have been widely linked to the activation of the NFκB transcription factor and in regulating cell survival during oxidative stress [17,21–23].
T19	2387-2531	Sentence	denotes	Another recently proposed PKD1 substrate is HSP27 [24], a small heat shock protein involved in regulating cell migration and cell survival [25].
T20	2532-2712	Sentence	denotes	An essential role for PKD enzymes in regulating class II histone deacetylases (HDACs), enzymes that repress MEF2-dependent gene transcription, has also been demonstrated [1,26–28].
T21	2713-3066	Sentence	denotes	To investigate the biological role of PKDs we have generated DT40 B cell lines that lack expression of one or more members of the PKD family [1], allowing us to investigate the function(s) of PKD isoforms following B cell antigen receptor (BCR) stimulation, as well addressing the issue of functional redundancy between the different PKD family members.
T22	3067-3158	Sentence	denotes	Previous studies have shown that PKDs are indispensable for HDAC regulation in B cells [1].
T23	3159-3244	Sentence	denotes	Herein we show that PKDs are also indispensable for HSP27 phosphorylation in B cells.
T24	3245-3312	Sentence	denotes	However, PKD-null DT40 B cells are viable and proliferate normally.
T25	3313-3514	Sentence	denotes	Moreover, loss of the entire cellular pool of PKD does not critically affect oxidative stress responses in B cells nor do PKD kinases play an essential role in regulating NFκB transcriptional activity.
T26	3515-3698	Sentence	denotes	Together, these findings reveal that in B lymphocytes, PKD kinases are not critical regulators of many of the cellular processes previously ascribed to them in other cellular systems.
T27	3700-3724	Sentence	denotes	2 Materials and methods
T28	3726-3789	Sentence	denotes	2.1 Cell culture, transient transfections and cell stimulation
T29	3790-3925	Sentence	denotes	The generation, culture and activation of PKD1−/−, PKD3−/− and PKD1/3−/− knockout DT40 B cell lines have been described previously [1].
T30	3926-4038	Sentence	denotes	Cells were lysed and protein extracts were analysed in Western blotting experiments as previously described [1].
T31	4039-4117	Sentence	denotes	Chloramphenicol acetyl transferase assays have been described previously [29].
T32	4119-4137	Sentence	denotes	2.2 sIgM staining
T33	4138-4335	Sentence	denotes	DT40 B cells (2 × 106 cells per point) were resuspended in 200 μl buffer (RPMI 1640 media, 1% foetal calf serum) containing anti-chicken M1 monoclonal antibody conjugated to FITC for 20 min on ice.
T34	4336-4421	Sentence	denotes	The cells were washed twice and fluorescent intensity was analysed by flow cytometry.
T35	4422-4528	Sentence	denotes	All results shown are representative of at two to four independent experiments unless otherwise indicated.
T36	4530-4540	Sentence	denotes	3 Results
T37	4542-4633	Sentence	denotes	3.1 Loss of HSP27 phosphorylation in DT40 B cells lacking expression of PKD family kinases
T38	4634-4825	Sentence	denotes	DT40 B cells express two PKD isoforms, PKD1 and PKD3, and as previously described we have recently generated DT40 B cell lines that lack expression of either PKD1 or PKD3 or both enzymes [1].
T39	4826-5011	Sentence	denotes	In generating the double knockout cell lines we targeted the PKD1 loci in a PKD3−/− cell line that expressed a Flag-PKD3 transgene under the control of a doxycycline-inducible promoter.
T40	5012-5283	Sentence	denotes	Hence, in the presence of doxycycline, Flag-PKD3 expression in PKD1/3 double knockout cells is comparable to endogenous PKD3 present in wild-type DT40 cells and removal of doxycycline from the culture media for 5 days results in a completely null PKD phenotype (Fig. 1A).
T41	5284-5521	Sentence	denotes	Previously, we have demonstrated that phosphorylation and nuclear exclusion of class II histone deacetylases (HDACs) during BCR engagement is defective in PKD1/3−/− B cells and can restored upon re-expression of a single PKD isoform [1].
T42	5522-5756	Sentence	denotes	The small heat shock protein HSP27 has recently been proposed as a PKD1 substrate [24] and we accordingly assessed whether PKD-null DT40 cells have defective phosphorylation of HSP27 on serine 82, the proposed PKD1 substrate sequence.
T43	5757-5883	Sentence	denotes	We initially investigated the regulation of HSP27 phosphorylation in single knockout DT40 B cells lacking either PKD1 or PKD3.
T44	5884-6045	Sentence	denotes	As shown in Fig. 1B, activation of the BCR or treatment with the DAG-mimetic PdBu increased the levels of HSP27 phosphorylation at S82 in wild-type DT40 B cells.
T45	6046-6180	Sentence	denotes	BCR and phorbol ester signals were also able to increase HSP27 phosphorylation in PKD1 or PKD3 single knockout DT40 B cells (Fig. 1B).
T46	6181-6319	Sentence	denotes	However, BCR- and phorbol ester-induced phosphorylation of HSP27 on S82 was abolished in B cells that lacked both PKD1 and PKD3 (Fig. 1C).
T47	6320-6501	Sentence	denotes	Significantly, doxycycline-induced expression of the Flag-PKD3 transgene in the double knockout cells was sufficient to restore normal regulation of HSP27 phosphorylation (Fig. 1C).
T48	6502-6683	Sentence	denotes	In contrast, expression of a kinase-deficient PKD3 mutant protein in the double knockout cells was not able to restore BCR- or phorbol ester-induced HSP27 phosphorylation (Fig. 1D).
T49	6684-6816	Sentence	denotes	Hence, PKD3 as well as PKD1 can regulate HSP27 phosphorylation and in DT40 B cells they are functionally redundant as HSP27 kinases.
T50	6818-6915	Sentence	denotes	3.2 Cellular proliferation and survival in DT40 B cells lacking expression of PKD family kinases
T51	6916-7028	Sentence	denotes	PKD enzymes have previously been linked to the regulation of cell proliferation and survival (reviewed in [20]).
T52	7029-7192	Sentence	denotes	To investigate the effect that loss of PKD kinases had on B cell survival and/or proliferation we cultured wild-type and PKD-null cells in the presence (PKD1/3−/−:
T53	7193-7278	Sentence	denotes	Flag-PKD3+ve) or absence (PKD1/3−/−) of doxycycline and monitored exponential growth.
T54	7279-7432	Sentence	denotes	As shown in Fig. 2A, PKD1/3−/− cells proliferated exponentially and re-expression of Flag-PKD3 in these cells had no impact on the rate of proliferation.
T55	7433-7586	Sentence	denotes	Furthermore, the viability of PKD1/3−/− B cells during routine culturing was not significantly different from that of wild-type B cells (data not shown).
T56	7587-7974	Sentence	denotes	It was noted that the population doubling time of PKD1/3−/− cells was slightly slower than that of wild type DT40 cells (12.7 ± 2.8 h versus 10.2 ± 0.4 h) but the failure of PKD3 re-expression to modify the proliferation rate of PKD1/3−/− cells suggests that these small differences were most likely the result of clonal variation and were not caused specifically by loss of PKD enzymes.
T57	7975-8082	Sentence	denotes	Thus, PKD family enzymes are not essential for regulating basal survival and proliferation of DT40 B cells.
T58	8083-8274	Sentence	denotes	PKD enzymes, specifically PKD1 and PKD2, have previously been linked to a protective role against oxidative stress-induced injury in 3T3 fibroblast, HeLa and epithelial cell lines [17,30–32].
T59	8275-8416	Sentence	denotes	We therefore addressed the role of PKD family kinases in regulating B cell survival in response to oxidative stress and other stress stimuli.
T60	8417-8805	Sentence	denotes	As shown in Fig. 2B, loss of PKD1/3 expression had no significant impact on the survival of DT40 B cells in response to mitochondrial stress stimuli (H2O2 or serum deprivation); DNA damaging agents (etoposide or doxorubicin); ER pathway stress due to calcium overload (thapsigargin) or following prolonged treatment with phorbol esters or Trichostatin A, an inhibitor of class I/II HDACs.
T61	8806-8935	Sentence	denotes	Thus, PKD kinases do not play an essential role in regulating B cell survival in response to a range of different stress stimuli.

ICD10

Id	Subject	Object	Predicate	Lexical cue
T3136	5537-5542	http://purl.bioontology.org/ontology/ICD10/R57.9	denotes	shock
T1482	2456-2461	http://purl.bioontology.org/ontology/ICD10/R57.9	denotes	shock
T4612	8206-8212	http://purl.bioontology.org/ontology/ICD10/T14.9	denotes	injury

events-check-again

Id	Subject	Object	Predicate	Lexical cue	Negation	Speculation
T3563	5132-5136	Protein	denotes	PKD3
T3564	5439-5443	Protein	denotes	PKD1
T3565	5444-5445	Protein	denotes	3
T3566	5551-5556	Protein	denotes	HSP27
T3567	5589-5593	Protein	denotes	PKD1
T3568	5670-5679	Negative_regulation	denotes	defective
T3569	5680-5695	Phosphorylation	denotes	phosphorylation
T3570	5699-5704	Protein	denotes	HSP27
T3571	5708-5717	Entity	denotes	serine 82
T3572	5732-5736	Protein	denotes	PKD1
T3573	5787-5797	Regulation	denotes	regulation		true
T3574	5801-5806	Protein	denotes	HSP27
T3575	5807-5822	Phosphorylation	denotes	phosphorylation
T3576	5855-5862	Negative_regulation	denotes	lacking
T3577	5855-5862	Negative_regulation	denotes	lacking
T3578	5870-5874	Protein	denotes	PKD1
T3579	5878-5882	Protein	denotes	PKD3
T3580	5966-5975	Positive_regulation	denotes	increased
T3581	5990-5995	Protein	denotes	HSP27
T3582	5996-6011	Phosphorylation	denotes	phosphorylation
T3583	6015-6018	Entity	denotes	S82
T3584	6094-6102	Positive_regulation	denotes	increase
T3585	6103-6108	Protein	denotes	HSP27
T3586	6109-6124	Phosphorylation	denotes	phosphorylation
T3587	6128-6132	Protein	denotes	PKD1
T3588	6136-6140	Protein	denotes	PKD3
T3589	6148-6156	Negative_regulation	denotes	knockout
T3590	6148-6156	Negative_regulation	denotes	knockout
T3591	6213-6220	Positive_regulation	denotes	induced
T3592	6221-6236	Phosphorylation	denotes	phosphorylation
T3593	6240-6245	Protein	denotes	HSP27
T3594	6249-6252	Entity	denotes	S82
T3595	6257-6266	Negative_regulation	denotes	abolished
T3596	6257-6266	Negative_regulation	denotes	abolished
T3597	6283-6289	Negative_regulation	denotes	lacked
T3598	6283-6289	Negative_regulation	denotes	lacked
T3599	6295-6299	Protein	denotes	PKD1
T3600	6304-6308	Protein	denotes	PKD3
T3601	6347-6354	Positive_regulation	denotes	induced
T3602	6355-6365	Gene_expression	denotes	expression
T3603	6378-6382	Protein	denotes	PKD3
T3604	6426-6447	Positive_regulation	denotes	sufficient to restore
T3605	6455-6465	Regulation	denotes	regulation
T3606	6469-6474	Protein	denotes	HSP27
T3607	6475-6490	Phosphorylation	denotes	phosphorylation
T3608	6515-6525	Gene_expression	denotes	expression
T3609	6538-6547	Negative_regulation	denotes	deficient
T3610	6548-6552	Protein	denotes	PKD3
T3611	6613-6620	Positive_regulation	denotes	restore	true
T3612	6643-6650	Positive_regulation	denotes	induced
T3613	6651-6656	Protein	denotes	HSP27
T3614	6657-6672	Phosphorylation	denotes	phosphorylation
T3615	6691-6695	Protein	denotes	PKD3
T3616	6707-6711	Protein	denotes	PKD1
T3617	6716-6724	Regulation	denotes	regulate
T3618	6716-6724	Regulation	denotes	regulate
T3619	6725-6730	Protein	denotes	HSP27
T3620	6731-6746	Phosphorylation	denotes	phosphorylation
T3621	6802-6807	Protein	denotes	HSP27
T4751	7182-7186	Protein	denotes	PKD1
T4752	7187-7188	Protein	denotes	3
T4753	7198-7202	Protein	denotes	PKD3
T4754	7219-7223	Protein	denotes	PKD1
T4755	7224-7225	Protein	denotes	3
T4756	7300-7304	Protein	denotes	PKD1
T4757	7305-7306	Protein	denotes	3
T4758	7347-7360	Gene_expression	denotes	re-expression
T4759	7369-7373	Protein	denotes	PKD3
T4760	7463-7467	Protein	denotes	PKD1
T4761	7468-7469	Protein	denotes	3
T4762	7637-7641	Protein	denotes	PKD1
T4763	7642-7643	Protein	denotes	3
T4764	7761-7765	Protein	denotes	PKD3
T4765	7766-7779	Gene_expression	denotes	re-expression
T4766	7816-7820	Protein	denotes	PKD1
T4767	7821-7822	Protein	denotes	3
T4768	8109-8113	Protein	denotes	PKD1
T4769	8118-8122	Protein	denotes	PKD2
T4770	8438-8442	Negative_regulation	denotes	loss
T4771	8438-8442	Negative_regulation	denotes	loss
T4772	8446-8450	Protein	denotes	PKD1
T4773	8451-8452	Protein	denotes	3
T4774	8453-8463	Gene_expression	denotes	expression
T4775	8453-8463	Gene_expression	denotes	expression
T425	678-686	Positive_regulation	denotes	required
T426	690-698	Regulation	denotes	regulate
T427	699-704	Protein	denotes	HSP27
T428	705-720	Phosphorylation	denotes	phosphorylation
T1575	1131-1135	Protein	denotes	PKD1
T1576	1137-1141	Protein	denotes	PKD2
T1577	1146-1150	Protein	denotes	PKD3
T1578	1755-1762	Binding	denotes	binding
T1579	1775-1786	Positive_regulation	denotes	contributes
T1580	1790-1794	Protein	denotes	PKD1
T1581	1795-1805	Positive_regulation	denotes	activation
T1582	2413-2417	Protein	denotes	PKD1
T1583	2431-2436	Protein	denotes	HSP27
T1584	2640-2644	Protein	denotes	MEF2
T1585	3193-3206	Positive_regulation	denotes	indispensable
T1586	3211-3216	Protein	denotes	HSP27
T1587	3217-3232	Phosphorylation	denotes	phosphorylation
T3540	4547-4551	Negative_regulation	denotes	Loss		true
T3541	4555-4560	Protein	denotes	HSP27
T3542	4561-4576	Phosphorylation	denotes	phosphorylation
T3543	4647-4654	Gene_expression	denotes	express
T3544	4647-4654	Gene_expression	denotes	express
T3545	4673-4677	Protein	denotes	PKD1
T3546	4682-4686	Protein	denotes	PKD3
T3547	4766-4770	Negative_regulation	denotes	lack
T3548	4766-4770	Negative_regulation	denotes	lack
T3549	4771-4781	Gene_expression	denotes	expression
T3550	4771-4781	Gene_expression	denotes	expression
T3551	4792-4796	Protein	denotes	PKD1
T3552	4800-4804	Protein	denotes	PKD3
T3553	4887-4891	Protein	denotes	PKD1
T3554	4902-4906	Protein	denotes	PKD3
T3555	4925-4934	Gene_expression	denotes	expressed
T3556	4942-4946	Protein	denotes	PKD3
T3557	5056-5060	Protein	denotes	PKD3
T3558	5061-5071	Gene_expression	denotes	expression
T3559	5075-5079	Protein	denotes	PKD1
T3560	5080-5081	Protein	denotes	3
T3561	5082-5097	Negative_regulation	denotes	double knockout
T3562	5082-5097	Negative_regulation	denotes	double knockout
T1851	3832-3836	Protein	denotes	PKD1
T1852	3841-3845	Protein	denotes	PKD3
T1853	3853-3857	Protein	denotes	PKD1
T1854	3858-3859	Protein	denotes	3
T1855	3863-3871	Negative_regulation	denotes	knockout
T1856	3863-3871	Negative_regulation	denotes	knockout
T1857	3863-3871	Negative_regulation	denotes	knockout
T1858	3863-3871	Negative_regulation	denotes	knockout
R2918	T3541	T3542	themeOf	HSP27,phosphorylation
R2925	T3542	T3540	themeOf	phosphorylation,Loss
R2926	T3545	T3543	themeOf	PKD1,express
R2927	T3546	T3544	themeOf	PKD3,express
R2929	T3550	T3548	themeOf	expression,lack
R2930	T3551	T3550	themeOf	PKD1,expression
R2941	T3587	T3590	themeOf	PKD1,knockout
R2942	T3588	T3589	themeOf	PKD3,knockout
R2944	T3591	T3595	themeOf	induced,abolished
R2945	T3591	T3596	themeOf	induced,abolished
R2946	T3592	T3591	themeOf	phosphorylation,induced
R2948	T3594	T3592	themeOf	S82,phosphorylation
R2949	T3594	T3593	partOf	S82,HSP27
R2950	T3597	T3596	causeOf	lacked,abolished
R2951	T3598	T3595	causeOf	lacked,abolished
R2952	T3599	T3598	themeOf	PKD1,lacked
R2953	T3600	T3597	themeOf	PKD3,lacked
R2954	T3601	T3604	causeOf	induced,sufficient to restore
R2955	T3602	T3601	themeOf	expression,induced
R2957	T3603	T3602	themeOf	PKD3,expression
R2958	T3605	T3604	themeOf	regulation,sufficient to restore
R2959	T3606	T3607	themeOf	HSP27,phosphorylation
R2960	T3607	T3605	themeOf	phosphorylation,regulation
R2962	T3608	T3611	causeOf	expression,restore
R2963	T3610	T3608	themeOf	PKD3,expression
R2964	T3610	T3609	themeOf	PKD3,deficient
R2966	T3612	T3611	themeOf	induced,restore
R2967	T3613	T3614	themeOf	HSP27,phosphorylation
R2968	T3614	T3612	themeOf	phosphorylation,induced
R2969	T3615	T3618	causeOf	PKD3,regulate
R2971	T3616	T3617	causeOf	PKD1,regulate
R2973	T3619	T3620	themeOf	HSP27,phosphorylation
R2974	T3620	T3618	themeOf	phosphorylation,regulate
R2975	T3620	T3617	themeOf	phosphorylation,regulate
R3843	T4759	T4758	themeOf	PKD3,re-expression
R3844	T4764	T4765	themeOf	PKD3,re-expression
R3845	T4772	T4774	themeOf	PKD1,expression
R3846	T4773	T4775	themeOf	3,expression
R3847	T4774	T4771	themeOf	expression,loss
R3848	T4775	T4770	themeOf	expression,loss
R410	T426	T425	themeOf	regulate,required
R411	T427	T428	themeOf	HSP27,phosphorylation
R412	T428	T426	themeOf	phosphorylation,regulate
R1400	T1578	T1579	causeOf	binding,contributes
R1402	T1580	T1578	themeOf	PKD1,binding
R1403	T1580	T1581	themeOf	PKD1,activation
R1404	T1581	T1579	themeOf	activation,contributes
R1405	T1586	T1587	themeOf	HSP27,phosphorylation
R1406	T1587	T1585	themeOf	phosphorylation,indispensable
R2928	T3549	T3547	themeOf	expression,lack
R2931	T3579	T3577	themeOf	PKD3,lacking
R2932	T3552	T3549	themeOf	PKD3,expression
R2933	T3582	T3580	themeOf	phosphorylation,increased
R2934	T3556	T3555	themeOf	PKD3,expressed
R2935	T3583	T3582	themeOf	S82,phosphorylation
R2936	T3583	T3581	partOf	S82,HSP27
R2937	T3585	T3586	themeOf	HSP27,phosphorylation
R2938	T3586	T3584	themeOf	phosphorylation,increase
R2939	T3557	T3558	themeOf	PKD3,expression
R2940	T3559	T3562	themeOf	PKD1,double knockout
R2943	T3560	T3561	themeOf	3,double knockout
R2947	T3569	T3568	themeOf	phosphorylation,defective
R2956	T3571	T3569	themeOf	serine 82,phosphorylation
R2961	T3571	T3570	partOf	serine 82,HSP27
R2965	T3574	T3575	themeOf	HSP27,phosphorylation
R2970	T3575	T3573	themeOf	phosphorylation,regulation
R2972	T3578	T3576	themeOf	PKD1,lacking
R1592	T1851	T1855	themeOf	PKD1,knockout
R1593	T1852	T1856	themeOf	PKD3,knockout
R1594	T1853	T1857	themeOf	PKD1,knockout
R1595	T1854	T1858	themeOf	3,knockout

bionlp-st-ge-2016-reference-tees

Id	Subject	Object	Predicate	Lexical cue
T429	0-24	Protein	denotes	Protein kinase D enzymes
T430	100-104	Protein	denotes	NFκB
T431	81-99	Regulation	denotes	receptor-regulated
T432	180-196	Protein	denotes	protein kinase D
T433	198-201	Protein	denotes	PKD
T434	226-229	Protein	denotes	PKD
T435	294-298	Protein	denotes	PKDs
T436	336-365	Protein	denotes	class II histone deacetylases
T437	514-545	Protein	denotes	protein kinase D family kinases
T438	567-596	Protein	denotes	class II histone deacetylases
T439	553-563	Regulation	denotes	regulation
T440	664-668	Protein	denotes	PKDs
T441	699-704	Protein	denotes	HSP27
T442	705-720	Phosphorylation	denotes	phosphorylation
T443	690-698	Regulation	denotes	regulate
T444	678-686	Positive_regulation	denotes	required
T445	805-816	Protein	denotes	PKD enzymes
T446	907-911	Protein	denotes	NFκB
T447	955-978	Protein	denotes	B cell antigen receptor
T448	824-832	Regulation	denotes	regulate
T449	986-990	Protein	denotes	PKDs
T1588	1058-1074	Protein	denotes	protein kinase D
T1589	1076-1079	Protein	denotes	PKD
T1590	1081-1111	Protein	denotes	serine/threonine kinase family
T1591	1131-1135	Protein	denotes	PKD1
T1592	1137-1141	Protein	denotes	PKD2
T1593	1146-1150	Protein	denotes	PKD3
T1594	1189-1192	Protein	denotes	PKD
T1595	1206-1209	Protein	denotes	PKD
T1596	1168-1175	Gene_expression	denotes	express
T1597	1240-1249	Gene_expression	denotes	expressed
T1598	1412-1416	Protein	denotes	PKDs
T1599	1444-1448	Protein	denotes	PLCγ
T1600	1543-1560	Protein	denotes	protein kinase Cs
T1601	1562-1565	Protein	denotes	PKC
T1602	1647-1658	Protein	denotes	PKD kinases
T1603	1509-1519	Positive_regulation	denotes	stimulates
T1604	1509-1519	Positive_regulation	denotes	stimulates
T1605	1572-1585	Phosphorylation	denotes	phosphorylate
T1606	1572-1585	Phosphorylation	denotes	phosphorylate
T1607	1702-1713	Protein	denotes	PKD enzymes
T1608	1766-1769	Protein	denotes	DAG
T1609	1790-1794	Protein	denotes	PKD1
T1610	1856-1867	Protein	denotes	PKD enzymes
T1611	1755-1762	Binding	denotes	binding
T1612	1795-1805	Positive_regulation	denotes	activation
T1613	1844-1852	Localization	denotes	location
T1614	1775-1786	Positive_regulation	denotes	contributes
T1615	1821-1831	Regulation	denotes	regulating
T1616	1889-1892	Protein	denotes	PKD
T1617	2135-2139	Protein	denotes	PKDs
T1618	2062-2072	Gene_expression	denotes	Expression
T1619	2125-2134	Positive_regulation	denotes	activated
T1620	2125-2134	Positive_regulation	denotes	activated
T1621	2239-2243	Protein	denotes	PKDs
T1622	2293-2318	Protein	denotes	NFκB transcription factor
T1623	2275-2285	Positive_regulation	denotes	activation
T1624	2413-2417	Protein	denotes	PKD1
T1625	2554-2565	Protein	denotes	PKD enzymes
T1626	2580-2609	Protein	denotes	class II histone deacetylases
T1627	2611-2616	Protein	denotes	HDACs
T1628	2640-2644	Protein	denotes	MEF2
T1629	2569-2579	Regulation	denotes	regulating
T1630	2569-2579	Regulation	denotes	regulating
T1631	2751-2755	Protein	denotes	PKDs
T1632	2843-2846	Protein	denotes	PKD
T1633	2905-2908	Protein	denotes	PKD
T1634	2928-2951	Protein	denotes	B cell antigen receptor
T1635	2953-2956	Protein	denotes	BCR
T1636	3047-3065	Protein	denotes	PKD family members
T1637	3100-3104	Protein	denotes	PKDs
T1638	3127-3131	Protein	denotes	HDAC
T1639	3132-3142	Regulation	denotes	regulation
T1640	3179-3183	Protein	denotes	PKDs
T1641	3211-3216	Protein	denotes	HSP27
T1642	3217-3232	Phosphorylation	denotes	phosphorylation
T1643	3193-3206	Positive_regulation	denotes	indispensable
T1644	3254-3257	Protein	denotes	PKD
T1645	3359-3362	Protein	denotes	PKD
T1646	3435-3446	Protein	denotes	PKD kinases
T1647	3484-3488	Protein	denotes	NFκB
T1648	3323-3327	Negative_regulation	denotes	loss
T1649	3570-3581	Protein	denotes	PKD kinases
T3622	4555-4560	Protein	denotes	HSP27
T3623	4580-4586	Protein	denotes	DT40 B
T3624	4615-4625	Protein	denotes	PKD family
T3625	4561-4576	Phosphorylation	denotes	phosphorylation
T3626	4601-4611	Gene_expression	denotes	expression
T3627	0-4551	Negative_regulation	denotes	Protein kinase D enzymes are dispensable for proliferation, survival and antigen receptor-regulated NFκB activity in vertebrate B-cells Abstract To investigate the importance of protein kinase D (PKD) enzymes we generated a PKD-null DT40 B-lymphocyte cell line. Previously we have shown that PKDs have an essential role in regulating class II histone deacetylases in DT40 B-cells [Matthews, S.A., Liu, P., Spitaler, M., Olson, E.N., McKinsey, T.A., Cantrell, D.A. and Scharenberg, A.M. (2006) Essential role for protein kinase D family kinases in the regulation of class II histone deacetylases in B lymphocytes. Mol. Cell Biol. 26, 1569–1577]. We now show that PKDs are also required to regulate HSP27 phosphorylation in DT40 B-cells. However, in contrast to previous observations in other cell types, PKD enzymes do not regulate basic cellular processes such as proliferation or survival responses, nor NFκB transcriptional activity downstream of the B cell antigen receptor. Thus, PKDs have a selective role in DT40 B-cell biology. 1 Introduction The protein kinase D (PKD) serine/threonine kinase family has three members: PKD1, PKD2 and PKD3. Most cell types express at least two PKD isoforms but PKD enzymes are especially highly expressed in haematopoietic cells, where they are activated in response to antigen receptors stimulation [2,3]. A conserved signalling pathway linking antigen receptors to PKDs involves the activation of PLCγ and the subsequent production of diacylglycerol (DAG) which stimulates classical and/or novel protein kinase Cs (PKC) that phosphorylate two key regulatory serine residues in the activation loop of PKD kinases [3–6]. The N-terminal regulatory region of PKD enzymes contains a DAG binding domain and direct binding of DAG also contributes to PKD1 activation [7] as well as regulating the spatial location of PKD enzymes within cells [8–12]. PKD enzymes have been proposed to regulate numerous cellular functions, including cell proliferation [13–16], anti-apoptotic signals [17,18] and thymocyte development [19]. Expression of mutant catalytically inactive and constitutively activated PKDs can also modify Golgi function, cell adhesion and cell motility (reviewed in [20]). In particular, PKDs have been widely linked to the activation of the NFκB transcription factor and in regulating cell survival during oxidative stress [17,21–23]. Another recently proposed PKD1 substrate is HSP27 [24], a small heat shock protein involved in regulating cell migration and cell survival [25]. An essential role for PKD enzymes in regulating class II histone deacetylases (HDACs), enzymes that repress MEF2-dependent gene transcription, has also been demonstrated [1,26–28]. To investigate the biological role of PKDs we have generated DT40 B cell lines that lack expression of one or more members of the PKD family [1], allowing us to investigate the function(s) of PKD isoforms following B cell antigen receptor (BCR) stimulation, as well addressing the issue of functional redundancy between the different PKD family members. Previous studies have shown that PKDs are indispensable for HDAC regulation in B cells [1]. Herein we show that PKDs are also indispensable for HSP27 phosphorylation in B cells. However, PKD-null DT40 B cells are viable and proliferate normally. Moreover, loss of the entire cellular pool of PKD does not critically affect oxidative stress responses in B cells nor do PKD kinases play an essential role in regulating NFκB transcriptional activity. Together, these findings reveal that in B lymphocytes, PKD kinases are not critical regulators of many of the cellular processes previously ascribed to them in other cellular systems. 2 Materials and methods 2.1 Cell culture, transient transfections and cell stimulation The generation, culture and activation of PKD1−/−, PKD3−/− and PKD1/3−/− knockout DT40 B cell lines have been described previously [1]. Cells were lysed and protein extracts were analysed in Western blotting experiments as previously described [1]. Chloramphenicol acetyl transferase assays have been described previously [29]. 2.2 sIgM staining DT40 B cells (2 × 106 cells per point) were resuspended in 200 μl buffer (RPMI 1640 media, 1% foetal calf serum) containing anti-chicken M1 monoclonal antibody conjugated to FITC for 20 min on ice. The cells were washed twice and fluorescent intensity was analysed by flow cytometry. All results shown are representative of at two to four independent experiments unless otherwise indicated. 3 Results 3.1 Loss
T3628	4593-4600	Negative_regulation	denotes	lacking
T3629	4634-4640	Protein	denotes	DT40 B
T3630	4659-4662	Protein	denotes	PKD
T3631	4673-4677	Protein	denotes	PKD1
T3632	4682-4686	Protein	denotes	PKD3
T3633	4792-4796	Protein	denotes	PKD1
T3634	4800-4804	Protein	denotes	PKD3
T3635	4647-4654	Gene_expression	denotes	express
T3636	4647-4654	Gene_expression	denotes	express
T3637	4647-4654	Gene_expression	denotes	express
T3638	4771-4781	Gene_expression	denotes	expression
T3639	4771-4781	Gene_expression	denotes	expression
T3640	4766-4770	Negative_regulation	denotes	lack
T3641	4766-4770	Negative_regulation	denotes	lack
T3642	4887-4896	Protein	denotes	PKD1 loci
T3643	4902-4906	Protein	denotes	PKD3
T3644	4937-4941	Protein	denotes	Flag
T3645	4942-4946	Protein	denotes	PKD3
T3646	4925-4934	Gene_expression	denotes	expressed
T3647	4925-4934	Gene_expression	denotes	expressed
T3648	5051-5055	Protein	denotes	Flag
T3649	5056-5060	Protein	denotes	PKD3
T3650	5075-5081	Protein	denotes	PKD1/3
T3651	5132-5136	Protein	denotes	PKD3
T3652	5061-5071	Gene_expression	denotes	expression
T3653	5061-5071	Gene_expression	denotes	expression
T3654	5363-5392	Protein	denotes	class II histone deacetylases
T3655	5394-5399	Protein	denotes	HDACs
T3656	5408-5411	Protein	denotes	BCR
T3657	5439-5443	Protein	denotes	PKD1
T3658	5322-5337	Phosphorylation	denotes	phosphorylation
T3659	5322-5337	Phosphorylation	denotes	phosphorylation
T3660	5412-5422	Binding	denotes	engagement
T3661	5465-5473	Positive_regulation	denotes	restored
T3662	5465-5473	Positive_regulation	denotes	restored
T3663	5526-5550	Protein	denotes	small heat shock protein
T3664	5551-5556	Protein	denotes	HSP27
T3665	5589-5593	Protein	denotes	PKD1
T3666	5645-5648	Protein	denotes	PKD
T3667	5699-5704	Protein	denotes	HSP27
T3668	5732-5736	Protein	denotes	PKD1
T3669	5708-5714	Entity	denotes	serine
T3670	5680-5695	Phosphorylation	denotes	phosphorylation
T3671	5670-5679	Negative_regulation	denotes	defective
T3672	5801-5806	Protein	denotes	HSP27
T3673	5870-5874	Protein	denotes	PKD1
T3675	5807-5822	Phosphorylation	denotes	phosphorylation
T3676	5855-5862	Negative_regulation	denotes	lacking
T3677	5855-5862	Negative_regulation	denotes	lacking
T3678	5787-5797	Regulation	denotes	regulation
T3679	5896-5899	Protein	denotes	Fig
T3680	5923-5926	Protein	denotes	BCR
T3681	5990-5995	Protein	denotes	HSP27
T3682	5905-5915	Positive_regulation	denotes	activation
T3683	5966-5975	Positive_regulation	denotes	increased
T3684	5996-6011	Phosphorylation	denotes	phosphorylation
T3685	6046-6049	Protein	denotes	BCR
T3686	6103-6108	Protein	denotes	HSP27
T3687	6128-6132	Protein	denotes	PKD1
T3688	6136-6140	Protein	denotes	PKD3
T3689	6109-6124	Phosphorylation	denotes	phosphorylation
T3690	6148-6156	Negative_regulation	denotes	knockout
T3691	6148-6156	Negative_regulation	denotes	knockout
T3692	6094-6102	Positive_regulation	denotes	increase
T3693	6190-6193	Protein	denotes	BCR
T3694	6240-6245	Protein	denotes	HSP27
T3695	6295-6299	Protein	denotes	PKD1
T3696	6304-6308	Protein	denotes	PKD3
T3697	6310-6317	Protein	denotes	Fig. 1C
T3698	6249-6252	Entity	denotes	S82
T3699	6283-6289	Negative_regulation	denotes	lacked
T3700	6283-6289	Negative_regulation	denotes	lacked
T3701	6283-6289	Negative_regulation	denotes	lacked
T3702	6221-6236	Phosphorylation	denotes	phosphorylation
T3703	6221-6236	Phosphorylation	denotes	phosphorylation
T3704	6257-6266	Negative_regulation	denotes	abolished
T3705	6257-6266	Negative_regulation	denotes	abolished
T3706	6373-6377	Protein	denotes	Flag
T3707	6378-6382	Protein	denotes	PKD3
T3708	6469-6474	Protein	denotes	HSP27
T3709	6355-6365	Gene_expression	denotes	expression
T3710	6355-6365	Gene_expression	denotes	expression
T3711	6475-6490	Phosphorylation	denotes	phosphorylation
T3712	6335-6354	Positive_regulation	denotes	doxycycline-induced
T3713	6335-6354	Positive_regulation	denotes	doxycycline-induced
T3714	6455-6465	Regulation	denotes	regulation
T3715	6440-6447	Positive_regulation	denotes	restore
T3716	6548-6552	Protein	denotes	PKD3
T3717	6651-6656	Protein	denotes	HSP27
T3718	6515-6525	Gene_expression	denotes	expression
T3719	6531-6547	Negative_regulation	denotes	kinase-deficient
T3720	6657-6672	Phosphorylation	denotes	phosphorylation
T3721	6613-6620	Positive_regulation	denotes	restore
T3722	6637-6650	Positive_regulation	denotes	ester-induced
T3723	6691-6695	Protein	denotes	PKD3
T3724	6707-6711	Protein	denotes	PKD1
T3725	6725-6730	Protein	denotes	HSP27
T3726	6802-6815	Protein	denotes	HSP27 kinases
T3727	6731-6746	Phosphorylation	denotes	phosphorylation
T3728	6716-6724	Regulation	denotes	regulate
T3729	6716-6724	Regulation	denotes	regulate
T4793	7463-7467	Protein	denotes	PKD1
T4794	7637-7641	Protein	denotes	PKD1
T4795	7761-7765	Protein	denotes	PKD3
T4796	7816-7820	Protein	denotes	PKD1
T4797	7962-7973	Protein	denotes	PKD enzymes
T4798	7766-7779	Gene_expression	denotes	re-expression
T4799	7954-7958	Negative_regulation	denotes	loss
T4800	7981-7999	Protein	denotes	PKD family enzymes
T4801	8069-8075	Protein	denotes	DT40 B
T4802	8083-8094	Protein	denotes	PKD enzymes
T4803	8109-8113	Protein	denotes	PKD1
T4804	8118-8122	Protein	denotes	PKD2
T4805	8310-8328	Protein	denotes	PKD family kinases
T4806	8429-8432	Protein	denotes	Fig
T4807	8446-8450	Protein	denotes	PKD1
T4808	8451-8452	Protein	denotes	3
T4809	8509-8515	Protein	denotes	DT40 B
T4810	8794-8804	Protein	denotes	I/II HDACs
T4811	8453-8463	Gene_expression	denotes	expression
T4812	8453-8463	Gene_expression	denotes	expression
T4813	8775-8784	Negative_regulation	denotes	inhibitor
T4814	8438-8442	Negative_regulation	denotes	loss
T4815	8438-8442	Negative_regulation	denotes	loss
T4816	8812-8823	Protein	denotes	PKD kinases
T4776	6897-6907	Protein	denotes	PKD family
T4777	6883-6893	Gene_expression	denotes	expression
T4778	6875-6882	Negative_regulation	denotes	lacking
T4779	6916-6919	Protein	denotes	PKD
T4780	7068-7079	Protein	denotes	PKD kinases
T4781	7150-7153	Protein	denotes	PKD
T4782	7182-7186	Protein	denotes	PKD1
T4783	7193-7197	Protein	denotes	Flag
T4784	7198-7202	Protein	denotes	PKD3
T4785	7219-7223	Protein	denotes	PKD1
T4786	7060-7064	Negative_regulation	denotes	loss
T4787	7291-7294	Protein	denotes	Fig
T4788	7300-7304	Protein	denotes	PKD1
T4789	7364-7368	Protein	denotes	Flag
T4790	7369-7373	Protein	denotes	PKD3
T4791	7347-7360	Gene_expression	denotes	re-expression
T4792	7347-7360	Gene_expression	denotes	re-expression
T3674	5878-5882	Protein	denotes	PKD3
T1859	3832-3836	Protein	denotes	PKD1
T1860	3841-3845	Protein	denotes	PKD3
T1861	3853-3857	Protein	denotes	PKD1
T1862	4039-4073	Protein	denotes	Chloramphenicol acetyl transferase
T2020	4262-4297	Protein	denotes	anti-chicken M1 monoclonal antibody
R1413	T1609	T1612	themeOf	PKD1,activation
R1414	T1610	T1613	themeOf	PKD enzymes,location
R1415	T1611	T1614	causeOf	binding,contributes
R1416	T1612	T1614	themeOf	activation,contributes
R1417	T1613	T1615	themeOf	location,regulating
R1418	T1617	T1618	themeOf	PKDs,Expression
R1419	T1617	T1619	themeOf	PKDs,activated
R1420	T1618	T1620	themeOf	Expression,activated
R1421	T1622	T1623	themeOf	NFκB transcription factor,activation
R1422	T1626	T1629	themeOf	class II histone deacetylases,regulating
R1423	T1627	T1630	themeOf	HDACs,regulating
R1424	T1638	T1639	themeOf	HDAC,regulation
R1425	T1640	T1643	causeOf	PKDs,indispensable
R1426	T1641	T1642	themeOf	HSP27,phosphorylation
R1427	T1642	T1643	themeOf	phosphorylation,indispensable
R1428	T1645	T1648	themeOf	PKD,loss
R2976	T3625	T3627	themeOf	phosphorylation,"Protein kinase D enzymes are dispensable for proliferation, survival and antigen receptor-regulated NFκB activity in vertebrate B-cells Abstract To investigate the importance of protein kinase D (PKD) enzymes we generated a PKD-null DT40 B-lymphocyte cell line. Previously we have shown that PKDs have an essential role in regulating class II histone deacetylases in DT40 B-cells [Matthews, S.A., Liu, P., Spitaler, M., Olson, E.N., McKinsey, T.A., Cantrell, D.A. and Scharenberg, A.M. (2006) Essential role for protein kinase D family kinases in the regulation of class II histone deacetylases in B lymphocytes. Mol. Cell Biol. 26, 1569–1577]. We now show that PKDs are also required to regulate HSP27 phosphorylation in DT40 B-cells. However, in contrast to previous observations in other cell types, PKD enzymes do not regulate basic cellular processes such as proliferation or survival responses, nor NFκB transcriptional activity downstream of the B cell antigen receptor. Thus, PKDs have a selective role in DT40 B-cell biology. 1 Introduction The protein kinase D (PKD) serine/threonine kinase family has three members: PKD1, PKD2 and PKD3. Most cell types express at least two PKD isoforms but PKD enzymes are especially highly expressed in haematopoietic cells, where they are activated in response to antigen receptors stimulation [2,3]. A conserved signalling pathway linking antigen receptors to PKDs involves the activation of PLCγ and the subsequent production of diacylglycerol (DAG) which stimulates classical and/or novel protein kinase Cs (PKC) that phosphorylate two key regulatory serine residues in the activation loop of PKD kinases [3–6]. The N-terminal regulatory region of PKD enzymes contains a DAG binding domain and direct binding of DAG also contributes to PKD1 activation [7] as well as regulating the spatial location of PKD enzymes within cells [8–12]. PKD enzymes have been proposed to regulate numerous cellular functions, including cell proliferation [13–16], anti-apoptotic signals [17,18] and thymocyte development [19]. Expression of mutant catalytically inactive and constitutively activated PKDs can also modify Golgi function, cell adhesion and cell motility (reviewed in [20]). In particular, PKDs have been widely linked to the activation of the NFκB transcription factor and in regulating cell survival during oxidative stress [17,21–23]. Another recently proposed PKD1 substrate is HSP27 [24], a small heat shock protein involved in regulating cell migration and cell survival [25]. An essential role for PKD enzymes in regulating class II histone deacetylases (HDACs), enzymes that repress MEF2-dependent gene transcription, has also been demonstrated [1,26–28]. To investigate the biological role of PKDs we have generated DT40 B cell lines that lack expression of one or more members of the PKD family [1], allowing us to investigate the function(s) of PKD isoforms following B cell antigen receptor (BCR) stimulation, as well addressing the issue of functional redundancy between the different PKD family members. Previous studies have shown that PKDs are indispensable for HDAC regulation in B cells [1]. Herein we show that PKDs are also indispensable for HSP27 phosphorylation in B cells. However, PKD-null DT40 B cells are viable and proliferate normally. Moreover, loss of the entire cellular pool of PKD does not critically affect oxidative stress responses in B cells nor do PKD kinases play an essential role in regulating NFκB transcriptional activity. Together, these findings reveal that in B lymphocytes, PKD kinases are not critical regulators of many of the cellular processes previously ascribed to them in other cellular systems. 2 Materials and methods 2.1 Cell culture, transient transfections and cell stimulation The generation, culture and activation of PKD1−/−, PKD3−/− and PKD1/3−/− knockout DT40 B cell lines have been described previously [1]. Cells were lysed and protein extracts were analysed in Western blotting experiments as previously described [1]. Chloramphenicol acetyl transferase assays have been described previously [29]. 2.2 sIgM staining DT40 B cells (2 × 106 cells per point) were resuspended in 200 μl buffer (RPMI 1640 media, 1% foetal calf serum) containing anti-chicken M1 monoclonal antibody conjugated to FITC for 20 min on ice. The cells were washed twice and fluorescent intensity was analysed by flow cytometry. All results shown are representative of at two to four independent experiments unless otherwise indicated. 3 Results 3.1 Loss"
R2977	T3626	T3628	themeOf	expression,lacking
R2978	T3630	T3635	themeOf	PKD,express
R2979	T3631	T3636	themeOf	PKD1,express
R2980	T3622	T3625	themeOf	HSP27,phosphorylation
R2981	T3632	T3637	themeOf	PKD3,express
R2982	T3624	T3626	themeOf	PKD family,expression
R2983	T3633	T3638	themeOf	PKD1,expression
R2984	T3634	T3639	themeOf	PKD3,expression
R2985	T3638	T3640	themeOf	expression,lack
R2986	T3674	T3677	themeOf	PKD3,lacking
R2987	T3675	T3678	themeOf	phosphorylation,regulation
R2988	T3639	T3641	themeOf	expression,lack
R2989	T3644	T3646	themeOf	Flag,expressed
R2990	T3680	T3682	themeOf	BCR,activation
R2991	T3681	T3684	themeOf	HSP27,phosphorylation
R2992	T3682	T3683	themeOf	activation,increased
R2993	T3686	T3689	themeOf	HSP27,phosphorylation
R2994	T3645	T3647	themeOf	PKD3,expressed
R2995	T3648	T3652	themeOf	Flag,expression
R2999	T3649	T3653	themeOf	PKD3,expression
R3001	T3654	T3658	themeOf	class II histone deacetylases,phosphorylation
R3003	T3694	T3703	themeOf	HSP27,phosphorylation
R3004	T3694	T3698	partOf	HSP27,S82
R3005	T3695	T3699	themeOf	PKD1,lacked
R3006	T3696	T3700	themeOf	PKD3,lacked
R3007	T3697	T3701	themeOf	Fig. 1C,lacked
R3008	T3698	T3702	Site	S82,phosphorylation
R3009	T3698	T3703	Site	S82,phosphorylation
R3010	T3655	T3659	themeOf	HDACs,phosphorylation
R3011	T3702	T3704	themeOf	phosphorylation,abolished
R3012	T3656	T3660	themeOf	BCR,engagement
R3013	T3703	T3705	themeOf	phosphorylation,abolished
R3014	T3706	T3709	themeOf	Flag,expression
R3015	T3707	T3710	themeOf	PKD3,expression
R3016	T3658	T3661	themeOf	phosphorylation,restored
R3017	T3708	T3711	themeOf	HSP27,phosphorylation
R3018	T3709	T3712	themeOf	expression,doxycycline-induced
R3019	T3659	T3662	themeOf	phosphorylation,restored
R3020	T3667	T3670	themeOf	HSP27,phosphorylation
R3021	T3710	T3713	themeOf	expression,doxycycline-induced
R3022	T3711	T3714	themeOf	phosphorylation,regulation
R3023	T3714	T3715	themeOf	regulation,restore
R3024	T3716	T3718	themeOf	PKD3,expression
R3025	T3717	T3720	themeOf	HSP27,phosphorylation
R3026	T3667	T3669	partOf	HSP27,serine
R3027	T3718	T3719	themeOf	expression,kinase-deficient
R3028	T3718	T3721	causeOf	expression,restore
R3029	T3668	T3669	partOf	PKD1,serine
R3030	T3720	T3721	themeOf	phosphorylation,restore
R3031	T3720	T3722	themeOf	phosphorylation,ester-induced
R3032	T3669	T3670	Site	serine,phosphorylation
R3033	T3723	T3728	causeOf	PKD3,regulate
R3034	T3670	T3671	themeOf	phosphorylation,defective
R3035	T3724	T3729	causeOf	PKD1,regulate
R3036	T3725	T3727	themeOf	HSP27,phosphorylation
R3037	T3672	T3675	themeOf	HSP27,phosphorylation
R3038	T3727	T3728	themeOf	phosphorylation,regulate
R3039	T3727	T3729	themeOf	phosphorylation,regulate
R3040	T3673	T3676	themeOf	PKD1,lacking
R3849	T4776	T4777	themeOf	PKD family,expression
R3850	T4777	T4778	themeOf	expression,lacking
R3851	T4780	T4786	themeOf	PKD kinases,loss
R3852	T4789	T4791	themeOf	Flag,re-expression
R3853	T4790	T4792	themeOf	PKD3,re-expression
R3854	T4795	T4798	themeOf	PKD3,re-expression
R3855	T4797	T4799	themeOf	PKD enzymes,loss
R3856	T4807	T4811	themeOf	PKD1,expression
R3857	T4808	T4812	themeOf	3,expression
R3858	T4810	T4813	themeOf	I/II HDACs,inhibitor
R3859	T4811	T4814	themeOf	expression,loss
R3860	T4812	T4815	themeOf	expression,loss
R1401	T1594	T1596	themeOf	PKD,express
R1407	T1595	T1597	themeOf	PKD,expressed
R1408	T1600	T1603	themeOf	protein kinase Cs,stimulates
R1409	T1600	T1605	themeOf	protein kinase Cs,phosphorylate
R1410	T1601	T1604	themeOf	PKC,stimulates
R1411	T1601	T1606	themeOf	PKC,phosphorylate
R1412	T1608	T1611	themeOf	DAG,binding
R362	T430	T431	themeOf	NFκB,receptor-regulated
R363	T438	T439	themeOf	class II histone deacetylases,regulation
R364	T440	T443	causeOf	PKDs,regulate
R365	T440	T444	causeOf	PKDs,required
R366	T441	T442	themeOf	HSP27,phosphorylation
R367	T442	T443	themeOf	phosphorylation,regulate
R368	T443	T444	themeOf	regulate,required
R369	T445	T448	causeOf	PKD enzymes,regulate
R370	T447	T448	themeOf	B cell antigen receptor,regulate
R2996	T3687	T3690	themeOf	PKD1,knockout
R2997	T3688	T3691	themeOf	PKD3,knockout
R2998	T3689	T3692	themeOf	phosphorylation,increase
R3000	T3693	T3702	themeOf	BCR,phosphorylation
R3002	T3693	T3698	partOf	BCR,S82

bionlp-st-ge-2016-reference

Id	Subject	Object	Predicate	Lexical cue	Negation	Speculation
T2250	6651-6656	Protein	denotes	HSP27
T2251	6657-6672	Phosphorylation	denotes	phosphorylation
T2252	6691-6695	Protein	denotes	PKD3
T2253	6707-6711	Protein	denotes	PKD1
T2254	6716-6724	Regulation	denotes	regulate
T2255	6716-6724	Regulation	denotes	regulate
T2256	6725-6730	Protein	denotes	HSP27
T2257	6731-6746	Phosphorylation	denotes	phosphorylation
T2258	6802-6807	Protein	denotes	HSP27
T10	678-686	Positive_regulation	denotes	required
T11	690-698	Regulation	denotes	regulate
T12	699-704	Protein	denotes	HSP27
T13	705-720	Phosphorylation	denotes	phosphorylation
T2177	4547-4551	Negative_regulation	denotes	Loss		true
T2178	4555-4560	Protein	denotes	HSP27
T2179	4561-4576	Phosphorylation	denotes	phosphorylation
T2180	4647-4654	Gene_expression	denotes	express
T2181	4647-4654	Gene_expression	denotes	express
T2182	4673-4677	Protein	denotes	PKD1
T2183	4682-4686	Protein	denotes	PKD3
T2184	4766-4770	Negative_regulation	denotes	lack
T2185	4766-4770	Negative_regulation	denotes	lack
T2186	4771-4781	Gene_expression	denotes	expression
T2187	4771-4781	Gene_expression	denotes	expression
T2188	4792-4796	Protein	denotes	PKD1
T2189	4800-4804	Protein	denotes	PKD3
T2190	4887-4891	Protein	denotes	PKD1
T2191	4902-4906	Protein	denotes	PKD3
T2192	4925-4934	Gene_expression	denotes	expressed
T2193	4942-4946	Protein	denotes	PKD3
T2194	5056-5060	Protein	denotes	PKD3
T2195	5061-5071	Gene_expression	denotes	expression
T2196	5075-5079	Protein	denotes	PKD1
T2197	5080-5081	Protein	denotes	3
T2198	5082-5097	Negative_regulation	denotes	double knockout
T2199	5082-5097	Negative_regulation	denotes	double knockout
T2200	5132-5136	Protein	denotes	PKD3
T2201	5439-5443	Protein	denotes	PKD1
T2202	5444-5445	Protein	denotes	3
T2203	5551-5556	Protein	denotes	HSP27
T2204	5589-5593	Protein	denotes	PKD1
T2205	5670-5679	Negative_regulation	denotes	defective
T2206	5680-5695	Phosphorylation	denotes	phosphorylation
T2207	5699-5704	Protein	denotes	HSP27
T2208	5708-5717	Entity	denotes	serine 82
T2209	5732-5736	Protein	denotes	PKD1
T2210	5787-5797	Regulation	denotes	regulation		true
T2211	5801-5806	Protein	denotes	HSP27
T2212	5807-5822	Phosphorylation	denotes	phosphorylation
T2213	5855-5862	Negative_regulation	denotes	lacking
T2214	5855-5862	Negative_regulation	denotes	lacking
T2215	5870-5874	Protein	denotes	PKD1
T2216	5878-5882	Protein	denotes	PKD3
T2217	5966-5975	Positive_regulation	denotes	increased
T2218	5990-5995	Protein	denotes	HSP27
T2219	5996-6011	Phosphorylation	denotes	phosphorylation
T2220	6015-6018	Entity	denotes	S82
T2221	6094-6102	Positive_regulation	denotes	increase
T2222	6103-6108	Protein	denotes	HSP27
T2223	6109-6124	Phosphorylation	denotes	phosphorylation
T2224	6128-6132	Protein	denotes	PKD1
T2225	6136-6140	Protein	denotes	PKD3
T2226	6148-6156	Negative_regulation	denotes	knockout
T2227	6148-6156	Negative_regulation	denotes	knockout
T2228	6213-6220	Positive_regulation	denotes	induced
T2229	6221-6236	Phosphorylation	denotes	phosphorylation
T2230	6240-6245	Protein	denotes	HSP27
T2231	6249-6252	Entity	denotes	S82
T2232	6257-6266	Negative_regulation	denotes	abolished
T2233	6257-6266	Negative_regulation	denotes	abolished
T2234	6283-6289	Negative_regulation	denotes	lacked
T2235	6283-6289	Negative_regulation	denotes	lacked
T2236	6295-6299	Protein	denotes	PKD1
T2237	6304-6308	Protein	denotes	PKD3
T2238	6347-6354	Positive_regulation	denotes	induced
T2239	6355-6365	Gene_expression	denotes	expression
T2240	6378-6382	Protein	denotes	PKD3
T2241	6426-6447	Positive_regulation	denotes	sufficient to restore
T2242	6455-6465	Regulation	denotes	regulation
T2243	6469-6474	Protein	denotes	HSP27
T2244	6475-6490	Phosphorylation	denotes	phosphorylation
T2245	6515-6525	Gene_expression	denotes	expression
T2246	6538-6547	Negative_regulation	denotes	deficient
T2247	6548-6552	Protein	denotes	PKD3
T2248	6613-6620	Positive_regulation	denotes	restore	true
T2249	6643-6650	Positive_regulation	denotes	induced
T3783	7182-7186	Protein	denotes	PKD1
T3784	7187-7188	Protein	denotes	3
T3785	7198-7202	Protein	denotes	PKD3
T3786	7219-7223	Protein	denotes	PKD1
T3787	7224-7225	Protein	denotes	3
T3788	7300-7304	Protein	denotes	PKD1
T3789	7305-7306	Protein	denotes	3
T3790	7347-7360	Gene_expression	denotes	re-expression
T3791	7369-7373	Protein	denotes	PKD3
T3792	7463-7467	Protein	denotes	PKD1
T3793	7468-7469	Protein	denotes	3
T3794	7637-7641	Protein	denotes	PKD1
T3795	7642-7643	Protein	denotes	3
T3796	7761-7765	Protein	denotes	PKD3
T3797	7766-7779	Gene_expression	denotes	re-expression
T3798	7816-7820	Protein	denotes	PKD1
T3799	7821-7822	Protein	denotes	3
T3800	8109-8113	Protein	denotes	PKD1
T3801	8118-8122	Protein	denotes	PKD2
T3802	8438-8442	Negative_regulation	denotes	loss
T3803	8438-8442	Negative_regulation	denotes	loss
T3804	8446-8450	Protein	denotes	PKD1
T3805	8451-8452	Protein	denotes	3
T3806	8453-8463	Gene_expression	denotes	expression
T3807	8453-8463	Gene_expression	denotes	expression
T476	1131-1135	Protein	denotes	PKD1
T477	1137-1141	Protein	denotes	PKD2
T478	1146-1150	Protein	denotes	PKD3
T479	1755-1762	Binding	denotes	binding
T480	1775-1786	Positive_regulation	denotes	contributes
T481	1790-1794	Protein	denotes	PKD1
T482	1795-1805	Positive_regulation	denotes	activation
T483	2413-2417	Protein	denotes	PKD1
T484	2431-2436	Protein	denotes	HSP27
T485	2640-2644	Protein	denotes	MEF2
T486	3193-3206	Positive_regulation	denotes	indispensable
T487	3211-3216	Protein	denotes	HSP27
T488	3217-3232	Phosphorylation	denotes	phosphorylation
T1662	3832-3836	Protein	denotes	PKD1
T1663	3841-3845	Protein	denotes	PKD3
T1664	3853-3857	Protein	denotes	PKD1
T1665	3858-3859	Protein	denotes	3
T1666	3863-3871	Negative_regulation	denotes	knockout
T1667	3863-3871	Negative_regulation	denotes	knockout
T1668	3863-3871	Negative_regulation	denotes	knockout
T1669	3863-3871	Negative_regulation	denotes	knockout
R1883	T2253	T2254	causeOf	PKD1,regulate
R1884	T2256	T2257	themeOf	HSP27,phosphorylation
R1885	T2257	T2255	themeOf	phosphorylation,regulate
R1886	T2257	T2254	themeOf	phosphorylation,regulate
R371	T11	T10	themeOf	regulate,required
R372	T12	T13	themeOf	HSP27,phosphorylation
R373	T13	T11	themeOf	phosphorylation,regulate
R1839	T2186	T2184	themeOf	expression,lack
R1842	T2189	T2186	themeOf	PKD3,expression
R1843	T2193	T2192	themeOf	PKD3,expressed
R1844	T2194	T2195	themeOf	PKD3,expression
R1845	T2196	T2199	themeOf	PKD1,double knockout
R1846	T2197	T2198	themeOf	3,double knockout
R1847	T2206	T2205	themeOf	phosphorylation,defective
R1848	T2208	T2206	themeOf	serine 82,phosphorylation
R1849	T2208	T2207	partOf	serine 82,HSP27
R1850	T2211	T2212	themeOf	HSP27,phosphorylation
R1851	T2212	T2210	themeOf	phosphorylation,regulation
R1852	T2215	T2213	themeOf	PKD1,lacking
R1853	T2216	T2214	themeOf	PKD3,lacking
R1854	T2219	T2217	themeOf	phosphorylation,increased
R1855	T2220	T2219	themeOf	S82,phosphorylation
R1856	T2220	T2218	partOf	S82,HSP27
R1857	T2222	T2223	themeOf	HSP27,phosphorylation
R1858	T2223	T2221	themeOf	phosphorylation,increase
R1835	T2178	T2179	themeOf	HSP27,phosphorylation
R1836	T2179	T2177	themeOf	phosphorylation,Loss
R1837	T2182	T2180	themeOf	PKD1,express
R1838	T2183	T2181	themeOf	PKD3,express
R1840	T2187	T2185	themeOf	expression,lack
R1841	T2188	T2187	themeOf	PKD1,expression
R1859	T2224	T2227	themeOf	PKD1,knockout
R1860	T2225	T2226	themeOf	PKD3,knockout
R1861	T2228	T2232	themeOf	induced,abolished
R1862	T2228	T2233	themeOf	induced,abolished
R1863	T2229	T2228	themeOf	phosphorylation,induced
R1864	T2231	T2229	themeOf	S82,phosphorylation
R1865	T2231	T2230	partOf	S82,HSP27
R1866	T2234	T2233	causeOf	lacked,abolished
R1867	T2235	T2232	causeOf	lacked,abolished
R1868	T2236	T2235	themeOf	PKD1,lacked
R1869	T2237	T2234	themeOf	PKD3,lacked
R1870	T2238	T2241	causeOf	induced,sufficient to restore
R1871	T2239	T2238	themeOf	expression,induced
R1872	T2240	T2239	themeOf	PKD3,expression
R1873	T2242	T2241	themeOf	regulation,sufficient to restore
R1874	T2243	T2244	themeOf	HSP27,phosphorylation
R1875	T2244	T2242	themeOf	phosphorylation,regulation
R1876	T2245	T2248	causeOf	expression,restore
R1877	T2247	T2245	themeOf	PKD3,expression
R1878	T2247	T2246	themeOf	PKD3,deficient
R1879	T2249	T2248	themeOf	induced,restore
R1880	T2250	T2251	themeOf	HSP27,phosphorylation
R1881	T2251	T2249	themeOf	phosphorylation,induced
R1882	T2252	T2255	causeOf	PKD3,regulate
R3050	T3791	T3790	themeOf	PKD3,re-expression
R3051	T3796	T3797	themeOf	PKD3,re-expression
R3052	T3804	T3806	themeOf	PKD1,expression
R3053	T3805	T3807	themeOf	3,expression
R3054	T3806	T3803	themeOf	expression,loss
R3055	T3807	T3802	themeOf	expression,loss
R424	T479	T480	causeOf	binding,contributes
R425	T481	T479	themeOf	PKD1,binding
R426	T481	T482	themeOf	PKD1,activation
R427	T482	T480	themeOf	activation,contributes
R428	T487	T488	themeOf	HSP27,phosphorylation
R429	T488	T486	themeOf	phosphorylation,indispensable
R1433	T1662	T1666	themeOf	PKD1,knockout
R1434	T1663	T1667	themeOf	PKD3,knockout
R1435	T1664	T1668	themeOf	PKD1,knockout
R1436	T1665	T1669	themeOf	3,knockout

bionlp-st-ge-2016-uniprot

Id	Subject	Object	Predicate	Lexical cue
T1737	3853-3857	P98161	denotes	PKD1
T2676	4555-4560	P04792	denotes	HSP27
T2677	4673-4677	P98161	denotes	PKD1
T2678	4682-4686	Q99853	denotes	PKD3
T2679	4792-4796	P98161	denotes	PKD1
T2680	4800-4804	Q99853	denotes	PKD3
T2681	4887-4891	P98161	denotes	PKD1
T2682	4902-4906	Q99853	denotes	PKD3
T2683	4942-4946	Q99853	denotes	PKD3
T2684	5056-5060	Q99853	denotes	PKD3
T2685	5075-5079	P98161	denotes	PKD1
T2686	5132-5136	Q99853	denotes	PKD3
T2687	5408-5411	P11274	denotes	BCR
T2688	5439-5443	P98161	denotes	PKD1
T2689	5551-5556	P04792	denotes	HSP27
T2690	5589-5593	P98161	denotes	PKD1
T2691	5699-5704	P04792	denotes	HSP27
T2692	5732-5736	P98161	denotes	PKD1
T2693	5801-5806	P04792	denotes	HSP27
T4193	7182-7186	P98161	denotes	PKD1
T4194	7198-7202	Q99853	denotes	PKD3
T4195	7219-7223	P98161	denotes	PKD1
T4196	7300-7304	P98161	denotes	PKD1
T4197	7369-7373	Q99853	denotes	PKD3
T4198	7463-7467	P98161	denotes	PKD1
T4199	7637-7641	P98161	denotes	PKD1
T4200	7761-7765	Q99853	denotes	PKD3
T4201	7816-7820	P98161	denotes	PKD1
T4202	8109-8113	P98161	denotes	PKD1
T4203	8446-8450	P98161	denotes	PKD1
T4204	8643-8645	P03372	denotes	ER
T986	1131-1135	P98161	denotes	PKD1
T987	1146-1150	Q99853	denotes	PKD3
T988	1790-1794	P98161	denotes	PKD1
T989	2413-2417	P98161	denotes	PKD1
T990	2431-2436	P04792	denotes	HSP27
T991	2953-2956	P11274	denotes	BCR
T992	3211-3216	P04792	denotes	HSP27
T1735	3832-3836	P98161	denotes	PKD1
T1736	3841-3845	Q99853	denotes	PKD3
T204	699-704	P04792	denotes	HSP27
T2694	5870-5874	P98161	denotes	PKD1
T2695	5878-5882	Q99853	denotes	PKD3
T2696	5923-5926	P11274	denotes	BCR
T2697	5990-5995	P04792	denotes	HSP27
T2698	6046-6049	P11274	denotes	BCR
T2699	6086-6090	P00519	denotes	able
T2700	6103-6108	P04792	denotes	HSP27
T2701	6128-6132	P98161	denotes	PKD1
T2702	6136-6140	Q99853	denotes	PKD3
T2703	6190-6193	P11274	denotes	BCR
T2704	6240-6245	P04792	denotes	HSP27
T2705	6295-6299	P98161	denotes	PKD1
T2706	6304-6308	Q99853	denotes	PKD3
T2707	6378-6382	Q99853	denotes	PKD3
T2708	6469-6474	P04792	denotes	HSP27
T2709	6548-6552	Q99853	denotes	PKD3
T2710	6605-6609	P00519	denotes	able
T2711	6621-6624	P11274	denotes	BCR
T2712	6651-6656	P04792	denotes	HSP27
T2713	6691-6695	Q99853	denotes	PKD3
T2714	6707-6711	P98161	denotes	PKD1
T2715	6725-6730	P04792	denotes	HSP27
T2716	6802-6807	P04792	denotes	HSP27

test2

Id	Subject	Object	Predicate	Lexical cue	Negation	Speculation
T3755	7182-7186	Protein	denotes	PKD1
T3756	7187-7188	Protein	denotes	3
T5	678-686	Positive_regulation	denotes	required
T6	690-698	Regulation	denotes	regulate
T7	699-704	Protein	denotes	HSP27
T8	705-720	Phosphorylation	denotes	phosphorylation
T463	1131-1135	Protein	denotes	PKD1
T464	1137-1141	Protein	denotes	PKD2
T465	1146-1150	Protein	denotes	PKD3
T466	1755-1762	Binding	denotes	binding
T467	1775-1786	Positive_regulation	denotes	contributes
T468	1790-1794	Protein	denotes	PKD1
T469	1795-1805	Positive_regulation	denotes	activation
T470	2413-2417	Protein	denotes	PKD1
T471	2431-2436	Protein	denotes	HSP27
T472	2640-2644	Protein	denotes	MEF2
T473	3193-3206	Positive_regulation	denotes	indispensable
T474	3211-3216	Protein	denotes	HSP27
T475	3217-3232	Phosphorylation	denotes	phosphorylation
T1658	3832-3836	Protein	denotes	PKD1
T1659	3841-3845	Protein	denotes	PKD3
T1660	3853-3857	Protein	denotes	PKD1
T1661	3858-3859	Protein	denotes	3
T2103	4555-4560	Protein	denotes	HSP27		true
T2104	4561-4576	Phosphorylation	denotes	phosphorylation
T2105	4647-4654	Gene_expression	denotes	express
T2106	4673-4677	Protein	denotes	PKD1
T2107	4682-4686	Protein	denotes	PKD3
T2108	4766-4770	Negative_regulation	denotes	lack
T2109	4771-4781	Gene_expression	denotes	expression
T2110	4792-4796	Protein	denotes	PKD1
T2111	4800-4804	Protein	denotes	PKD3
T2112	4887-4891	Protein	denotes	PKD1
T2113	4902-4906	Protein	denotes	PKD3
T2114	4925-4934	Gene_expression	denotes	expressed
T2115	4942-4946	Protein	denotes	PKD3
T2116	5056-5060	Protein	denotes	PKD3
T2117	5061-5071	Gene_expression	denotes	expression		true
T2118	5075-5079	Protein	denotes	PKD1
T2119	5080-5081	Protein	denotes	3
T2120	5089-5097	Negative_regulation	denotes	knockout
T2121	5132-5136	Protein	denotes	PKD3
T2122	5439-5443	Protein	denotes	PKD1
T2123	5444-5445	Protein	denotes	3
T2124	5551-5556	Protein	denotes	HSP27
T2125	5589-5593	Protein	denotes	PKD1
T2126	5670-5679	Negative_regulation	denotes	defective
T2127	5680-5695	Phosphorylation	denotes	phosphorylation
T2128	5699-5704	Protein	denotes	HSP27
T2129	5708-5714	Entity	denotes	serine
T2130	5732-5736	Protein	denotes	PKD1
T2131	5787-5797	Regulation	denotes	regulation
T2132	5801-5806	Protein	denotes	HSP27
T2133	5807-5822	Phosphorylation	denotes	phosphorylation
T2134	5833-5841	Negative_regulation	denotes	knockout
T2135	5855-5862	Negative_regulation	denotes	lacking
T2136	5870-5874	Protein	denotes	PKD1
T2137	5878-5882	Protein	denotes	PKD3
T2138	5966-5975	Positive_regulation	denotes	increased
T2139	5990-5995	Protein	denotes	HSP27
T2140	5996-6011	Phosphorylation	denotes	phosphorylation	true
T2141	6015-6018	Entity	denotes	S82
T2142	6094-6102	Positive_regulation	denotes	increase
T2143	6103-6108	Protein	denotes	HSP27
T2144	6109-6124	Phosphorylation	denotes	phosphorylation
T2145	6128-6132	Protein	denotes	PKD1
T2146	6136-6140	Protein	denotes	PKD3
T2147	6148-6156	Negative_regulation	denotes	knockout
T2148	6213-6220	Positive_regulation	denotes	induced
T2149	6221-6236	Phosphorylation	denotes	phosphorylation
T2150	6240-6245	Protein	denotes	HSP27
T2151	6249-6252	Entity	denotes	S82
T2152	6257-6266	Negative_regulation	denotes	abolished
T2153	6283-6289	Negative_regulation	denotes	lacked
T2154	6295-6299	Protein	denotes	PKD1
T2155	6304-6308	Protein	denotes	PKD3
T2156	6347-6354	Positive_regulation	denotes	induced
T2157	6355-6365	Gene_expression	denotes	expression
T2158	6378-6382	Protein	denotes	PKD3
T2159	6455-6465	Regulation	denotes	regulation
T2160	6469-6474	Protein	denotes	HSP27
T2161	6475-6490	Phosphorylation	denotes	phosphorylation
T2162	6515-6525	Gene_expression	denotes	expression
T2163	6531-6537	Negative_regulation	denotes	kinase
T2164	6538-6547	Negative_regulation	denotes	deficient
T2165	6548-6552	Protein	denotes	PKD3
T2166	6613-6620	Positive_regulation	denotes	restore
T2167	6643-6650	Positive_regulation	denotes	induced
T2168	6651-6656	Protein	denotes	HSP27
T2169	6657-6672	Phosphorylation	denotes	phosphorylation
T2170	6691-6695	Protein	denotes	PKD3
T2171	6707-6711	Protein	denotes	PKD1
T2172	6716-6724	Regulation	denotes	regulate
T2173	6725-6730	Protein	denotes	HSP27
T2174	6731-6746	Phosphorylation	denotes	phosphorylation
T2175	6789-6798	Negative_regulation	denotes	redundant
T2176	6802-6807	Protein	denotes	HSP27
T3757	7198-7202	Protein	denotes	PKD3
T3758	7219-7223	Protein	denotes	PKD1
T3759	7224-7225	Protein	denotes	3
T3760	7300-7304	Protein	denotes	PKD1
T3761	7305-7306	Protein	denotes	3
T3762	7347-7349	Positive_regulation	denotes	re
T3763	7349-7350	Gene_expression	denotes	-
T3764	7350-7360	Gene_expression	denotes	expression
T3765	7369-7373	Protein	denotes	PKD3
T3766	7463-7467	Protein	denotes	PKD1
T3767	7468-7469	Protein	denotes	3
T3768	7637-7641	Protein	denotes	PKD1
T3769	7642-7643	Protein	denotes	3
T3770	7761-7765	Protein	denotes	PKD3
T3771	7766-7769	Gene_expression	denotes	re-
T3772	7769-7779	Gene_expression	denotes	expression
T3773	7816-7820	Protein	denotes	PKD1
T3774	7821-7822	Protein	denotes	3
T3775	8109-8113	Protein	denotes	PKD1
T3776	8118-8122	Protein	denotes	PKD2
T3777	8438-8442	Negative_regulation	denotes	loss
T3778	8446-8450	Protein	denotes	PKD1
T3779	8451-8452	Protein	denotes	3
T3780	8453-8463	Gene_expression	denotes	expression
T3781	8775-8784	Negative_regulation	denotes	inhibitor
R1820	T2154	T2153	themeOf	PKD1,lacked
R1821	T2155	T2153	themeOf	PKD3,lacked
R1822	T2157	T2156	themeOf	expression,induced
R1823	T2158	T2157	themeOf	PKD3,expression
R1825	T2161	T2159	themeOf	phosphorylation,regulation
R4	T6	T5	themeOf	regulate,required
R5	T7	T8	themeOf	HSP27,phosphorylation
R6	T8	T6	themeOf	phosphorylation,regulate
R419	T466	T467	causeOf	binding,contributes
R420	T468	T469	themeOf	PKD1,activation
R421	T469	T467	themeOf	activation,contributes
R422	T474	T475	themeOf	HSP27,phosphorylation
R423	T475	T473	themeOf	phosphorylation,indispensable
R1790	T2103	T2104	themeOf	HSP27,phosphorylation
R1791	T2106	T2105	themeOf	PKD1,express
R1792	T2107	T2105	themeOf	PKD3,express
R1793	T2109	T2108	themeOf	expression,lack
R1794	T2110	T2109	themeOf	PKD1,expression
R1795	T2111	T2109	themeOf	PKD3,expression
R1796	T2115	T2114	themeOf	PKD3,expressed
R1797	T2116	T2117	themeOf	PKD3,expression
R1798	T2118	T2120	themeOf	PKD1,knockout
R1799	T2127	T2126	themeOf	phosphorylation,defective
R1800	T2132	T2133	themeOf	HSP27,phosphorylation
R1801	T2133	T2131	themeOf	phosphorylation,regulation
R1802	T2134	T2131	causeOf	knockout,regulation
R1803	T2135	T2131	causeOf	lacking,regulation
R1804	T2136	T2135	themeOf	PKD1,lacking
R1805	T2137	T2135	themeOf	PKD3,lacking
R1806	T2139	T2140	themeOf	HSP27,phosphorylation
R1807	T2140	T2138	themeOf	phosphorylation,increased
R1808	T2141	T2140	themeOf	S82,phosphorylation
R1809	T2141	T2139	partOf	S82,HSP27
R1810	T2143	T2144	themeOf	HSP27,phosphorylation
R1811	T2144	T2142	themeOf	phosphorylation,increase
R1812	T2145	T2147	themeOf	PKD1,knockout
R1813	T2146	T2147	themeOf	PKD3,knockout
R1814	T2148	T2152	themeOf	induced,abolished
R1815	T2149	T2148	themeOf	phosphorylation,induced
R1816	T2149	T2152	themeOf	phosphorylation,abolished
R1817	T2151	T2149	themeOf	S82,phosphorylation
R1818	T2151	T2150	partOf	S82,HSP27
R1819	T2153	T2152	causeOf	lacked,abolished
R1824	T2160	T2161	themeOf	HSP27,phosphorylation
R1826	T2165	T2164	themeOf	PKD3,deficient
R1827	T2165	T2162	themeOf	PKD3,expression
R1828	T2167	T2166	themeOf	induced,restore
R1829	T2168	T2169	themeOf	HSP27,phosphorylation
R1830	T2169	T2167	themeOf	phosphorylation,induced
R1831	T2170	T2172	causeOf	PKD3,regulate
R1832	T2171	T2172	causeOf	PKD1,regulate
R1833	T2173	T2174	themeOf	HSP27,phosphorylation
R1834	T2174	T2172	themeOf	phosphorylation,regulate
R3047	T3778	T3780	themeOf	PKD1,expression
R3048	T3779	T3780	themeOf	3,expression
R3049	T3780	T3777	themeOf	expression,loss

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