> top > projects > BioNLP16_Messiy > docs > PMC:3585731 > annotations
BioNLP16_Messiy  

PMC:3585731 JSONTXT 30 Projects

Annnotations TAB TSV DIC JSON TextAE

Id Subject Object Predicate Lexical cue
T452 4-13 Regulation denotes Regulates
T441 14-18 Protein denotes TNFα
T451 19-28 Gene_expression denotes Synthesis
T442 43-47 Protein denotes RIP1
T453 55-65 Positive_regulation denotes Activation
T443 443-473 Protein denotes receptor interacting protein-1
T444 475-479 Protein denotes RIP1
T445 588-592 Protein denotes TNFα
T454 593-603 Gene_expression denotes production
T446 647-651 Protein denotes RIP1
T447 807-811 Protein denotes RIP1
T448 973-977 Protein denotes RIP1
T449 1024-1028 Protein denotes TNFα
T450 1149-1153 Protein denotes TNFα
T455 1154-1164 Gene_expression denotes production
T2122 2132-2136 Protein denotes RIP1
T2123 2141-2145 Protein denotes RIP3
T2124 2757-2761 Protein denotes RIP1
T2125 2766-2770 Protein denotes RIP3
T2126 2964-2968 Protein denotes RIP1
T2127 3015-3020 Protein denotes c-Jun
T2128 3180-3184 Protein denotes TNFα
T2143 3185-3194 Gene_expression denotes synthesis
T2142 3196-3206 Positive_regulation denotes activation
T2141 3231-3240 Positive_regulation denotes induction
T2129 3308-3312 Protein denotes RIP1
T6660 3308-3312 Protein denotes RIP1
T6694 3313-10126 Regulation denotes kinase dependent activation of JNK and TNFα production has recently been described to be independent of its role in necroptosis [15]. Curiously, Akt kinase, a key pro-survival molecule and a well-established inhibitor of apoptotic cell death, has also recently been linked to necroptosis in L929 cells [16], where insulin-dependent activation of Akt was suggested to promote necroptosis by suppressing autophagy. This conclusion was unexpected, since several reports from different groups, including ours, have established that autophagy promotes, rather than suppresses, zVAD.fmk-induced necroptosis in L929 cells [11], [14], [17]. This raised the possibility that Akt controls more general mechanisms that contribute to the execution of necroptosis. Furthermore, the key question of whether insulin-dependent Akt activity solely provides an environment conducive for necroptosis or if Akt activation is an intrinsic component of necroptosis signaling that is linked to RIP1 kinase has not been explored. In this study, we expanded these observations to delineate the specific contributions and molecular ordering of the Akt and JNK pathways downstream from RIP1 kinase during necroptosis. Our data reveal that Akt is activated through RIP1 kinase-dependent Thr308 phosphorylation during necroptosis in multiple cell types. Furthermore, we found that downstream Akt signaling through mTORC1 and S6 contributes to the activation of necroptosis and TNFα production. We found that the Akt pathway serves as a critical link between RIP1 kinase and JNK activation in L929 cells. Further data suggested that in multiple other cell types including FADD deficient Jurkat cells, RAW and J774.1 macrophage cell lines, and mouse lung fibroblasts Akt provides a key link to TNFα production, but is dispensible for cell death per se. Overall, our results reveal a specific and novel role for the Akt pathway in regulated necrosis and necrosis-associated inflammatory signaling. Results Basic Fibroblast Growth Factor Promotes Necroptosis in L929 Cells It has been established that mouse fibrosarcoma L929 cells undergo necroptotic cell death following stimulation with TNFα [10], [17]. In addition, inhibition of caspase-8 activity alone, either through siRNA knockdown or by using the pan-caspase inhibitor, zVAD.fmk, is sufficient to trigger necroptosis in these cells [10], [14]. Interestingly, while necroptosis was initially identified as a back-up form of cell death triggered by pro-apoptotic stimuli in the presence of apoptosis inhibitors [17], recent analysis of physiological cell death during mouse development has suggested that the loss of apoptotic regulators, such as caspase-8 and FADD [18], [19], [20], leads to robust induction of necroptosis and death of E10.5 embryos even though apoptosis is not normally induced in wild type embryos. These data are reminiscent of the observations in L929 cells where the loss of caspase activity in healthy cells is sufficient to trigger necroptosis and prompted us to explore the extrinsic or intrinsic cellular factors that promote necroptosis once caspase-8 activity, which cleaves and inactivates RIP1 kinase and the RIP1 deubiquitinase CYLD [21], [22], is removed in L929 cells. Consistent with a previous report [16], we found that serum starvation of L929 cells prevented necroptosis in response to zVAD.fmk (Fig. 1A). The addition of growth factors, such as bFGF, restored zVAD.fmk induced death under serum free conditions (Fig. 1B). Interestingly, this does not reflect a generic requirement for growth factor signaling, as only some growth factors (bFGF and IGF-1, but not EGF and PDGF) promoted death (Fig. 1B). Furthermore, growth factor-dependent necroptosis required the inhibition of caspase activity, as bFGF alone did not induce cell death (Fig. 1C). In contrast, TNFα triggered necroptosis equally efficiently in the absence of serum (Fig. 1A), suggesting that either growth factors and zVAD.fmk or TNFα are required for necroptotic death in L929 cells. 10.1371/journal.pone.0056576.g001 Figure 1 bFGF and IGF-1 promote necroptosis in concert with zVAD.fmk. (A) L929 cells were treated with TNFα or zVAD.fmk under normal serum (10% FBS) or serum free conditions. Cell viability was determined after 24 hr using the CellTiter-Glo Viability assay. The concentrations of all necroptosis-inducing agents are listed in the Materials and Methods section or indicated in the figures. (B) Cells were treated with zVAD.fmk, the indicated growth factors, and Nec-1 under serum free conditions for 24 hrs followed by measurement of cell viability. (C) Cells under serum free conditions were treated with FGF, zVAD.fmk, or both for 24 hrs followed by viability assay. (D) Cell death was induced by zVAD.fmk or TNFα under full serum condition in the presence of 2 µM PD173074 and 20 µM PD166866. In all graphs, average±SD was plotted. Previously we described the development of 7-Cl-O-Nec-1 (Nec-1) as a potent and selective inhibitor of RIP1 kinase and necroptosis (Fig. S1A) [23], [24]. Recently, its selectivity has been further validated against a panel of more than 400 human kinases [15]. This inhibitor efficiently blocked growth factor/zVAD.fmk-induced necroptosis under serum free conditions in L929 cells and both zVAD.fmk and TNFα-induced necroptosis under full serum conditions (Fig. 1B, S1B). To further validate the role of RIP1, we used an inactive analog, 7-Cl-O-Nec-1i (Nec-1i) (Fig. S1A), which contains an extra N-methyl group that leads to almost complete loss of RIP1 kinase inhibitory activity in vitro [23]. Nec-1i was unable to protect L929 cell death under serum condtions treated with zVAD.fmk or TNFα (Fig. S1B) or serum free conditions treated with bFGF/zVAD.fmk (Fig. S1C). This confirms that RIP1 kinase is responsible for necroptosis in L929 cells under both serum and serum free conditions. We next examined whether bFGF contributes to zVAD.fmk-induced necroptosis under normal serum conditions (10% FBS). We used two bFGF receptor tyrosine kinase inhibitors (PD173074 and PD166866), and determined that inhibition of bFGF signaling strongly inhibited zVAD.fmk-induced necroptosis under normal serum conditions (Fig. 1D). In contrast, neither bFGF receptor inhibitor was able to attenuate TNFα-induced necroptosis (Fig. 1D), consistent with growth factors being dispensable for this pathway (Fig. 1A). Overall, these data suggest that the induction of necroptosis by zVAD.fmk is promoted by bFGF under both serum and serum free conditions. The induction of necroptosis, however, is not a simple consequence of growth factor signaling since not all growth factors allowed death to occur. Instead, specific signaling events mediated by particular growth factors appear to contribute to necroptotic death. RIP1 Kinase-dependent
T2130 3352-3356 Protein denotes TNFα
T2144 3357-3367 Gene_expression denotes production
T2131 3627-3634 Protein denotes insulin
T2132 4106-4113 Protein denotes insulin
T2133 4284-4288 Protein denotes RIP1
T2134 4472-4476 Protein denotes RIP1
T2146 4532-4541 Positive_regulation denotes activated
T15242 4550-4554 Protein denotes RIP1
T2135 4550-4554 Protein denotes RIP1
T15275 4555-4571 Regulation denotes kinase-dependent
T2147 4555-4571 Regulation denotes kinase-dependent
T15277 4579-4594 Phosphorylation denotes phosphorylation
T2145 4579-4594 Phosphorylation denotes phosphorylation
T2136 4709-4711 Protein denotes S6
T2148 4731-4741 Positive_regulation denotes activation
T2137 4761-4765 Protein denotes TNFα
T2149 4766-4776 Gene_expression denotes production
T2138 4842-4846 Protein denotes RIP1
T2139 4955-4959 Protein denotes FADD
T2150 4960-4969 Negative_regulation denotes deficient
T2151 5068-5072 Positive_regulation denotes link
T2140 5076-5080 Protein denotes TNFα
T2152 5081-5091 Gene_expression denotes production
T4048 5289-5319 Protein denotes Basic Fibroblast Growth Factor
T4049 5472-5476 Protein denotes TNFα
T4077 5502-5512 Negative_regulation denotes inhibition
T4050 5516-5525 Protein denotes caspase-8
T4078 5949-5953 Negative_regulation denotes loss
T4051 5987-5996 Protein denotes caspase-8
T4052 6001-6005 Protein denotes FADD
T4053 6411-6420 Protein denotes caspase-8
T4054 6461-6465 Protein denotes RIP1
T4055 6481-6500 Protein denotes RIP1 deubiquitinase
T4056 6501-6505 Protein denotes CYLD
T4057 6726-6730 Protein denotes bFGF
T4058 6920-6924 Protein denotes bFGF
T4059 6929-6934 Protein denotes IGF-1
T4060 6944-6947 Protein denotes EGF
T4061 7081-7085 Protein denotes bFGF
T4062 7142-7146 Protein denotes TNFα
T4063 7278-7282 Protein denotes TNFα
T28702 7377-7381 Protein denotes bFGF
T28703 7386-7391 Protein denotes IGF-1
T28704 7471-7475 Protein denotes TNFα
T28705 8080-8084 Protein denotes TNFα
T4079 8294-8303 Negative_regulation denotes inhibitor
T4064 8307-8311 Protein denotes RIP1
T4065 8606-8610 Protein denotes TNFα
T4066 8707-8711 Protein denotes RIP1
T4067 8853-8857 Protein denotes RIP1
T4068 8992-8996 Protein denotes TNFα
T4069 9046-9050 Protein denotes bFGF
T4070 9091-9095 Protein denotes RIP1
T4071 9217-9221 Protein denotes bFGF
T4072 9319-9348 Protein denotes bFGF receptor tyrosine kinase
T4080 9349-9359 Negative_regulation denotes inhibitors
T4073 9419-9423 Protein denotes bFGF
T4074 9544-9557 Protein denotes bFGF receptor
T4081 9558-9567 Negative_regulation denotes inhibitor
T4075 9590-9594 Protein denotes TNFα
T4082 9595-9602 Positive_regulation denotes induced
T4076 9792-9796 Protein denotes bFGF
T6661 10595-10599 Protein denotes bFGF
T6662 10603-10608 Protein denotes IGF-1
T6663 10750-10754 Protein denotes TNFα
T6664 10870-10874 Protein denotes TNFα
T6665 11088-11092 Protein denotes TNFα
T6695 11093-11100 Positive_regulation denotes induced
T29142 11193-11200 Positive_regulation denotes induced
T29136 11217-11221 Protein denotes TNFα
T29137 11268-11272 Protein denotes TNFα
T29143 11499-11510 Gene_expression denotes transfected
T29138 11516-11520 Protein denotes Akt1
T29139 11522-11526 Protein denotes Akt2
T29140 11532-11543 Protein denotes Akt3 siRNAs
T29141 11585-11589 Protein denotes TNFα
T6697 12035-12044 Negative_regulation denotes knockdown
T6666 12061-12065 Protein denotes Akt1
T6667 12070-12074 Protein denotes Akt2
T6696 12121-12128 Positive_regulation denotes induced
T6668 12150-12154 Protein denotes TNFα
T6698 12169-12179 Gene_expression denotes expression
T6669 12183-12187 Protein denotes Akt3
T6670 12241-12251 Protein denotes Akt3 siRNA
T6671 12555-12559 Protein denotes TNFα
T6672 12725-12729 Protein denotes TNFα
T6700 12977-12985 Positive_regulation denotes increase
T6699 12993-13008 Phosphorylation denotes phosphorylation
T6673 13021-13024 Protein denotes p46
T6674 13029-13032 Protein denotes p54
T6675 13073-13078 Protein denotes c-Jun
T29671 13220-13224 Protein denotes RIP1
T29676 13225-13241 Regulation denotes kinase-dependent
T29677 13242-13257 Phosphorylation denotes phosphorylation
T29672 13338-13342 Protein denotes TNFα
T29673 13460-13464 Protein denotes bFGF
T29674 13624-13628 Protein denotes bFGF
T29675 13632-13636 Protein denotes bFGF
T6676 13745-13749 Protein denotes RIP1
T6702 13757-13766 Negative_regulation denotes inhibitor
T6703 13786-13795 Negative_regulation denotes prevented
T6701 13800-13808 Positive_regulation denotes increase
T6677 13819-13822 Protein denotes Akt
T6704 13823-13838 Phosphorylation denotes phosphorylation
T6678 14014-14018 Protein denotes TNFα
T6705 14046-14053 Regulation denotes changes
T6679 14089-14094 Protein denotes c-Jun
T6706 14175-14183 Positive_regulation denotes increase
T6680 14187-14192 Protein denotes c-Jun
T6681 14402-14406 Protein denotes RIP1
T6682 14698-14702 Protein denotes RIP1
T6683 14869-14873 Protein denotes bFGF
T6684 15030-15035 Protein denotes c-Jun
T6685 15158-15162 Protein denotes bFGF
T6686 15181-15185 Protein denotes bFGF
T6687 15453-15458 Protein denotes c-Jun
T6688 15493-15497 Protein denotes bFGF
T6689 15502-15506 Protein denotes bFGF
T6707 15585-15592 Negative_regulation denotes blocked
T6690 15597-15601 Protein denotes bFGF
T6708 15607-15615 Positive_regulation denotes increase
T6691 15741-15745 Protein denotes RIP1
T6692 16165-16169 Protein denotes RIP1
T6693 16449-16453 Protein denotes RIP1
T7929 16463-16467 Protein denotes TNFα
T7930 16584-16588 Protein denotes TNFα
T7931 16687-16691 Protein denotes TNFα
T7940 16692-16699 Positive_regulation denotes induced
T7932 16843-16847 Protein denotes TNFα
T7933 16976-16980 Protein denotes TNFα
T7934 17069-17073 Protein denotes TNFα
T7935 17133-17137 Protein denotes TNFα
T7942 17175-17183 Positive_regulation denotes increase
T7941 17191-17206 Phosphorylation denotes phosphorylation
T7936 17218-17223 Protein denotes c-Jun
T7939 17377-17381 Protein denotes RIP1
T7937 17476-17480 Protein denotes Ask1
T7938 17517-17521 Protein denotes RIP1
T8727 17698-17702 Protein denotes RIP1
T8733 17703-17719 Regulation denotes kinase-dependent
T8734 17720-17728 Positive_regulation denotes increase
T8732 17772-17783 Positive_regulation denotes contributes
T8728 18244-18248 Protein denotes TNFα
T8729 18252-18256 Protein denotes bFGF
T8730 18462-18466 Protein denotes bFGF
T8731 18501-18505 Protein denotes bFGF
T29968 19117-19121 Protein denotes bFGF
T29969 19237-19241 Protein denotes TNFα
T29970 19249-19253 Protein denotes bFGF
T29971 19503-19507 Protein denotes bFGF
T29972 19738-19742 Protein denotes bFGF
T10502 20088-20096 Positive_regulation denotes increase
T10501 20104-20119 Phosphorylation denotes phosphorylation
T10459 20216-20231 Protein denotes double minute 2
T10460 20233-20237 Protein denotes MDM2
T10461 20273-20304 Protein denotes p70 ribosomal protein S6 Kinase
T10462 20306-20312 Protein denotes p70S6K
T10463 20315-20317 Protein denotes S6
T10464 20345-20350 Protein denotes FoxO1
T10465 20352-20357 Protein denotes FoxO4
T10466 20359-20363 Protein denotes MDM2
T10471 20414-20420 Protein denotes FoxO3a
T10467 20422-20428 Protein denotes GSK-3α
T10468 20429-20430 Protein denotes β
T10469 20432-20438 Protein denotes p70S6K
T10470 20457-20459 Protein denotes S6
T10472 20629-20635 Protein denotes pFoxO1
T10503 20697-20705 Positive_regulation denotes increase
T10473 21124-21128 Protein denotes RIP1
T30570 21290-21294 Protein denotes TNFα
T30571 21390-21394 Protein denotes bFGF
T30572 21398-21402 Protein denotes bFGF
T30573 21551-21555 Protein denotes TNFα
T30574 21764-21768 Protein denotes TNFα
T10504 22209-22218 Negative_regulation denotes inhibitor
T10474 22241-22247 Protein denotes Raptor
T10475 22616-22620 Protein denotes TNFα
T10505 22621-22628 Positive_regulation denotes induced
T10506 22683-22693 Positive_regulation denotes activation
T10476 22697-22709 Protein denotes p70S6 kinase
T10477 22747-22749 Protein denotes S6
T10507 22912-22921 Negative_regulation denotes inhibitor
T10478 22925-22931 Protein denotes p70S6K
T10508 23005-23010 Negative_regulation denotes block
T10479 23011-23013 Protein denotes S6
T10509 23014-23029 Phosphorylation denotes phosphorylation
T10510 23059-23068 Negative_regulation denotes knockdown
T10480 23072-23074 Protein denotes S6
T10481 23167-23173 Protein denotes p70S6K
T10482 23174-23176 Protein denotes S6
T10483 23422-23426 Protein denotes RIP1
T10511 23467-23479 Positive_regulation denotes upregulation
T10484 23483-23487 Protein denotes TNFα
T10513 23635-23644 Positive_regulation denotes increased
T10512 23645-23654 Gene_expression denotes synthesis
T10485 23658-23662 Protein denotes TNFα
T10514 23747-23757 Positive_regulation denotes contribute
T10486 23761-23765 Protein denotes TNFα
T10515 23766-23775 Gene_expression denotes synthesis
T10487 23795-23799 Protein denotes RIP1
T10488 23822-23826 Protein denotes TNFα
T10489 23864-23868 Protein denotes TNFα
T10516 23881-23890 Positive_regulation denotes increased
T10490 23929-23933 Protein denotes RIP1
T10491 23974-23978 Protein denotes bFGF
T10518 23985-23994 Positive_regulation denotes triggered
T10517 24000-24009 Positive_regulation denotes induction
T10492 24013-24017 Protein denotes TNFα
T10523 24166-24178 Positive_regulation denotes upregulation
T10493 24182-24186 Protein denotes TNFα
T10521 24215-24224 Positive_regulation denotes increased
T10522 24349-24357 Positive_regulation denotes increase
T10519 24361-24370 Positive_regulation denotes autocrine
T10494 24371-24375 Protein denotes TNFα
T10520 24376-24385 Gene_expression denotes synthesis
T30913 24454-24463 Positive_regulation denotes autocrine
T30905 24464-24468 Protein denotes TNFα
T30914 24469-24478 Gene_expression denotes synthesis
T30906 24574-24578 Protein denotes bFGF
T30907 24654-24659 Protein denotes mTNFα
T30908 24690-24693 Protein denotes 18S
T30915 24719-24726 Positive_regulation denotes induced
T30909 24742-24746 Protein denotes TNFα
T30910 24848-24852 Protein denotes TNFα
T30911 24973-24977 Protein denotes TNFα
T30912 25020-25025 Protein denotes mTNFα
T10524 25146-25156 Positive_regulation denotes contribute
T10526 25164-25176 Positive_regulation denotes upregulation
T10495 25180-25184 Protein denotes TNFα
T10525 25279-25286 Negative_regulation denotes reduced
T10496 25287-25291 Protein denotes TNFα
T10497 25349-25353 Protein denotes RIP1
T10529 25362-25372 Negative_regulation denotes inhibitors
T10528 25380-25386 Regulation denotes effect
T10498 25404-25408 Protein denotes TNFα
T10499 25463-25467 Protein denotes bFGF
T10530 25570-25578 Positive_regulation denotes increase
T10500 25582-25586 Protein denotes TNFα
T10527 25587-25596 Gene_expression denotes synthesis
T12480 25689-25698 Regulation denotes regulator
T12467 25702-25706 Protein denotes TNFα
T12479 25707-25716 Gene_expression denotes synthesis
T12481 25830-25838 Positive_regulation denotes increase
T12468 25842-25846 Protein denotes TNFα
T12485 25927-25936 Negative_regulation denotes Knockdown
T12469 25953-25957 Protein denotes Akt1
T12470 25962-25966 Protein denotes Akt2
T12484 25970-25980 Negative_regulation denotes inhibition
T12482 26000-26010 Negative_regulation denotes suppressed
T12471 26057-26062 Protein denotes c-Jun
T12483 26063-26078 Phosphorylation denotes phosphorylation
T12472 26143-26147 Protein denotes RIP1
T12486 26156-26166 Positive_regulation denotes activation
T12489 26204-26213 Negative_regulation denotes inhibited
T12490 26246-26254 Positive_regulation denotes increase
T12473 26258-26262 Protein denotes bFGF
T12488 26272-26279 Positive_regulation denotes induced
T12474 26288-26293 Protein denotes c-Jun
T12487 26294-26309 Phosphorylation denotes phosphorylation
T12492 26322-26331 Negative_regulation denotes Knockdown
T12476 26359-26365 Protein denotes p70S6K
T12493 26392-26402 Negative_regulation denotes attenuated
T12491 26430-26438 Positive_regulation denotes increase
T12475 26450-26455 Protein denotes c-Jun
T12494 26456-26471 Phosphorylation denotes phosphorylation
T12477 26550-26553 Protein denotes S6K
T12478 26583-26587 Protein denotes RIP1
T13254 26691-26695 Protein denotes PDK1
T13261 27044-27058 Phosphorylation denotes phosphorylated
T13255 27083-27128 Protein denotes 3-phosphoinositide dependent protein kinase-1
T13256 27130-27134 Protein denotes PDK1
T13262 27311-27320 Positive_regulation denotes dependent
T13257 27333-27337 Protein denotes PDK1
T13263 27339-27349 Negative_regulation denotes Inhibition
T13258 27362-27366 Protein denotes PDK1
T13264 27533-27542 Negative_regulation denotes knockdown
T13259 27546-27550 Protein denotes PDK1
T13260 27652-27656 Protein denotes PDK1
T15271 27853-27863 Gene_expression denotes expressing
T15228 27896-27900 Protein denotes Akt1
T15229 27902-27909 Protein denotes Myr-Akt
T15230 27948-27953 Protein denotes K179M
T15231 28024-28028 Protein denotes RIP1
T15232 28096-28100 Protein denotes Akt1
T15233 28102-28109 Protein denotes Myr-Akt
T15272 28416-28426 Gene_expression denotes expressing
T15234 28427-28434 Protein denotes Myr-Akt
T15235 28713-28720 Protein denotes Myr-Akt
T15236 28734-28739 Protein denotes K179M
T15237 28826-28833 Protein denotes Myr-Akt
T15238 29030-29035 Protein denotes c-Jun
T15273 29036-29051 Phosphorylation denotes phosphorylation
T15274 29066-29078 Positive_regulation denotes upregulation
T15239 29082-29086 Protein denotes TNFα
T15240 29384-29388 Protein denotes RIP1
T15241 29493-29497 Protein denotes RIP1
T31702 30397-30401 Protein denotes TNFα
T31703 30468-30471 Protein denotes 18S
T31704 30699-30703 Protein denotes TNFα
T15243 30828-30835 Protein denotes Myr-Akt
T15276 30855-30864 Positive_regulation denotes increased
T15244 30865-30872 Protein denotes Myr-Akt
T15278 30986-30995 Positive_regulation denotes increased
T15279 31005-31015 Gene_expression denotes expression
T15245 31019-31026 Protein denotes Myr-Akt
T15246 31040-31045 Protein denotes K179M
T15247 31232-31239 Protein denotes Myr-Akt
T15248 31268-31274 Protein denotes p70S6K
T15249 31276-31278 Protein denotes S6
T15250 31290-31295 Protein denotes FoxO4
T15280 31308-31322 Phosphorylation denotes phosphorylated
T15282 31375-31390 Phosphorylation denotes phosphorylation
T15251 31394-31399 Protein denotes FoxO1
T15252 31404-31408 Protein denotes MDM2
T15283 31427-31436 Positive_regulation denotes increased
T15281 31501-31516 Phosphorylation denotes phosphorylation
T15253 31520-31527 Protein denotes Myr-Akt
T15254 31653-31657 Protein denotes TNFα
T15284 31658-31668 Gene_expression denotes production
T15285 31697-31706 Gene_expression denotes expressed
T15255 31707-31714 Protein denotes Myr-Akt
T15286 32276-32285 Negative_regulation denotes reduction
T15256 32315-32318 Protein denotes Akt
T15257 32771-32783 Protein denotes S473D mutant
T15287 32795-32809 Phosphorylation denotes phosphorylated
T15258 32896-32901 Protein denotes S473A
T15259 32963-32968 Protein denotes S473A
T15288 32998-33012 Phosphorylation denotes phosphorylated
T15260 33133-33137 Protein denotes PDK1
T15290 33249-33258 Positive_regulation denotes promoting
T15289 33271-33286 Phosphorylation denotes phosphorylation
T15261 33298-33303 Protein denotes c-Jun
T15262 33309-33313 Protein denotes TNFα
T15263 33339-33344 Protein denotes T308D
T15291 33419-33434 Phosphorylation denotes phosphorylation
T15264 33583-33588 Protein denotes T308D
T15265 33690-33697 Protein denotes Myr-Akt
T15266 33742-33747 Protein denotes FoxO1
T15267 33749-33754 Protein denotes Foxo4
T15268 33756-33760 Protein denotes MDM2
T15269 33766-33772 Protein denotes p70S6K
T15292 34004-34011 Positive_regulation denotes promote
T15270 34012-34016 Protein denotes TNFα
T15293 34017-34026 Gene_expression denotes synthesis
T17873 34082-34086 Protein denotes TNFα
T17892 34087-34097 Gene_expression denotes Production
T17874 34149-34153 Protein denotes RIP1
T17875 34303-34343 Protein denotes Fas-associated protein with death domain
T17876 34345-34349 Protein denotes FADD
T17877 34509-34513 Protein denotes TNFα
T17878 34571-34575 Protein denotes RIP1
T17879 34718-34722 Protein denotes TNFα
T17895 34740-34749 Positive_regulation denotes increased
T17893 34813-34822 Negative_regulation denotes inhibited
T17880 34831-34835 Protein denotes RIP1
T17894 34844-34854 Negative_regulation denotes inhibitors
T17898 34980-34990 Regulation denotes regulation
T17896 34994-35003 Positive_regulation denotes autocrine
T17882 35004-35008 Protein denotes TNFα
T17897 35009-35018 Gene_expression denotes synthesis
T17881 35131-35135 Protein denotes RIP1
T32189 35268-35279 Positive_regulation denotes contributes
T32190 35283-35292 Positive_regulation denotes autocrine
T32177 35293-35297 Protein denotes TNFα
T32191 35298-35308 Gene_expression denotes production
T32178 35333-35337 Protein denotes FADD
T32192 35338-35347 Negative_regulation denotes deficient
T32179 35379-35383 Protein denotes TNFα
T32180 35421-35425 Protein denotes TNFα
T32181 35476-35479 Protein denotes 18S
T32182 35631-35635 Protein denotes TNFα
T32183 35785-35789 Protein denotes TNFα
T32184 35817-35821 Protein denotes TNFα
T32193 35881-35891 Gene_expression denotes expressing
T32188 35908-35912 Protein denotes Akt1
T32185 35916-35920 Protein denotes Akt2
T32186 35952-35956 Protein denotes TNFα
T32187 35984-35988 Protein denotes TNFα
T17883 36243-36247 Protein denotes TNFα
T17901 36326-36334 Negative_regulation denotes restored
T17884 36335-36339 Protein denotes TNFα
T17899 36345-36355 Transcription denotes production
T17885 36371-36375 Protein denotes TNFα
T17900 36380-36388 Negative_regulation denotes zVAD.fmk
T17902 36511-36521 Gene_expression denotes expressing
T17886 36533-36537 Protein denotes Akt1
T17887 36541-36545 Protein denotes Akt2
T17888 36591-36595 Protein denotes TNFα
T17889 36645-36649 Protein denotes RIP1
T17903 36650-36659 Positive_regulation denotes dependent
T17890 36660-36664 Protein denotes TNFα
T17904 36670-36682 Positive_regulation denotes upregulation
T17905 36791-36799 Positive_regulation denotes critical
T17891 36814-36818 Protein denotes TNFα
T17906 36819-36828 Gene_expression denotes synthesis
T19491 37024-37028 Protein denotes RIP1
T19492 37118-37122 Protein denotes RIP1
T19493 37374-37378 Protein denotes RIP1
T19517 37686-37696 Gene_expression denotes Expression
T19494 37750-37757 Protein denotes Myr-Akt
T19518 37823-37833 Gene_expression denotes expression
T19495 37837-37844 Protein denotes Myr-Akt
T19496 37913-37917 Protein denotes RIP1
T19519 37918-37934 Regulation denotes kinase-dependent
T32435 38138-38142 Protein denotes RIP1
T32436 38297-38301 Protein denotes RIP1
T32439 38346-38351 Positive_regulation denotes leads
T32437 38355-38359 Protein denotes TNFα
T32438 38360-38369 Gene_expression denotes synthesis
T19497 39170-39174 Protein denotes RIP1
T19498 39297-39301 Protein denotes TNFα
T19520 39302-39311 Gene_expression denotes synthesis
T19521 39569-39578 Negative_regulation denotes inhibited
T19499 39579-39583 Protein denotes TNFα
T19522 39584-39594 Gene_expression denotes production
T19524 39789-39798 Negative_regulation denotes inhibitor
T19523 39808-39815 Negative_regulation denotes inhibit
T19500 39820-39833 Protein denotes p110δ subunit
T19501 39851-39855 Protein denotes PDK1
T19502 40119-40123 Protein denotes JNK1
T19503 40128-40132 Protein denotes JNK2
T19526 40170-40180 Negative_regulation denotes inhibition
T19528 40184-40193 Negative_regulation denotes knockdown
T19504 40197-40201 Protein denotes JNK1
T19505 40202-40203 Protein denotes 2
T19525 40251-40261 Negative_regulation denotes inhibiting
T19527 40266-40281 Phosphorylation denotes phosphorylation
T19506 40285-40290 Protein denotes c-Jun
T19530 40371-40380 Negative_regulation denotes reduction
T19507 40384-40388 Protein denotes TNFα
T19529 40389-40399 Gene_expression denotes production
T19508 40627-40632 Protein denotes c-Jun
T19509 40662-40666 Protein denotes TNFα
T19531 40667-40676 Gene_expression denotes synthesis
T19510 40735-40740 Protein denotes c-Jun
T19532 40747-40756 Negative_regulation denotes knockdown
T19533 40872-40881 Positive_regulation denotes autocrine
T19511 40882-40886 Protein denotes TNFα
T19534 40887-40897 Gene_expression denotes production
T19535 40899-40908 Positive_regulation denotes dependent
T19512 40912-40917 Protein denotes c-Jun
T19536 41054-41062 Positive_regulation denotes increase
T19513 41087-41092 Protein denotes c-Jun
T19514 41140-41144 Protein denotes TNFα
T19515 41267-41272 Protein denotes c-Jun
T19537 41407-41422 Phosphorylation denotes phosphorylation
T19516 41426-41431 Protein denotes c-Jun
T23082 41723-41727 Protein denotes RIP1
T23083 41867-41871 Protein denotes RIP1
T23084 41994-41998 Protein denotes TNFα
T23123 41999-42008 Gene_expression denotes synthesis
T23124 42215-42225 Gene_expression denotes Expression
T23125 42251-42268 Binding denotes membrane-targeted
T23085 42269-42276 Protein denotes Myr-Akt
T23126 42323-42333 Gene_expression denotes expression
T23086 42337-42344 Protein denotes Myr-Akt
T23127 42409-42419 Positive_regulation denotes activation
T23087 42431-42435 Protein denotes TNFα
T23128 42436-42445 Gene_expression denotes synthesis
T23088 42457-42461 Protein denotes RIP1
T23129 42462-42478 Regulation denotes kinase-dependent
T23089 43036-43040 Protein denotes RIP1
T23090 43115-43119 Protein denotes RIP1
T23091 43486-43490 Protein denotes RIP1
T23092 43508-43512 Protein denotes PDK1
T23093 43645-43649 Protein denotes RIP1
T23094 43762-43769 Protein denotes Myr-Akt
T23130 44068-44083 Phosphorylation denotes phosphorylation
T23095 44094-44098 Protein denotes RIP1
T23131 44230-44237 Positive_regulation denotes promote
T23096 44257-44261 Protein denotes TNFα
T23132 44262-44271 Gene_expression denotes synthesis
T23097 44983-44987 Protein denotes TNFα
T23098 45130-45134 Protein denotes TNFα
T23099 45456-45460 Protein denotes RIP1
T23100 45667-45672 Protein denotes FoxO1
T23101 45673-45674 Protein denotes 4
T23102 45680-45684 Protein denotes MDM2
T23103 46185-46189 Protein denotes RIP1
T23104 46851-46855 Protein denotes TNFα
T23133 46968-46975 Positive_regulation denotes induced
T23105 46979-46983 Protein denotes TNFα
T23106 47343-47347 Protein denotes RIP1
T23107 47593-47597 Protein denotes FADD
T23135 47598-47607 Negative_regulation denotes deficient
T23134 47688-47696 Positive_regulation denotes increase
T23108 47700-47704 Protein denotes TNFα
T23136 47705-47714 Gene_expression denotes synthesis
T23109 47861-47865 Protein denotes RIP1
T23137 47893-47902 Positive_regulation denotes mediating
T23138 47907-47917 Gene_expression denotes production
T23110 47921-47925 Protein denotes TNFα
T23140 48673-48683 Positive_regulation denotes activation
T23111 48687-48691 Protein denotes TNFα
T23139 48692-48701 Gene_expression denotes synthesis
T23112 48705-48709 Protein denotes RIP1
T23141 48928-48932 Negative_regulation denotes loss
T23113 48949-48953 Protein denotes RIP1
T23114 48954-48958 Protein denotes RIP3
T23115 48980-48984 Protein denotes FADD
T23116 48989-48998 Protein denotes caspase-8
T23117 49069-49073 Protein denotes TNFα
T23142 49074-49084 Gene_expression denotes production
T23144 49213-49222 Positive_regulation denotes increased
T23143 49223-49233 Gene_expression denotes production
T23118 49237-49241 Protein denotes TNFα
T23119 49582-49586 Protein denotes RIP1
T23120 49591-49595 Protein denotes RIP3
T23145 49596-49603 Positive_regulation denotes mediate
T23121 49672-49676 Protein denotes RIP1
T23122 49785-49789 Protein denotes RIP1
T23146 49797-49807 Negative_regulation denotes inhibitors
T25528 50802-50806 Protein denotes TNFα
T25529 50825-50829 Protein denotes bFGF
T25530 50842-50845 Protein denotes EGF
T25531 50882-50887 Protein denotes IGF-1
T25657 50989-50997 Protein denotes Myr-Akt1
T25658 51056-51069 Protein denotes Myr-Akt1-FLAG
T25659 51201-51209 Protein denotes Myr-Akt1
T26083 52217-52221 Protein denotes TNFα
T26084 52302-52305 Protein denotes 18S
T26085 52392-52396 Protein denotes TNFα
T26086 52443-52447 Protein denotes TNFα
T26087 52493-52496 Protein denotes 18S
T26088 52541-52544 Protein denotes 18S
T26332 52603-52607 Protein denotes FADD
T26333 52608-52617 Negative_regulation denotes deficient
T26889 54009-54013 Protein denotes Akt1
T26890 54035-54039 Protein denotes Akt2
T26891 54061-54065 Protein denotes Akt3
T26892 54113-54117 Protein denotes PDK1
T26893 54176-54181 Protein denotes Mapk8
T26894 54203-54208 Protein denotes Mapk9
T26895 54402-54406 Protein denotes TNFα
T27325 54647-54651 Protein denotes TNFα
T27326 54806-54810 Protein denotes TNFα
T27861 56096-56104 Protein denotes Myr-Akt1
T28225 57079-57083 Protein denotes TNFα
T28226 57096-57100 Protein denotes TNFα
T28379 57427-57434 Protein denotes Myr-Akt
T28380 57439-57457 Binding denotes immunoprecipitated
R10009 T15228 T15271 themeOf Akt1,expressing
R10010 T15229 T15271 themeOf Myr-Akt,expressing
R10011 T15230 T15271 themeOf K179M,expressing
R10012 T15234 T15272 themeOf Myr-Akt,expressing
R10013 T15238 T15273 themeOf c-Jun,phosphorylation
R10014 T15239 T15274 themeOf TNFα,upregulation
R10015 T15242 T15277 themeOf RIP1,phosphorylation
R10016 T15243 T15277 themeOf Myr-Akt,phosphorylation
R10017 T15245 T15279 themeOf Myr-Akt,expression
R10018 T15248 T15280 themeOf p70S6K,phosphorylated
R10019 T15249 T15280 themeOf S6,phosphorylated
R10020 T15250 T15280 themeOf FoxO4,phosphorylated
R10021 T15251 T15282 themeOf FoxO1,phosphorylation
R10022 T15252 T15282 themeOf MDM2,phosphorylation
R10023 T15253 T15281 themeOf Myr-Akt,phosphorylation
R10024 T15254 T15284 themeOf TNFα,production
R10025 T15255 T15285 themeOf Myr-Akt,expressed
R10026 T15256 T15286 themeOf Akt,reduction
R10027 T15257 T15287 themeOf S473D mutant,phosphorylated
R10028 T15259 T15288 themeOf S473A,phosphorylated
R10029 T15261 T15290 themeOf c-Jun,promoting
R10030 T15261 T15289 themeOf c-Jun,phosphorylation
R10031 T15263 T15291 themeOf T308D,phosphorylation
R10032 T15270 T15293 themeOf TNFα,synthesis
R10033 T15277 T15275 themeOf phosphorylation,kinase-dependent
R10034 T15277 T15275 themeOf phosphorylation,kinase-dependent
R10035 T15277 T15276 themeOf phosphorylation,increased
R10036 T15277 T15276 themeOf phosphorylation,increased
R10037 T15279 T15278 themeOf expression,increased
R10038 T15282 T15283 themeOf phosphorylation,increased
R10039 T15282 T15283 themeOf phosphorylation,increased
R10040 T15289 T15290 themeOf phosphorylation,promoting
R10041 T15293 T15292 themeOf synthesis,promote
R11996 T17873 T17892 themeOf TNFα,Production
R11997 T17879 T17893 themeOf TNFα,inhibited
R11998 T17879 T17895 themeOf TNFα,increased
R11999 T17880 T17894 themeOf RIP1,inhibitors
R12000 T17882 T17897 themeOf TNFα,synthesis
R12001 T17884 T17899 themeOf TNFα,production
R12002 T17885 T17900 themeOf TNFα,zVAD.fmk
R12003 T17886 T17902 themeOf Akt1,expressing
R12004 T17887 T17902 themeOf Akt2,expressing
R12005 T17889 T17903 causeOf RIP1,dependent
R12006 T17890 T17904 themeOf TNFα,upregulation
R12007 T17891 T17906 themeOf TNFα,synthesis
R12008 T17897 T17898 themeOf synthesis,regulation
R12009 T17897 T17896 themeOf synthesis,autocrine
R12010 T17899 T17901 themeOf production,restored
R12011 T17904 T17903 themeOf upregulation,dependent
R12012 T17906 T17905 themeOf synthesis,critical
R12974 T19494 T19517 themeOf Myr-Akt,Expression
R12975 T19495 T19518 themeOf Myr-Akt,expression
R12976 T19496 T19519 themeOf RIP1,kinase-dependent
R12977 T19498 T19520 themeOf TNFα,synthesis
R12978 T19499 T19522 themeOf TNFα,production
R12979 T19500 T19523 themeOf p110δ subunit,inhibit
R12980 T19504 T19526 themeOf JNK1,inhibition
R12981 T19504 T19528 themeOf JNK1,knockdown
R12982 T19505 T19526 themeOf 2,inhibition
R12983 T19505 T19528 themeOf 2,knockdown
R12984 T19506 T19527 themeOf c-Jun,phosphorylation
R12985 T19507 T19529 themeOf TNFα,production
R12986 T19509 T19531 themeOf TNFα,synthesis
R12987 T19510 T19532 themeOf c-Jun,knockdown
R12988 T19511 T19534 themeOf TNFα,production
R12989 T19512 T19535 causeOf c-Jun,dependent
R12990 T19513 T19536 themeOf c-Jun,increase
R12991 T19516 T19537 themeOf c-Jun,phosphorylation
R12992 T19522 T19521 themeOf production,inhibited
R12993 T19523 T19524 themeOf inhibit,inhibitor
R12994 T19526 T19528 themeOf inhibition,knockdown
R12995 T19526 T19528 themeOf inhibition,knockdown
R12996 T19527 T19525 themeOf phosphorylation,inhibiting
R12997 T19529 T19530 themeOf production,reduction
R12998 T19534 T19533 themeOf production,autocrine
R12999 T19534 T19535 themeOf production,dependent
R1453 T2128 T2143 themeOf TNFα,synthesis
R1455 T2130 T2144 themeOf TNFα,production
R1456 T2135 T2145 themeOf RIP1,phosphorylation
R1458 T2137 T2149 themeOf TNFα,production
R1461 T2139 T2150 themeOf FADD,deficient
R1462 T2140 T2152 themeOf TNFα,production
R1463 T2143 T2142 themeOf synthesis,activation
R1464 T2143 T2141 themeOf synthesis,induction
R1466 T2145 T2146 themeOf phosphorylation,activated
R1467 T2145 T2147 themeOf phosphorylation,kinase-dependent
R1469 T2149 T2148 themeOf production,activation
R1471 T2152 T2151 themeOf production,link
R15457 T23084 T23123 themeOf TNFα,synthesis
R15458 T23085 T23124 themeOf Myr-Akt,Expression
R15459 T23085 T23125 themeOf Myr-Akt,membrane-targeted
R15460 T23086 T23126 themeOf Myr-Akt,expression
R15461 T23087 T23128 themeOf TNFα,synthesis
R15462 T23088 T23129 themeOf RIP1,kinase-dependent
R15463 T23095 T23130 themeOf RIP1,phosphorylation
R15464 T23096 T23132 themeOf TNFα,synthesis
R15465 T23105 T23133 themeOf TNFα,induced
R15466 T23107 T23135 themeOf FADD,deficient
R15467 T23108 T23136 themeOf TNFα,synthesis
R15468 T23110 T23138 themeOf TNFα,production
R15469 T23111 T23139 themeOf TNFα,synthesis
R15470 T23114 T23141 themeOf RIP3,loss
R15471 T23117 T23142 themeOf TNFα,production
R15472 T23118 T23143 themeOf TNFα,production
R15473 T23120 T23145 themeOf RIP3,mediate
R15474 T23122 T23146 themeOf RIP1,inhibitors
R15475 T23128 T23127 themeOf synthesis,activation
R15476 T23132 T23131 themeOf synthesis,promote
R15477 T23136 T23134 themeOf synthesis,increase
R15478 T23138 T23137 themeOf production,mediating
R15479 T23139 T23140 themeOf synthesis,activation
R15480 T23143 T23144 themeOf production,increased
R18022 T26332 T26333 themeOf FADD,deficient
R19680 T28379 T28380 themeOf Myr-Akt,immunoprecipitated
R20189 T29136 T29142 themeOf TNFα,induced
R20190 T29138 T29143 themeOf Akt1,transfected
R20191 T29139 T29143 themeOf Akt2,transfected
R20568 T29671 T29677 themeOf RIP1,phosphorylation
R20569 T29677 T29676 themeOf phosphorylation,kinase-dependent
R21359 T30905 T30914 themeOf TNFα,synthesis
R21360 T30909 T30915 themeOf TNFα,induced
R21361 T30914 T30913 themeOf synthesis,autocrine
R22236 T32177 T32191 themeOf TNFα,production
R22237 T32178 T32192 themeOf FADD,deficient
R22238 T32185 T32193 themeOf Akt2,expressing
R22239 T32188 T32193 themeOf Akt1,expressing
R22240 T32191 T32189 themeOf production,contributes
R22241 T32191 T32190 themeOf production,autocrine
R22327 T32437 T32438 themeOf TNFα,synthesis
R22328 T32438 T32439 themeOf synthesis,leads
R2745 T4050 T4077 themeOf caspase-8,inhibition
R2746 T4051 T4078 themeOf caspase-8,loss
R2747 T4064 T4079 themeOf RIP1,inhibitor
R2748 T4072 T4080 themeOf bFGF receptor tyrosine kinase,inhibitors
R2749 T4074 T4081 themeOf bFGF receptor,inhibitor
R2750 T4075 T4082 themeOf TNFα,induced
R4478 T6660 T6694 themeOf RIP1,"kinase dependent activation of JNK and TNFα production has recently been described to be independent of its role in necroptosis [15]. Curiously, Akt kinase, a key pro-survival molecule and a well-established inhibitor of apoptotic cell death, has also recently been linked to necroptosis in L929 cells [16], where insulin-dependent activation of Akt was suggested to promote necroptosis by suppressing autophagy. This conclusion was unexpected, since several reports from different groups, including ours, have established that autophagy promotes, rather than suppresses, zVAD.fmk-induced necroptosis in L929 cells [11], [14], [17]. This raised the possibility that Akt controls more general mechanisms that contribute to the execution of necroptosis. Furthermore, the key question of whether insulin-dependent Akt activity solely provides an environment conducive for necroptosis or if Akt activation is an intrinsic component of necroptosis signaling that is linked to RIP1 kinase has not been explored. In this study, we expanded these observations to delineate the specific contributions and molecular ordering of the Akt and JNK pathways downstream from RIP1 kinase during necroptosis. Our data reveal that Akt is activated through RIP1 kinase-dependent Thr308 phosphorylation during necroptosis in multiple cell types. Furthermore, we found that downstream Akt signaling through mTORC1 and S6 contributes to the activation of necroptosis and TNFα production. We found that the Akt pathway serves as a critical link between RIP1 kinase and JNK activation in L929 cells. Further data suggested that in multiple other cell types including FADD deficient Jurkat cells, RAW and J774.1 macrophage cell lines, and mouse lung fibroblasts Akt provides a key link to TNFα production, but is dispensible for cell death per se. Overall, our results reveal a specific and novel role for the Akt pathway in regulated necrosis and necrosis-associated inflammatory signaling. Results Basic Fibroblast Growth Factor Promotes Necroptosis in L929 Cells It has been established that mouse fibrosarcoma L929 cells undergo necroptotic cell death following stimulation with TNFα [10], [17]. In addition, inhibition of caspase-8 activity alone, either through siRNA knockdown or by using the pan-caspase inhibitor, zVAD.fmk, is sufficient to trigger necroptosis in these cells [10], [14]. Interestingly, while necroptosis was initially identified as a back-up form of cell death triggered by pro-apoptotic stimuli in the presence of apoptosis inhibitors [17], recent analysis of physiological cell death during mouse development has suggested that the loss of apoptotic regulators, such as caspase-8 and FADD [18], [19], [20], leads to robust induction of necroptosis and death of E10.5 embryos even though apoptosis is not normally induced in wild type embryos. These data are reminiscent of the observations in L929 cells where the loss of caspase activity in healthy cells is sufficient to trigger necroptosis and prompted us to explore the extrinsic or intrinsic cellular factors that promote necroptosis once caspase-8 activity, which cleaves and inactivates RIP1 kinase and the RIP1 deubiquitinase CYLD [21], [22], is removed in L929 cells. Consistent with a previous report [16], we found that serum starvation of L929 cells prevented necroptosis in response to zVAD.fmk (Fig. 1A). The addition of growth factors, such as bFGF, restored zVAD.fmk induced death under serum free conditions (Fig. 1B). Interestingly, this does not reflect a generic requirement for growth factor signaling, as only some growth factors (bFGF and IGF-1, but not EGF and PDGF) promoted death (Fig. 1B). Furthermore, growth factor-dependent necroptosis required the inhibition of caspase activity, as bFGF alone did not induce cell death (Fig. 1C). In contrast, TNFα triggered necroptosis equally efficiently in the absence of serum (Fig. 1A), suggesting that either growth factors and zVAD.fmk or TNFα are required for necroptotic death in L929 cells. 10.1371/journal.pone.0056576.g001 Figure 1 bFGF and IGF-1 promote necroptosis in concert with zVAD.fmk. (A) L929 cells were treated with TNFα or zVAD.fmk under normal serum (10% FBS) or serum free conditions. Cell viability was determined after 24 hr using the CellTiter-Glo Viability assay. The concentrations of all necroptosis-inducing agents are listed in the Materials and Methods section or indicated in the figures. (B) Cells were treated with zVAD.fmk, the indicated growth factors, and Nec-1 under serum free conditions for 24 hrs followed by measurement of cell viability. (C) Cells under serum free conditions were treated with FGF, zVAD.fmk, or both for 24 hrs followed by viability assay. (D) Cell death was induced by zVAD.fmk or TNFα under full serum condition in the presence of 2 µM PD173074 and 20 µM PD166866. In all graphs, average±SD was plotted. Previously we described the development of 7-Cl-O-Nec-1 (Nec-1) as a potent and selective inhibitor of RIP1 kinase and necroptosis (Fig. S1A) [23], [24]. Recently, its selectivity has been further validated against a panel of more than 400 human kinases [15]. This inhibitor efficiently blocked growth factor/zVAD.fmk-induced necroptosis under serum free conditions in L929 cells and both zVAD.fmk and TNFα-induced necroptosis under full serum conditions (Fig. 1B, S1B). To further validate the role of RIP1, we used an inactive analog, 7-Cl-O-Nec-1i (Nec-1i) (Fig. S1A), which contains an extra N-methyl group that leads to almost complete loss of RIP1 kinase inhibitory activity in vitro [23]. Nec-1i was unable to protect L929 cell death under serum condtions treated with zVAD.fmk or TNFα (Fig. S1B) or serum free conditions treated with bFGF/zVAD.fmk (Fig. S1C). This confirms that RIP1 kinase is responsible for necroptosis in L929 cells under both serum and serum free conditions. We next examined whether bFGF contributes to zVAD.fmk-induced necroptosis under normal serum conditions (10% FBS). We used two bFGF receptor tyrosine kinase inhibitors (PD173074 and PD166866), and determined that inhibition of bFGF signaling strongly inhibited zVAD.fmk-induced necroptosis under normal serum conditions (Fig. 1D). In contrast, neither bFGF receptor inhibitor was able to attenuate TNFα-induced necroptosis (Fig. 1D), consistent with growth factors being dispensable for this pathway (Fig. 1A). Overall, these data suggest that the induction of necroptosis by zVAD.fmk is promoted by bFGF under both serum and serum free conditions. The induction of necroptosis, however, is not a simple consequence of growth factor signaling since not all growth factors allowed death to occur. Instead, specific signaling events mediated by particular growth factors appear to contribute to necroptotic death. RIP1 Kinase-dependent"
R4479 T6665 T6695 themeOf TNFα,induced
R4480 T6666 T6697 themeOf Akt1,knockdown
R4481 T6667 T6697 themeOf Akt2,knockdown
R4482 T6668 T6696 themeOf TNFα,induced
R4483 T6669 T6698 themeOf Akt3,expression
R4484 T6673 T6699 themeOf p46,phosphorylation
R4485 T6674 T6699 themeOf p54,phosphorylation
R4486 T6675 T6699 themeOf c-Jun,phosphorylation
R4487 T6676 T6702 themeOf RIP1,inhibitor
R4488 T6677 T6704 themeOf Akt,phosphorylation
R4489 T6679 T6705 themeOf c-Jun,changes
R4490 T6680 T6706 themeOf c-Jun,increase
R4491 T6690 T6708 themeOf bFGF,increase
R4492 T6699 T6700 themeOf phosphorylation,increase
R4493 T6699 T6700 themeOf phosphorylation,increase
R4494 T6699 T6700 themeOf phosphorylation,increase
R4495 T6701 T6703 themeOf increase,prevented
R4496 T6702 T6703 causeOf inhibitor,prevented
R4497 T6704 T6701 themeOf phosphorylation,increase
R4498 T6708 T6707 themeOf increase,blocked
R469 T441 T451 themeOf TNFα,Synthesis
R470 T442 T453 themeOf RIP1,Activation
R471 T445 T454 themeOf TNFα,production
R472 T450 T455 themeOf TNFα,production
R473 T451 T452 themeOf Synthesis,Regulates
R5193 T7931 T7940 themeOf TNFα,induced
R5194 T7936 T7941 themeOf c-Jun,phosphorylation
R5195 T7941 T7942 themeOf phosphorylation,increase
R5748 T8727 T8734 themeOf RIP1,increase
R5749 T8734 T8732 themeOf increase,contributes
R5750 T8734 T8733 themeOf increase,kinase-dependent
R6777 T10459 T10501 themeOf double minute 2,phosphorylation
R6778 T10460 T10501 themeOf MDM2,phosphorylation
R6779 T10461 T10501 themeOf p70 ribosomal protein S6 Kinase,phosphorylation
R6780 T10462 T10501 themeOf p70S6K,phosphorylation
R6781 T10463 T10501 themeOf S6,phosphorylation
R6782 T10472 T10503 themeOf pFoxO1,increase
R6783 T10474 T10504 themeOf Raptor,inhibitor
R6784 T10475 T10505 themeOf TNFα,induced
R6785 T10476 T10506 themeOf p70S6 kinase,activation
R6786 T10478 T10507 themeOf p70S6K,inhibitor
R6787 T10479 T10509 themeOf S6,phosphorylation
R6788 T10480 T10510 themeOf S6,knockdown
R6789 T10484 T10511 themeOf TNFα,upregulation
R6790 T10485 T10512 themeOf TNFα,synthesis
R6791 T10486 T10515 themeOf TNFα,synthesis
R6792 T10489 T10516 themeOf TNFα,increased
R6793 T10490 T10516 themeOf RIP1,increased
R6794 T10492 T10517 themeOf TNFα,induction
R6795 T10493 T10523 themeOf TNFα,upregulation
R6796 T10494 T10520 themeOf TNFα,synthesis
R6797 T10495 T10526 themeOf TNFα,upregulation
R6798 T10496 T10525 themeOf TNFα,reduced
R6799 T10497 T10529 themeOf RIP1,inhibitors
R6800 T10498 T10528 themeOf TNFα,effect
R6801 T10500 T10527 themeOf TNFα,synthesis
R6802 T10501 T10502 themeOf phosphorylation,increase
R6803 T10501 T10502 themeOf phosphorylation,increase
R6804 T10501 T10502 themeOf phosphorylation,increase
R6805 T10501 T10502 themeOf phosphorylation,increase
R6806 T10501 T10502 themeOf phosphorylation,increase
R6807 T10509 T10508 themeOf phosphorylation,block
R6808 T10512 T10513 themeOf synthesis,increased
R6809 T10515 T10514 themeOf synthesis,contribute
R6810 T10517 T10518 themeOf induction,triggered
R6811 T10520 T10522 themeOf synthesis,increase
R6812 T10520 T10519 themeOf synthesis,autocrine
R6813 T10523 T10521 themeOf upregulation,increased
R6814 T10526 T10524 themeOf upregulation,contribute
R6815 T10527 T10530 themeOf synthesis,increase
R8272 T12467 T12479 themeOf TNFα,synthesis
R8273 T12468 T12481 themeOf TNFα,increase
R8274 T12469 T12484 themeOf Akt1,inhibition
R8275 T12469 T12485 themeOf Akt1,Knockdown
R8276 T12470 T12485 themeOf Akt2,Knockdown
R8277 T12471 T12483 themeOf c-Jun,phosphorylation
R8278 T12472 T12486 themeOf RIP1,activation
R8279 T12473 T12490 themeOf bFGF,increase
R8280 T12474 T12487 themeOf c-Jun,phosphorylation
R8281 T12475 T12494 themeOf c-Jun,phosphorylation
R8282 T12476 T12492 themeOf p70S6K,Knockdown
R8283 T12479 T12480 themeOf synthesis,regulator
R8284 T12483 T12482 themeOf phosphorylation,suppressed
R8285 T12483 T12482 themeOf phosphorylation,suppressed
R8286 T12483 T12482 themeOf phosphorylation,suppressed
R8287 T12484 T12485 themeOf inhibition,Knockdown
R8288 T12485 T12482 causeOf Knockdown,suppressed
R8289 T12485 T12482 causeOf Knockdown,suppressed
R8290 T12485 T12482 causeOf Knockdown,suppressed
R8291 T12487 T12488 themeOf phosphorylation,induced
R8292 T12490 T12489 themeOf increase,inhibited
R8293 T12491 T12493 themeOf increase,attenuated
R8294 T12494 T12491 themeOf phosphorylation,increase
R8801 T13255 T13261 themeOf 3-phosphoinositide dependent protein kinase-1,phosphorylated
R8802 T13256 T13261 themeOf PDK1,phosphorylated
R8803 T13257 T13262 themeOf PDK1,dependent
R8804 T13258 T13263 themeOf PDK1,Inhibition
R8805 T13259 T13264 themeOf PDK1,knockdown