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bionlp-st-ge-2016-uniprot

2_test

{"project":"2_test","denotations":[{"id":"21399639-20041225-74765175","span":{"begin":140,"end":141},"obj":"20041225"},{"id":"21399639-20041225-74765175","span":{"begin":140,"end":141},"obj":"20041225"},{"id":"21399639-12657048-74765176","span":{"begin":948,"end":950},"obj":"12657048"},{"id":"21399639-12657048-74765176","span":{"begin":948,"end":950},"obj":"12657048"},{"id":"21399639-20041225-74765177","span":{"begin":1135,"end":1136},"obj":"20041225"},{"id":"21399639-20041225-74765177","span":{"begin":1135,"end":1136},"obj":"20041225"}],"text":"NleH1 steers the IKKβ substrate specificities\nNleH1 is an autophosphorylated serine-threonine kinase, which depends on the lysine 159 (K159)9. 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). 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. Wild-type NleH1 expression significantly reduced TNF-induced RPS3 S209 phosphorylation, whereas the K159A mutant failed to do so (Fig. 7b). Thus NleH1 kinase activity is required to protect RPS3 from IKKβ-mediated phosphorylation.\nCitrobacter 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. 9). We assayed RPS3 S209 phosphorylation in HeLa cells infected with different C. rodentium strains. In uninfected cells, TNF-treatment stimulated a ∼3.5-fold increase in RPS3 S209 phosphorylation (Fig. 7c). Such augmentation of RPS3 phosphorylation was reduced by about 60% by wild-type C. rodentium infection (Fig. 7c). However, RPS3 phosphorylation was enhanced when cells were infected with a C. rodentium strain lacking NleH (Fig. 7c, ΔnleH). 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. 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). Collectively, these results demonstrate that NleH1 kinase activity is required to block RPS3 S209 phosphorylation.\nWe 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). 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). RPS3 nuclear accumulation was substantially retarded by infecting cells with wild-type E. coli O157:H7 (Fig. 7d). 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). 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). Thus, during infection NleH1 is sufficiently potent to inhibit RPS3 nuclear translocation even in cells expressing constitutively-activated IKKβ.\nWe examined whether NleH1 could directly phosphorylate IKKβ thus inhibiting IKKβ-mediated RPS3 S209 phosphorylation. 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. However, we did not observe any detectable 32P incorporation in IKKβ (Supplementary Fig. 13), thus ruling out this possibility.\nWe then tested the hypothesis that NleH1 could alter the IKKβ substrate specificities. To this end, we performed in vitro kinase assays using both CK2 and IKK substrates for IKKβ. 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. 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). Control experiments revealed no NleH1-mediated phosphorylation or autophosphorylation of RPS3 or GST-IκBα (Fig. 7e). 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."}

pmc-enju-pas

bionlp-st-ge-2016-coref

{"project":"bionlp-st-ge-2016-coref","denotations":[{"id":"T15644","span":{"begin":3753,"end":3755},"obj":"Anaphor"},{"id":"T15645","span":{"begin":3669,"end":3673},"obj":"Antecedent"},{"id":"T15640","span":{"begin":751,"end":753},"obj":"Anaphor"},{"id":"T15641","span":{"begin":669,"end":676},"obj":"Antecedent"},{"id":"T15642","span":{"begin":2080,"end":2099},"obj":"Anaphor"},{"id":"T15643","span":{"begin":2018,"end":2023},"obj":"Antecedent"}],"relations":[{"id":"R12627","pred":"boundBy","subj":"T15640","obj":"T15641"},{"id":"R12628","pred":"boundBy","subj":"T15642","obj":"T15643"},{"id":"R12629","pred":"boundBy","subj":"T15644","obj":"T15645"}],"namespaces":[{"prefix":"_base","uri":"https://bionlp.dbcls.jp/ontology/ge.owl#"}],"text":"NleH1 steers the IKKβ substrate specificities\nNleH1 is an autophosphorylated serine-threonine kinase, which depends on the lysine 159 (K159)9. 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). 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. Wild-type NleH1 expression significantly reduced TNF-induced RPS3 S209 phosphorylation, whereas the K159A mutant failed to do so (Fig. 7b). Thus NleH1 kinase activity is required to protect RPS3 from IKKβ-mediated phosphorylation.\nCitrobacter 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. 9). We assayed RPS3 S209 phosphorylation in HeLa cells infected with different C. rodentium strains. In uninfected cells, TNF-treatment stimulated a ∼3.5-fold increase in RPS3 S209 phosphorylation (Fig. 7c). Such augmentation of RPS3 phosphorylation was reduced by about 60% by wild-type C. rodentium infection (Fig. 7c). However, RPS3 phosphorylation was enhanced when cells were infected with a C. rodentium strain lacking NleH (Fig. 7c, ΔnleH). 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. 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). Collectively, these results demonstrate that NleH1 kinase activity is required to block RPS3 S209 phosphorylation.\nWe 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). 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). RPS3 nuclear accumulation was substantially retarded by infecting cells with wild-type E. coli O157:H7 (Fig. 7d). 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). 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). Thus, during infection NleH1 is sufficiently potent to inhibit RPS3 nuclear translocation even in cells expressing constitutively-activated IKKβ.\nWe examined whether NleH1 could directly phosphorylate IKKβ thus inhibiting IKKβ-mediated RPS3 S209 phosphorylation. 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. However, we did not observe any detectable 32P incorporation in IKKβ (Supplementary Fig. 13), thus ruling out this possibility.\nWe then tested the hypothesis that NleH1 could alter the IKKβ substrate specificities. To this end, we performed in vitro kinase assays using both CK2 and IKK substrates for IKKβ. 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. 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). Control experiments revealed no NleH1-mediated phosphorylation or autophosphorylation of RPS3 or GST-IκBα (Fig. 7e). 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."}

bionlp-st-ge-2016-spacy-parsed

GO-BP

GO-MF

{"project":"GO-MF","denotations":[{"id":"T16729","span":{"begin":468,"end":483},"obj":"http://purl.obolibrary.org/obo/GO_0016301"},{"id":"T16730","span":{"begin":779,"end":794},"obj":"http://purl.obolibrary.org/obo/GO_0016301"},{"id":"T16731","span":{"begin":1621,"end":1636},"obj":"http://purl.obolibrary.org/obo/GO_0016301"},{"id":"T16732","span":{"begin":1987,"end":2002},"obj":"http://purl.obolibrary.org/obo/GO_0016301"},{"id":"T16733","span":{"begin":1068,"end":1087},"obj":"http://purl.obolibrary.org/obo/GO_0045289"},{"id":"T16734","span":{"begin":1068,"end":1087},"obj":"http://purl.obolibrary.org/obo/GO_0047077"},{"id":"T16735","span":{"begin":1068,"end":1087},"obj":"http://purl.obolibrary.org/obo/GO_0047712"},{"id":"T16736","span":{"begin":1068,"end":1087},"obj":"http://purl.obolibrary.org/obo/GO_0050248"},{"id":"T16737","span":{"begin":1068,"end":1087},"obj":"http://purl.obolibrary.org/obo/GO_0050397"},{"id":"T16738","span":{"begin":3611,"end":3614},"obj":"http://purl.obolibrary.org/obo/GO_0008384"},{"id":"T16739","span":{"begin":3778,"end":3781},"obj":"http://purl.obolibrary.org/obo/GO_0008384"},{"id":"T16740","span":{"begin":3972,"end":3975},"obj":"http://purl.obolibrary.org/obo/GO_0008384"}],"text":"NleH1 steers the IKKβ substrate specificities\nNleH1 is an autophosphorylated serine-threonine kinase, which depends on the lysine 159 (K159)9. 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). 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. Wild-type NleH1 expression significantly reduced TNF-induced RPS3 S209 phosphorylation, whereas the K159A mutant failed to do so (Fig. 7b). Thus NleH1 kinase activity is required to protect RPS3 from IKKβ-mediated phosphorylation.\nCitrobacter 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. 9). We assayed RPS3 S209 phosphorylation in HeLa cells infected with different C. rodentium strains. In uninfected cells, TNF-treatment stimulated a ∼3.5-fold increase in RPS3 S209 phosphorylation (Fig. 7c). Such augmentation of RPS3 phosphorylation was reduced by about 60% by wild-type C. rodentium infection (Fig. 7c). However, RPS3 phosphorylation was enhanced when cells were infected with a C. rodentium strain lacking NleH (Fig. 7c, ΔnleH). 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. 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). Collectively, these results demonstrate that NleH1 kinase activity is required to block RPS3 S209 phosphorylation.\nWe 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). 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). RPS3 nuclear accumulation was substantially retarded by infecting cells with wild-type E. coli O157:H7 (Fig. 7d). 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). 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). Thus, during infection NleH1 is sufficiently potent to inhibit RPS3 nuclear translocation even in cells expressing constitutively-activated IKKβ.\nWe examined whether NleH1 could directly phosphorylate IKKβ thus inhibiting IKKβ-mediated RPS3 S209 phosphorylation. 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. However, we did not observe any detectable 32P incorporation in IKKβ (Supplementary Fig. 13), thus ruling out this possibility.\nWe then tested the hypothesis that NleH1 could alter the IKKβ substrate specificities. To this end, we performed in vitro kinase assays using both CK2 and IKK substrates for IKKβ. 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. 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). Control experiments revealed no NleH1-mediated phosphorylation or autophosphorylation of RPS3 or GST-IκBα (Fig. 7e). 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."}

GO-CC

{"project":"GO-CC","denotations":[{"id":"T16741","span":{"begin":621,"end":626},"obj":"http://purl.obolibrary.org/obo/GO_0005623"},{"id":"T16742","span":{"begin":1184,"end":1189},"obj":"http://purl.obolibrary.org/obo/GO_0005623"},{"id":"T16743","span":{"begin":1250,"end":1255},"obj":"http://purl.obolibrary.org/obo/GO_0005623"},{"id":"T16744","span":{"begin":1505,"end":1510},"obj":"http://purl.obolibrary.org/obo/GO_0005623"},{"id":"T16745","span":{"begin":2487,"end":2492},"obj":"http://purl.obolibrary.org/obo/GO_0005623"},{"id":"T16746","span":{"begin":2686,"end":2691},"obj":"http://purl.obolibrary.org/obo/GO_0005623"},{"id":"T16747","span":{"begin":2807,"end":2812},"obj":"http://purl.obolibrary.org/obo/GO_0005623"},{"id":"T16748","span":{"begin":2953,"end":2958},"obj":"http://purl.obolibrary.org/obo/GO_0005623"},{"id":"T16749","span":{"begin":4317,"end":4321},"obj":"http://purl.obolibrary.org/obo/GO_0018995"}],"text":"NleH1 steers the IKKβ substrate specificities\nNleH1 is an autophosphorylated serine-threonine kinase, which depends on the lysine 159 (K159)9. 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). 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. Wild-type NleH1 expression significantly reduced TNF-induced RPS3 S209 phosphorylation, whereas the K159A mutant failed to do so (Fig. 7b). Thus NleH1 kinase activity is required to protect RPS3 from IKKβ-mediated phosphorylation.\nCitrobacter 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. 9). We assayed RPS3 S209 phosphorylation in HeLa cells infected with different C. rodentium strains. In uninfected cells, TNF-treatment stimulated a ∼3.5-fold increase in RPS3 S209 phosphorylation (Fig. 7c). Such augmentation of RPS3 phosphorylation was reduced by about 60% by wild-type C. rodentium infection (Fig. 7c). However, RPS3 phosphorylation was enhanced when cells were infected with a C. rodentium strain lacking NleH (Fig. 7c, ΔnleH). 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. 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). Collectively, these results demonstrate that NleH1 kinase activity is required to block RPS3 S209 phosphorylation.\nWe 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). 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). RPS3 nuclear accumulation was substantially retarded by infecting cells with wild-type E. coli O157:H7 (Fig. 7d). 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). 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). Thus, during infection NleH1 is sufficiently potent to inhibit RPS3 nuclear translocation even in cells expressing constitutively-activated IKKβ.\nWe examined whether NleH1 could directly phosphorylate IKKβ thus inhibiting IKKβ-mediated RPS3 S209 phosphorylation. 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. However, we did not observe any detectable 32P incorporation in IKKβ (Supplementary Fig. 13), thus ruling out this possibility.\nWe then tested the hypothesis that NleH1 could alter the IKKβ substrate specificities. To this end, we performed in vitro kinase assays using both CK2 and IKK substrates for IKKβ. 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. 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). Control experiments revealed no NleH1-mediated phosphorylation or autophosphorylation of RPS3 or GST-IκBα (Fig. 7e). 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."}

sentences

events-check-again

bionlp-st-ge-2016-reference-tees

bionlp-st-ge-2016-reference

test2

PMC:3062687 / 19647-23992 JSONTXT

Annnotations TAB JSON ListView MergeView

PMC:3062687 / 19647-23992 JSON TXT