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:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Extracellular protein kinase activity is demonstrated in intact cultured porcine aortic endothelial cells and is characterised. When cells were incubated with [gamma-32P]ATP (1 microM) a major cell surface protein, corresponding to 115 kDa, and at least four serum proteins (19, 21, 55 and 126 kDa) became phosphorylated. Protein kinase activity is released by intact endothelial cells, which is not due to cell damage, as judged by various cell viability parameters (e.g., release of marker enzymes, trypan blue exclusion). The activity of the protein kinase released amounted to 170 fmol/min per mg endothelial cell protein with phosvitin as substrate, which represents 9% of the total cellular phosvitin protein kinase activity. Repetitive incubation of endothelial cells substantially decreased phosvitin-kinase release. Exo-protein kinase is not influenced by cAMP and cGMP but is effectively inhibited by heparin (EC50, 0.3 microgram/ml). The findings clearly demonstrate: (1) exo-protein kinase is released by intact porcine aortic endothelial cells; (2) substrates of this enzyme are endothelial surface proteins and serum proteins.
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PMID:Exo-protein kinase release from intact cultured aortic endothelial cells. 150 3

Regulation of prostaglandin (PG) E2 receptors was investigated in a 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate-solubilized fraction from the synaptic membrane of porcine temporal cortex. The fraction was preincubated with exogenous protein kinases, and then the binding of PGE2 was measured. PGE2 binding was increased approximately twofold by pretreatment with the catalytic subunit of cyclic AMP-dependent protein kinase (A kinase) or calmodulin-dependent protein kinase II but not by that with protein kinase C. The increase was dependent on the ATP concentration, with ED50 values being close to the Km values of these protein kinases. Protein kinase inhibitors specific for A kinase and for calmodulin-dependent protein kinase II abolished the effect in a dose-dependent manner, with IC50 values being similar to those reported. Further study using the catalytic subunit of A kinase revealed that the maximal binding capacity apparently increased without affecting the affinity and the rate constants for association and dissociation. On the other hand, acid phosphatase treatment reduced the binding activity to the level of nonspecific binding. In addition, treatment by A kinase did not affect the binding of guanosine 5'-(3-thiotriphosphate) by the GTP-binding proteins and the activation of adenylate cyclase mediated by stimulatory guanine nucleotide-binding regulatory protein, and therefore the phosphorylation is believed to occur on the receptor protein. The results suggest that the PGE2 receptor can take active phosphorylated and inactive dephosphorylated forms, of which only the phosphorylated one can bind PGE2.
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PMID:Regulation of prostaglandin E2 receptor binding activity in porcine temporal cortex by protein phosphorylation. 165 90

Protein kinase c-beta-like immunoreactivity was studied in the adrenal gland of adult rats and at different pre- and postnatal stages of development (E17-P21) with an antibody specific to both the beta 1 and beta 2 subtypes of the kinase. In the adult rat adrenal gland, the immunoreactivity was seen in numerous nerve fibres in the adrenal medulla both in bundles and individually forming occasionally dense networks around chromaffin cell groups. Several protein kinase c-beta-immunoreactive fibres were also observed transversing the adrenal cortex towards the medulla. No remaining immunoreactive fibres two weeks after transection of the splanchnic nerve could be seen; nor was any immunoreactivity observed in the chromaffin cells of the adrenal medulla or in the cortical cells, but some faintly immunoreactive ganglion cells were detected in the adrenal medulla. The amount and distribution of protein kinase c-beta-like immunoreactivity in the fetal and developing adrenals was very similar to that seen in the adrenal glands of adult rats. On the basis of its localization, the beta-subtype of protein kinase c does not appear to be directly involved in the release of catecholamines from the adrenal medulla, but it might have a role in the regulation of neurotransmitter release from preganglionic cholinergic neurons.
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PMID:Protein kinase c-beta-like immunoreactivity in the developing and adult rat adrenal gland. 168 Aug 34

Although most studies of protein phosphorylation have focused on intracellular protein kinases, evidence for protein kinase activity on the surface of several types of cells has been described. Evidence was recently provided for the existence of ecto-protein kinase activity on the surface of human neutrophils. Evidence for three distinct ecto-protein kinase activities was detected, one that phosphorylates endogenous surface proteins, one that phosphorylates exogenous substrates in a cAMP-independent manner and is released in the presence of substrate, and a low level of activity of one that phosphorylates exogenous Kemptide in a cAMP-dependent manner. To begin to elucidate its role in neutrophil function, we have characterized several properties of the releasable ecto-protein kinase activity on human neutrophils. This enzyme activity was inhibited by impermeant stilbene disulfonic acids, which are known to alter neutrophil function, as well as by impermeant sulfhydryl reactive agents. Enzyme activity was detectable at physiologic concentrations of Mg2+, but was higher in the presence of Mn2+. Protein kinase activity was strongly inhibited by heparin, whereas trifluoperazine, cAMP, and cGMP had little effect on kinase activity. Protein kinase activity was selectively removed from the cell surface by incubation with the ecto-kinase substrates casein and phosvitin, but the enzyme was not released by phosphatidylinositol-specific phospholipase C. Repeated exposure of neutrophils to substrate depleted ecto-protein kinase activity from the cell surface, but activity was rapidly restored by incubation in buffer lacking substrate. The released protein kinase had a Km for ATP of approximately 0.5 microM and a pH maximum between 7.0 and 7.5. At least four ecto-protein kinase substrates were detected in serum; vitronectin was identified as one of these substrates by immunoprecipitation studies. Although the exact role of ecto-protein kinase activity in neutrophil function remains undefined, the identification of vitronectin as a serum substrate suggests that it interacts with a physiologically important substrate.
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PMID:Characterization of human neutrophil ecto-protein kinase activity released by kinase substrates. 171 14

We examined the role of protein kinase-C in contractile responses of small arteries of the rat by stimulating and inhibiting protein kinase-C with phorbol myristate acetate and staurosporine, respectively. The experiments were performed in isolated mesenteric resistance arteries that had been sympathectomized and mounted for recording of isometric force development. Phorbol myristate acetate (i) at concentrations lower than 3 nM increased sensitivity for the contractile effect of potassium, but not for the effect of noradrenaline or BAY-K8644, (ii) at concentrations higher than 30 nM increased the sensitivity of depolarized vessels to extracellular calcium and (iii) at concentrations higher than 30 nM induced a contractile effect that depended on the presence of extracellular calcium and that was reduced by the calcium antagonist felodipine. Neither the phorbol ester nor staurosporine affected contractile responses to caffeine in calcium-free solution. Staurosporine (10 nM) reduced the response of resistance arteries to potassium but not to noradrenaline. These results are in agreement with direct observations by others that protein kinase-C plays a role in the activation of voltage-operated calcium channels. Protein kinase-C could participate in this way in electro-mechanical coupling in resistance arterial smooth muscle and, when strongly activated, sensitize the contractile apparatus to calcium.
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PMID:Effects of a phorbol ester and staurosporine on electro- and pharmacomechanical coupling in a resistance artery. 178 1

Tyrosine protein kinase present in the membrane fraction of bovine cerebral cortex were extracted and chromatographically fractionated. The activity associated with tyrosine protein kinases was fully extracted from the membranes by 1% sodium cholate and eluted in two peaks (I and II) during chromatography of protein extracts on DEAE-Toyopearl in the presence of sodium cholate. The predominant in cerebral cortex membrane tyrosine protein kinase of peak I (about 75% of the total activity) was purified 1930-fold by gel filtration on Sephacryl S-300, chromatography on hexyl- and phenyl-Sepharose and by rechromatography on DEAE-Toyopearl. The amount of the enzyme prepared from 250 g of bovine brain was 20 micrograms, the enzyme yield and specific activity being 3.8% and 3.9 nmol/mg protein/min, respectively. The purified protein kinase of peak I represents a protein with Mr of 62-63,000 (p62) capable of being autophosphorylated in the presence of [gamma-32P]. Protein kinase p62 phosphorylates enolase, tubulin and calpactin I as well as model substrates in the series: histone H5 greater than poly(G, T)n greater than or equal to histone H2A greater than poly(G, A, T)n, histone H4 greater than caseins, histones H1 and H2B, poly(G, A, L, T)n. The enzyme is specific for Mn2+ at the optimal concentration about 1 mM. The KmMn-ATP is 0.3 microM; Km for histone H5 and poly(G, T)n are 0.45 mg/ml and 0.06 mg/ml, respectively. The protein kinase p62 activity is inhibited by NaCl (IC50 approximately 75-100 mM) as well as by quercetin, adriamycin and lasalocid (IC50 approximately 14-34, 23 and 90 microM, respectively). It is concluded that protein kinase p62 is analogous to the c-src gene protein kinase.
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PMID:[Tyrosine protein kinase from cattle cerebral cortex: purification, characteristics, protein substrates for phosphorylation and inhibitors of activity]. 180 85

Protein kinase assays that use recombinant pp90rsk as a substrate were developed in an attempt to identify growth-regulated enzymes responsible for the phosphorylation and activation of pp90rsk S6 phosphotransferase activity. With this assay we have ientified a pp60v-src-, growth factor-, phorbol ester-, and vanadate-regulated serine/threonine protein kinase activity that is not related to two other cofactor-independent, growth-regulated protein kinases, pp70-S6 protein kinase and pp90rsk. The pp90rsk-protein kinase activity (referred to as rsk-kinase) is also not related to cofactor-dependent signal transducing protein kinases such as the cyclic AMP-dependent protein kinases, members of the protein kinase C family, or other Ca2(+)-dependent protein kinases. In vitro, partially purified rsk-kinase phosphorylates several of the sites (serine and threonine) that are phosphorylated in growth-stimulated cultured cells. A detailed examination of the mitogen-regulated activation kinetics of rsk-kinase and pp90rsk activities demonstrated that they are coordinately regulated. In addition, protein kinase C is not absolutely required for epidermal and fibroblast growth factor-stimulated activation of rsk-kinase, whereas, like pp90rsk, platelet-derived growth factor- and vanadate-stimulated rsk-kinase activity exhibits a greater dependence on protein kinase C-mediated signal transduction. The characterization and future purification of the rsk-kinase(s) will improve our understanding of the early signaling events regulating cell growth.
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PMID:Coordinate regulation of pp90rsk and a distinct protein-serine/threonine kinase activity that phosphorylates recombinant pp90rsk in vitro. 182 43

IL-1 treatment of human endothelial cells leads to the rapid phosphorylation of a Mr = 29,000 (P29) set of proteins to 18 times that of control cultures. Approximately 80% of the phosphorylated P29 (pP29) disappeared within 60 min although the remaining component was stable and remained for at least another 2 h. IL-1R antagonist protein blocked phosphorylation completely. Secondary treatment of IL-1 failed to increase the level of pP29 above that remaining after 1 h although other unrelated agonists that stimulated pP29 generation could. Removal of the cytokine and incubation of the cells in agonist-free medium for 2 h resulted in the total loss of the remaining pP29. Readdition of IL-1 2 h after washout restimulated P29 phosphorylation but only back to the lower level. Maximum rephosphorylation could not be attained until 16 h after IL-1 removal. Protein kinase inhibitors 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine and staurosporine, the calcium chelators bis(2-amino-5-methylphenoxy)ethane-N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester and EGTA, and the calmodulin inhibitor N-(6-aminohexyl)-1-naphthalene-sulfonamide had no effect on IL-I-induced phosphorylation. However, when cultures were treated with the protein phosphatase inhibitor okadaic acid alone, the level of pP29 increased after 1 h and the presence of okadaic acid during prolonged IL-1 treatment blocked the decline in pP29. The protein synthesis inhibitors puromycin, emetine, and cycloheximide also blocked the decline in pP29 during IL-1 treatment. These data suggest that IL-1-stimulated P29 phosphorylation is made up of two components, one susceptible to prolonged down-regulation even in the absence of the cytokine and one refractory to desensitization but that remains active only in the presence of IL-1. IL-1-induced changes in pP29 levels may be dependent on the relative activities of protein kinase and protein phosphatase activities.
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PMID:Phosphorylation of an Mr = 29,000 protein by IL-1 is susceptible to partial down-regulation after endothelial cell activation. 203 50

5,6-Dichloro-1-(beta-D-ribofuranosyl)benzimidazole (DiCl-RB) is a powerful inhibitor of casein kinase-2 (CK-2) [Zandomeni, R. et al. (1986) J. Biol. Chem. 261, 3414-3420]. Here a series of 17 analogues of DiCl-RB has been employed for studying the specificity and the mode of action of this family of CK-2 inhibitors. The two halogen substituents on the benzene ring are shown to play a prominent role in inhibition, the 5,6-dibromo derivative (DiBr-RB) being fivefold more effective than DiCl-RB (Ki = 2 microM, with GTP as substrate), whereas the difluoro derivative (DiF-RB) is nearly as ineffective as unsubstituted 1-(beta-D-ribofuranosyl)benzimidazole. On the other hand, although some modifications of the ribose group significantly decrease the inhibitory efficiency, the sugar moiety is not strictly required, since dichlorobenzimidazole itself (DiCl-Bz) is an inhibitor almost as good as DiCl-RB. Inhibition of CK-2 by DiCl-RB and by its analogues, DiCl-Bz included, is of the competitive type with respect to the nucleotide substrate, the Ki values being lower with GTP than with ATP. The Ki values of the most potent inhibitor, DiBr-RB, with ATP and GTP, are 6 microM and 2 microM, respectively, denoting an affinity for the enzyme higher than that of the physiological substrates, ATP and GTP. DiBr-RB has been assayed for its inhibitory capacity toward several protein kinase other than CK-2. Protein kinase-C, cAMP-dependent protein kinase, the Ser/Thr protein kinase expressed by Pseudorabies virus, and four different tyrosine protein kinases from spleen, proved insensitive to DiBr-RB concentrations capable of almost entirely suppressing the activity of rat liver and maize seedling CK-2. Casein kinase-1 however is nearly as sensitive as CK-2 to DiBr-RB. Inhibition of CK-1 is also of the competitive type with respect to ATP (Ki = 14 microM). Although the inhibitory spectrum of CK-1 by the various analogues is reminiscent of that observed with CK-2, a remarkable difference is revealed by 5'-phosphorylation of ribose which increases the Ki with CK-2 while decreasing that with CK-1.
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PMID:Ribofuranosyl-benzimidazole derivatives as inhibitors of casein kinase-2 and casein kinase-1. 210 15

NIH 3T3 fibroblasts were transfected with the chloramphenicol-acetyltransferase (CAT) gene under the control of the SV40 early promoter, which can be stimulated by IL-1. CAT activity in cell lysates and PGE2 release in the supernatants were measured in control and stimulated cell cultures in parallel. Human IL-1 beta (180 pM) and human rTNF-alpha (3 nM) significantly stimulated both CAT activity and PGE2 release. The combined incubation of the two cytokines resulted in a synergistic effect on PGE2 release. The addition of AA (30 microM) greatly stimulated PGE2 release without affecting CAT activity. Similarly, drugs interfering with AA metabolism were without effect on CAT activity although profoundly reducing PGE2 release. Forskolin (0.1 microM) did not modify either parameter. The glucocorticoid fluocinolone (20 nM) was able to decrease both parameters. Protein kinase inhibitors H7 (5-50 microM) and sphingosine (50 microM) inhibited only IL-1-induced CAT activity, whereas H8 (5-50 microM) and HA1004 (50 microM) were ineffective on both parameters. PMA (0.5 microM) and R59 022, a diacylglycerol kinase inhibitor (10 microM), did not modify either control or IL-1-induced CAT activity. IL-1-stimulated PGE2 release was potentiated by PMA, although this effect was not inhibited by H7. The data suggest that: 1) in NIH 3T3 cells the activation of AA metabolism by IL-1 is not involved in IL-1-induced gene expression; 2) protein kinase C activity is required but not sufficient for IL-1-induced gene expression; and 3) PMA may stimulate AA metabolism by a mechanism in part independent of protein kinase activity.
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PMID:Induction of gene expression by IL-1 in NIH 3T3 cells. Possible requirement of protein kinase C activity and independence from arachidonic acid metabolism. 212 35


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