Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.5.1.18 (glutathione S-transferase)
 22,582 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Apoptosis is an essential mechanism for the maintenance of somatic tissues, and when dysregulated can lead to numerous pathological conditions. G proteins regulate apoptosis in addition to other cellular functions, but the roles of specific G proteins in apoptosis signaling are not well characterized. Galpha12 stimulates protein phosphatase 2A (PP2A), a serine/threonine phosphatase that modulates essential signaling pathways, including apoptosis. Herein, we examined whether Galpha12 regulates apoptosis in epithelial cells. Inducible expression of Galpha12 or constitutively active (QL)alpha12 in Madin-Darby canine kidney cells led to increased apoptosis with expression of QLalpha12, but not Galpha12. Inducing QLalpha12 led to degradation of the anti-apoptotic protein Bcl-2 (via the proteasome pathway), increased JNK activity, and up-regulated IkappaBalpha protein levels, a potent stimulator of apoptosis. Furthermore, the QLalpha12-stimulated activation of JNK was blocked by inhibiting PP2A. To characterize endogenous Galpha12 signaling pathways, non-transfected MDCK-II and HEK293 cells were stimulated with thrombin. Thrombin activated endogenous Galpha12 (confirmed by GST-tetratricopeptide repeat (TPR) pull-downs) and stimulated apoptosis in both cell types. The mechanisms of thrombin-stimulated apoptosis through endogenous Galpha12 were nearly identical to the mechanisms identified in QLalpha12-MDCK cells and included loss of Bcl-2, JNK activation, and up-regulation of IkappaBalpha. Knockdown of the PP2A catalytic subunit in HEK293 cells inhibited thrombin-stimulated apoptosis, prevented JNK activation, and blocked Bcl-2 degradation. In summary, Galpha12 has a major role in regulating epithelial cell apoptosis through PP2A and JNK activation leading to loss of Bcl-2 protein expression. Targeting these pathways in vivo may lead to new therapeutic strategies for a variety of disease processes.
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PMID:Galpha12 stimulates apoptosis in epithelial cells through JNK1-mediated Bcl-2 degradation and up-regulation of IkappaBalpha. 1756 96

The Cdc34 E2 ubiquitin (Ub) conjugating enzyme catalyzes polyubiquitination of a substrate recruited by the Skp1-Cullin 1-F-box protein-ROC1 E3 Ub ligase. Using mutagenesis studies, we now show that human Cdc34 employs distinct sites to coordinate the transfer of Ub to a substrate and the assembly of polyubiquitin chains. Mutational disruption of the conserved charged stretch (residues 143 to 153) or the acidic loop residues D102 and D103 led to accumulation of monoubiquitinated IkappaBalpha while failing to yield polyubiquitin chains, due to a catalytic defect in Ub-Ub ligation. These results suggest an ability of human Cdc34 to position the attacking Ub for assembly of polyubiquitin chains. Analysis of Cdc34N85Q and Cdc34S138A revealed severe defects of these mutants in both poly- and monoubiquitination of IkappaBalpha, supporting a role for N85 in stabilizing the oxyanion and in coordinating, along with S138, the attacking lysine for catalysis. Finally, Cdc34S95D and Cdc34(E108A/E112A) abolished both poly- and monoubiquitination of IkappaBalpha. Unexpectedly, the catalytic defects of these mutants in di-Ub synthesis can be rescued by fusion of a glutathione S-transferase moiety at E2's N terminus. These findings support the hypothesis that human Cdc34 S95 and E108/E112 are required to position the donor Ub optimally for catalysis, in a manner that might depend on E2 dimerization.
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PMID:Human Cdc34 employs distinct sites to coordinate attachment of ubiquitin to a substrate and assembly of polyubiquitin chains. 1769 85

In this study, we have identified protein kinase A-interacting protein 1 (AKIP1) as a binding partner of NF-kappaB p65 subunit, and AKIP1 enhances the NF-kappaB-mediated gene expression. AKIP1 is a nuclear protein and known to interact with the catalytic subunit of PKA (PKAc). We identified AKIP1 by a yeast two-hybrid screen using the N terminus region of p65 as bait. The interaction between AKIP1 and p65 was confirmed by glutathione S-transferase pull-down assay in vitro and immunoprecipitation-Western blotting assay in vivo. We found that the PKAc was present in the AKIP1.p65 complex and enhanced the transcriptional activity of NF-kappaB by phosphorylating p65. In a transient luciferase assay, AKIP1 cotransfection efficiently increased the transcriptional activity of NF-kappaB induced by phorbol 12-myristate 13-acetate (PMA). When AKIP1 was knocked down by RNA interference, the PMA-mediated NF-kappaB-dependent gene expression was abolished, indicating a physiological role of AKIP1. We found that PKAc, which is maintained in an inactive form by binding to IkappaBalpha and NF-kappaB in resting cells, was activated by PMA-induced signaling and could phosphorylate p65. Overexpression of AKIP1 increased the PKAc binding to p65 and enhanced the PKAc-mediated phosphorylation of p65 at Ser-276. Interestingly, this p65 phosphorylation promoted nuclear translocation of p65 and enhanced NF-kappaB transcription. In fact, we observed that AKIP1 colocalized with p65 within the cells and appeared to retain p65 in nucleus. These findings indicate a positive role of AKIP1 in NF-kappaB signaling and suggest a novel mechanism by which AKIP1 augments the transcriptional competence of NF-kappaB.
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PMID:AKIP1 enhances NF-kappaB-dependent gene expression by promoting the nuclear retention and phosphorylation of p65. 1817 62

Type I interferons (IFNs) play critical roles in the host defense by modulating gene expression through the IFN-dependent activation of STAT and NFkappaB transcription factors. Previous studies established that IFN activates NFkappaB through a classical NFkappaB pathway that results in IkappaBalpha degradation and formation of p50-containing NFkappaB complexes, as well as an alternative pathway that involves NFkappaB-inducing kinase and TRAF2, which results in the formation of p52-containing NFkappaB complexes. In this study, we examined the interaction of TRAF proteins with the type I IFN receptor. We found that TRAF2 was directly coupled to the signal-transducing IFNAR1 subunit of the IFN receptor. By immunoprecipitation, overexpression of epitope-tagged IFNAR1 constructs, and glutathione S-transferase pulldown experiments, we demonstrate that TRAF2 rapidly binds to the IFNAR1 subunit of the IFN receptor upon IFN binding. The membrane proximal half of the IFNAR1 subunit was found to directly bind TRAF2. Moreover, analysis of mouse embryo fibroblasts derived from TRAF2 knock-out mice demonstrated that TRAF2 plays a critical role in the activation of the alternative NFkappaB pathway by IFN, but not the classical NFkappaB pathway, as well as in the antiviral action of IFN. Our results place TRAF2 directly in the signaling pathway transduced through the IFNAR1 subunit of the IFN receptor. These findings provide an important insight into the molecular mechanisms by which IFN generates signals to induce its biological effects.
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PMID:The role of TRAF2 binding to the type I interferon receptor in alternative NF kappaB activation and antiviral response. 1836 56

TNFalpha activated NF-kappaB and associated regulatory factors including IKK are strongly implicated in a variety of hematological and solid tumor malignancies. We show that tautomycetin (TC) specifically inhibits activation of NF-kappaB among the three TNFalpha effectors (NF-kappaB, JNK and caspase). TC inhibited T-loop phosphorylation of IKKalpha and IKKbeta, thereby preventing degradation of the NF-kappaB inhibitor, IkappaBalpha. Co-immunoprecipitation experiments revealed that the catalytic subunit of PP1 (PP1C) was involved in the IKK complex. Pull-down analysis using recombinant GST-TNFalpha, showed that PP1C was recruited to TNFR1 together with IKK complex, RIP and TAK1 upon stimulus. These results suggest that the PP1 positively regulates the TNFalpha-induced NF-kappaB pathway at the level of IKK activation. Thus, TC might be used therapeutically to suppress the TNFalpha/NF-kappaB pathway.
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PMID:Tautomycetin suppresses the TNFalpha/NF-kappaB pathway via inhibition of IKK activation. 1894 66

Tumor necrosis factor alpha (TNF-alpha) activates the nuclear factor kappaB (NF-kappaB) signaling pathway that regulates expression of many cellular factors playing important roles in innate immune responses and inflammation in infected hosts. Poxviruses employ many strategies to inhibit NF-kappaB activation in cells. In this report, we describe a poxvirus host range protein, CP77, which blocked NF-kappaB activation by TNF-alpha. Immunofluorescence analyses revealed that nuclear translocation of NF-kappaB subunit p65 protein in TNF-alpha-treated HeLa cells was blocked by CP77. CP77 did so without blocking IkappaBalpha phosphorylation, suggesting that upstream kinase activation was not affected by CP77. Using GST pull-down, we showed that CP77 bound to the NF-kappaB subunit p65 through the N-terminal six-ankyrin-repeat region in vitro. CP77 also bound to Cullin-1 and Skp1 of the SCF complex through a C-terminal 13-amino-acid F-box-like sequence. Both regions of CP77 are required to block NF-kappaB activation. We thus propose a model in which poxvirus CP77 suppresses NF-kappaB activation by two interactions: the C-terminal F-box of CP77 binding to the SCF complex and the N-terminal six ankyrins binding to the NF-kappaB subunit p65. In this way, CP77 attenuates innate immune response signaling in cells. Finally, we expressed CP77 or a CP77 F-box deletion protein from a vaccinia virus host range mutant (VV-hr-GFP) and showed that either protein was able to rescue the host range defect, illustrating that the F-box region, which is important for NF-kappaB modulation and binding to SCF complex, is not required for CP77's host range function. Consistently, knocking down the protein level of NF-kappaB did not relieve the growth restriction of VV-hr-GFP in HeLa cells.
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PMID:Poxvirus host range protein CP77 contains an F-box-like domain that is necessary to suppress NF-kappaB activation by tumor necrosis factor alpha but is independent of its host range function. 1921 46


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