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)

The Ste20p protein kinase was immunopurified from yeast cells and analyzed in an in vitro assay system. Ste20p immune complexes exhibited autophosphorylating activity at serine and threonine residues and specifically phosphorylated a bacterially expressed glutathione S-transferase (GST) fusion of Ste11p (a mitogen-activated protein or extracellular signal-regulated kinase kinase (MEK) kinase homologue) at serine and threonine residues. In contrast, GST fusions either of Ste7p (a MEK homologue) or the beta-subunit of the mating response G-protein and immunoprecipitated Ste5p were not phosphorylated by the Ste20p immune complexes. Myelin basic protein was identified as an excellent in vitro substrate, whereas histone H1 was only poorly phosphorylated. Evidence was obtained that autophosphorylation might play a regulatory role for the in vitro kinase activity. The in vitro activity was found to be Ca(2+)-independent. Both the in vivo and in vitro activities were abolished by mutational changes of either the conserved lysine residue 649 within the ATP binding site or threonine 777 between the catalytic subdomains VII and VIII. Wild-type Ste20p and the catalytically inactive T777A mutant were identified as phosphoproteins in vivo. The phosphorylation occurred at serine and threonine residues independent of pheromone stimulation. Based on the genetically determined significance of Ste20p in pheromone signal transduction and on our in vitro studies, we propose the model that Ste20p represents a yeast MEK kinase kinase whose function is to link G-protein-coupled receptors through G beta gamma to a mitogen-activated protein kinase module.
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PMID:Molecular characterization of Ste20p, a potential mitogen-activated protein or extracellular signal-regulated kinase kinase (MEK) kinase kinase from Saccharomyces cerevisiae. 760 57

The cyclin-dependent kinase (Cdk) inhibitor p21SDI1/WAF1/CIP1 has been found to be involved in cell senescence, cell cycle arrest, and differentiation. p21SDI1 inhibits the activity of several Cdks, in contrast to other inhibitors such as p15INK4B and p16INK4A, which act on specific cyclin-Cdk complexes. Of interest were reports that p21SDI1 also bound proliferating cell nuclear antigen (PCNA), an auxiliary protein for DNA polymerase delta, and inhibited DNA replication but not DNA repair in vitro. To better understand the function of this interaction in vivo, we first determined the region of p21SDI1 that was needed for PCNA binding. Analysis of deletion mutants of p21SDI1, which covered the majority of the protein, revealed that deletion of either amino acids 142-147 or 149-154 resulted in loss of ability to bind a glutathione S-transferase-PCNA fusion protein. Site-directed mutagenesis in this region led to the identification of the PCNA binding motif RQXXMTXFYXXXR and demonstrated that mutation of either amino acid Met-147 or Phe-150 resulted in almost complete ablation of PCNA binding. Interestingly, when we determined DNA synthesis inhibitory activity of deletion mutants or point mutants that were unable to bind Cdk2 and/or PCNA, we found that loss of binding to PCNA did not affect inhibitory activity, whereas lack of Cdk2 binding greatly reduced the same. This result suggests that the primary mechanism for inhibition of DNA synthesis by p21SDI1 occurs via inhibition of Cdk activity.
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PMID:The C-terminal region of p21SDI1/WAF1/CIP1 is involved in proliferating cell nuclear antigen binding but does not appear to be required for growth inhibition. 761 95

FK506-binding proteins (FKBPs) have been identified as the cellular receptors of the immunosuppressive drugs FK506 and rapamycin. Recently, we cloned a 25-kDa FKBP family member (FKBP25) and found that FKBP25 contains a nuclear localization sequence and several potential casein kinase II phosphorylation sites. It has been previously shown that phosphorylation of proteins by casein kinase II can enhance their nuclear localization. Here we demonstrate that FKBP25 is localized to the nucleus and that a glutathione S-transferase fusion protein of FKBP25 (GST-FKBP25) can be phosphorylated by casein kinase II. Also a stable FKBP25/casein kinase II complex was formed when the GST-FKBP25 fusion protein was incubated either with purified casein kinase II or with cell lysates. Furthermore, when GST-FKBP25 was incubated with nuclear lysates, nucleolin, a major nuclear substrate of casein kinase II, was found associated with the GST-FKBP25/casein kinase II complex. Casein kinase II phosphorylation of several cytosolic and nuclear substrates, including nucleolin, appears to be important for the regulation of cell growth. The interaction of FKBP25 with casein kinase II may regulate these functions.
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PMID:The 25-kDa FK506-binding protein is localized in the nucleus and associates with casein kinase II and nucleolin. 768 29

Activation of protein kinase enzyme activity by Ca2+ and diacylglycerol or phorbol esters is a feature of certain isoforms of protein kinase C (PKC). Although the binding sites of phorbol ester on the regulatory domain of PKC have been extensively studied, little is known about the actual mechanisms of Ca2+ binding and how this leads to enzyme activation. We previously reported that high affinity binding of 45Ca2+ to the regulatory domain of PKC beta 1, expressed as a GST fusion protein in Escherichia coli, is dependent on the presence of phosphatidylserine (PS) or 12-O-tetradecanoylphorbol-13-acetate (TPA). In the present study we have used this system to further analyze Ca2+ binding. Using various deletions, we found that different domains in the regulatory domain of PKC beta 1 are involved in TPA-induced Ca2+ binding, depending on whether or not PS was also present in the binding assay. In addition, Ca2+ binding in the presence of TPA alone displayed very different kinetics than Ca2+ binding in the presence of TPA and PS. Scatchard analysis indicated that in the presence of TPA, the Kd value for Ca2+ binding was 51.9 microM. However, in the presence of both TPA and PS, the Kd value dropped to 0.23 microM. These results provide direct evidence that TPA activates certain isoforms of PKC by enhancing PS-dependent Ca2+ binding, thus decreasing the Kd value for Ca2+ binding to a physiological level.
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PMID:The phorbol ester TPA markedly enhances the binding of calcium to the regulatory domain of protein kinase C beta 1 in the presence of phosphatidylserine. 772 72

The MPS1 gene has been previously identified by a mutant allele that shows defects in spindle pole body (SPB) duplication and cell cycle control. The SPB is the centrosome-equivalent organelle in the yeast Saccharomyces cerevisiae, and it nucleates all the microtubules in the cell. We report the isolation of the MPS1 gene, which encodes an essential protein kinase homolog. The MPS1 open reading frame has been fused to those that encode the LexA protein or the GST protein and both of these constructs function in yeast. The fusion proteins have been affinity-purified from yeast extracts and the GST chimeric protein has been found to be a phosphoprotein. Both proteins have been used to demonstrate intrinsic in vitro protein kinase activity of Mps1p against exogenous substrates and itself (autophosphorylation). A mutation predicted to abolish kinase function not only eliminates in vitro protein kinase activity, but also behaves like a null mutation in vivo, suggesting that kinase activity contributes to the essential function of the protein. Phosphoamino acid analysis of substrates phosphorylated by Mps1p indicates that this kinase can phosphorylate serine, threonine and tyrosine residues, identifying Mps1p as a dual specificity protein kinase.
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PMID:Yeast spindle pole body duplication gene MPS1 encodes an essential dual specificity protein kinase. 773 18

Mitogen-activated protein kinase kinase kinase (MEKK1) is a serine-threonine kinase that regulates sequential protein kinase pathways involving stress-activated protein kinases and mitogen-activated protein kinases. MEKK1 is activated in response to growth factor stimulation of cells and by expression of activated Ras. We demonstrate that the kinase domain of MEKK1 (MEKKCOOH) binds to GST-RasV12 in a GTP-dependent manner. Purified bacterially expressed MEKKCOOH binds to GST-RasV12(GTP gamma S) (GTP gamma S is guanosine 5'-3-O-(thio)triphosphate), demonstrating a direct interaction of the two proteins. A Ras effector domain peptide blocks the binding of MEKKCOOH to GST-RasV12(GTP gamma S). MEKKCOOH complexed with GST-RasV12(GTP gamma S) is capable of phosphorylating MEK1. These findings indicate that MEKK1 directly binds Ras.GTP. Thus, Ras interacts with protein kinases of both the Raf and MEKK families.
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PMID:Direct interaction between Ras and the kinase domain of mitogen-activated protein kinase kinase kinase (MEKK1). 774 23

We have identified two components of a new protein kinase signaling cascade, MAPK/ERK kinase 5 (MEK5) and extracellular signal-regulated kinase 5 (ERK5). The MEK5 cDNA was isolated by degenerate PCR and encodes a 444-amino acid protein, which has approximately 40% identity to known MEKs. ERK5 was identified by a specific interaction with the MEK5 mutants S311A/T315A and K195M in the yeast two-hybrid system. The proteins were found to interact in an in vitro binding assay as well. ERK5 did not interact with MEK1 or MEK2. ERK5 is predicted to contain 815 amino acids and is approximately twice the size of all known ERKs. The C terminus of ERK5 has sequences which suggest that it may be targeted to the cytoskeleton. Sequences located in the N terminus of MEK5 may be important in coupling GTPase signaling molecules to the MEK5 protein kinase cascade. Both MEK5 and ERK5 are expressed in many adult tissue and are abundant in heart and skeletal muscle. A recombinant GST-ERK5 kinase domain displays autophosphorylation on Ser/Thr and Tyr residues.
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PMID:Components of a new human protein kinase signal transduction pathway. 775 17

Human, Drosophila melanogaster, and Caenorhabditis elegans cDNA clones encoding homologues of a serine(threonine) protein kinase (EC 2.7.1.37) (designated Ndr protein kinase) have been isolated and sequenced. The human and Drosophila cDNAs predict polypeptides of 54 kDa and 52 kDa, respectively, which share approximately 80% amino acid similarity. Northern analysis of human tissues revealed a ubiquitously expressed 3.9-kb transcript. Recombinant GST-Ndr underwent intramolecular autophosphorylation on serine and threonine residues in vitro but failed to transphosphorylate several standard protein kinase substrates. Transfection of the human cDNA into COS-1 cells resulted in the appearance of an intense nuclear staining in cells analyzed by indirect immunofluorescence; deletion mutagenesis identified a short basic peptide, KRKAETWKRNRR, responsible for the nuclear accumulation of Ndr. Thus, Ndr is a conserved and widely expressed nuclear protein kinase. The closest known relative of this previously uncharacterized kinase is Dbf2, a budding yeast protein kinase required for the completion of nuclear division.
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PMID:Molecular cloning and characterization of a conserved nuclear serine(threonine) protein kinase. 776 41

Srp1p, the protein encoded by SRP1 of Saccharomyces cerevisiae, is a nuclear-pore-associated protein. Its Xenopus homolog, importin, was recently shown to be an essential component required for nuclear localization signal (NLS)-dependent binding of karyophilic proteins to the nuclear envelope [Gorlich, D., Prehn, S., Laskey, R. A. & Hartman, E. (1994) Cell 79, 767-778]. We have discovered a protein kinase whose activity is stimulated by Srp1p (Srp1p fused to glutathione S-transferase and expressed in Escherichia coli) and is detected by phosphorylation of Srp1p and of a 36-kDa protein, a component of the protein kinase complex. The enzyme, called Srp1p kinase, is a protein-serine kinase and was found in extracts in two related complexes of approximately 180 kDa and 220 kDa. The second complex, when purified, contained four protein components including the 36-kDa protein. We observed that, upon purification of the kinase, phosphorylation of Srp1p became very weak, while activation of phosphorylation of the 36-kDa protein by Srp1p remained unaltered. Significantly, NLS peptides and the nuclear proteins we have tested greatly stimulated phosphorylation of Srp1p, suggesting that Srp1p, complexed with karyophilic proteins carrying an NLS, is the in vivo substrate of this protein kinase.
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PMID:Isolation of a yeast protein kinase that is activated by the protein encoded by SRP1 (Srp1p) and phosphorylates Srp1p complexed with nuclear localization signal peptides. 776 67

The binding of granulocyte-macrophage colony stimulating factor (GM-CSF) to its receptor stimulates JAK2 protein kinase activation, protein phosphorylation, and JAK2 association with the beta c chain of the GM-CSF receptor. To better understand how different domains of the JAK2 function to regulate association and phosphorylation of the beta c receptor, the minimal portion of the beta c receptor necessary for JAK2 binding has been determined. Using glutathione S-transferase (GST) fusion proteins expressing different portions of the membrane-proximal domain of the beta c chain, we demonstrate that JAK2 binds to amino acids 458-495, but showed little binding to fusion proteins containing amino acids 483-559, 483-530, or 458-484. The GST-beta c 458-495 bound equally well to the wild type (WT) JAK2, a carboxyl-terminal deletion of JAK2 removing the protein kinase domain (amino acids 1000-1129), and a deletion of the kinase-like domain (amino acids 523-746). However, an amino-terminal JAK2 deletion (amino acids 2-239) markedly reduced binding to this GST-beta c. Far Western blotting demonstrated that a GST fusion protein containing amino acids 1-294 of JAK2, but not fusion proteins containing amino acids 295-522, 523-746, or 747-1127, bound GST-beta c 458-559. When the JAK2 WT and deletions were transiently expressed along with the alpha and beta c subunits of the GM-CSF receptor and the cells were treated with GM-CSF, the following results were obtained: 1) WT JAK2 phosphorylated the beta c subunit in a GM-CSF-dependent manner, 2) the kinase-like domain deletion phosphorylated the beta c subunit, and 3) both the kinase domain deletion and the amino-terminal deletion failed to stimulate phosphorylation of the beta c subunit. Therefore, phosphorylation of the beta c subunit requires the binding of JAK2 through its amino terminus.
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PMID:The amino-terminal portion of the JAK2 protein kinase is necessary for binding and phosphorylation of the granulocyte-macrophage colony-stimulating factor receptor beta c chain. 777 38


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