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)

The indole carbazole staurosporine is an extraordinarily potent antiproliferative agent that inhibits the growth of cultured mammalian cells at concentrations of less than 1 nM. The antiproliferative activity of staurosporine is attributed to its potent inhibition of diverse protein kinases, but the mechanism of staurosporine inhibition has not been elucidated for any protein kinase. Protein kinase C (PKC) is a family of Ca(2+)- and phosphatidylserine-dependent protein kinases that are activated in vivo by the second messenger diacylglycerol. A fully active, Ca(2+)- and phosphatidylserine-independent, catalytic fragment of PKC that contains only the catalytic domain of the enzyme can be produced by limited proteolysis. Previous studies indicated that staurosporine inhibits PKC by binding its catalytic domain. In this study, we define the kinetics of inhibition by staurosporine of a catalytic fragment of rat brain PKC-gamma and of a catalytic fragment generated from a rat brain PKC-alpha/PKC-beta mixture. Our kinetic results provide evidence that staurosporine inhibits PKC by binding to a site of the catalytic domain other than the ATP substrate and protein substrate binding sites. Staurosporine inhibition appears to entail binding at a conserved site in the catalytic domain of PKC, because staurosporine inhibited rat brain PKC-alpha, PKC-beta, and PKC-gamma, as well as the catalytic fragments of PKC-beta and PKC-gamma, with similar protencies. The kinetics of inhibition of the catalytic fragment of PKC-gamma were uncompetitive with respect to histone III-S, providing evidence that the binding of histone III-S at the active site of the catalytic fragment precedes the binding of staurosporine to the enzyme. Taken in the context of previous mechanistic studies of PKC-catalyzed histone III-S phosphorylation, these results provide evidence that staurosporine binds to a complex of PKC, MgATP, and histone III-S, thereby forming a complex that cannot break down to products. In addition, the inhibitory kinetics observed when the ATP concentration was varied provided evidence that staurosporine reduces the affinity of MgATP for the catalytic fragment of PKC-gamma. Thus, the kinetics of inhibition of the catalytic fragment of PKC-gamma by staurosporine provide evidence that staurosporine inhibits PKC by a mixed mechanism.
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PMID:Kinetic analysis of protein kinase C inhibition by staurosporine: evidence that inhibition entails inhibitor binding at a conserved region of the catalytic domain but not competition with substrates. 153 15

The molecular heterogeneity of protein kinase C (PKC) is now widely documented. In our first report, we characterized the rat lacrimal gland PKC along with a phorbol 12-myristate 13-acetate (PMA)-activated and phospholipid-independent protein kinase activity [Mauduit P., Zoukhri D. and Rossignol B. (1989) Fedn Eur. biochem. Socs Lett. 252, 5-11. In this work, we show that when the rat lacrimal gland cytosolic fraction is chromatographed on hydroxyapatite, only one peak of PKC activity can be detected. Comparison with a rat brain cytosolic fraction indicated that it is PKC-alpha which is expressed in the rat lacrimal gland. This result was confirmed by the use of polyclonal antibodies raised against rat brain PKC-alpha, beta and gamma isoforms. We also provide evidence that free arachidonic acid activates PKC, as does PMA, in a calcium and phospholipid-free system.
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PMID:The rat lacrimal gland expresses the alpha isoform of PKC. Further evidence for the PMA-activated and phospholipid-independent protein kinase activity. 157 Dec 2

The expression of members of the Ca2+ and phospholipid-dependent protein kinase (PKC) family were studied in murine Swiss 3T3 cells. In addition to PKC-alpha, the presence of immunoreactive PKC-delta, -epsilon, and zeta was detected. Treatment with 500 nM 12-0-tetradecanoylphorbol-13-acetate (TPA) led to the down-regulation of alpha, delta, and epsilon isoforms, but not that of zeta. Higher concentrations of TPA similarly had no effect on the level of PKC-zeta. In contrast to PKC-alpha, the membrane localization of PKC-delta, -epsilon, and -zeta was not enhanced by extraction in Ca(2+)-containing buffers, whereas acute TPA treatment increased membrane association of PKC-alpha, -delta, and -epsilon but not that of PKC-zeta.
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PMID:Identification of multiple PKC isoforms in Swiss 3T3 cells: differential down-regulation by phorbol ester. 163 59

Expression of rat protein kinase C-delta (PKC-delta) and PKC-zeta in insect cells using recombinant baculovirus resulted in the production of proteins with a molecular size of approximately 76 kD and 78 kD, respectively, as determined by immunoblotting with subtype-specific antisera. Although the PKC-zeta cDNA encoded for 592 amino acids, a 76 kD protein was also generated by in vitro transcription/translation. Extracts of cells expressing PKC-delta were able to bind phorbol ester to levels comparable to extracts of cells expressing PKC-alpha. No phorbol ester binding was, however, detected in insect cell extracts expressing PKC-zeta. However, similar levels of protein kinase activity were detected in lysates of cells expressing PKC-delta or PKC-zeta when protamine sulfate was used as exogenous substrate. Compared to protamine sulfate, both, myelin basic protein (MBP) or histone, were poor substrates for PKC-delta and PKC-zeta. In contrast to PKC-zeta, the PKC-delta enzyme activity phosphorylated MBP or histone in a phosphatidylserine-(PS)/diacylglycerol(DG)-dependent manner, albeit not to the same extent as PKC-alpha. Lack of stimulation of the enzyme activity of PKC-zeta by PS/DG, was confirmed by endogenous phosphorylation of insect cell proteins by PKC-zeta, whereas several insect cell proteins were phosphorylated by PKC-delta in a PS/DG-dependent manner, including a protein of 78 kD. Our data demonstrate that the 76 kD PKC-zeta, in contrast to PKC-delta, is unable to bind phorbol esters and displays a protein kinase activity that is independent of PS or PS/DG. In addition, staurosporine was about 2-4 order of magnitudes less effective in inhibiting the protein kinase activities of PKC-delta and PKC-zeta when compared to PKC-alpha.
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PMID:Expression and partial characterization of rat protein kinase C-delta and protein kinase C-zeta in insect cells using recombinant baculovirus. 164 61

NPC 15437 inhibited protein kinase C (PKC) activity and [3H]phorbol 12,13-dibutyrate (PDBu) binding to the enzyme in a concentration-dependent manner (IC50 values, 19 +/- 2 microM and 23 +/- 4 microM, respectively). No inhibition of cAMP-dependent protein kinase A (PKA) or calcium/calmodulin-dependent myosin light chain kinase (MLCK) was observed. A detailed kinetic analysis of the interaction of NPC 15437 and a homogeneous preparation of PKC-alpha revealed a competitive type of inhibition with respect to activation of the enzyme by both phorbol 12-myristate 13-acetate (PMA) (Ki = 5 +/- 3 microM) and phosphatidylserine (PS) (Ki = 12 +/- 4 microM). Mixed inhibition (predominantly of the non-competitive type), with respect to activation of the enzyme by calcium, was also observed. These studies indicate that NPC 15437 is a selective inhibitor of PKC, interacting at the regulatory region of the molecule. NPC 15437 inhibited phorbol ester-induced ear edema in mouse (IC50 = 175 micrograms/ear) demonstrating the ability of NPC 15437 to inhibit PKC-mediated activity in intact cells.
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PMID:2,6-Diamino-N-([1-oxotridecyl)-2-piperidinyl]methyl)hexanamide (NPC 15437): a selective inhibitor of protein kinase C. 179 19

The human promyelocytic leukemia cell line HL-60 differentiates in vitro when treated with various inducers. It has previously been shown that protein kinase C (PKC) isozymes are modulated during granulocytic differentiation of HL-60 cells induced by dimethyl sulfoxide or retinoic acid (M. Makowske, R. Ballester, Y. Cayre, and O.M. Rosen, J. Biol. Chem., 263: 3402-3410, 1988; K. Hashimoto, A. Kishimoto, H. Aihara, I. Yasuda, K. Mikawa, and Y. Nishizuka, FEBS Left., 263: 31-34, 1990). HL-60 responds to 1 alpha, 25-dihydroxyvitamin D3 (1,25-(OH)2D3) or to 12-O-tetradecanoylphorbol-13-acetate by giving rise to monocytic cells. In the present study, we demonstrate that treatment of HL-60 cells with 1,25-(OH)2D3 causes dramatic increases in PKC-alpha and PKC-beta protein levels detected by immunoblotting with PKC isoform-specific antibodies and in Ca(2+)- and phospholipid-dependent protein kinase activity. We also observed a transient increase in the steady-state levels of PKC-alpha and PKC-beta mRNA species in Northern blotting experiments, with maximal induction occurring 48 h after addition of 1,25-(OH)2D3. Analyses of 1,25-(OH)2D3-induced PKC mRNA expression by nuclear run-on transcription experiments suggest that the observed increases in PKC mRNA levels may occur by a posttranscriptional mechanism(s). In contrast to the transient increases in PKC mRNA levels, the increases in PKC Mr 80,000 protein species and in PKC enzyme activity were progressive in HL-60 cells treated with 1,25-(OH)2D3 between 1 and 5 days, thus implying the existence of a further up-regulation of PKC proteins occurring at the translational and/or posttranslational levels.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:1 alpha,25-dihydroxyvitamin D3-induced regulation of protein kinase C gene expression during HL-60 cell differentiation. 186 31

Normal human melanocytes, unlike malignant melanomas, require the presence of phorbol ester for growth in culture. Because protein kinase C (PKC) represents the intracellular receptor for phorbol esters, we investigated a possible correlation between expression of PKC and tumor progression in the melanocytic system. The results failed to show expression of PKC-alpha, -beta or -gamma in normal human melanocytes. However, PKC-alpha was expressed in primary and metastatic melanomas; even though antisense oligodeoxynucleotides targeted against different mRNA regions of human PKC-alpha, and H7, an inhibitor of PKC, did not display significant growth-inhibitory effects. A similar pattern of expression was detected with respect to the expression of cAMP-dependent protein kinase (PKA). Normal human melanocytes did not reveal expression of either of the known catalytic or regulatory subunits of human PKA, whereas primary and metastatic melanomas demonstrated expression of the PKA-specific subunits C alpha and RI alpha.
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PMID:Differential expression of protein kinase C and cAMP-dependent protein kinase in normal human melanocytes and malignant melanomas. 239 22

MRL-lpr mice are severely impaired in the Fas pathway of apoptosis induction. We here evaluate another pathway of apoptosis induction in MRL-lpr mice which is protein kinase C (PKC) dependent. Despite the defect of the Fas pathway, apoptosis developed during culture in vitro in splenic T lymphocytes from MRL-lpr mice more extensively than in T lymphocytes from MRL-(+/+) mice. Apoptosis induction in the former cells was then found to be greatly promoted by PKC inhibitor H-7, and partially prevented by PKC activator phorbol 12-myristate 13-acetate (PMA). High sensitivity to H-7, but not to PKA inhibitor HA 1004, of these cells for apoptosis induction was confirmed by detailed time course and dose-dependency experiments of the drug effect. Population analysis showed that both CD4+ T lymphocytes and CD8+ T lymphocytes from MRL-lpr mice were highly sensitive to H-7, whereas CD8+ T lymphocytes, but not CD4+ T lymphocytes, from MRL-(+/+) mice were susceptible to the reagent. Interestingly, B220+ Thy-1+ CD4-CD8- T lymphocytes from MRL-lpr mice were most sensitive to H-7 for apoptosis induction. Correspondingly, the membrane-translocated activated PKC-alpha level in splenic T lymphocytes from MRL-lpr was more extensively up-regulated by PMA than in splenic T lymphocytes from MRL-(+/+). These results suggest that some signal consistently activates PKC in MRL-lpr T lymphocytes, and this event is needed for survival of these cells. On the other hand, CD4+ CD8+ thymocytes were deleted by apoptosis in culture with PMA, whether these thymocytes were from MRL-lpr mice or MRL-(+/+) mice. This finding suggested that the apoptosis induction pathway linked to PKC activation is intact in CD4+ CD8+ thymocytes from the Fas-defective MRL-lpr mice. We conclude from these results that the PKC-dependent signal pathways for either cell death or cell activation are intact or even accelerated in lpr mice, which could both compensate for the loss of the Fas pathway and promote the generation of autoreactive T lymphocytes.
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PMID:Elucidation of the protein kinase C-dependent apoptosis pathway in distinct subsets of T lymphocytes in MRL-lpr/lpr mice. 748 61

The effects of phorbol ester and forskolin on the net phosphorylation and turnover of P0 phosphate groups was studied in normal and experimentally diabetic rats. In sciatic nerve segments isolated from normal rats and incubated with [32P]-inorganic phosphate, phosphorylation of the major peripheral myelin protein, P0, was increased 2-5 fold in a time and dose-dependent manner by phorbol 12,13 dibutyrate (PDB). This increase was blocked by the protein kinase inhibitors, H-7 and staurosporine. Both the basal and PDB-stimulated phosphorylation of P0 were significantly greater in segments of sciatic nerve from streptozotocin-induced diabetic rats. Prolonged exposure of nerve segments to PDB abolished the stimulated phosphorylation of P0 and immunoblots of nerve proteins revealed a decrease in the content of the protein kinase C alpha-isoform. The adenylate cyclase activator, forskolin, had no effect on the PDB-stimulated phosphorylation of P0 in normal nerve but decreased phosphorylation in diabetic nerve. To measure turnover of P0 phosphate groups, nerves were incubated with 32P and incorporated label was then chased in radioactivity-free medium for up to 4 hours. P0 from normal nerve prelabeled under basal conditions lost 25% of its radioactivity during this time. In contrast, nearly all of the additional phosphate groups prelabeled in the presence of PDB disappeared after 2 hours of chase. P0 phosphate groups from diabetic nerve displayed similar turnover kinetics. When forskolin was added to the chase medium, the turnover of P0 phosphate moieties was accelerated in normal, but not in diabetic nerve.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:P0 phosphorylation in nerves from normal and diabetic rats: role of protein kinase C and turnover of phosphate groups. 752 47

We have recently shown that expression of specific protein kinase C (PKC) isoforms correlates with cell fate in neural chicken embryo cells. Therefore we investigated the effects of PKC activation by phorbol esters on acquisition of the astrocytic phenotype, using cultured embryonic cortical astrocytes, derived from 15-day-old chick embryos (E15CH), as a model. Short term treatment with the phorbol ester 12-tetradecanoylphorbol-13-acetate (TPA), which activates PKC-alpha/beta in E15CH, caused association of PKC with the cytoskeleton. In vitro kinase assays of cytoskeleton-associated PKC demonstrated phosphorylation of many cytoskeletal proteins. Phosphorylation was blocked by protein kinase inhibitors (H8), and enhanced by phosphatase inhibitors (calyculin A). Among these PKC substrates, a most prominent 60-kDa protein was identified as vimentin. Assembly of vimentin into the cytoskeleton depends on cell type and state of differentiation. To establish that TPA (PKC) regulates assembly of vimentin into the cytoskeleton of astrocytes, we used pulse-chase (20/5 min) labeling with [35S]methionine, and immunoprecipitations with an anti-vimentin mAb from extractable and cytoskeletal fractions. These studies revealed that 20 min treatment with TPA leads to a 3-fold increase in the rate of newly synthesized full-length vimentin assembly (posttranslational assembly). Furthermore, TPA increased cotranslational assembly of vimentin. The protein kinase A activator forskolin, did not have such effects on vimentin assembly. Long-term TPA treatment, which correlates with a prolonged phospholipase D (PLD) activation, was mitogenic and caused dramatic changes in the morphology of astrocytes. In addition these fibrous, polarized astrocytes had decreased activity of the astrocyte specific enzyme, glutamine synthetase, but had increased abundance of vimentin protein. These studies provide biochemical evidence on acquisition of a different astrocytic phenotype after activation of the PKC/PLD pathway, in the chick embryo. Therefore PKC and PLD activation is pivotal for the acquisition and maintenance of phenotypes in chick embryonic astrocytes.
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PMID:Phorbol esters and PKC signaling regulate proliferation, vimentin cytoskeleton assembly and glutamine synthetase activity of chick embryo cerebrum astrocytes in culture. 755 27


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