Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.1 (protein kinase)
 81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Rat liver microsomes contain type-1 S6 phosphatase (acting on the serine residues phosphorylated by protein kinase A) and type-1 phosphorylase phosphatase activities. The main aim of this study has been to characterize the microsomal S6 phosphatase activity and to compare its properties with those of the phosphorylase phosphatase activity in the same microsomal preparation. The specific activities of both microsomal S6 phosphatase and phosphorylase phosphatase were 1.6- to 1.7-fold higher in the smooth endoplasmic reticulum than in the rough sarcoplasmic reticulum. Both phosphatase activities were inhibited to a similar extent by MgCl2 (10 mM) and NaF (22 mM), were completely suppressed by glycerophosphate (80 mM) and ZnCl2(10 mM), and were stimulated by MnCl2(1 mM). When analyzed by gel filtration on Sephadex G-100 superfine, both phosphatase activities eluted as broad peaks, stretching from the void volume to 45-60 kDa. The microsomal S6 phosphatase and phosphorylase phosphatase activities also displayed the following distinct characteristics: (a) Mn2+ stimulated the S6 phosphatase activity 2.9-fold more than the phosphorylase phosphatase activity, (b) limited trypsin digestion of microsomal preparations increased the phosphorylase phosphatase activity by 1.5- to 2-fold, but decreased the S6 phosphatase activity by 50%, (c) a synthetic peptide analog of S6 (S6229-239) (200 microM), which did not act as a substrate for the microsomal S6 phosphatase and did not affect its activity, inhibited the microsomal phosphorylase phosphatase activity by about 50%, and (d) the elution profile of the phosphorylase phosphatase activity was markedly broader than that of the S6 phosphatase activity. A series of in vivo studies showed that streptozotocin-diabetes and insulin replacement therapy as well as ip injection of insulin or vanadate, which modified the microsomal S6 phosphatase activity, had no statistically significant effects on the microsomal phosphorylase phosphatase activity. Taken together, these results suggest that the microsomal S6 phosphatase and phosphorylase phosphatase activities are due to two distinct enzyme populations.
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PMID:A comparative study of the microsomal S6 phosphatase and phosphorylase phosphatase activities in rat liver. 165 55

This investigation was carried out to determine if prolongation of ethanol-induced sleep by divalent cations is mediated by calmodulin (CaM) and biogenic amine. The effects of CaM antagonist, W-7:[N-(6-Aminohexyl)-5-chloro-1-naphthalenesulfonamide], serotonin (5-HT) synthesizing enzyme inhibitor, p-chlorophenylalanine (PCPA), and catecholamine synthesizing enzyme inhibitor, alpha-methyltyrosine (alpha MPT) on ethanol-induced sleeping time enhanced by divalent cations were studied in ddY male mice. The ethanol-induced sleeping time was increased by 70, 200, 180, 70, and 45% by intraventricular (IVT) injection of CaCl2 (10 mumol/kg), MnCl2 (15 mumol/kg), ZnCl2 (2.5 mumol/kg), CdCl2 (1 mumol/kg), and HgCl2 (1 mumol/kg), respectively, compared to the saline group. On the other hand, when mice were treated IVT with W-7 and their divalent cation, the sleeping time induced by ethanol was decreased compared to that of the cation without W-7 treated mice. Also, when mice were injected simultaneously with either PCPA or alpha MPT and CaCl2, ZnCl2, CdCl2, or HgCl2, the ethanol-induced sleeping time was less compared to those given saline together with their cation, respectively. These results would suggest a probable mechanism in which Ca++, Zn++, Cd++, and Hg++ prolong ethanol-induced sleeping time by activating biogenic amine synthesizing enzymes through cerebral CaM and CaM-dependent protein kinase.
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PMID:The ability of divalent cations to enhance ethanol-induced sleeping time. 293 5

A phosphorylated regulatory subunit of cyclic AMP-dependent protein kinase (type II) was purified to homogeneity from inorganic [32P]phosphate-injected rats. A new method of measuring the phosphorylation reaction was developed. It was found that this regulatory subunit was phosphorylated in cells and comprised 60, 82 and 55% of the total regulatory subunit in brain, heart and liver cytosol fractions from rats, respectively. Dephosphorylation was stimuated by cyclic nucleotides. The Ka values for cyclic AMP and cyclic IMP were 0.30 and 1.0 microM, respectively. Purified phosphoprotein phosphatase could dephosphorylate the regulatory subunit and this reaction was also stimulated by cyclic nucleotides with similar Ka values. The inhibitors of phosphoprotein phosphatase, NaF and ZnCl2, protected against dephosphorylation unless ADP or cyclic AMP were present.
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PMID:Phosphorylation and dephosphorylation of the regulatory subunit of cyclic 3',5'-monophosphate-dependent protein kinase (type II) in vivo and in vitro. 624 93

Calmodulin-dependent protein phosphatase of bovine brain exhibited a pH optimum of 7 and appeared to require sulfhydryl groups for activity. Phosphatase activity was inhibited by both NaF and ZnCl2, but was stimulated approximately 2-fold by MnCl2. The enzyme exhibited broad substrate specificity, dephosphorylating casein, troponin I, protamine, histone, and phosvitin, and was not phosphorylated by cAMP-dependent protein kinase. With 32P-labeled casein as a substrate, phosphatase was activated 15-fold by calmodulin; the dissociation constant of phosphatase for calmodulin was 30 nM. Activation of the enzyme by calmodulin as a function of Ca2+ was highly cooperative; the Hill coefficient was 4.9. At a saturating concentration of calmodulin, half-maximal activation of phosphatase was obtained at 0.3 microM Ca2+. Calmodulin increased the Vmax from 1.7 to 41 nmol mg protein-1 min-1 with no significant change in its Km. Formation of a Ca2+-dependent complex between calmodulin and the phosphatase was demonstrated by a calmodulin-Sepharose affinity column, gel-filtration chromatography, and sedimentation on a sucrose density gradient. The rate of formation and dissociation of the calmodulin X phosphatase complex was rapid and readily reversible in response to changes in Ca2+ concentration. The calmodulin X phosphatase complex consists of 1 mol of calmodulin and 1 mol of phosphatase.
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PMID:Characterization of bovine brain calmodulin-dependent protein phosphatase. 633 19

The mitogen-activated protein (MAP) kinases are serine-threonine protein kinases that are activated by tyrosine and threonine phosphorylation by the dual specificity protein kinase MEK (MAP kinase/ERK kinase). The present report describes the purification to near homogeneity and characterization of a protein tyrosine phosphatase from Xenopus laevis eggs that dephosphorylates MAP kinase phosphorylated by MEK. Bacterially expressed Xenopus MAP kinase phosphorylated by purified Xenopus MEK was used as substrate throughout the purification. The purification procedure included anion-exchange, cation-exchange, gel filtration, heparin-Sepharose, and chromatography on a column of thiophosphorylated MAP kinase-Sepharose, resulting in a > 3000-fold purification. Upon analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a protein of 47 kDa correlated with activity. The phosphatase showed absolute specificity toward phosphotyrosine and no activity toward phosphothreonyl-phosphoseryl residues of MAP kinase. The pH optimum of the enzyme was 7.0 with a Km of 9.0 microM for phosphorylated MAP kinase. The phosphatase was inhibited by ammonium molybdate (IC50, 2 microM), vanadate (IC50, 250 microM), millimolar concentrations of MnCl2, ZnCl2 and p-nitrophenylphosphate but not by okadaic acid or microcystin. This tyrosine phosphatase may be involved in deactivating MAP kinase in vivo.
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PMID:Purification and characterization of a mitogen-activated protein kinase tyrosine phosphatase from Xenopus eggs. 822 71

Pituitary adenylate cyclase-activating polypeptide (PACAP) is the most potent non-cholinergic neurotransmitter to stimulate catecholamine secretion from rat chromaffin cells; however, the mechanism of action is not clear. We used amperometric detection of exocytosis and indo-1 monitoring of [Ca2+]i to identify PACAP actions in cultured chromaffin cells. PACAP (100 nM) required external Ca2+ to evoke secretion. However, unlike nicotine and KCl which caused immediate and relatively brief secretion, PACAP has a latency of 6.8 +/- 0.96 s to the first secretory response and secretion continued for up to 2 min. PACAP elevation of [Ca2+]i showed similar latency and often remained above base line for several minutes following a brief exposure. ZnCl2 (100 microM) selectively inhibited PACAP-stimulated secretion and [Ca2+]i with little effect on nicotine-evoked responses. Nifedipine (10 microM) had little effect on PACAP-evoked secretion but inhibited nicotine-evoked secretion by more than 80%, while omega-conotoxin (100 nM) failed to affect either agonist. PACAP-stimulated cAMP levels required 5 s to significantly increase, consistent with the latency of exocytotic and Ca2+ responses. Forskolin (10 microM) caused responses similar to PACAP. PACAP-evoked exocytosis was blocked by the protein kinase A inhibitor adenosine 3'5'-cyclic monophosphorothioate Rp-diastereomer (Rp-cAMPS). These data showed that PACAP stimulates exocytosis by a mechanism distinctly different from cholinergic transmitters that appears to involve cAMP-mediated Ca2+ influx. Differences in receptor coupling mechanisms and pharmacology of Ca2+ entry stimulated by cholinergic and peptidergic agonists support the idea that the peptidergic system maintains catecholamine secretion under conditions where the cholinergic system desensitizes or otherwise fails.
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PMID:A non-cholinergic transmitter, pituitary adenylate cyclase-activating polypeptide, utilizes a novel mechanism to evoke catecholamine secretion in rat adrenal chromaffin cells. 863 54

Our previous data demonstrated that Ras activation is necessary and sufficient for transforming growth factor beta (TGFbeta)-mediated Erk1 activation, and is partially required for the inhibition of cyclin-dependent kinase 2 (Cdk2) activity, cyclin A expression and DNA synthesis by TGFbeta (KM Mulder and SL Morris, J. Biol. Chem., 267: 5029-5031, 1992; MT Hartsough and KM Mulder, J. Biol. Chem., 270: 7117-7124, 1995; and MT Hartsough et al., J. Biol. Chem., 271: 22368-22375, 1996). Here, we examined the kinetics and role of Ras in TGFbeta3-mediated effects on specific G1 cell cycle components in TGFbeta-sensitive (4-1) and TGFbeta-resistant (4-6) intestinal epithelial cells (IEC's). Our results indicate that inactivation of Ras by stable, inducible expression of a dominant-negative mutant of Ras (RasN17) completely abrogated the ability of TGFbeta3 to up-regulate both CKI's. In contrast, the ability of TGFbeta3 to up-regulate p27Kip1 and p21Cip1 was maintained in ZnCl2-treated control cells. Inactivation of Ras also completely blocked the rapid TGFbeta-mediated increase in new synthesis of p27Kip1 protein. Moreover, up-regulation of p21Cip1 protein levels and new synthesis of p27Kip1, as well as the association of these CKI's with Cdk2, preceded the decrease in Cdk2 activity by TGFbeta. Collectively, our results suggest that p21Cip1 and p27Kip1 are upstream effectors of the TGFbeta-mediated inhibition of Cdk2 activity in IEC 4-1 cells, and demonstrate that Ras activation is obligatory for TGFbeta-mediated up-regulation of these CKIs in untransformed epithelial cells.
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PMID:Blockade of TGFbeta3 up-regulation of p27Kip1 and p21Cip1 by expression of RasN17 in epithelial cells. 967 13

The involvement of the double-stranded RNA-activated protein kinase PKR in the regulation of the myogenic process was investigated. For this purpose, the murine myogenic cell line C2C12 was used. The cells were first cultivated in either growth medium or differentiation medium (DM), and the activation of PKR during differentiation was determined by monitoring its enzymatic activity and by immunoblot analysis. A significant increase in both parameters was detected already at 24 h in DM, whereas in cells grown in growth medium, the increase was evident only after 96 h, when spontaneous differentiation was observed in highly crowded cultures. Consequently, we established the direct effect of PKR activation on the myogenic process. C2C12 cells were transfected with an expression vector harboring a cDNA molecule encoding human PKR fused to the inducible metallothionein promoter. One of the clones (clone 8) expressing high levels of PKR was selected and further analyzed. In the presence of ZnCl2, which activates the promoter, the rate of cell growth of the transfected cells was clearly reduced compared to that of wild-type C2C12 cells transfected with only the neomycin-resistant gene (C2-NEO). In addition, altered morphology with partial fusion was observed. Biochemically, an increase in creatine kinase activity accompanied by an increased rate of expression of the myogenic protein troponin T and the myogenic transcription factors myoD and myogenin was detected in clone 8 cells exposed to ZnCl2. Most importantly, an induction in the level of cyclin-dependent kinase inhibitor p21WAF1 and an increase in the level of the underphosphorylated active form of the tumor suppressor protein pRb concomitant with the down-regulation of cyclin D1 and c-myc were also evident in the transfected clones. These changes were similar to those observed in normal C2C12 cells cultivated in DM. We conclude that PKR is an important regulatory protein participating in the myogenic process.
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PMID:Involvement of PKR in the regulation of myogenesis. 1009 34

Ecto-protein kinases (ecto-PK) are surface constituents of many, if not all, animal cell types; normal, transformed or malignant. The occurrence of ecto-PK on the surface of human leukemia cell lines was described [Paas, Y., Fishelson, Z., 1995. Shedding of tyrosine and serine/threonine ecto-PK from human leukemic cells. Arch. Biochem. Biophys. 316 780-788.]. These ecto-PKs have been shown to phosphorylate several exogenous substrates, including the complement C9 protein, an essential component of the terminal complement system. C9 is phosphorylated by ecto-PK of K562 cells on serine residue(s). Phosphorylation occurs in the N-terminal C9a portion produced by cleavage of phosphorylated C9 with human alpha-thrombin. C9 polymers generated upon incubation of C9 with ZnCl2 do not serve as substrates for the K562 ecto-PK. In contrast, unfolded C9, obtained by reduction and alkylation, serves as a superior substrate for the K562 ecto-PK. Native C9 phosphorylation produced a rather low stoichiometry of incorporated phosphate (around 3%) per C9. Despite that, the phosphorylated C9 expressed reduced hemolytic activity. The complement-sensitive variant of K562 (K562/S) did not express the C9 phosphorylating activity. Various PK inhibitors tested failed to block C9 phosphorylation. Only heparin and 2,3-diphosphoglycerate (dpGA) prevented C9 phosphorylation, indicating that the ecto-PK is related to the casein kinase CK2. C9 can be phosphorylated by ecto-PK from other tumor cells, including Jurkat, SK-OV-3 and BT-474. These results suggest that extracellular phosphorylation of C9 may serve as a protective mechanism against complement in tumor cells.
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PMID:Phosphorylation of the complement component, C9, by an ecto-protein kinase of human leukemic cells. 1040 78

Zinc ions have both essential and toxic effects on mammalian cells. Here we report the ability of zinc to act as an inducer of apoptosis in C6 rat glioma cells. Incubation with 150 to 300 microM ZnCl2 caused cell death that was characterized as apoptotic by internucleosomal DNA fragmentation, formation of apoptotic bodies, nuclear fragmentation and breakdown of the mitochondrial membrane potential. On the other hand, zinc deprivation by the membrane permeable chelator TPEN [N,N,N',N',-tetrakis (2-pyridyl-methyl)-ethylenediamine] also induced programmed death in this cell line, indicating the existence of intracellular zinc levels below and above which apoptosis is induced. Zinc-induced apoptosis in C6 cells was independent of major signaling pathways (protein kinase C, mitogen activated protein kinase and guanylate cyclase) and protein synthesis, but was increased by facilitating zinc uptake with the ionophore pyrithione. Lanthanum(III)chloride was also able to increase the net zinc uptake, but nevertheless apoptotic features and zinc toxicity were reduced. Remarkably, lanthanum suppressed the zinc-induced breakdown of the mitochondrial membrane potential. We conclude that in C6 cells lanthanum acts in two different ways, as a promoter of net zinc uptake and as a suppressor of zinc-induced apoptosis.
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PMID:Zinc induces apoptosis that can be suppressed by lanthanum in C6 rat glioma cells. 1159 4


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