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T94 0-118 Sentence denotes IKKβ phosphorylation regulates RPS3 nuclear translocation and NF-κB function during Escherichia coli O157:H7 infection
T1 0-118 Sentence denotes IKKβ phosphorylation regulates RPS3 nuclear translocation and NF-κB function during Escherichia coli O157:H7 infection
T2 121-129 Sentence denotes Abstract
T95 130-275 Sentence denotes NF-κB is a major gene regulator in immune responses and ribosomal protein S3 (RPS3) is an NF-κB subunit that directs specific gene transcription.
T3 130-275 Sentence denotes NF-κB is a major gene regulator in immune responses and ribosomal protein S3 (RPS3) is an NF-κB subunit that directs specific gene transcription.
T96 276-343 Sentence denotes However, it is unknown how RPS3 nuclear translocation is regulated.
T4 276-343 Sentence denotes However, it is unknown how RPS3 nuclear translocation is regulated.
T97 344-482 Sentence denotes Here we report that IKKβ phosphorylation of serine 209 (S209) was crucial for RPS3 nuclear localization in response to activating stimuli.
T5 344-482 Sentence denotes Here we report that IKKβ phosphorylation of serine 209 (S209) was crucial for RPS3 nuclear localization in response to activating stimuli.
T98 483-760 Sentence denotes Moreover, the foodborne pathogen Escherichia coli O157:H7 virulence protein NleH1 specifically inhibited RPS3 S209 phosphorylation and blocked RPS3 function, thereby promoting bacterial colonization and diarrhea but decreasing mortality in a gnotobiotic piglet infection model.
T6 483-760 Sentence denotes Moreover, the foodborne pathogen Escherichia coli O157:H7 virulence protein NleH1 specifically inhibited RPS3 S209 phosphorylation and blocked RPS3 function, thereby promoting bacterial colonization and diarrhea but decreasing mortality in a gnotobiotic piglet infection model.
T99 761-937 Sentence denotes Thus, the IKKβ-dependent modification of a specific amino acid in RPS3 promotes specific NF-κB functions that underlie the molecular pathogenetic mechanisms of E. coli O157:H7.
T7 761-937 Sentence denotes Thus, the IKKβ-dependent modification of a specific amino acid in RPS3 promotes specific NF-κB functions that underlie the molecular pathogenetic mechanisms of E. coli O157:H7.
T779 939-1134 Sentence denotes Nuclear Factor-kappa B (NF-κB) regulates crucial cellular functions and diverse stimuli activate this pleiotropic transcription factor, which in turn regulates a vast array of genetic targets1-3.
T8 939-1134 Sentence denotes Nuclear Factor-kappa B (NF-κB) regulates crucial cellular functions and diverse stimuli activate this pleiotropic transcription factor, which in turn regulates a vast array of genetic targets1-3.
T780 1135-1247 Sentence denotes The best-known mammalian NF-κB subunits are Rel proteins, including RelA (p65), RelB, c-Rel, p50, and p52 (refs.
T9 1135-1247 Sentence denotes The best-known mammalian NF-κB subunits are Rel proteins, including RelA (p65), RelB, c-Rel, p50, and p52 (refs.
T781 1248-1254 Sentence denotes 4, 5).
T10 1248-1254 Sentence denotes 4, 5).
T782 1255-1382 Sentence denotes However, we recently demonstrated that ribosomal protein S3 (RPS3) is a key non-Rel subunit of certain native NF-κB complexes6.
T11 1255-1382 Sentence denotes However, we recently demonstrated that ribosomal protein S3 (RPS3) is a key non-Rel subunit of certain native NF-κB complexes6.
T783 1383-1563 Sentence denotes RPS3 is defined as a “specifier” subunit of NF-κB, because it facilitates high affinity DNA binding thus determining the regulatory specificity of NF-κB for selected target genes7.
T12 1383-1563 Sentence denotes RPS3 is defined as a “specifier” subunit of NF-κB, because it facilitates high affinity DNA binding thus determining the regulatory specificity of NF-κB for selected target genes7.
T784 1564-1822 Sentence denotes RPS3 regulation of NF-κB governs key physiological processes, including immunoglobulin κ light chain gene expression and receptor editing in B cells6, 8, cytokine production in T cells6, and in host defense against enterohemorrhagic Escherichia coli (EHEC)9.
T13 1564-1822 Sentence denotes RPS3 regulation of NF-κB governs key physiological processes, including immunoglobulin κ light chain gene expression and receptor editing in B cells6, 8, cytokine production in T cells6, and in host defense against enterohemorrhagic Escherichia coli (EHEC)9.
T785 1823-2044 Sentence denotes In particular, the E. coli O157:H7 type III secretion system (T3SS) effector protein NleH1 selectively blocks NF-κB target gene transcription by attenuating RPS3 nuclear translocation, without affecting p65 localization9.
T14 1823-2044 Sentence denotes In particular, the E. coli O157:H7 type III secretion system (T3SS) effector protein NleH1 selectively blocks NF-κB target gene transcription by attenuating RPS3 nuclear translocation, without affecting p65 localization9.
T15 2045-2141 Sentence denotes Nonetheless, how specific NF-κB activating signals induce RPS3 nuclear translocation is unknown.
T786 2045-2211 Sentence denotes Nonetheless, how specific NF-κB activating signals induce RPS3 nuclear translocation is unknown. Extra-ribosomal functions have been ascribed to ribosomal proteins10.
T16 2142-2211 Sentence denotes Extra-ribosomal functions have been ascribed to ribosomal proteins10.
T787 2212-2337 Sentence denotes Besides binding RNA within the 40S ribosomal subunit, RPS3 participates in transcription6, DNA repair11, 12, and apoptosis13.
T17 2212-2337 Sentence denotes Besides binding RNA within the 40S ribosomal subunit, RPS3 participates in transcription6, DNA repair11, 12, and apoptosis13.
T788 2338-2404 Sentence denotes Whether or not RPS3 is phosphorylated had been controversial14-18.
T18 2338-2404 Sentence denotes Whether or not RPS3 is phosphorylated had been controversial14-18.
T789 2405-2593 Sentence denotes Since kinase cascades play a critical role in NF-κB regulation, we tested whether RPS3 is phosphorylated in the context of NF-κB activation and sought to identify the responsible kinase19.
T19 2405-2593 Sentence denotes Since kinase cascades play a critical role in NF-κB regulation, we tested whether RPS3 is phosphorylated in the context of NF-κB activation and sought to identify the responsible kinase19.
T20 2594-2704 Sentence denotes Additionally, we aimed to define a regulatory role for the C-terminal tail of RPS3 whose function was unknown.
T790 2594-2809 Sentence denotes Additionally, we aimed to define a regulatory role for the C-terminal tail of RPS3 whose function was unknown. Here we show that the Inhibitor of κB (IκB) kinase beta (IKKβ) phosphorylated RPS3 at serine 209 (S209).
T21 2705-2809 Sentence denotes Here we show that the Inhibitor of κB (IκB) kinase beta (IKKβ) phosphorylated RPS3 at serine 209 (S209).
T791 2810-2954 Sentence denotes RPS3 S209 phosphorylation enhanced its association with importin-α, mediating RPS3 entry into the karyopherin pathway for nuclear translocation.
T22 2810-2954 Sentence denotes RPS3 S209 phosphorylation enhanced its association with importin-α, mediating RPS3 entry into the karyopherin pathway for nuclear translocation.
T792 2955-3115 Sentence denotes Furthermore, the E. coli NleH1 effector specifically inhibited RPS3 S209 revealing how E. coli O157:H7 inhibits this important innate immune response mechanism.
T23 2955-3115 Sentence denotes Furthermore, the E. coli NleH1 effector specifically inhibited RPS3 S209 revealing how E. coli O157:H7 inhibits this important innate immune response mechanism.
T24 3117-3124 Sentence denotes Results
T2011 3126-3178 Sentence denotes RPS3 phosphorylation in response to NF-κB activation
T25 3126-3178 Sentence denotes RPS3 phosphorylation in response to NF-κB activation
T2012 3179-3338 Sentence denotes To test whether RPS3 is phosphorylated during NF-κB activation, we performed 32P-labeling experiments in tumor necrosis factor (TNF)-stimulated HEK 293T cells.
T26 3179-3338 Sentence denotes To test whether RPS3 is phosphorylated during NF-κB activation, we performed 32P-labeling experiments in tumor necrosis factor (TNF)-stimulated HEK 293T cells.
T2013 3339-3520 Sentence denotes While RPS3 was scarcely phosphorylated in unstimulated cells, we observed a marked increase in 32P-incorporation after TNF stimulation despite no increase in RPS3 protein (Fig. 1a).
T27 3339-3520 Sentence denotes While RPS3 was scarcely phosphorylated in unstimulated cells, we observed a marked increase in 32P-incorporation after TNF stimulation despite no increase in RPS3 protein (Fig. 1a).
T2014 3521-3712 Sentence denotes To determine which RPS3 residues were phosphorylated, we immunoprecipitated RPS3 from either resting or stimulated cells and performed immunoblotting with phosphorylation-specific antibodies.
T28 3521-3712 Sentence denotes To determine which RPS3 residues were phosphorylated, we immunoprecipitated RPS3 from either resting or stimulated cells and performed immunoblotting with phosphorylation-specific antibodies.
T2015 3713-3966 Sentence denotes Both TNF and phorbol myristate acetate/ionomycin (PMA+I) stimulated rapid phosphorylation and degradaion of IκBα within 5 min which was accompanied by RPS3 phosphorylation on serine residues (Fig. 1b and data not shown), similar to the in vivo labeling.
T29 3713-3966 Sentence denotes Both TNF and phorbol myristate acetate/ionomycin (PMA+I) stimulated rapid phosphorylation and degradaion of IκBα within 5 min which was accompanied by RPS3 phosphorylation on serine residues (Fig. 1b and data not shown), similar to the in vivo labeling.
T2016 3967-4042 Sentence denotes We did not detect tyrosine- or threonine-phosphorylation of RPS3 (Fig. 1b).
T30 3967-4042 Sentence denotes We did not detect tyrosine- or threonine-phosphorylation of RPS3 (Fig. 1b).
T2634 4044-4069 Sentence denotes RPS3 and IKKβ interaction
T31 4044-4069 Sentence denotes RPS3 and IKKβ interaction
T2635 4070-4310 Sentence denotes The activation of the inhibitor of κB kinase (IKK), consisting of a regulatory subunit IKKγ and two catalytic subunits, IKKα and IKKβ, is critical for the phosphorylation and dispatch of the inhibitory IκBs and the liberation of NF-κB20-22.
T32 4070-4310 Sentence denotes The activation of the inhibitor of κB kinase (IKK), consisting of a regulatory subunit IKKγ and two catalytic subunits, IKKα and IKKβ, is critical for the phosphorylation and dispatch of the inhibitory IκBs and the liberation of NF-κB20-22.
T2636 4311-4488 Sentence denotes Given that RPS3 can be found in the cytoplasmic p65-p50-IκBα inhibitory complex in resting cells6, we hypothesized that activated IKKβ might also bind to and phosphorylate RPS3.
T33 4311-4488 Sentence denotes Given that RPS3 can be found in the cytoplasmic p65-p50-IκBα inhibitory complex in resting cells6, we hypothesized that activated IKKβ might also bind to and phosphorylate RPS3.
T2637 4489-4567 Sentence denotes First, we found that ectopically expressed IKKβ and RPS3 interacted (Fig. 1c).
T34 4489-4567 Sentence denotes First, we found that ectopically expressed IKKβ and RPS3 interacted (Fig. 1c).
T2638 4568-4774 Sentence denotes We next examined resting Jurkat cells and detected a modest endogenous IKKβ-RPS3 interaction (Fig. 1d), potentially accounting for the basal NF-κB transcription required for cell proliferation and survival.
T35 4568-4774 Sentence denotes We next examined resting Jurkat cells and detected a modest endogenous IKKβ-RPS3 interaction (Fig. 1d), potentially accounting for the basal NF-κB transcription required for cell proliferation and survival.
T2639 4775-4939 Sentence denotes RPS3-IKKβ association was clearly augmented upon TNF stimulation, peaking at 10 min. (Fig. 1d), following similar kinetics to RPS3 serine phosphorylation (Fig. 1b).
T36 4775-4939 Sentence denotes RPS3-IKKβ association was clearly augmented upon TNF stimulation, peaking at 10 min. (Fig. 1d), following similar kinetics to RPS3 serine phosphorylation (Fig. 1b).
T2640 4940-5021 Sentence denotes By contrast, there was no detectable interaction between RPS3 and IKKα (Fig. 1d).
T37 4940-5021 Sentence denotes By contrast, there was no detectable interaction between RPS3 and IKKα (Fig. 1d).
T3532 5023-5070 Sentence denotes IKKβ is required for RPS3 nuclear translocation
T38 5023-5070 Sentence denotes IKKβ is required for RPS3 nuclear translocation
T3533 5071-5319 Sentence denotes To examine whether the RPS3-IKKβ interaction is required for RPS3 nuclear translocation, we knocked down IKKα or IKKβ expression with siRNAs (Supplementary Fig. 1) and then observed stimulation-induced RPS3 nuclear migration by confocal microscopy.
T39 5071-5319 Sentence denotes To examine whether the RPS3-IKKβ interaction is required for RPS3 nuclear translocation, we knocked down IKKα or IKKβ expression with siRNAs (Supplementary Fig. 1) and then observed stimulation-induced RPS3 nuclear migration by confocal microscopy.
T3534 5320-5455 Sentence denotes Both TNF and PMA+I triggered RPS3 nuclear translocation in Jurkat cells transfected with a scrambled nonspecific (NS) siRNA (Fig. 2a)6.
T40 5320-5455 Sentence denotes Both TNF and PMA+I triggered RPS3 nuclear translocation in Jurkat cells transfected with a scrambled nonspecific (NS) siRNA (Fig. 2a)6.
T3535 5456-5540 Sentence denotes RPS3 nuclear translocation was only slightly, if at all, impaired by IKKα-silencing.
T41 5456-5540 Sentence denotes RPS3 nuclear translocation was only slightly, if at all, impaired by IKKα-silencing.
T3536 5541-5650 Sentence denotes Conversely, knockdown of IKKβ attenuated 60-70% of RPS3 nuclear accumulation following stimulation (Fig. 2a).
T42 5541-5650 Sentence denotes Conversely, knockdown of IKKβ attenuated 60-70% of RPS3 nuclear accumulation following stimulation (Fig. 2a).
T3537 5651-5815 Sentence denotes Immunoblotting of nuclear fractions confirmed that full expression of IKKβ, but not IKKα, was necessary for activation-induced RPS3 nuclear translocation (Fig. 2b).
T43 5651-5815 Sentence denotes Immunoblotting of nuclear fractions confirmed that full expression of IKKβ, but not IKKα, was necessary for activation-induced RPS3 nuclear translocation (Fig. 2b).
T3538 5816-5924 Sentence denotes Control immunoblots revealed that p65 nuclear translocation was blocked under the same conditions (Fig. 2b).
T44 5816-5924 Sentence denotes Control immunoblots revealed that p65 nuclear translocation was blocked under the same conditions (Fig. 2b).
T3539 5925-6087 Sentence denotes We next examined the nuclear translocation of RPS3 in cells ectopically expressing either kinase-dead (SSAA) or constitutively-active (SSEE) mutant IKKβ proteins.
T45 5925-6087 Sentence denotes We next examined the nuclear translocation of RPS3 in cells ectopically expressing either kinase-dead (SSAA) or constitutively-active (SSEE) mutant IKKβ proteins.
T3540 6088-6209 Sentence denotes As expected, the SSEE, but not SSAA, mutant of IKKβ induced NF-κB-dependent luciferase reporter activity (Fig. 2c, left).
T46 6088-6209 Sentence denotes As expected, the SSEE, but not SSAA, mutant of IKKβ induced NF-κB-dependent luciferase reporter activity (Fig. 2c, left).
T3541 6210-6402 Sentence denotes Whereas RPS3 remained cytosolic in IKKβ (SSAA)-expressing cells (Fig. 2c, right), a substantial proportion of RPS3 translocated to the nucleus in cells expressing IKKβ (SSEE) (Fig. 2c, right).
T47 6210-6402 Sentence denotes Whereas RPS3 remained cytosolic in IKKβ (SSAA)-expressing cells (Fig. 2c, right), a substantial proportion of RPS3 translocated to the nucleus in cells expressing IKKβ (SSEE) (Fig. 2c, right).
T3542 6403-6586 Sentence denotes The percentage of cells containing detectable nuclear RPS3 increased 5-fold in IKKβ (SSEE)-expressing cells, but not in IKKβ (SSAA)-expressing ones (Fig. 2d and Supplementary Fig. 2).
T48 6403-6586 Sentence denotes The percentage of cells containing detectable nuclear RPS3 increased 5-fold in IKKβ (SSEE)-expressing cells, but not in IKKβ (SSAA)-expressing ones (Fig. 2d and Supplementary Fig. 2).
T3543 6587-6706 Sentence denotes Thus, IKKβ activity is necessary and sufficient for RPS3 nuclear translocation in response to NF-κB activating stimuli.
T49 6587-6706 Sentence denotes Thus, IKKβ activity is necessary and sufficient for RPS3 nuclear translocation in response to NF-κB activating stimuli.
T5142 6708-6755 Sentence denotes IκBα degradation and RPS3 nuclear translocation
T50 6708-6755 Sentence denotes IκBα degradation and RPS3 nuclear translocation
T5143 6756-6824 Sentence denotes Importin-α regulates the nuclear import of NF-κB Rel subunits23, 24.
T51 6756-6824 Sentence denotes Importin-α regulates the nuclear import of NF-κB Rel subunits23, 24.
T5144 6825-6977 Sentence denotes RPS3 harbors a nuclear localization signal (NLS) sequence and its nuclear translocation occurs in parallel to, but independently of, p65 translocation6.
T52 6825-6977 Sentence denotes RPS3 harbors a nuclear localization signal (NLS) sequence and its nuclear translocation occurs in parallel to, but independently of, p65 translocation6.
T5145 6978-7046 Sentence denotes We envisioned that RPS3 could also utilize the importin-α/β pathway.
T53 6978-7046 Sentence denotes We envisioned that RPS3 could also utilize the importin-α/β pathway.
T5146 7047-7177 Sentence denotes Consistent with this notion, RPS3 association with importin-α, but not importin-β, was enhanced in TNF–stimulated cells (Fig. 3a).
T54 7047-7177 Sentence denotes Consistent with this notion, RPS3 association with importin-α, but not importin-β, was enhanced in TNF–stimulated cells (Fig. 3a).
T5147 7178-7299 Sentence denotes Therefore, we examined whether RPS3 binding to importin-α is essential for nuclear translocation during NF-κB activation.
T55 7178-7299 Sentence denotes Therefore, we examined whether RPS3 binding to importin-α is essential for nuclear translocation during NF-κB activation.
T5148 7300-7515 Sentence denotes Since IκBα degradation is a prerequisite to unmask the NLS of p65, and both RPS3 and IκBα bind to p65 in the cytoplasmic inhibitory complex, we tested whether IκBα degradation is required for the liberation of RPS3.
T56 7300-7515 Sentence denotes Since IκBα degradation is a prerequisite to unmask the NLS of p65, and both RPS3 and IκBα bind to p65 in the cytoplasmic inhibitory complex, we tested whether IκBα degradation is required for the liberation of RPS3.
T5149 7516-7695 Sentence denotes We measured the association of RPS3 with importin-α in 293T cells overexpressing wild-type IκBα or an IκBα mutant (SSAA) resistant to IKKβ-induced phosphorylation and degradation.
T57 7516-7695 Sentence denotes We measured the association of RPS3 with importin-α in 293T cells overexpressing wild-type IκBα or an IκBα mutant (SSAA) resistant to IKKβ-induced phosphorylation and degradation.
T5150 7696-7851 Sentence denotes In cells transfected with wild-type IκBα, TNF stimulation augmented the interaction of RPS3 and importin-α to a similar degree as in non-transfected cells.
T58 7696-7851 Sentence denotes In cells transfected with wild-type IκBα, TNF stimulation augmented the interaction of RPS3 and importin-α to a similar degree as in non-transfected cells.
T5151 7852-7977 Sentence denotes By contrast, we observed that the RPS3-importin-α association was abolished by the presence of non-degradable IκBα (Fig. 3b).
T59 7852-7977 Sentence denotes By contrast, we observed that the RPS3-importin-α association was abolished by the presence of non-degradable IκBα (Fig. 3b).
T5152 7978-8166 Sentence denotes To examine whether IκBα is the only cytoplasmic barrier precluding RPS3 nuclear translocation, we measured both RPS3-importin-α association and nuclear RPS3 after reducing IκBα expression.
T60 7978-8166 Sentence denotes To examine whether IκBα is the only cytoplasmic barrier precluding RPS3 nuclear translocation, we measured both RPS3-importin-α association and nuclear RPS3 after reducing IκBα expression.
T5153 8167-8282 Sentence denotes Compared with nonspecific siRNA, siRNA targeting of IκBα completely depleted IκBα in Jurkat cells (Fig. 3c, input).
T61 8167-8282 Sentence denotes Compared with nonspecific siRNA, siRNA targeting of IκBα completely depleted IκBα in Jurkat cells (Fig. 3c, input).
T5154 8283-8410 Sentence denotes Nevertheless, the RPS3-importin-α association was not augmented (Fig. 3c), nor was significant nuclear RPS3 detected (Fig. 3d).
T62 8283-8410 Sentence denotes Nevertheless, the RPS3-importin-α association was not augmented (Fig. 3c), nor was significant nuclear RPS3 detected (Fig. 3d).
T5155 8411-8723 Sentence denotes Moreover, cells treated with sodium pervanadate (Pv) to induce IκBα degradation through an IKK-independent mechanism25-27 did not show increased association between RPS3 and importin-α (Fig. 3e and Supplementary Fig. 3b) or nuclear accumulation of RPS3, despite complete IκBα degradation (Supplementary Fig. 3c).
T63 8411-8723 Sentence denotes Moreover, cells treated with sodium pervanadate (Pv) to induce IκBα degradation through an IKK-independent mechanism25-27 did not show increased association between RPS3 and importin-α (Fig. 3e and Supplementary Fig. 3b) or nuclear accumulation of RPS3, despite complete IκBα degradation (Supplementary Fig. 3c).
T5156 8724-8892 Sentence denotes We further examined whether a subsequent NF-κB activation signal independently promotes the importin-α association and nuclear transport of RPS3 after IκBα degradation.
T64 8724-8892 Sentence denotes We further examined whether a subsequent NF-κB activation signal independently promotes the importin-α association and nuclear transport of RPS3 after IκBα degradation.
T5157 8893-9042 Sentence denotes We found that TNF stimulation following Pv treatment was required for the RPS3-importin-α association, comparable to TNF stimulation alone (Fig. 3e).
T65 8893-9042 Sentence denotes We found that TNF stimulation following Pv treatment was required for the RPS3-importin-α association, comparable to TNF stimulation alone (Fig. 3e).
T5158 9043-9200 Sentence denotes Thus, IκBα phosphorylation and degradation itself is required but not sufficient to cause RPS3 association with importin-α followed by nuclear translocation.
T66 9043-9200 Sentence denotes Thus, IκBα phosphorylation and degradation itself is required but not sufficient to cause RPS3 association with importin-α followed by nuclear translocation.
T5159 9201-9285 Sentence denotes Rather, an additional signal, potentially IKKβ phosphorylation of RPS3, is required.
T67 9201-9285 Sentence denotes Rather, an additional signal, potentially IKKβ phosphorylation of RPS3, is required.
T7141 9287-9325 Sentence denotes IKKβ phosphorylates RPS3 at serine 209
T68 9287-9325 Sentence denotes IKKβ phosphorylates RPS3 at serine 209
T7142 9326-9520 Sentence denotes Although originally defined as the kinase that phosphorylates IκB19, IKKβ also phosphorylates unrelated substrates including 14-3-3β and Bcl10, which lack the IKK consensus motif (DpSGYXpS/T)28.
T69 9326-9520 Sentence denotes Although originally defined as the kinase that phosphorylates IκB19, IKKβ also phosphorylates unrelated substrates including 14-3-3β and Bcl10, which lack the IKK consensus motif (DpSGYXpS/T)28.
T7143 9521-9591 Sentence denotes We therefore hypothesized that IKKβ could directly phosphorylate RPS3.
T70 9521-9591 Sentence denotes We therefore hypothesized that IKKβ could directly phosphorylate RPS3.
T7144 9592-9910 Sentence denotes By in vitro kinase assays using recombinant IKK and RPS3 proteins, we observed strong incorporation of 32P in autophosphorylatd IKKα and IKKβ (Fig. 4a, lanes 2-7) as well as phosporylated GST-IκBα (1-54) (Supplementary Fig. 4), but not the GST protein alone (Fig. 4a, lanes 3 and 6), when either IKKα or IKKβ was used.
T71 9592-9910 Sentence denotes By in vitro kinase assays using recombinant IKK and RPS3 proteins, we observed strong incorporation of 32P in autophosphorylatd IKKα and IKKβ (Fig. 4a, lanes 2-7) as well as phosporylated GST-IκBα (1-54) (Supplementary Fig. 4), but not the GST protein alone (Fig. 4a, lanes 3 and 6), when either IKKα or IKKβ was used.
T7145 9911-10028 Sentence denotes We discovered that GST-RPS3 could be phosphorylated by IKKβ, but not IKKα, in vitro (Fig. 4a, compare lanes 4 and 7).
T72 9911-10028 Sentence denotes We discovered that GST-RPS3 could be phosphorylated by IKKβ, but not IKKα, in vitro (Fig. 4a, compare lanes 4 and 7).
T7146 10029-10200 Sentence denotes To identify the RPS3 amino acid residue(s) phosphorylated by IKKβ, we performed liquid chromatography-tandem mass spectrometry analyses using in vitro phosphorylated RPS3.
T73 10029-10200 Sentence denotes To identify the RPS3 amino acid residue(s) phosphorylated by IKKβ, we performed liquid chromatography-tandem mass spectrometry analyses using in vitro phosphorylated RPS3.
T7147 10201-10295 Sentence denotes The results indicated that IKKβ phosphorylated S209, located in the RPS3 C-terminus (Fig. 4b).
T74 10201-10295 Sentence denotes The results indicated that IKKβ phosphorylated S209, located in the RPS3 C-terminus (Fig. 4b).
T7148 10296-10558 Sentence denotes RPS3 amino acid sequence alignment revealed that S209 is conserved in many species throughout phylogeny with the exception of Caenorhabditis elegans and Schizosaccharomyces pombe, two organisms that do not possess the NF-κB signal pathway (Supplementary Fig. 5).
T75 10296-10558 Sentence denotes RPS3 amino acid sequence alignment revealed that S209 is conserved in many species throughout phylogeny with the exception of Caenorhabditis elegans and Schizosaccharomyces pombe, two organisms that do not possess the NF-κB signal pathway (Supplementary Fig. 5).
T7149 10559-10721 Sentence denotes To verify biochemically that S209 is an IKKβ substrate, we performed 32P-labeling in vitro kinase assays with recombinant wild-type or S209A mutant RPS3 proteins.
T76 10559-10721 Sentence denotes To verify biochemically that S209 is an IKKβ substrate, we performed 32P-labeling in vitro kinase assays with recombinant wild-type or S209A mutant RPS3 proteins.
T7150 10722-10831 Sentence denotes Compared with the wild-type protein, the S209A mutation reduced IKKβ–mediated RPS3 phosphorylation (Fig. 4c).
T77 10722-10831 Sentence denotes Compared with the wild-type protein, the S209A mutation reduced IKKβ–mediated RPS3 phosphorylation (Fig. 4c).
T7151 10832-10958 Sentence denotes There might be alternative phosphorylation site(s) under these conditions given modest residual phosphorylated RPS3 (Fig. 4c).
T78 10832-10958 Sentence denotes There might be alternative phosphorylation site(s) under these conditions given modest residual phosphorylated RPS3 (Fig. 4c).
T7152 10959-11130 Sentence denotes RPS3 S209 does not fall within a conventional IKK recognition motif, but rather resides in a sequence motif (XXXpS/TXXE), potentially recognized by casein kinase II (CK2).
T79 10959-11130 Sentence denotes RPS3 S209 does not fall within a conventional IKK recognition motif, but rather resides in a sequence motif (XXXpS/TXXE), potentially recognized by casein kinase II (CK2).
T7153 11131-11291 Sentence denotes Although IKKβ kinase can display a CK2-like phosphorylation specificity29, no CK2 protein was detectable in our recombinant IKK proteins (Supplementary Fig. 6).
T80 11131-11291 Sentence denotes Although IKKβ kinase can display a CK2-like phosphorylation specificity29, no CK2 protein was detectable in our recombinant IKK proteins (Supplementary Fig. 6).
T7154 11292-11430 Sentence denotes Thus, RPS3 S209 phosphorylation was due to the alternate specificity of the IKKβ kinase rather than any trace amount of CK2 bound to IKKs.
T81 11292-11430 Sentence denotes Thus, RPS3 S209 phosphorylation was due to the alternate specificity of the IKKβ kinase rather than any trace amount of CK2 bound to IKKs.
T7155 11431-11624 Sentence denotes To determine whether S209 is the critical site at which IKKβ phosphorylates RPS3 in living cells, we transfected the wild-type or S209A mutant Flag-RPS3 alone, or together with IKKβ into cells.
T82 11431-11624 Sentence denotes To determine whether S209 is the critical site at which IKKβ phosphorylates RPS3 in living cells, we transfected the wild-type or S209A mutant Flag-RPS3 alone, or together with IKKβ into cells.
T7156 11625-11864 Sentence denotes Indeed, we observed that overexpressing IKKβ enhanced Flag-RPS3 phosphorylation, but phosphorylation was effectively eliminated by alanine substitution indicating that S209 is the predominant target site for IKKβ phosphorylation (Fig. 4d).
T83 11625-11864 Sentence denotes Indeed, we observed that overexpressing IKKβ enhanced Flag-RPS3 phosphorylation, but phosphorylation was effectively eliminated by alanine substitution indicating that S209 is the predominant target site for IKKβ phosphorylation (Fig. 4d).
T7157 11865-12032 Sentence denotes We next generated a phospho-S209 RPS3 antibody and confirmed that endogenous RPS3 was phosphorylated at S209 in a time-dependent manner upon TNF stimulation (Fig. 4e).
T84 11865-12032 Sentence denotes We next generated a phospho-S209 RPS3 antibody and confirmed that endogenous RPS3 was phosphorylated at S209 in a time-dependent manner upon TNF stimulation (Fig. 4e).
T7158 12033-12114 Sentence denotes Thus, the RPS3 C-terminal tail potentially contains an important regulatory site.
T85 12033-12114 Sentence denotes Thus, the RPS3 C-terminal tail potentially contains an important regulatory site.
T9155 12116-12162 Sentence denotes Phosphorylation of RPS3 and its NF-κB function
T86 12116-12162 Sentence denotes Phosphorylation of RPS3 and its NF-κB function
T9156 12163-12283 Sentence denotes We next examined whether S209 phosphorylation plays a role in the nuclear translocation of RPS3 during NF-κB activation.
T87 12163-12283 Sentence denotes We next examined whether S209 phosphorylation plays a role in the nuclear translocation of RPS3 during NF-κB activation.
T9157 12284-12551 Sentence denotes Subcellular fractions from either wild-type or S209A mutant RPS3-transfected cells were prepared and blotted for heat-shock protein 90 (hsp90), a cytoplasmic protein, and poly (ADP-ribose) polymerase (PARP), a nuclear protein, confirming a clean separation (Fig. 5a).
T88 12284-12551 Sentence denotes Subcellular fractions from either wild-type or S209A mutant RPS3-transfected cells were prepared and blotted for heat-shock protein 90 (hsp90), a cytoplasmic protein, and poly (ADP-ribose) polymerase (PARP), a nuclear protein, confirming a clean separation (Fig. 5a).
T9158 12552-12645 Sentence denotes As expected, PMA+I stimulation triggered wild-type Flag-RPS3 nuclear translocation (Fig. 5a).
T89 12552-12645 Sentence denotes As expected, PMA+I stimulation triggered wild-type Flag-RPS3 nuclear translocation (Fig. 5a).
T9159 12646-12715 Sentence denotes However, RPS3 (S209A) nuclear translocation was attenuated (Fig. 5a).
T90 12646-12715 Sentence denotes However, RPS3 (S209A) nuclear translocation was attenuated (Fig. 5a).
T9160 12716-12815 Sentence denotes We also tested the impact of activating NF-κB by overexpressing IKKβ on RPS3 nuclear translocation.
T91 12716-12815 Sentence denotes We also tested the impact of activating NF-κB by overexpressing IKKβ on RPS3 nuclear translocation.
T9161 12816-12993 Sentence denotes IKKβ overexpression activated NF-κB measured by luciferase assays (Supplementary Fig. 7), and also induced the nuclear translocation of wild-type, but not S209A, RPS3 (Fig. 5b).
T92 12816-12993 Sentence denotes IKKβ overexpression activated NF-κB measured by luciferase assays (Supplementary Fig. 7), and also induced the nuclear translocation of wild-type, but not S209A, RPS3 (Fig. 5b).
T9162 12994-13111 Sentence denotes These data suggest that S209 phosphorylation is critical for the NF-κB activation-induced RPS3 nuclear translocation.
T93 12994-13111 Sentence denotes These data suggest that S209 phosphorylation is critical for the NF-κB activation-induced RPS3 nuclear translocation.
T9163 13112-13400 Sentence denotes To examine the role of S209 phosphorylation of RPS3 to its NF-κB function6, 7, 30, we silenced endogenous RPS3 expression using an siRNA that targets the 3′ untranslated region (3′ UTR) of RPS3 mRNA, followed by complementation with either wild-type or S209A mutant RPS3 via transfection.
T94 13112-13400 Sentence denotes To examine the role of S209 phosphorylation of RPS3 to its NF-κB function6, 7, 30, we silenced endogenous RPS3 expression using an siRNA that targets the 3′ untranslated region (3′ UTR) of RPS3 mRNA, followed by complementation with either wild-type or S209A mutant RPS3 via transfection.
T9164 13401-13609 Sentence denotes As expected, RPS3 siRNA severely reduced endogenous RPS3 abundance compared to NS siRNA, but did not affect the robust expression of Flag-tagged RPS3 from a transfected construct lacking the 3′ UTR (Fig. 5c).
T95 13401-13609 Sentence denotes As expected, RPS3 siRNA severely reduced endogenous RPS3 abundance compared to NS siRNA, but did not affect the robust expression of Flag-tagged RPS3 from a transfected construct lacking the 3′ UTR (Fig. 5c).
T9165 13610-13726 Sentence denotes We also found that RPS3 knockdown reduced TNF-induced expression of an Ig κB-driven luciferase construct6 (Fig. 5d).
T96 13610-13726 Sentence denotes We also found that RPS3 knockdown reduced TNF-induced expression of an Ig κB-driven luciferase construct6 (Fig. 5d).
T9166 13727-13908 Sentence denotes The impaired luciferase signal caused by RPS3 deficiency was completely restored by transfecting wild-type, but not by S209A RPS3 (Fig. 5d), despite equivalent expression (Fig. 5c).
T97 13727-13908 Sentence denotes The impaired luciferase signal caused by RPS3 deficiency was completely restored by transfecting wild-type, but not by S209A RPS3 (Fig. 5d), despite equivalent expression (Fig. 5c).
T9167 13909-14179 Sentence denotes Moreover, the failure of S209A RPS3 to restore luciferase activity did not result from defective translation because the transient overexpression of green fluorescent protein (GFP) was comparable in cells complemented with wild-type or S029A RPS3 (Supplementary Fig. 8).
T98 13909-14179 Sentence denotes Moreover, the failure of S209A RPS3 to restore luciferase activity did not result from defective translation because the transient overexpression of green fluorescent protein (GFP) was comparable in cells complemented with wild-type or S029A RPS3 (Supplementary Fig. 8).
T9168 14180-14312 Sentence denotes Taken together, these data suggest that RPS3 S209 phosphorylation is critical for NF-κB activity involving the canonical Ig κB site.
T99 14180-14312 Sentence denotes Taken together, these data suggest that RPS3 S209 phosphorylation is critical for NF-κB activity involving the canonical Ig κB site.
T9169 14313-14496 Sentence denotes We next used chromatin immunoprecipitation to determine whether S209 phosphorylation affects RPS3 and p65 recruitment to specific κB sites in intact chromatin during NF-κB activation.
T100 14313-14496 Sentence denotes We next used chromatin immunoprecipitation to determine whether S209 phosphorylation affects RPS3 and p65 recruitment to specific κB sites in intact chromatin during NF-κB activation.
T9170 14497-14681 Sentence denotes In RPS3 knockdown cells, PMA+I stimulated the recruitment of ectopically expressed, Flag-tagged wild-type, but not S209A RPS3 to the κB sites of the NFKBIA and IL8 promoters (Fig. 5e).
T101 14497-14681 Sentence denotes In RPS3 knockdown cells, PMA+I stimulated the recruitment of ectopically expressed, Flag-tagged wild-type, but not S209A RPS3 to the κB sites of the NFKBIA and IL8 promoters (Fig. 5e).
T9171 14682-14808 Sentence denotes While expressing RPS3 S209A had no impact on p65 nuclear translocation, it substantially attenuated p65 recruitment (Fig. 5e).
T102 14682-14808 Sentence denotes While expressing RPS3 S209A had no impact on p65 nuclear translocation, it substantially attenuated p65 recruitment (Fig. 5e).
T9172 14809-15003 Sentence denotes Additional experiments revealed that p65 attraction to RPS3-independent NF-κB target gene promoters such as CD25 was increased (Supplementary Fig. 9), consistent with our previous observations6.
T103 14809-15003 Sentence denotes Additional experiments revealed that p65 attraction to RPS3-independent NF-κB target gene promoters such as CD25 was increased (Supplementary Fig. 9), consistent with our previous observations6.
T9173 15004-15151 Sentence denotes There was no significant Flag-RPS3 or p65 recruitment to ACTB promoter lacking κB sites (Fig. 5e), suggesting the recruitment was κB site-specific.
T104 15004-15151 Sentence denotes There was no significant Flag-RPS3 or p65 recruitment to ACTB promoter lacking κB sites (Fig. 5e), suggesting the recruitment was κB site-specific.
T9174 15152-15261 Sentence denotes Thus, the recruitment of RPS3 as well as the contingent recruitment of p65 to key promoters depended on S209.
T105 15152-15261 Sentence denotes Thus, the recruitment of RPS3 as well as the contingent recruitment of p65 to key promoters depended on S209.
T9175 15262-15528 Sentence denotes Interleukin 8 (IL-8) secretion induced by either T cell receptor (TCR) agonist stimulation or PMA+I was decreased as a consequence of reduced RPS3/p65 recruitment to the IL8 κB sites in the presence of S209A mutant compared to wild-type RPS3 (Supplementary Fig. 10).
T106 15262-15528 Sentence denotes Interleukin 8 (IL-8) secretion induced by either T cell receptor (TCR) agonist stimulation or PMA+I was decreased as a consequence of reduced RPS3/p65 recruitment to the IL8 κB sites in the presence of S209A mutant compared to wild-type RPS3 (Supplementary Fig. 10).
T9176 15529-15661 Sentence denotes However, cell surface CD25 expression was comparable between the wild-type and S209A RPS3 transfected cells (Supplementary Fig. 11).
T107 15529-15661 Sentence denotes However, cell surface CD25 expression was comparable between the wild-type and S209A RPS3 transfected cells (Supplementary Fig. 11).
T9177 15662-15795 Sentence denotes Therefore, RPS3 S209 phosphorylation by IKKβ is apparently required for RPS3 in directing NF-κB to a specific subset of target genes.
T108 15662-15795 Sentence denotes Therefore, RPS3 S209 phosphorylation by IKKβ is apparently required for RPS3 in directing NF-κB to a specific subset of target genes.
T11937 15797-15841 Sentence denotes NleH1 inhibits RPS3 phosphorylation in vitro
T109 15797-15841 Sentence denotes NleH1 inhibits RPS3 phosphorylation in vitro
T11938 15842-15944 Sentence denotes EHEC pathogens are important causative agents of both foodborne disease and pediatric renal failure31.
T110 15842-15944 Sentence denotes EHEC pathogens are important causative agents of both foodborne disease and pediatric renal failure31.
T11939 15945-16103 Sentence denotes EHEC utilize T3SS to inject effector proteins directly into intestinal epithelial cells32, a subset of which inhibit NF-κB-dependent innate responses9, 33-38.
T111 15945-16103 Sentence denotes EHEC utilize T3SS to inject effector proteins directly into intestinal epithelial cells32, a subset of which inhibit NF-κB-dependent innate responses9, 33-38.
T11940 16104-16253 Sentence denotes The E. coli O157:H7 EDL933 effector protein NleH1 binds to and attenuates RPS3 nuclear translocation, thus impairing RPS3-dependent NF-κB signaling9.
T112 16104-16253 Sentence denotes The E. coli O157:H7 EDL933 effector protein NleH1 binds to and attenuates RPS3 nuclear translocation, thus impairing RPS3-dependent NF-κB signaling9.
T11941 16254-16344 Sentence denotes We therefore hypothesized that NleH1 may function by inhibiting RPS3 S209 phosphorylation.
T113 16254-16344 Sentence denotes We therefore hypothesized that NleH1 may function by inhibiting RPS3 S209 phosphorylation.
T11942 16345-16488 Sentence denotes As expected, transfecting increasing amounts of NleH1-HA plasmid blocked TNFα-induced NF-κB activation in a dose-dependent manner (Fig. 6a-b)9.
T114 16345-16488 Sentence denotes As expected, transfecting increasing amounts of NleH1-HA plasmid blocked TNFα-induced NF-κB activation in a dose-dependent manner (Fig. 6a-b)9.
T11943 16489-16615 Sentence denotes Remarkably, NleH1 reduced both TNF-induced, as well as basal RPS3 phosphorylation to roughly 20% of vehicle control (Fig. 6c).
T115 16489-16615 Sentence denotes Remarkably, NleH1 reduced both TNF-induced, as well as basal RPS3 phosphorylation to roughly 20% of vehicle control (Fig. 6c).
T11944 16616-16793 Sentence denotes Expressing NleH1 does not interfere with either TNF-induced IKK activation or IκBα degradation, consistent with the lack of NleH1 impact on p65 nuclear translocation9 (Fig. 6c).
T116 16616-16793 Sentence denotes Expressing NleH1 does not interfere with either TNF-induced IKK activation or IκBα degradation, consistent with the lack of NleH1 impact on p65 nuclear translocation9 (Fig. 6c).
T11945 16794-17038 Sentence denotes To determine if NleH1 inhibits RPS3 phosphorylation, we infected HeLa cells with E. coli O157:H7 strains possessing or lacking either nleH1 (ΔnleH1) or with a strain lacking a functional T3SS unable to inject NleH1 into mammalian cells (ΔescN).
T117 16794-17038 Sentence denotes To determine if NleH1 inhibits RPS3 phosphorylation, we infected HeLa cells with E. coli O157:H7 strains possessing or lacking either nleH1 (ΔnleH1) or with a strain lacking a functional T3SS unable to inject NleH1 into mammalian cells (ΔescN).
T11946 17039-17166 Sentence denotes In uninfected cells, TNF-treatment stimulated a ∼7-fold increase in RPS3 S209 phosphorylation, peaking at 30 minutes (Fig. 6d).
T118 17039-17166 Sentence denotes In uninfected cells, TNF-treatment stimulated a ∼7-fold increase in RPS3 S209 phosphorylation, peaking at 30 minutes (Fig. 6d).
T11947 17167-17292 Sentence denotes By contrast, RPS3 S209 phosphorylation was substantially impaired in cells infected with wild-type E. coli O157:H7 (Fig. 6d).
T119 17167-17292 Sentence denotes By contrast, RPS3 S209 phosphorylation was substantially impaired in cells infected with wild-type E. coli O157:H7 (Fig. 6d).
T11948 17293-17411 Sentence denotes However, TNF-induced RPS3 S209 phosphorylation was unimpaired in cells infected with either ΔnleH1 or ΔescN (Fig. 6d).
T120 17293-17411 Sentence denotes However, TNF-induced RPS3 S209 phosphorylation was unimpaired in cells infected with either ΔnleH1 or ΔescN (Fig. 6d).
T11949 17412-17554 Sentence denotes We showed previously that wild-type, but not ΔnleH1 or ΔescN E. coli O157:H7 significantly attenuated TNF-induced RPS3 nuclear translocation9.
T121 17412-17554 Sentence denotes We showed previously that wild-type, but not ΔnleH1 or ΔescN E. coli O157:H7 significantly attenuated TNF-induced RPS3 nuclear translocation9.
T11950 17555-17793 Sentence denotes The parallel between RPS3 phosphorylation and its nuclear translocation during E. coli infection provides evidence in the context of an NF-κB-dependent disease process that RPS3 S209 phosphorylation is important for nuclear translocation.
T122 17555-17793 Sentence denotes The parallel between RPS3 phosphorylation and its nuclear translocation during E. coli infection provides evidence in the context of an NF-κB-dependent disease process that RPS3 S209 phosphorylation is important for nuclear translocation.
T11951 17794-17970 Sentence denotes Our discovery that NleH1 inhibits RPS3 S209 phosphorylation suggested that it should also blocks RPS3-dependent NF-κB target gene transcription (e.g. IL8, NFKBIA, and TNFAIP3).
T123 17794-17970 Sentence denotes Our discovery that NleH1 inhibits RPS3 S209 phosphorylation suggested that it should also blocks RPS3-dependent NF-κB target gene transcription (e.g. IL8, NFKBIA, and TNFAIP3).
T11952 17971-18162 Sentence denotes Indeed, these genes were only modestly upregulated in cells infected with wild-type E. coli O157:H7, but significantly induced in cells infected with either ΔnleH1 or ΔescN strains (Fig. 6e).
T124 17971-18162 Sentence denotes Indeed, these genes were only modestly upregulated in cells infected with wild-type E. coli O157:H7, but significantly induced in cells infected with either ΔnleH1 or ΔescN strains (Fig. 6e).
T11953 18163-18302 Sentence denotes In contrast, deleting nleH1 had no impact on the expression of RPS3-independent genes, including CD25 and TNFSF13B (Supplementary Fig. 12).
T125 18163-18302 Sentence denotes In contrast, deleting nleH1 had no impact on the expression of RPS3-independent genes, including CD25 and TNFSF13B (Supplementary Fig. 12).
T11954 18303-18514 Sentence denotes Together these results demonstrate that NleH1 specifically inhibits the protective immune response by directly blocking RPS3 S209 phosphorylation and thereby impairing critical RPS3-dependent NF-κB target genes.
T126 18303-18514 Sentence denotes Together these results demonstrate that NleH1 specifically inhibits the protective immune response by directly blocking RPS3 S209 phosphorylation and thereby impairing critical RPS3-dependent NF-κB target genes.
T13755 18516-18564 Sentence denotes NleH1 inhibits RPS3 S209 phosphorylation in vivo
T127 18516-18564 Sentence denotes NleH1 inhibits RPS3 S209 phosphorylation in vivo
T13756 18565-18748 Sentence denotes We previously utilized a gnotobiotic piglet infection model to determine that piglets infected with ΔnleH1 mutant died more rapidly than those infected with wild-type E. coli O157:H7.
T128 18565-18748 Sentence denotes We previously utilized a gnotobiotic piglet infection model to determine that piglets infected with ΔnleH1 mutant died more rapidly than those infected with wild-type E. coli O157:H7.
T13757 18749-18914 Sentence denotes Piglets infected with ΔnleH1 displayed clinical disease consistent with a robust inflammatory response, but with reduced bacterial colonization and little diarrhea9.
T129 18749-18914 Sentence denotes Piglets infected with ΔnleH1 displayed clinical disease consistent with a robust inflammatory response, but with reduced bacterial colonization and little diarrhea9.
T13758 18915-19122 Sentence denotes While seemingly paradoxical, based on our cell culture data (Fig. 6d), we hypothesized that NleH1 blocked RPS3 S209 phosphorylation in vivo, thereby preventing RPS3 nuclear translocation in infected piglets.
T130 18915-19122 Sentence denotes While seemingly paradoxical, based on our cell culture data (Fig. 6d), we hypothesized that NleH1 blocked RPS3 S209 phosphorylation in vivo, thereby preventing RPS3 nuclear translocation in infected piglets.
T13759 19123-19237 Sentence denotes We isolated piglet colons at necropsy, and subjected them to immunohistochemistry using our phospho-RPS3 antibody.
T131 19123-19237 Sentence denotes We isolated piglet colons at necropsy, and subjected them to immunohistochemistry using our phospho-RPS3 antibody.
T13760 19238-19481 Sentence denotes Consistent with in vitro data, piglets infected with wild-type E. coli O157:H7 exhibited diffuse and low intensity phospho-RPS3 staining, whereas in piglets infected with ΔnleH1 mutant, phospho-RPS3 expression was florid and intense (Fig. 6f).
T132 19238-19481 Sentence denotes Consistent with in vitro data, piglets infected with wild-type E. coli O157:H7 exhibited diffuse and low intensity phospho-RPS3 staining, whereas in piglets infected with ΔnleH1 mutant, phospho-RPS3 expression was florid and intense (Fig. 6f).
T13761 19482-19645 Sentence denotes These data demonstrate that NleH1 inhibits RPS3 S209 phosphorylation both in vitro and in vivo, which might benefit the bacterium in colonization and transmission.
T133 19482-19645 Sentence denotes These data demonstrate that NleH1 inhibits RPS3 S209 phosphorylation both in vitro and in vivo, which might benefit the bacterium in colonization and transmission.
T14904 19647-19692 Sentence denotes NleH1 steers the IKKβ substrate specificities
T134 19647-19692 Sentence denotes NleH1 steers the IKKβ substrate specificities
T14905 19693-19789 Sentence denotes NleH1 is an autophosphorylated serine-threonine kinase, which depends on the lysine 159 (K159)9.
T135 19693-19789 Sentence denotes NleH1 is an autophosphorylated serine-threonine kinase, which depends on the lysine 159 (K159)9.
T14906 19790-20091 Sentence denotes To explore the mechanism by which NleH1 inhibits RPS3 S209 phosphorylation, we first performed an in vitro kinase assay with purified wild-type His-NleH1 protein and a mutant His-NleH1 (K159A) protein, confirming that NleH1 is autophosphorylated and the K159A is an NleH1 kinase-dead mutant (Fig. 7a).
T136 19790-20091 Sentence denotes To explore the mechanism by which NleH1 inhibits RPS3 S209 phosphorylation, we first performed an in vitro kinase assay with purified wild-type His-NleH1 protein and a mutant His-NleH1 (K159A) protein, confirming that NleH1 is autophosphorylated and the K159A is an NleH1 kinase-dead mutant (Fig. 7a).
T14907 20092-20274 Sentence denotes To examine whether the kinase activity is required for NleH1 to inhibit IKKβ phosphorlyation of RPS3 on S209, we ectopically expressing either wild-type or K159A NleH1 in 293T cells.
T137 20092-20274 Sentence denotes To examine whether the kinase activity is required for NleH1 to inhibit IKKβ phosphorlyation of RPS3 on S209, we ectopically expressing either wild-type or K159A NleH1 in 293T cells.
T14908 20275-20414 Sentence denotes Wild-type NleH1 expression significantly reduced TNF-induced RPS3 S209 phosphorylation, whereas the K159A mutant failed to do so (Fig. 7b).
T138 20275-20414 Sentence denotes Wild-type NleH1 expression significantly reduced TNF-induced RPS3 S209 phosphorylation, whereas the K159A mutant failed to do so (Fig. 7b).
T14909 20415-20505 Sentence denotes Thus NleH1 kinase activity is required to protect RPS3 from IKKβ-mediated phosphorylation.
T139 20415-20505 Sentence denotes Thus NleH1 kinase activity is required to protect RPS3 from IKKβ-mediated phosphorylation.
T14910 20506-20781 Sentence denotes Citrobacter rodentium is a mouse pathogen that shares pathogenic strategies with E. coli 39, most notably for our investigation, C. rodentium NleH inhibited RPS3 nuclear translocation and RPS3-dependent NF-κB luciferase activity to an extent equivalent to E. coli NleH1 (ref.
T140 20506-20781 Sentence denotes Citrobacter rodentium is a mouse pathogen that shares pathogenic strategies with E. coli 39, most notably for our investigation, C. rodentium NleH inhibited RPS3 nuclear translocation and RPS3-dependent NF-κB luciferase activity to an extent equivalent to E. coli NleH1 (ref.
T14911 20782-20785 Sentence denotes 9).
T141 20782-20785 Sentence denotes 9).
T14912 20786-20882 Sentence denotes We assayed RPS3 S209 phosphorylation in HeLa cells infected with different C. rodentium strains.
T142 20786-20882 Sentence denotes We assayed RPS3 S209 phosphorylation in HeLa cells infected with different C. rodentium strains.
T14913 20883-20989 Sentence denotes In uninfected cells, TNF-treatment stimulated a ∼3.5-fold increase in RPS3 S209 phosphorylation (Fig. 7c).
T143 20883-20989 Sentence denotes In uninfected cells, TNF-treatment stimulated a ∼3.5-fold increase in RPS3 S209 phosphorylation (Fig. 7c).
T14914 20990-21103 Sentence denotes Such augmentation of RPS3 phosphorylation was reduced by about 60% by wild-type C. rodentium infection (Fig. 7c).
T144 20990-21103 Sentence denotes Such augmentation of RPS3 phosphorylation was reduced by about 60% by wild-type C. rodentium infection (Fig. 7c).
T14915 21104-21229 Sentence denotes However, RPS3 phosphorylation was enhanced when cells were infected with a C. rodentium strain lacking NleH (Fig. 7c, ΔnleH).
T145 21104-21229 Sentence denotes However, RPS3 phosphorylation was enhanced when cells were infected with a C. rodentium strain lacking NleH (Fig. 7c, ΔnleH).
T14916 21230-21396 Sentence denotes We further examined the role of NleH1 kinase activity using the C. rodentium ΔnleH strain as a background on which to express either wild-type or K159A E. coli NleH1.
T146 21230-21396 Sentence denotes We further examined the role of NleH1 kinase activity using the C. rodentium ΔnleH strain as a background on which to express either wild-type or K159A E. coli NleH1.
T14917 21397-21582 Sentence denotes Complementing ΔnleH mutant with wild-type NleH1 almost abolished TNF-induced RPS3 S209 phosphorylation whereas complementing with K159A failed to inhibit RPS3 phosphorylation (Fig. 7c).
T147 21397-21582 Sentence denotes Complementing ΔnleH mutant with wild-type NleH1 almost abolished TNF-induced RPS3 S209 phosphorylation whereas complementing with K159A failed to inhibit RPS3 phosphorylation (Fig. 7c).
T14918 21583-21697 Sentence denotes Collectively, these results demonstrate that NleH1 kinase activity is required to block RPS3 S209 phosphorylation.
T148 21583-21697 Sentence denotes Collectively, these results demonstrate that NleH1 kinase activity is required to block RPS3 S209 phosphorylation.
T14919 21698-21896 Sentence denotes We next examined whether the inhibitory activity of NleH1 is sufficiently robust to impair the strong nuclear translocation of RPS3 trigged by the constitutively-active IKKβ (IKKβ [SSEE]) (Fig. 2d).
T149 21698-21896 Sentence denotes We next examined whether the inhibitory activity of NleH1 is sufficiently robust to impair the strong nuclear translocation of RPS3 trigged by the constitutively-active IKKβ (IKKβ [SSEE]) (Fig. 2d).
T14920 21897-22067 Sentence denotes We found that ectopically expressing either wild-type or SSEE IKKβ proteins, triggered more RPS3 nuclear translocation than the kinase-dead IKKβ (SSAA) protein (Fig. 7d).
T150 21897-22067 Sentence denotes We found that ectopically expressing either wild-type or SSEE IKKβ proteins, triggered more RPS3 nuclear translocation than the kinase-dead IKKβ (SSAA) protein (Fig. 7d).
T14921 22068-22181 Sentence denotes RPS3 nuclear accumulation was substantially retarded by infecting cells with wild-type E. coli O157:H7 (Fig. 7d).
T151 22068-22181 Sentence denotes RPS3 nuclear accumulation was substantially retarded by infecting cells with wild-type E. coli O157:H7 (Fig. 7d).
T14922 22182-22349 Sentence denotes In contrast, infecting with either ΔnleH1 or ΔescN strains only slightly impaired RPS3 nuclear translocation in either IKKβ- or IKKβ (SSEE)-expressing cells (Fig. 7d).
T152 22182-22349 Sentence denotes In contrast, infecting with either ΔnleH1 or ΔescN strains only slightly impaired RPS3 nuclear translocation in either IKKβ- or IKKβ (SSEE)-expressing cells (Fig. 7d).
T14923 22350-22501 Sentence denotes As expected, E. coli infections did not affect the RPS3 nuclear translocation in IKKβ (SSAA)-expressing cells, where NF-κB signaling was low (Fig. 7d).
T153 22350-22501 Sentence denotes As expected, E. coli infections did not affect the RPS3 nuclear translocation in IKKβ (SSAA)-expressing cells, where NF-κB signaling was low (Fig. 7d).
T14924 22502-22647 Sentence denotes Thus, during infection NleH1 is sufficiently potent to inhibit RPS3 nuclear translocation even in cells expressing constitutively-activated IKKβ.
T154 22502-22647 Sentence denotes Thus, during infection NleH1 is sufficiently potent to inhibit RPS3 nuclear translocation even in cells expressing constitutively-activated IKKβ.
T14925 22648-22764 Sentence denotes We examined whether NleH1 could directly phosphorylate IKKβ thus inhibiting IKKβ-mediated RPS3 S209 phosphorylation.
T155 22648-22764 Sentence denotes We examined whether NleH1 could directly phosphorylate IKKβ thus inhibiting IKKβ-mediated RPS3 S209 phosphorylation.
T14926 22765-22974 Sentence denotes We performed in vitro kinase assays using immunoprecipitated Flag-IKKβ (K44A) as substrate and recombinant His-NleH1 as kinase, so that IKKβ autophosphorylation would not obscure NleH1-induced phosphorylation.
T156 22765-22974 Sentence denotes We performed in vitro kinase assays using immunoprecipitated Flag-IKKβ (K44A) as substrate and recombinant His-NleH1 as kinase, so that IKKβ autophosphorylation would not obscure NleH1-induced phosphorylation.
T14927 22975-23102 Sentence denotes However, we did not observe any detectable 32P incorporation in IKKβ (Supplementary Fig. 13), thus ruling out this possibility.
T157 22975-23102 Sentence denotes However, we did not observe any detectable 32P incorporation in IKKβ (Supplementary Fig. 13), thus ruling out this possibility.
T14928 23103-23189 Sentence denotes We then tested the hypothesis that NleH1 could alter the IKKβ substrate specificities.
T158 23103-23189 Sentence denotes We then tested the hypothesis that NleH1 could alter the IKKβ substrate specificities.
T14929 23190-23282 Sentence denotes To this end, we performed in vitro kinase assays using both CK2 and IKK substrates for IKKβ.
T159 23190-23282 Sentence denotes To this end, we performed in vitro kinase assays using both CK2 and IKK substrates for IKKβ.
T14930 23283-23451 Sentence denotes As expected, IKKβ phosphorylated RPS3 (Fig. 7e, lane 7) and GST-IκBα (1-54) protein (Fig. 7e, lane 9), demonstrating it harbors either CK2 or IKK substrate specificity.
T160 23283-23451 Sentence denotes As expected, IKKβ phosphorylated RPS3 (Fig. 7e, lane 7) and GST-IκBα (1-54) protein (Fig. 7e, lane 9), demonstrating it harbors either CK2 or IKK substrate specificity.
T14931 23452-23652 Sentence denotes Preincubation of IKKβ with NleH1 reduced IKKβ-mediated RPS3 phosphorylation, i.e. the CK2 kinase specificity, but not IKKβ-mediated GST-IκBα phosphorylation, i.e. the IKK kinase specificity (Fig. 7e).
T161 23452-23652 Sentence denotes Preincubation of IKKβ with NleH1 reduced IKKβ-mediated RPS3 phosphorylation, i.e. the CK2 kinase specificity, but not IKKβ-mediated GST-IκBα phosphorylation, i.e. the IKK kinase specificity (Fig. 7e).
T14932 23653-23769 Sentence denotes Control experiments revealed no NleH1-mediated phosphorylation or autophosphorylation of RPS3 or GST-IκBα (Fig. 7e).
T162 23653-23769 Sentence denotes Control experiments revealed no NleH1-mediated phosphorylation or autophosphorylation of RPS3 or GST-IκBα (Fig. 7e).
T14933 23770-23992 Sentence denotes Taken together, NleH1 blocks the CK2 substrate specificity of IKKβ thus inhibiting the IKKβ-mediated RPS3 S209 phosphorylation thus representing a novel strategy by E. coli O157:H7 to alter the host innate immune response.
T163 23770-23992 Sentence denotes Taken together, NleH1 blocks the CK2 substrate specificity of IKKβ thus inhibiting the IKKβ-mediated RPS3 S209 phosphorylation thus representing a novel strategy by E. coli O157:H7 to alter the host innate immune response.
T164 23994-24004 Sentence denotes Discussion
T18122 24005-24114 Sentence denotes RPS3 was previously demonstrated to function as an integral subunit conferring NF-κB regulatory specificity6.
T165 24005-24114 Sentence denotes RPS3 was previously demonstrated to function as an integral subunit conferring NF-κB regulatory specificity6.
T18123 24115-24252 Sentence denotes Here we sought to elucidate how NF-κB activation signaling triggers RPS3 to translocate and participate in NF-κB function in the nucleus.
T166 24115-24252 Sentence denotes Here we sought to elucidate how NF-κB activation signaling triggers RPS3 to translocate and participate in NF-κB function in the nucleus.
T18124 24253-24448 Sentence denotes We demonstrate that IKKβ-mediated RPS3 S209 phosphorylation represents a critical determinant in governing its nuclear import thus unveiling a novel mechanism behind NF-κB regulatory specificity.
T167 24253-24448 Sentence denotes We demonstrate that IKKβ-mediated RPS3 S209 phosphorylation represents a critical determinant in governing its nuclear import thus unveiling a novel mechanism behind NF-κB regulatory specificity.
T18125 24449-24573 Sentence denotes IKKβ is the major kinase that phosphorylates IκBs in the classical NF-κB pathway, leading to their subsequent degradation40.
T168 24449-24573 Sentence denotes IKKβ is the major kinase that phosphorylates IκBs in the classical NF-κB pathway, leading to their subsequent degradation40.
T18126 24574-24681 Sentence denotes Strikingly, RPS3 possesses not any consensus IKK motif; instead, S209 is centered in a consensus CK2 motif.
T169 24574-24681 Sentence denotes Strikingly, RPS3 possesses not any consensus IKK motif; instead, S209 is centered in a consensus CK2 motif.
T18127 24682-24855 Sentence denotes The recent observation that human IKKβ displayed CK2-like phosphorylation specificity29 coincides with our evidence that recombinant IKKβ, but not IKKα, phosphorylated RPS3.
T170 24682-24855 Sentence denotes The recent observation that human IKKβ displayed CK2-like phosphorylation specificity29 coincides with our evidence that recombinant IKKβ, but not IKKα, phosphorylated RPS3.
T18128 24856-25006 Sentence denotes We found this phosphorylation is a critical modulation for RPS3 nuclear translocation (via importin-α) and engagement in specific NF-κB transcription.
T171 24856-25006 Sentence denotes We found this phosphorylation is a critical modulation for RPS3 nuclear translocation (via importin-α) and engagement in specific NF-κB transcription.
T18129 25007-25299 Sentence denotes CK2 was previously shown to phosphorylate p65 and to bind to and phosphorylate IKKβ41-43, however, we ruled out the possibility that the IKKβ-bound CK2 could account for the observed RPS3 phosphorylation because no CK2 was detected in the IKKβ preparations used for the in vitro kinase assay.
T172 25007-25299 Sentence denotes CK2 was previously shown to phosphorylate p65 and to bind to and phosphorylate IKKβ41-43, however, we ruled out the possibility that the IKKβ-bound CK2 could account for the observed RPS3 phosphorylation because no CK2 was detected in the IKKβ preparations used for the in vitro kinase assay.
T18130 25300-25486 Sentence denotes Because RPS3 only harbors the CK2 motif and not a traditional IKK motif, this RPS3 regulatory function could explain why IKK harbors the alternative substrate phosphorylation capability.
T173 25300-25486 Sentence denotes Because RPS3 only harbors the CK2 motif and not a traditional IKK motif, this RPS3 regulatory function could explain why IKK harbors the alternative substrate phosphorylation capability.
T18131 25487-25686 Sentence denotes More importantly, our study has elucidated how RPS3 is biochemically integrated into NF-κB activation signaling in a manner that is pivotal for the pathogenesis of foodborne pathogen E. coli O157:H7.
T174 25487-25686 Sentence denotes More importantly, our study has elucidated how RPS3 is biochemically integrated into NF-κB activation signaling in a manner that is pivotal for the pathogenesis of foodborne pathogen E. coli O157:H7.
T18132 25687-25807 Sentence denotes IKKβ-mediated RPS3 S209 phosphorylation is a critical target modulated by this pathogen to subvert host NF-κB signaling.
T175 25687-25807 Sentence denotes IKKβ-mediated RPS3 S209 phosphorylation is a critical target modulated by this pathogen to subvert host NF-κB signaling.
T18133 25808-25973 Sentence denotes The bacterial effector NleH1 specifically binds to RPS3 once injected into host cells and profoundly suppresses NF-κB and its attendant protective immune responses9.
T176 25808-25973 Sentence denotes The bacterial effector NleH1 specifically binds to RPS3 once injected into host cells and profoundly suppresses NF-κB and its attendant protective immune responses9.
T18134 25974-26157 Sentence denotes Our data now show that NleH1 selectively inhibits RPS3 phosphorylation, thus retarding its nuclear translocation and subsequent NF-κB function, without altering other NF-κB signaling.
T177 25974-26157 Sentence denotes Our data now show that NleH1 selectively inhibits RPS3 phosphorylation, thus retarding its nuclear translocation and subsequent NF-κB function, without altering other NF-κB signaling.
T18135 26158-26294 Sentence denotes Although NleH1 did not directly phosphorylate IKKβ, its kinase activity was required to inhibit IKKβ-mediated RPS3 S209 phosphorylation.
T178 26158-26294 Sentence denotes Although NleH1 did not directly phosphorylate IKKβ, its kinase activity was required to inhibit IKKβ-mediated RPS3 S209 phosphorylation.
T18136 26295-26524 Sentence denotes Many bacteria pathogens have products that target key kinases to inactivate them in host cells, whereas E. coli O157:H7 employed NleH1 to steer the substrate specificity of IKKβ thus specifically fine-tuning host NF-κB signaling.
T179 26295-26524 Sentence denotes Many bacteria pathogens have products that target key kinases to inactivate them in host cells, whereas E. coli O157:H7 employed NleH1 to steer the substrate specificity of IKKβ thus specifically fine-tuning host NF-κB signaling.
T18137 26525-26637 Sentence denotes This could represent a novel strategy to fine-tune host NF-κB signaling that could be shared by other pathogens.
T180 26525-26637 Sentence denotes This could represent a novel strategy to fine-tune host NF-κB signaling that could be shared by other pathogens.
T18138 26638-26738 Sentence denotes These data provide new insights into the poorly understood action mechanism for most T3SS effectors.
T181 26638-26738 Sentence denotes These data provide new insights into the poorly understood action mechanism for most T3SS effectors.
T18139 26739-26923 Sentence denotes NleH1 attenuates the transcription of RPS3-dependent, but not all, NF-κB target genes, in particular those genes associated with acute proinflammatory responses, including IL8 and TNF.
T182 26739-26923 Sentence denotes NleH1 attenuates the transcription of RPS3-dependent, but not all, NF-κB target genes, in particular those genes associated with acute proinflammatory responses, including IL8 and TNF.
T18140 26924-27159 Sentence denotes In contrast, NleH1 does not block NF-κB p65 nuclear translocation, which suggests that certain p65-dependent but RPS3-independent NF-κB target genes might thus be beneficial for E. coli O157:H7 to replicate and disseminate in the host.
T183 26924-27159 Sentence denotes In contrast, NleH1 does not block NF-κB p65 nuclear translocation, which suggests that certain p65-dependent but RPS3-independent NF-κB target genes might thus be beneficial for E. coli O157:H7 to replicate and disseminate in the host.
T18141 27160-27347 Sentence denotes By selectively inhibiting RPS3 and its attendant NF-κB function with NleH1, the pathogen achieves the ability to increase colonization and diarrhea yet limiting the mortality of the host.
T184 27160-27347 Sentence denotes By selectively inhibiting RPS3 and its attendant NF-κB function with NleH1, the pathogen achieves the ability to increase colonization and diarrhea yet limiting the mortality of the host.
T18142 27348-27605 Sentence denotes This seemingly paradoxical combination of effects make sense when one considers that increased bacterial load and diarrhea together with survival of the infected host would promote the spreading of the bacteria among a population of susceptible individuals.
T185 27348-27605 Sentence denotes This seemingly paradoxical combination of effects make sense when one considers that increased bacterial load and diarrhea together with survival of the infected host would promote the spreading of the bacteria among a population of susceptible individuals.
T18143 27606-27744 Sentence denotes Such complex and paradoxical pathological effects that influence the spread of disease are often poorly understood at the molecular level.
T186 27606-27744 Sentence denotes Such complex and paradoxical pathological effects that influence the spread of disease are often poorly understood at the molecular level.
T18144 27745-27981 Sentence denotes Our data elucidate how alterations in selective NF-κB function, achieved by impeding RPS3, but not altering p65 nuclear translocation, can influence specific cytokines that affect bacterial colonization, diarrhea diseases and mortality.
T187 27745-27981 Sentence denotes Our data elucidate how alterations in selective NF-κB function, achieved by impeding RPS3, but not altering p65 nuclear translocation, can influence specific cytokines that affect bacterial colonization, diarrhea diseases and mortality.
T18145 27982-28180 Sentence denotes It may be fruitful in attempting to understand other infectious and autoimmune diseases involving NF-κB to consider selective effects of subunits such as RPS3 in addition to global NF-κB inhibition.
T188 27982-28180 Sentence denotes It may be fruitful in attempting to understand other infectious and autoimmune diseases involving NF-κB to consider selective effects of subunits such as RPS3 in addition to global NF-κB inhibition.
T189 28182-28189 Sentence denotes Methods
T20201 28191-28209 Sentence denotes Cells and Reagents
T190 28191-28209 Sentence denotes Cells and Reagents
T20202 28210-28401 Sentence denotes Jurkat E6.1, HEK293T and HeLa cells were cultured in RPMI 1640 and DMEM supplemented with 10% fetal calf serum, 2 mM glutamine, and 100 U/ml each of penicillin and streptomycin, respectively.
T191 28210-28401 Sentence denotes Jurkat E6.1, HEK293T and HeLa cells were cultured in RPMI 1640 and DMEM supplemented with 10% fetal calf serum, 2 mM glutamine, and 100 U/ml each of penicillin and streptomycin, respectively.
T192 28402-29063 Sentence denotes IκBα (C-21, sc-371), p65 (C-20, sc-372), and phospho-threonine (H-2, sc-5267) antibodies were from Santa Cruz Biotechnology; β-actin (AC-15, A5441), Flag (M2, F3165), HA (HA-7, H3663), importin-α (IM-75, I1784), and importin-β (31H4, I2534) antibodies were from Sigma; PARP (C2-10, 556362), IKKα (B78-1, 556532), and IKKβ (24, 611254) antibodies were from BD Pharmingen; CK2α (31, 611610) and Hsp90 (68, 610418) antibodies were from BD Transduction Laboratories; phospho-IκBα (5A5, 9246S) and phospho-IKKα/β (16A6, 2697S) antibodies were from Cell Signaling Technology; phospho-serine (AB1603) and phospho-tyrosine (4G10, 05-777) antibodies were from Millipore.
T20203 28402-29130 Sentence denotes IκBα (C-21, sc-371), p65 (C-20, sc-372), and phospho-threonine (H-2, sc-5267) antibodies were from Santa Cruz Biotechnology; β-actin (AC-15, A5441), Flag (M2, F3165), HA (HA-7, H3663), importin-α (IM-75, I1784), and importin-β (31H4, I2534) antibodies were from Sigma; PARP (C2-10, 556362), IKKα (B78-1, 556532), and IKKβ (24, 611254) antibodies were from BD Pharmingen; CK2α (31, 611610) and Hsp90 (68, 610418) antibodies were from BD Transduction Laboratories; phospho-IκBα (5A5, 9246S) and phospho-IKKα/β (16A6, 2697S) antibodies were from Cell Signaling Technology; phospho-serine (AB1603) and phospho-tyrosine (4G10, 05-777) antibodies were from Millipore. The rabbit polyclonal RPS3 antiserum was as described previously6.
T193 29064-29130 Sentence denotes The rabbit polyclonal RPS3 antiserum was as described previously6.
T20204 29131-29296 Sentence denotes The rabbit polyclonal antibody specific for S209 phosphorylated RPS3 was generated and affinity purified by Primm Biotech using the peptide NH2-CKPLPDHV(Sp)IVE-COOH.
T194 29131-29296 Sentence denotes The rabbit polyclonal antibody specific for S209 phosphorylated RPS3 was generated and affinity purified by Primm Biotech using the peptide NH2-CKPLPDHV(Sp)IVE-COOH.
T20515 29298-29316 Sentence denotes Plasmid Constructs
T195 29298-29316 Sentence denotes Plasmid Constructs
T196 29317-29406 Sentence denotes The Flag-IKKβ (SSEE), Flag-IKKβ (SSAA), and HA-IκBα (SSAA) constructs were provided by C.
T20516 29317-29476 Sentence denotes The Flag-IKKβ (SSEE), Flag-IKKβ (SSAA), and HA-IκBα (SSAA) constructs were provided by C. Wu (NCI, Bethesda) and U. Siebenlist (NIAID, Bethesda), respectively.
T197 29407-29432 Sentence denotes Wu (NCI, Bethesda) and U.
T198 29433-29476 Sentence denotes Siebenlist (NIAID, Bethesda), respectively.
T20517 29477-29553 Sentence denotes The HA-IκBα and IKKβ (K44A)-Flag plasmids were purchased from Addgene44, 45.
T199 29477-29553 Sentence denotes The HA-IκBα and IKKβ (K44A)-Flag plasmids were purchased from Addgene44, 45.
T20518 29554-29643 Sentence denotes The Flag-RPS3, GST-RPS3, HA-RPS3, VN-HA, NleH1-HA plasmids were described previously6, 9.
T200 29554-29643 Sentence denotes The Flag-RPS3, GST-RPS3, HA-RPS3, VN-HA, NleH1-HA plasmids were described previously6, 9.
T20519 29644-29877 Sentence denotes The point mutants of RPS3 were generated by site-directed mutagenesis using the Quick Change Kit (Stratagene) with primers forward 5′-CTGCCTGACCACGTGGCCATTGTGGAACCCAAA-3′ and reverse 5′-TTTGGGTTCCACAATGGCCACGTGGTCAGGCAG-3′ for S209A.
T201 29644-29877 Sentence denotes The point mutants of RPS3 were generated by site-directed mutagenesis using the Quick Change Kit (Stratagene) with primers forward 5′-CTGCCTGACCACGTGGCCATTGTGGAACCCAAA-3′ and reverse 5′-TTTGGGTTCCACAATGGCCACGTGGTCAGGCAG-3′ for S209A.
T20520 29878-29922 Sentence denotes All mutants were verified by DNA sequencing.
T202 29878-29922 Sentence denotes All mutants were verified by DNA sequencing.
T20843 29924-29944 Sentence denotes 32P in vivo Labeling
T203 29924-29944 Sentence denotes 32P in vivo Labeling
T20844 29945-30067 Sentence denotes HEK 293T cells were labeled with 2 mCi/ml 32P-orthophosphate (Perkin Elmer) in phosphate-free medium (Invitrogen) for 2 h.
T204 29945-30067 Sentence denotes HEK 293T cells were labeled with 2 mCi/ml 32P-orthophosphate (Perkin Elmer) in phosphate-free medium (Invitrogen) for 2 h.
T20845 30068-30165 Sentence denotes Cells were then left untreated or treated with TNF (50 ng/ml, R&D Systems) for indicated periods.
T205 30068-30165 Sentence denotes Cells were then left untreated or treated with TNF (50 ng/ml, R&D Systems) for indicated periods.
T20846 30166-30246 Sentence denotes Cell lysates were prepared and used for immunoprecipitations with RPS3 antibody.
T206 30166-30246 Sentence denotes Cell lysates were prepared and used for immunoprecipitations with RPS3 antibody.
T20989 30248-30269 Sentence denotes In Vitro Kinase Assay
T207 30248-30269 Sentence denotes In Vitro Kinase Assay
T20990 30270-30380 Sentence denotes Kinase-active recombinant IKKβ and IKKα proteins were purchased from Active Motif and Millipore, respectively.
T208 30270-30380 Sentence denotes Kinase-active recombinant IKKβ and IKKα proteins were purchased from Active Motif and Millipore, respectively.
T20991 30381-30521 Sentence denotes Bacterially purified glutathione S-transferase (GST), GST-IκBα (1-54), wild type, mutant GST-RPS3, or RPS3 proteins were used as substrates.
T209 30381-30521 Sentence denotes Bacterially purified glutathione S-transferase (GST), GST-IκBα (1-54), wild type, mutant GST-RPS3, or RPS3 proteins were used as substrates.
T20992 30522-30588 Sentence denotes The in vitro kinase assay was performed as previously described29.
T210 30522-30588 Sentence denotes The in vitro kinase assay was performed as previously described29.
T20993 30589-30907 Sentence denotes Briefly, enzyme (100 ng) and substrate (2 μg) were co-incubated in IKK reaction buffer (25 mM Tris–HCl [pH 8.0], 50 mM KCl, 10 mM MgCl2, 1 mM DTT, 1 mM Na3VO4, 1 mM ATP) or NleH1 reaction buffer (50 mM Tris-HCl [pH 7.6], 5 mM MgCl2, 1 mM DTT, 1 mM ATP) with 0.5 μCi 32P-γ-ATP (GE Healthcare) added at 37 °C for 30 min.
T211 30589-30907 Sentence denotes Briefly, enzyme (100 ng) and substrate (2 μg) were co-incubated in IKK reaction buffer (25 mM Tris–HCl [pH 8.0], 50 mM KCl, 10 mM MgCl2, 1 mM DTT, 1 mM Na3VO4, 1 mM ATP) or NleH1 reaction buffer (50 mM Tris-HCl [pH 7.6], 5 mM MgCl2, 1 mM DTT, 1 mM ATP) with 0.5 μCi 32P-γ-ATP (GE Healthcare) added at 37 °C for 30 min.
T20994 30908-30982 Sentence denotes The reactions were resolved by SDS–PAGE and visualized by autoradiography.
T212 30908-30982 Sentence denotes The reactions were resolved by SDS–PAGE and visualized by autoradiography.
T21363 30984-31001 Sentence denotes LC-MS/MS Analysis
T213 30984-31001 Sentence denotes LC-MS/MS Analysis
T21364 31002-31155 Sentence denotes GST or GST-RPS3 was incubated with recombinant IKKβ protein as described above in an in vitro kinase assay reaction conducted without 32P-γ-ATP labeling.
T214 31002-31155 Sentence denotes GST or GST-RPS3 was incubated with recombinant IKKβ protein as described above in an in vitro kinase assay reaction conducted without 32P-γ-ATP labeling.
T21365 31156-31261 Sentence denotes The reaction was separated by SDS-PAGE, and the protein gel was stained with Colloidal Blue (Invitrogen).
T215 31156-31261 Sentence denotes The reaction was separated by SDS-PAGE, and the protein gel was stained with Colloidal Blue (Invitrogen).
T21366 31262-31428 Sentence denotes The corresponding protein fragments were excised and subjected to trypsin digestion and LC-MS/MS at the Yale Cancer Center Mass Spectrometry Resource (New Haven, CT).
T216 31262-31428 Sentence denotes The corresponding protein fragments were excised and subjected to trypsin digestion and LC-MS/MS at the Yale Cancer Center Mass Spectrometry Resource (New Haven, CT).
T21548 31430-31451 Sentence denotes RNAi and Transfection
T217 31430-31451 Sentence denotes RNAi and Transfection
T21549 31452-31661 Sentence denotes The siRNA (sense-strand sequence) IKKα, 5′-AUGACAGAGAAUGAUCAUGUUCUGC -3′; IKKβ, 5′-GCAGCAAGGAGAACAGAGGUUAAUA -3′; IκBα, 5′-GAGCUCCGAGACUUUCGAGGAAAUA -3′; RPS3-3′ UTR, 5′-GGAUGUUGCUCUCUAAAGACC -3′ (Invitrogen).
T218 31452-31661 Sentence denotes The siRNA (sense-strand sequence) IKKα, 5′-AUGACAGAGAAUGAUCAUGUUCUGC -3′; IKKβ, 5′-GCAGCAAGGAGAACAGAGGUUAAUA -3′; IκBα, 5′-GAGCUCCGAGACUUUCGAGGAAAUA -3′; RPS3-3′ UTR, 5′-GGAUGUUGCUCUCUAAAGACC -3′ (Invitrogen).
T21550 31662-31772 Sentence denotes Transient transfection of siRNA and DNA constructs into Jurkat cells and 293T cells was described previously6.
T219 31662-31772 Sentence denotes Transient transfection of siRNA and DNA constructs into Jurkat cells and 293T cells was described previously6.
T21706 31774-31799 Sentence denotes Subcellular Fractionation
T220 31774-31799 Sentence denotes Subcellular Fractionation
T21707 31800-31896 Sentence denotes Subcellular fractionation was performed by differential centrifugation as previously described6.
T221 31800-31896 Sentence denotes Subcellular fractionation was performed by differential centrifugation as previously described6.
T21708 31897-32103 Sentence denotes Briefly, cells were resuspended in ice-cold Buffer A (10 mM HEPES pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 0.1 mM EDTA, 0.5 mM DTT, 0.4 % NP-40, 0.5 mM PMSF, complete protease inhibitor cocktail) at 4 °C for 5 min.
T222 31897-32103 Sentence denotes Briefly, cells were resuspended in ice-cold Buffer A (10 mM HEPES pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 0.1 mM EDTA, 0.5 mM DTT, 0.4 % NP-40, 0.5 mM PMSF, complete protease inhibitor cocktail) at 4 °C for 5 min.
T21709 32104-32212 Sentence denotes Lysates were centrifuged at 4 °C, 500 × g for 3 min, and supernatants were collected as cytosolic fractions.
T223 32104-32212 Sentence denotes Lysates were centrifuged at 4 °C, 500 × g for 3 min, and supernatants were collected as cytosolic fractions.
T21710 32213-32404 Sentence denotes Pellets were incubated in Buffer C (20 mM HEPES pH7.9, 420 mM NaCl, 1.5 mM MgCl2, 25% glycerol, 0.5 mM PMSF, 0.2 mM EDTA, 0.5 mM DTT, complete protease inhibitor cocktail) at 4 °C for 10 min.
T224 32213-32404 Sentence denotes Pellets were incubated in Buffer C (20 mM HEPES pH7.9, 420 mM NaCl, 1.5 mM MgCl2, 25% glycerol, 0.5 mM PMSF, 0.2 mM EDTA, 0.5 mM DTT, complete protease inhibitor cocktail) at 4 °C for 10 min.
T21711 32405-32508 Sentence denotes Supernatants were collected as nuclear fractions following a centrifuge at 4 °C, 13,800 × g for 10 min.
T225 32405-32508 Sentence denotes Supernatants were collected as nuclear fractions following a centrifuge at 4 °C, 13,800 × g for 10 min.
T22036 32510-32541 Sentence denotes Luciferase Reporter Gene Assays
T226 32510-32541 Sentence denotes Luciferase Reporter Gene Assays
T22037 32542-32614 Sentence denotes Luciferase reporter gene assays were performed as previously described6.
T227 32542-32614 Sentence denotes Luciferase reporter gene assays were performed as previously described6.
T22038 32615-32801 Sentence denotes Briefly, cells were cotransfected at a ratio of 10:1 with various promoter-driven firefly luciferase constructs to the Renilla luciferase pTKRL plasmid, together with indicated plasmids.
T228 32615-32801 Sentence denotes Briefly, cells were cotransfected at a ratio of 10:1 with various promoter-driven firefly luciferase constructs to the Renilla luciferase pTKRL plasmid, together with indicated plasmids.
T22039 32802-32884 Sentence denotes Cells were cultured for 1–2 days and then stimulated in triplicate before harvest.
T229 32802-32884 Sentence denotes Cells were cultured for 1–2 days and then stimulated in triplicate before harvest.
T22040 32885-32947 Sentence denotes Lysates were analyzed using the Dual-Luciferase Kit (Promega).
T230 32885-32947 Sentence denotes Lysates were analyzed using the Dual-Luciferase Kit (Promega).
T22214 32949-32985 Sentence denotes Chromatin Immunoprecipitation (ChIP)
T231 32949-32985 Sentence denotes Chromatin Immunoprecipitation (ChIP)
T22215 32986-33037 Sentence denotes ChIP assays was performed as previously described6.
T232 32986-33037 Sentence denotes ChIP assays was performed as previously described6.
T22216 33038-33167 Sentence denotes The primers used to amplify the promoter region adjacent to the κB sites of IL8 and NFKBIA, as well as ACTB have been described6.
T233 33038-33167 Sentence denotes The primers used to amplify the promoter region adjacent to the κB sites of IL8 and NFKBIA, as well as ACTB have been described6.
T22324 33169-33198 Sentence denotes Immunofluorescence Microscopy
T234 33169-33198 Sentence denotes Immunofluorescence Microscopy
T22325 33199-33258 Sentence denotes Confocal microscopy was performed as previously described6.
T235 33199-33258 Sentence denotes Confocal microscopy was performed as previously described6.
T22326 33259-33356 Sentence denotes Briefly, cells were fixed with 4 % paraformaldehyde in PBS and then Cellspin mounted onto slides.
T236 33259-33356 Sentence denotes Briefly, cells were fixed with 4 % paraformaldehyde in PBS and then Cellspin mounted onto slides.
T237 33357-33643 Sentence denotes The fixed cells were then permeabilized with 0.05 % Triton X-100 in PBS and stained with FITC-conjugated rabbit anti-RPS3 antibodies (Primm Biotech), or AlexaFluor 594-conjugated rat anti-Flag antibodies (BD) for 40 min together with 1 μg/ml of Hoechst 33342 (Sigma) for 5 min at 25 °C.
T22327 33357-33739 Sentence denotes The fixed cells were then permeabilized with 0.05 % Triton X-100 in PBS and stained with FITC-conjugated rabbit anti-RPS3 antibodies (Primm Biotech), or AlexaFluor 594-conjugated rat anti-Flag antibodies (BD) for 40 min together with 1 μg/ml of Hoechst 33342 (Sigma) for 5 min at 25 °C. The slides were then rinsed with PBS three times and cover mounted for fluorescence microscopy.
T238 33644-33739 Sentence denotes The slides were then rinsed with PBS three times and cover mounted for fluorescence microscopy.
T22542 33741-33775 Sentence denotes Immunoprecipitation and immunoblot
T239 33741-33775 Sentence denotes Immunoprecipitation and immunoblot
T22543 33776-34105 Sentence denotes The cells were harvested and lysed on ice by 0.4 ml of the modified RIPA buffer (50 mM Tris-HCl [pH 7.4], 1% NP-40, 0.25% Na-deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1 mM Na3VO4, 1 mM NaF) supplemented with 1 × protease inhibitor cocktail (Roche) and 1 × phosphatase inhibitor cocktail set I (EMD Biosciences) for 30 min.
T240 33776-34105 Sentence denotes The cells were harvested and lysed on ice by 0.4 ml of the modified RIPA buffer (50 mM Tris-HCl [pH 7.4], 1% NP-40, 0.25% Na-deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1 mM Na3VO4, 1 mM NaF) supplemented with 1 × protease inhibitor cocktail (Roche) and 1 × phosphatase inhibitor cocktail set I (EMD Biosciences) for 30 min.
T22544 34106-34197 Sentence denotes The lysates were centrifuged at 10,000 × g at 4 °C for 10 min to remove insoluble material.
T241 34106-34197 Sentence denotes The lysates were centrifuged at 10,000 × g at 4 °C for 10 min to remove insoluble material.
T242 34198-34403 Sentence denotes After normalizing protein concentrations, lysates were subjected to immunoprecipitation by adding 10 mg/ml appropriate antibody plus 30 ml of protein G-agarose (Roche), and rotated for at least 2 h at 4°C.
T22545 34198-34551 Sentence denotes After normalizing protein concentrations, lysates were subjected to immunoprecipitation by adding 10 mg/ml appropriate antibody plus 30 ml of protein G-agarose (Roche), and rotated for at least 2 h at 4°C. The precipitates were washed at least five times with cold lysis buffer followed by separation by SDS-PAGE under reduced and denaturing conditions.
T243 34404-34551 Sentence denotes The precipitates were washed at least five times with cold lysis buffer followed by separation by SDS-PAGE under reduced and denaturing conditions.
T22546 34552-34697 Sentence denotes Nitrocellulose membranes were blocked in 5 % nonfat milk in 0.1 % PBS-Tween 20 (PBS-T), probed with specific antibodies as described previously6.
T244 34552-34697 Sentence denotes Nitrocellulose membranes were blocked in 5 % nonfat milk in 0.1 % PBS-Tween 20 (PBS-T), probed with specific antibodies as described previously6.
T22547 34698-34868 Sentence denotes For immunoblotting of phosphorylated proteins, gels were transferred to methanol-treated polyvinylidene chloride membranes, retreated with methanol, and dried for 30 min.
T245 34698-34868 Sentence denotes For immunoblotting of phosphorylated proteins, gels were transferred to methanol-treated polyvinylidene chloride membranes, retreated with methanol, and dried for 30 min.
T22548 34869-35026 Sentence denotes Blots were blocked in 5 % bovine serum albumin in 0.1 % Tris buffered saline-Tween 20 (TBS-T), and probed with specific antibodies as described previously46.
T246 34869-35026 Sentence denotes Blots were blocked in 5 % bovine serum albumin in 0.1 % Tris buffered saline-Tween 20 (TBS-T), and probed with specific antibodies as described previously46.
T22549 35027-35129 Sentence denotes Bands were imaged by the Super Signaling system (Pierce) according to the manufacturer's instructions.
T247 35027-35129 Sentence denotes Bands were imaged by the Super Signaling system (Pierce) according to the manufacturer's instructions.
T23103 35131-35136 Sentence denotes ELISA
T248 35131-35136 Sentence denotes ELISA
T23104 35137-35338 Sentence denotes The amount of IL-8 present in supernatants collected from Jurkat cell culture was measured using a Human Interleukin-8 ELISA Ready-SET-Go kit (eBioscience) according to the manufacturer's instructions.
T249 35137-35338 Sentence denotes The amount of IL-8 present in supernatants collected from Jurkat cell culture was measured using a Human Interleukin-8 ELISA Ready-SET-Go kit (eBioscience) according to the manufacturer's instructions.
T23193 35340-35355 Sentence denotes Cell Infections
T250 35340-35355 Sentence denotes Cell Infections
T23194 35356-35451 Sentence denotes HeLa cells were infected with E. coli O157:H7 or C. rodentium strains as described previously9.
T251 35356-35451 Sentence denotes HeLa cells were infected with E. coli O157:H7 or C. rodentium strains as described previously9.
T23239 35453-35473 Sentence denotes Immunohistochemistry
T252 35453-35473 Sentence denotes Immunohistochemistry
T23240 35474-35562 Sentence denotes Gnotobiotic piglets were infected with E. coli O157:H7 strains as described previously9.
T253 35474-35562 Sentence denotes Gnotobiotic piglets were infected with E. coli O157:H7 strains as described previously9.
T23241 35563-35638 Sentence denotes Spiral colon specimens were collected at necropsy and embedded in paraffin.
T254 35563-35638 Sentence denotes Spiral colon specimens were collected at necropsy and embedded in paraffin.
T23242 35639-35752 Sentence denotes Paraffin sectioning and immunohistochemical staining using phospho-RPS3 antibody were performed by Histoserv Inc.
T255 35639-35752 Sentence denotes Paraffin sectioning and immunohistochemical staining using phospho-RPS3 antibody were performed by Histoserv Inc.
T256 35754-35776 Sentence denotes Supplementary Material
T257 35777-35778 Sentence denotes 1