Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.5.1.18 (glutathione S-transferase)
22,582 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Raf-1 is a serine/threonine kinase which is essential in cell growth and differentiation. Tyrosine kinase oncogenes and receptors and p21ras can activate Raf-1, and recent studies have suggested that Raf-1 functions upstream of MEK (MAP/ERK kinase), which phosphorylates and activates ERK. To determine whether or not Raf-1 directly activates MEK, we developed an in vitro assay with purified recombinant proteins. Epitope-tagged versions of Raf-1 and MEK and kinase-inactive mutants of each protein were expressed in Sf9 cells, and ERK1 was purified as a glutathione S-transferase fusion protein from bacteria. Raf-1 purified from Sf9 cells which had been coinfected with v-src or v-ras was able to phosphorylate kinase-active and kinase-inactive MEK. A kinase-inactive version of Raf-1 purified from cells that had been coinfected with v-src or v-ras was not able to phosphorylate MEK. Raf-1 phosphorylation of MEK activated it, as judged by its ability to stimulate the phosphorylation of myelin basic protein by glutathione S-transferase-ERK1. We conclude that MEK is a direct substrate of Raf-1 and that the activation of MEK by Raf-1 is due to phosphorylation by Raf-1, which is sufficient for MEK activation. We also tested the ability of protein kinase C to activate Raf-1 and found that, although protein kinase C phosphorylation of Raf-1 was able to stimulate its autokinase activity, it did not stimulate its ability to phosphorylate MEK.
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PMID:Reconstitution of the Raf-1-MEK-ERK signal transduction pathway in vitro. 841 57

Association of the condensin multiprotein complex with chromatin is required for chromosome condensation at mitosis. What regulates condensin targeting to chromatin is largely unknown. We previously showed that the nuclear A kinase-anchoring protein, AKAP95, is implicated in chromosome condensation. We demonstrate here that AKAP95 acts as a targeting protein for human chromosome-associated protein (hCAP)-D2/Eg7, a component of the human condensin complex, to chromosomes. In HeLa cell mitotic extract, AKAP95 redistributes from the nuclear matrix to chromatin. When association of AKAP95 with chromatin is prevented, the chromatin does not condense. Condensation is rescued by a recombinant AKAP95 peptide containing the 306 COOH-terminal amino acids of AKAP95. Recombinant AKAP95 binds chromatin and elicits recruitment of Eg7 to chromosomes in a concentration-dependent manner. Amount of Eg7 recruited correlates with extent of chromosome condensation: resolution into distinct chromosomes is obtained only when near-endogenous levels of Eg7 are recruited. Eg7 and AKAP95 immunofluorescently colocalize to the central region of methanol-fixed metaphase chromosomes. GST pull-down data also suggest that AKAP95 recruits several condensin subunits. The results implicate AKAP95 as a receptor that assists condensin targeting to chromosomes.
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PMID:A kinase-anchoring protein (AKAP)95 recruits human chromosome-associated protein (hCAP)-D2/Eg7 for chromosome condensation in mitotic extract. 1079 67

Inhibitor 1 (I-1) is a protein inhibitor of protein phosphatase 1 (PP1), a major eukaryotic Ser/Thr phosphatase. Nonphosphorylated I-1 is inactive, whereas phosphorylated I-1 is a potent PP1 inhibitor. I-1 is phosphorylated in vivo on Thr(35) and Ser(67). Thr(35) is phosphorylated by cAMP-dependent protein kinase (A kinase), and Thr(35)-phosphorylated I-1 inhibits PP1. Until now the kinase that phosphorylates Ser(67) had not been identified and the physiological role of Ser(67) phosphorylation was unknown. In this study we detected a high level of kinase activity in brain extract when a glutathione S-transferase (GST) fusion I-1 mutant containing an Ala substituted for Thr(35) [GST-I-1(T35A)] was used as the substrate. GST-I-1(T35A) kinase and neuronal cdc2-like protein kinase (NCLK) in the brain extract could not be separated from each other by a series of sequential chromatographies. GST-I-1(T35A) kinase immunoprecipitated with anti-NCLK antibody from kinase-active column fractions. Purified NCLK-phosphorylated GST-I-1(T35A) and I-1 (0.7 mole of phosphate per mole of I-1). HPLC phosphopeptide mapping, amino acid sequencing, and site-directed mutagenesis determined that NCLK phosphorylates Ser(67) of I-1. NCLK-phosphorylated I-1 and I-1(T35A) inhibited PP1 with IC(50) values approximately 9.5 and 13. 8 nM, respectively. When compared, A kinase-phosphorylated I-1 was only approximately 1.2 times more inhibitory than NCLK-phosphorylated I-1. Our data indicate that NCLK is a potential in vivo I-1 kinase and that Thr(35) and Ser(67) phosphorylation independently activate I-1.
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PMID:Ser67-phosphorylated inhibitor 1 is a potent protein phosphatase 1 inhibitor. 1081 8

Protein kinase A (PKA) is targeted to discrete subcellular locations close to its intended substrates through interaction with A kinase-anchoring proteins (AKAPs). Ion channels represent a diverse and important group of kinase substrates, and it has been shown that membrane targeting of PKA through association with AKAPs facilitates PKA-mediated phosphorylation and regulation of several classes of ion channel. Here, we investigate the effect of AKAP79, a membrane-associated multivalent-anchoring protein, upon the function and modulation of the strong inwardly rectifying potassium channel, Kir2.1. Functionally, the presence of AKAP79 enhanced the response of Kir2.1 to elevated intracellular cAMP, suggesting a requirement for a pool of PKA anchored close to the channel. Antibodies directed against a hemagglutinin epitope tag on Kir2.1 coimmunoprecipitated AKAP79, indicating that the two proteins exist together in a complex within intact cells. In support of this, glutathione S-transferase fusion proteins of both the intracellular N and C domains of Kir2.1 isolated AKAP79 from cell lysates, while glutathione S-transferase alone failed to interact with AKAP79. Together, these findings suggest that AKAP79 associates directly with the Kir2.1 ion channel and may serve to anchor kinase enzymes in close proximity to key channel phosphorylation sites.
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PMID:Targeting of an A kinase-anchoring protein, AKAP79, to an inwardly rectifying potassium channel, Kir2.1. 1128 23

We have previously reported that the Jak2 tyrosine kinase but not Jak1 is tyrosine phosphorylated in the absence of IL-3 in Bcr-Abl positive M3.16 cells, which are rendered IL-3 independent by BCR-ABL gene expression. We have explored the involvement of Jak2 tyrosine phosphorylation in Bcr-Abl oncogenic effects. Our results indicate that Jak2 became tyrosine-phosphorylated in a number of cell lines expressing Bcr-Abl, when maintained in medium lacking IL-3, whereas Bcr-Abl negative cells lacked Jak2 tyrosine phosphorylation. Jak2 was poorly tyrosine-phosphorylated in cells expressing the SH2 deletion mutant of Bcr-Abl compared to either wild-type Bcr-Abl or its SH3 deletion mutant. Moreover, tyrosine phosphorylation of Jak2 by Bcr-Abl was inhibited by the Abl tyrosine kinase inhibitor, STI 571, in a dose-dependent manner. This inhibition of Bcr-Abl kinase by the drug did not interfere with the ability of Jak2 and Bcr-Abl to form a complex. Studies with deletion mutants of Bcr-Abl indicated that the C-terminal domain of Abl within Bcr-Abl was involved in complex formation with Jak2. Similarly, GST-Abl pull-down assays confirmed the strong binding to Jak2 by the C-terminus of Abl. Jak2 peptide substrate studies indicated that the Bcr-Abl and Abl tyrosine kinases specifically phosphorylated Y1007 of Jak2 but only poorly phosphorylated Y1008. Phosphorylation of Y1007 of Jak2 is known to be critical for its tyrosine kinase activation. Tyrosine residue 1007 of Jak2 was phosphorylated in 32Dp210 cells as measured by Western blotting with a phosphotyrosine 1007 sequence-specific antibody. A kinase-inactive Jak2 mutant blocked the colony forming ability of K562 cells. Tumor formation of K562 cells in nude mice was similarly inhibited by this kinase-inactive Jak2 mutant. This inhibition was independent of Stat5 tyrosine phosphorylation. Furthermore, tyrosine-phosphorylated Jak2 was detected in blood cells from CML patients in blast crisis but not in a normal marrow sample. In summary, these findings provide strong evidence that the Jak2 tyrosine kinase is a critical factor in Bcr-Abl malignant transformation.
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PMID:Involvement of Jak2 tyrosine phosphorylation in Bcr-Abl transformation. 1159 27

Parathyroid hormone inhibits sodium-phosphate cotransport in proximal renal tubule cells through activation of several kinases. We tested the hypothesis that the activity of these kinases was coordinated by an A kinase anchoring protein (AKAP) by demonstrating that the type II sodium-phosphate cotransporter (NaPi-4) physically associated with an AKAP and that this association was necessary for regulation of phosphate transport by parathyroid hormone. Immunoprecipitation with anti-NaPi-4 antiserum and glutathione S-transferase pull-down with GST-NaPi-4 showed that NaPi-4 associated with AKAP79, protein kinase A catalytic and regulatory subunits, and the parathyroid hormone receptor in opossum kidney cells. When the regulatory subunit of protein kinase A was uncoupled from the AKAP by a competing peptide, parathyroid hormone lost the ability to inhibit phosphate transport. This result was confirmed by co-transfecting HEK293 cells with the sodium-phosphate cotransporter and wild type AKAP, a mutant AKAP79, or the empty vector. 8-Bromo-cAMP was able to inhibit phosphate transport in cells expressing the wild type AKAP79 but not empty vector or mutant AKAP79. We conclude that parathyroid hormone inhibits proximal renal tubule sodium-phosphate cotransport through a signaling complex dependent upon an AKAP.
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PMID:Parathyroid hormone regulation of type II sodium-phosphate cotransporters is dependent on an A kinase anchoring protein. 1249 50

A kinase activity that phosphorylated myelin basic protein in vitro was detected in RalA and RalB immunoprecipitates from human platelets. Protein-protein interaction studies using recombinant GST-RalA, GST-RalB and GST-cH-Ras confirmed that the kinase specifically associates with the Ral GTPase. The Ral Interacting Protein 1 (RIP1), a GTPase Activating Protein (GAP) for Cdc42 and Rac1, was found to be the preferred substrate for the Ral Interacting Kinase (RIK). Phosphoamino acid analysis demonstrated that RIK phosphorylated serine residue in RIP1. The Ral-RIK interaction was not dependent on the guanine nucleotide status of Ral. RIK was detected in a variety of rat tissues with testis containing the highest and skeletal muscle the lowest activity. In-gel kinase renaturation assay using RIP1 as the substrate demonstrated that the kinase activity was associated with polypeptides of molecular mass of approximately 36-40 kDa and was detected in most rat tissues with a prominent 38 kDa band in testis and a 40 kDa band in brain. Human platelets contained a single band of approximately 36 kDa. RIK was distinct from MAPKs, CDKs, cyclic AMP dependent protein kinase and Ca2+/calmodulin dependent kinases. To demonstrate in vivo interaction, the endogenous Ral-RIK complex was isolated using a calmodulin affinity column. The Ral-RIK complex co-eluted from this column upon washing with a 13 residue peptide that encompasses the calmodulin-binding domain in RalA. The data suggest that RIK is a serine specific kinase that phosphorylates RIP1 and is constitutively associated with Ral. The current study provides additional support for a link between Ral and the Cdc42/Rac1 signalling pathways in the cell.
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PMID:A serine kinase associates with the RAL GTPase and phosphorylates RAL-interacting protein 1. 1694 34

Myosin VI (MVI) is a unique motor protein moving towards the minus end of actin filaments unlike other known myosins. Its important role has recently been postulated for striated muscle and myogenic cells. Since MVI functions through interactions of C-terminal globular tail (GT) domain with tissue specific partners, we performed a search for MVI partners in myoblasts and myotubes using affinity chromatography with GST-tagged MVI-GT domain as a bait. A kinase anchoring protein 9 (AKAP9), a regulator of PKA activity, was identified by means of mass spectrometry as a possible MVI interacting partner both in undifferentiated and differentiating myoblasts and in myotubes. Coimmunoprecipitation and proximity ligation assay confirmed that both proteins could interact. MVI and AKAP9 colocalized at Rab5 containing early endosomes. Similarly to MVI, the amount of AKAP9 decreased during myoblast differentiation. However, in MVI-depleted cells, both cAMP and PKA levels were increased and a change in the MVI motor-dependent AKAP9 distribution was observed. Moreover, we found that PKA phosphorylated MVI-GT domain, thus implying functional relevance of MVI-AKAP9 interaction. We postulate that this novel interaction linking MVI with the PKA pathway could be important for targeting AKAP9-PKA complex within cells and/or providing PKA to phosphorylate MVI tail domain.
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PMID:A Kinase Anchoring Protein 9 Is a Novel Myosin VI Binding Partner That Links Myosin VI with the PKA Pathway in Myogenic Cells. 2596 Oct 40

Overexpression of Aurora A kinase occurs in certain types of cancer, and therefore results in chromosome instability and phosphorylation-mediated ubiquitylation and degradation of p53 for tumorigenesis. The high-risk subtype human papillomavirus (HPV)16 early oncoprotein E6 is a major contributor inducing host cell immortalization and transformation through interaction with a number of cellular factors. In the present study, co-immunoprecipitation, glutathione S-transferase pull-down and immunostaining were used to show that HPV16 E6 and Aurora A bind to each other in vivo and in vitro. Western blotting and reverse transcription-polymerase chain reaction were used to reveal that HPV16 E6 inhibited cell apoptosis by stabilizing Aurora A expression. The present study may report a new mechanism for the involvement of HPV16 E6 in carcinogenesis, as HPV16 E6 elevates Aurora A expression and the latter may be a common target for oncogenic viruses that result in cell carcinogenesis.
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PMID:HPV16 E6 upregulates Aurora A expression. 2744 42

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