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)

We examined effects of an isoquinolinesulfonamide derivative, KN-62, on human ovarian cancer cells, NOS3AR, that are resistant to Adriamycin (ADR). MTT assay revealed that 10 microM KN-62 overcame the resistance. KN-62 had little effect on GST activity. In studies on the intracellular accumulation of ADR, KN-62 increased the ADR contents in the resistant cells close to the level seen in the sensitive cells. These results suggest that the reversal of the resistance against ADR in ovarian cancer cells by KN-62 is mainly due to higher accumulation of ADR in NOS3AR cells. Furthermore, we detected Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) in NOS3AR cells since KN-62 is a specific inhibitor of the kinase. In this paper, we discussed on modulation of ADR-resistance by KN-62.
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PMID:Effect of KN-62, Ca2+/calmodulin-dependent protein kinase II inhibitor, on adriamycin resistance of human ovarian cancer cells. 748 93

We report that the C-terminal domain of skeletal muscle dystrophin expressed as a fusion protein with glutathione S-transferase (designated GST-CT-1) is a substrate for Ca2+/calmodulin-dependent phosphorylation and dephosphorylation. GST-CT-1 and GST-CT-1F (GST-CT-1 truncated by 20-25 residues) were phosphorylated by Ca2+/calmodulin-dependent protein kinase II (CaM kinase II). The stoichiometries of phosphorylation by CaM kinase II were 1.65 mol of Pi/mol of GST-CT-1 and 0.39 mol of Pi/mol of GST-CT-1F, respectively, suggesting that the principal site(s) of phosphorylation is (are) located in the C-terminal 20-25 residues that are missing from GST-CT-1F. The GST-CT-1 fusion protein was phosphorylated on both serine and threonine residues, whereas GST-CT-1F was phosphorylated only on serine. CaM kinase II-phosphorylated GST-CT-1 and GST-CT-1F were efficiently dephosphorylated by calcineurin, a Ca2+/calmodulin-dependent protein phosphatase (type 2B protein phosphatase). Importantly, calcineurin was found to be associated with a purified sarcolemmal membrane preparation enriched in dystrophin. Type 2A protein phosphatase isolated from smooth muscle (SMP-I) and its catalytic subunit (SMP-ic) also dephosphorylated GST-CT-1, but were less active toward these substrates than was calcineurin. Type 2C phosphatase (SMP-II) and type 1 protein phosphatases [SMP-III, SMP-IV, and myosin-associated phosphatase (PP1M) of smooth muscle and skeletal muscle protein phosphatase 1c] were ineffective in dephosphorylating the C-terminal region of dystrophin.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Characterization of the recombinant C-terminal domain of dystrophin: phosphorylation by calmodulin-dependent protein kinase II and dephosphorylation by type 2B protein phosphatase. 772 17

A specific antiserum against the human m3-muscarinic receptor subtype was made by subcloning a variant region of the third intracellular loop of the m3-receptor (Ser345-Leu463) into a bacterial expression plasmid that produced a fusion protein with glutathione S-transferase. In immunoblot studies this anti-serum identified the human m3-receptor expressed in transfected Chinese hamster ovary (CHO) cells (CHO-m3 cells, 1343 fmol/mg protein) as a diffuse band at approximately 97-110 kDa. In vivo labeling of the ATP pool in CHO-m3 cells with [32P]orthophosphate followed by immunoprecipitation of solubilized m3-receptors revealed that the unstimulated receptor existed in a phosphorylated form. Incubation of CHO-m3 cells with the cholinergic agonist carbachol (1 mM) increased the phosphorylated state of the receptor dramatically, primarily at serine. The time course for agonist-dependent phosphorylation was very rapid occurring within seconds of agonist addition and was maintained for at least 30 min. The muscarinic antagonist atropine (10 microM) inhibited agonist-stimulated phosphorylation. Neither forskolin (10 microM) nor the calcium ionophore, ionomycin (1 microM), had any effect on the state of phosphorylation of the m3-receptor, eliminating a role for cAMP-dependent protein kinase and Ca2+/calmodulin-dependent protein kinase in the agonist-dependent phosphorylation of m3-receptors. 4 beta-Phorbol 12 beta-myristate 13 alpha-acetate (100 nM) did increase m3-receptor phosphorylation, an effect that was inhibited by the selective protein kinase C inhibitor RO-318220 (10 microM). However, agonist-stimulated m3-receptor phosphorylation was not inhibited by RO-318220 indicating that protein kinase C was not involved in agonist-induced m3-receptor phosphorylation. In conclusion the phosphorylation of m3-receptors, in vivo, was increased following the application of muscarinic agonist or PMA. The response to agonist was mediated via a kinase distinct from protein kinase C, protein kinase A and Ca2+/calmodulin dependent protein kinase, whereas the effect of 4 beta-phorbol 12 beta-myristate 13 alpha-acetate was mediated by protein kinase C.
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PMID:Rapid agonist-mediated phosphorylation of m3-muscarinic receptors revealed by immunoprecipitation. 848 62

The CLK1 gene of Saccharomyces cerevisiae encodes a 610-residue protein kinase that resembles known type II Ca2+/calmodulin-dependent protein kinases (CaM kinases), including the CMK1 and CMK2 gene products from the same yeast. The Clk1 kinase domain is preceded by a 162-residue N-terminal extension, followed by a 132-residue C-terminal extension (which contains a basic segment resembling known calmodulin-binding sites) and is as similar to mammalian CaM kinase (38% identity to rat CaM kinase alpha) as it is to yeast CaM kinase (37% identity to Cmk2). However, Clk1 shares 52% identity with Rck1, another putative protein kinase encoded in the S. cerevisiae genome. Clk1 tagged with a c-myc epitope (expressed in yeast) and a GST-Clk1 fusion (expressed in bacteria) underwent autophosphorylation and phosphorylated an exogenous substrate (yeast protein synthesis elongation factor 2), primarily on Ser. Neither Clk1 activity was stimulated by purified yeast calmodulin (CMD1 gene product), with or without Ca2+; no association of Clk1 with Cmd1 was detectable by other methods. C-terminally truncated Clk1(Delta487-610) was growth-inhibitory when overexpressed, whereas catalytically inactive Clk1(K201R Delta487-610) was not, suggesting that the C terminus is a negative regulatory domain. Using immunofluorescence, Clk1 was localized to the cytosol and excluded from the nucleus. A clk1Delta mutant, a clk1Delta rck1Delta double mutant, a clk1Delta cmk1Delta cmk2Delta triple mutant, and a clk1Delta rck1Delta cmk1Delta cmk2Delta quadruple mutant were all viable and manifested no other overt growth phenotype.
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PMID:Identification and characterization of the CLK1 gene product, a novel CaM kinase-like protein kinase from the yeast Saccharomyces cerevisiae. 893 41

Ca2+/calmodulin-dependent protein kinase II (CaM-KII) regulates numerous physiological functions, including neuronal synaptic plasticity through the phosphorylation of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type glutamate receptors. To identify proteins that may interact with and modulate CaM-KII function, a yeast two-hybrid screen was performed by using a rat brain cDNA library. This screen identified a unique clone of 1.4 kb, which encoded a 79-aa brain-specific protein that bound the catalytic domain of CaM-KII alpha and beta and potently inhibited kinase activity with an IC50 of 50 nM. The inhibitory protein (CaM-KIIN), and a 28-residue peptide derived from it (CaM-KIINtide), was highly selective for inhibition of CaM-KII with little effect on CaM-KI, CaM-KIV, CaM-KK, protein kinase A, or protein kinase C. CaM-KIIN interacted only with activated CaM-KII (i. e., in the presence of Ca2+/CaM or after autophosphorylation) by using glutathione S-transferase/CaM-KIIN precipitations as well as coimmunoprecipitations from rat brain extracts or from HEK293 cells cotransfected with both constructs. Colocalization of CaM-KIIN with activated CaM-KII was demonstrated in COS-7 cells transfected with green fluorescent protein fused to CaM-KIIN. In COS-7 cells phosphorylation of transfected alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type glutamate receptors by CaM-KII, but not by protein kinase C, was blocked upon cotransfection with CaM-KIIN. These results characterize a potent and specific cellular inhibitor of CaM-KII that may have an important role in the physiological regulation of this key protein kinase.
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PMID:Characterization of a calmodulin kinase II inhibitor protein in brain. 972

A growing body of evidence indicates that regulation of protein-serine/threonine phosphatase 2A (PP2A) involves its association with other cellular and viral proteins in multiprotein complexes. PP2A-containing protein complexes may exist that contribute to PP2A's important regulatory role in many cellular processes. To identify such protein complexes, PP2A was partially purified from rat brain soluble extracts following treatment with a reversible cross-linker to stabilize large molecular size forms of PP2A. Compared with native (uncross-linked) PP2A, cross-linked PP2A revealed an enrichment of p70 S6 kinase and two p21-activated kinases (PAK1 and PAK3) in the PP2A complex, indicating these kinases may associate with PP2A. The existence of protein kinase-PP2A complexes in rat brain soluble extracts was further substantiated by the following results: 1) independent immunoprecipitation of the kinases revealed that PP2A co-precipitated with p70 S6 kinase and the two PAK isoforms; 2) glutathione S-transferase fusion proteins of p70 S6 kinase and PAK3 each isolated PP2A; and 3) PAK3 and p70 S6 kinase bound to microcystin-Sepharose (an affinity resin for PP2A-PP1). Cumulatively, these findings provide evidence for association of PP2A with p70 S6 kinase, PAK1, and PAK3 in the context of the cellular environment. Moreover, together with the recent reports describing associations of PP2A with Ca2+/calmodulin-dependent protein kinase IV (Westphal, R. S., Anderson, K. A., Means, A. R., and Wadzinski, B. E. (1998) Science 280, 1258-1261) and casein kinase IIalpha (Heriche, J. K., Lebrin, F., Rabilloud, T., Leroy, D., Chambaz, E. M., and Goldberg, Y. (1997) Science 276, 952-955), the present data provide compelling evidence for the existence of protein kinase-PP2A signaling modules as a new paradigm for the control of various intracellular signaling cascades.
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PMID:Identification of kinase-phosphatase signaling modules composed of p70 S6 kinase-protein phosphatase 2A (PP2A) and p21-activated kinase-PP2A. 987 3

Mammalian Ca2+/CaM-dependent protein kinase kinase (CaM-KK) has been identified and cloned as an activator for two kinases, CaM kinase I (CaM-KI) and CaM kinase IV (CaM-KIV), and a recent report (Yano, S., Tokumitsu, H., and Soderling, T. R. (1998) Nature 396, 584-587) demonstrates that CaM-KK can also activate and phosphorylate protein kinase B (PKB). In this study, we identify a CaM-KK from Caenorhabditis elegans, and comparison of its sequence with the mammalian CaM-KK alpha and beta shows a unique Arg-Pro (RP)-rich insert in their catalytic domains relative to other protein kinases. Deletion of the RP-domain resulted in complete loss of CaM-KIV activation activity and physical interaction of CaM-KK with glutathione S-transferase-CaM-KIV (T196A). However, CaM-KK autophosphorylation and phosphorylation of a synthetic peptide substrate were normal in the RP-domain mutant. Site-directed mutagenesis of three conserved Arg in the RP- domain of CaM-KK confirmed that these positive charges are important for CaM-KIV activation. The RP- domain deletion mutant also failed to fully activate and phosphorylate CaM-KI, but this mutant was indistinguishable from wild-type CaM-KK for the phosphorylation and activation of PKB. These results indicate that the RP-domain in CaM-KK is critical for recognition of downstream CaM-kinases but not for its catalytic activity (i.e. autophosphorylation) and PKB activation.
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PMID:Substrate recognition by Ca2+/Calmodulin-dependent protein kinase kinase. Role of the arg-pro-rich insert domain. 1033 83

This study reports the cloning and characterization of a cDNA encoding elongation factor 1-alpha (EF1alpha) from the yeast Schizosaccharomyces pombe. The cDNA was cloned from an Schizosaccharomyces pombe expression library by a two-hybrid selection for clones encoding calmodulin (CaM)-binding proteins. The predicted protein is highly homologous to mammalian EF1alpha, indicating a strong tendency towards conservation of the primary amino acid sequence. The protein was expressed as a glutathione S-transferase fusion in both bacteria and in Schizosaccharomyces pombe. The bacterial protein was shown by solution assay to compete with CaM kinase II for CaM. The CaM binding domain was localized to the C-terminus of the protein by this method. Expression of full-length EF1alpha in vivo caused an increase in cell cycle length and a decreased rate of growth as evidenced by a lack of elongated cells in slowly dividing cultures. This effect appears to involve CaM binding because a truncation mutant version of EF1alpha lacking the CaM binding domain did not cause cell cycle delay.
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PMID:Cloning of a Schizosaccharomyces pombe homologue of elongation factor 1 alpha by two-hybrid selection of calmodulin-binding proteins. 1059 5

The function of synapsin I is regulated by phosphorylation of the molecule at multiple sites; among them, the Ser(603) residue (site 3) is considered to be a pivotal site targeted by Ca(2+)/calmodulin-dependent kinase II (CaMKII). Although phosphorylation of the Ser(603) residue responds to several kinds of stimuli, it is unlikely that many or all of the stimuli activate the CaMKII-involved pathway. Among the several stimulants tested in PC12 cells, bradykinin evoked the phosphorylation of Ser(603) without inducing the autophosphorylation of CaMKII, which was determined using phosphorylation site-specific antibodies against phospho-Ser(603)-synapsin I (pS603-Syn I-Ab) and phospho-Thr(286/287)-CaMKII. The bradykinin-evoked phosphorylation of Ser(603) was not suppressed by the CaMKII inhibitor KN62, whereas high KCl-evoked phosphorylation was accompanied by CaMKII autophosphorylation and inhibited by KN62. Thus, we attempted to identify Ser(603) kinase(s) besides CaMKII. We consequently detected four and three fractions with Ca(2+)/calmodulin-independent Ser(603) kinase activity on the DEAE column chromatography of bovine brain homogenate and PC12 cell lysate, respectively, two of which were purified and identified by amino acid sequence of proteolytic fragments as p21-activated kinase (PAK) 1 and PAK3. The immunoprecipitants from bovine brain homogenate with anti-PAK1 and PAK3 antibodies incorporated (32)P into synapsin I in a Cdc42/GTPgammaS-dependent manner, and its phosphorylation site was confirmed as Ser(603) using pS603-Syn I-Ab. Additionally, recombinant GST-PAK2 could phosphorylate the Ser(603) residue in the presence of Cdc42/GTPgammaS. Finally, we confirmed by immunocytochemical analysis that the transfection of constitutively active rat alphaPAK (PAK1) in PC12 cells evokes the phosphorylation of Ser(603) even in the resting mutant cells and enhances it in the bradykinin-stimulated cells, whereas that of dominant-negative alphaPAK quenches the phosphorylation. These results raise the possibility that Ser(603) on synapsin I is alternatively phosphorylated by PAKs, not only by CaMKII, in neuronal cells in response to some stimulants.
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PMID:Synapsin I is phosphorylated at Ser603 by p21-activated kinases (PAKs) in vitro and in PC12 cells stimulated with bradykinin. 1223 6

We investigated the relationship between Arc (activity-regulated cytoskeleton-associated protein) and Ca(2+)/calmodulin-dependent protein kinase II (CaM kinase II). Arc and CaM kinase II were concentrated in the postsynaptic density. These proteins were accumulated after electroconvulsive treatment. Arc increased about 2.5-fold within 30 min and was maintained at this level for 8h after the stimulation. CaM kinase II also increased within 30 min and remained at this level for at least 24h. The interaction of Arc with CaM kinase II was demonstrated using GST-Arc fusion protein, and confirmed in neuroblastoma cells by immunoprecipitation. We examined the function of Arc by introducing Arc cDNA into neuroblastoma cells expressing CaM kinase II. The cells expressing both Arc and CaM kinase II had longer neurites than those expressing CaM kinase II alone. Arc itself did not promote neurite outgrowth. The growth of neurites by Arc was completely blocked by treatment with KN62, an inhibitor of CaM kinases. These results indicated that Arc potentiated the action of CaM kinase II for neurite extension.
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PMID:Interaction of Arc with CaM kinase II and stimulation of neurite extension by Arc in neuroblastoma cells expressing CaM kinase II. 1463 Mar 44


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