Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.13 (protein kinase C)
 49,245 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In preparations of synaptic terminals (synaptosomes) isolated from rat brain, the activity of phospholipase A2 (PLA2), a phospholipid hydrolase that serves a central function in signal transduction, was inhibited in a Ca(2+)-dependent manner by incubation with 60 mM K+ or with the Ca(2+)-selective ionophore ionomycin. Reversal by alkaline phosphatase treatment suggested that this inhibitory effect resulted from phosphorylation of a synaptosomal protein substrate. When lysed synaptosomes were incubated with Ca2+/calmodulin (CaM), purified Ca2+/CAM-dependent protein kinase II (Ca2+/CaM-dependent PK II) and ATP, PLA2 activity in lysates was nearly abolished within 10 min. This effect was accompanied by a marked decrease in the Vmax of the enzyme and little or no change in the Km. Furthermore, Ca2+/CaM with ATP but without exogenous Ca2+/CaM-dependent PK II partially inhibited PLA2 activity, and this effect was prevented by treating the lysates with a selective peptide inhibitor of Ca2+/CaM-dependent PK II. In contrast, incubation of intact synaptosomes with 4 beta-phorbol 12-myristate 13-acetate or of lysed synaptosomes with purified protein kinase C had little or no effect on PLA2 activity. The results strongly suggest that the Ca(2+)-dependent inhibition of PLA2 activity observed in intact nerve endings was produced by activation of the multifunctional Ca2+/CaM-dependent PK II. A membrane-permeable adenylyl cyclase activator, forskolin, enhanced PLA2 activity in intact synaptosomes, and cAMP-dependent protein kinase potentiated PLA2 activity in lysed synaptosomes. Furthermore, another broad-spectrum protein kinase present in synaptic terminals, casein kinase II, also potentiated PLA2 activity in lysed synaptosomes. The effects of both protein kinases were associated with a decrease in Km and no change in Vmax. The results suggest that PLA2 activity in synaptic terminals is subject to bidirectional control by distinct signal transduction pathways. Moreover, mutually antagonistic effects of the Ca2+/CaM-dependent PK II and PLA2 pathways provide a possible molecular mechanism for bidirectional modulation of neurotransmitter release.
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PMID:Bidirectional control of phospholipase A2 activity by Ca2+/calmodulin-dependent protein kinase II, cAMP-dependent protein kinase, and casein kinase II. 165 Apr 81

We investigated the effects of hypergravity on DNA synthesis and alkaline phosphatase (ALP) activity in cloned osteoblast-like cells, MC3T3-E1. Hypergravity (5 x g) stimulated DNA synthesis in these cells in a time-dependent manner and increased it approximately up to 150% of that of the control (1 x g). 12-O-Tetra-decanoylphorbol-13-acetate (TPA), a protein kinase C activator, and insulin-like growth factor I (IGF-I) enhanced DNA synthesis additively with hypergravity (5 x g). An increase in ALP activity induced by 10% fetal calf serum (FCS) was suppressed by hypergravity (2 x g, 5 x g). Five x g completely suppressed the increase in ALP activity. TPA and hypergravity (2 x g) suppressed the increase in ALP activity induced by FCS additively. Hypergravity (5 x g) showed no significant effect on cAMP nor cGMP production in these cells, but increased prostaglandin E2 (PGE2) production. Exogenous PGE2 stimulated DNA synthesis in these cells but had little effect on 10% FCS-induced ALP activity. These results suggest that hypergravity stimulates proliferation but suppresses differentiation of osteoblast-like cells through a pathway independent of the activation of protein kinase C and the production of cyclic nucleotides, and that hypergravity and IGF-I stimulate proliferation of these cells through an independent signal transduction pathway. Moreover, our data strongly suggest that PGE2 mediates the signalling of hypergravity on the proliferation of osteoblast-like cells.
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PMID:Effects of hypergravity on proliferation and differentiation of osteoblast-like cells. 165 Nov 38

The present study characterizes the inhibitory effects of nodularin, a recently isolated hepatotoxic compound from the cyanobacterium Nodularia spumigena, on type 1 (PP1), type 2A, (PP2A), type 2B (PP2B), and type 2C (PP2C) protein phosphatases. Both PP2A and PP1 were potently inhibited (IC50 = 0.026 and 1.8 nM, respectively) by nodularin, whereas PP2B was inhibited to a lesser extent (IC50 = 8.7 microM). Nodularin had no apparent effect on PP2C, alkaline phosphatase, acid phosphatase, insulin receptor tyrosine kinase, protein kinase A, phosphorylase kinase, or protein kinase C. In a whole-cell extract of T51B liver cells, nodularin inhibited PP1 and PP2A activity with a potency similar to that seen with their purified catalytic subunits. Thus, due to the high specificity of nodularin for PP2A and PP1, this hepatotoxin may prove to be useful as a probe for distinguishing the activity of these protein phosphatases in cell extracts.
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PMID:Cyanobacterial nodularin is a potent inhibitor of type 1 and type 2A protein phosphatases. 165 93

A number of cell-surface proteins are anchored by a phosphatidylinositol (PI)-glycan moiety. These proteins can be released by PI-specific phospholipases C (PI-PLC). Decay-accelerating factor (DAF) is such a cell-surface protein that protects cells from inadvertent complement attack by binding to and inactivating C3 and C5 convertases. We have studied the regulation of DAF synthesis in human umbilical vein endothelial cells (HUVEC), a cell that has the highest level of surface DAF among those human cells that have been studied. HUVEC DAF was measured by immunoradiometric assay of detergent extracts and of cell supernatants after treatment of cells with a bacterial (Bacillus thuringiensis) PI-PLC. Eighty percent of the HUVEC DAF (4 to 8 x 10(5) molecules/cell) was released by exogenously added PI-PLC, indicating that it is predominantly PI-anchored. The level of PI-PLC-sensitive HUVEC DAF was increased three- to fourfold by overnight treatment of cultures with the protein kinase C activators, PMA (1 to 10 nM), phorbol-12,13-dibutyrate (10 to 100 nM), and teleocidin A (1 to 10 nM) under conditions where cell number, protein, and lactate dehydrogenase remain unchanged. This DAF synthesis was blocked by the protein kinase C inhibitor K-252a in a dose-dependent manner (ED50 = 0.06 microM). The biologically inactive phorbols, 4-alpha-phorbol-12 myristate-13-acetate (1 microM) and 4-alpha-phorbol-12, 13-didecanoate (1 microM) did not increase DAF levels. The newly expressed DAF in PMA-stimulated cells was still largely PI-anchored. In contrast, another PI-anchored protein, alkaline phosphatase, was not altered by PMA treatment, demonstrating that the PMA effect is not uniform among all surface proteins. The increased expression of DAF only was evident 8 h after PMA addition and was blocked by the RNA and protein synthesis inhibitors, actinomycin D and cycloheximide, indicating that both transcription and translation are required for DAF synthesis induced by phorbol esters. It is concluded that protein kinase C activators cause selective induction of endothelial cell DAF and that DAF synthesis involves protein kinase C activation.
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PMID:Phorbol esters increase synthesis of decay-accelerating factor, a phosphatidylinositol-anchored surface protein, in human endothelial cells. 168 81

Levels of the G-protein alpha-subunits alpha-Gi-2, alpha-Gi-3 and the 42 kDa, form of alpha-Gs were markedly decreased in hepatocyte membranes from streptozotocin-diabetic animals as compared with normals. In contrast, no detectable changes in alpha-Gi subunits were seen in liver plasma membranes of streptozotocin-diabetic animals, although levels of the 45 kDa form of Gs were increased. G-protein beta subunits in plasma membranes were unaffected by diabetes induction. Analysis of whole-liver RNA indicated that the induction of diabetes had little effect on transcript levels of Gi-3, caused an increase in Gs transcripts and decreased transcript number for Gi-2, albeit to a much lesser extent than was observed upon analysis of hepatocyte RNA. In both hepatocyte and liver plasma membranes, immunoblot analysis showed that levels of the catalytic unit of adenylate cyclase were increased upon induction of diabetes. Under basal conditions, alpha-Gi-2 from hepatocytes of diabetic animals was found to be both phosphorylated to a greater extent than alpha-Gi-2 isolated from hepatocytes of normal animals, and furthermore was resistant to any further phosphorylation upon challenge of hepatocytes with angiotensin, vasopressin or the phorbol ester 12-O-tetradecanoylphorbol 13-acetate. Treatment of isolated plasma membranes from normal, but not diabetic, animals with purified protein kinase C caused the phosphorylation of alpha-Gi-2. Treatment of membranes from diabetic animals with alkaline phosphatase caused the dephosphorylation of alpha-Gi-2 and rendered it susceptible to subsequent phosphorylation with protein kinase C. Low concentrations of the non-hydrolysable GTP analogue guanylyl 5'-imidodiphosphate inhibited adenylate cyclase activity in both hepatocyte and liver plasma membranes from normal, but not diabetic, animals.
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PMID:Diabetes-induced alterations in the expression, functioning and phosphorylation state of the inhibitory guanine nucleotide regulatory protein Gi-2 in hepatocytes. 170 Jul

A variety of signal transduction pathways contribute to the regulation of transcription in mammalian cells. Several of these pathways ultimately rely upon the interaction of transcription factors with genetic sequences termed response elements in the promoter regions of some genes. The biochemical mechanisms that control the levels and state of activation of transcription factors are poorly understood. However, specific phosphorylation events mediated by protein kinase C, growth factor receptor-linked tyrosine kinases, and protein kinase A clearly participate in the regulation of these signal transduction pathways. To understand the relationship between activation and/or inhibition of these pathways and regulation of gene expression controlled by specific response elements, cell lines were prepared containing the TPA response element (TRE), serum response element (SRE), or cyclic AMP response element (CRE) fused to a gene encoding a secretable form of alkaline phosphatase (SEAP). These TRE-SEAP, SRE-SEAP, and CRE-SEAP cells exhibit dramatic increases in alkaline phosphatase (AP) activity following exposure to TPA, PDGF, or forskolin. Down regulation of protein kinase C or inhibition of tyrosine kinase activity blocked the stimulation of AP activity caused by TPA or PDGF. These cell lines can be used to characterize existing inhibitors, and to identify new agents that affect specific signal transduction pathways in mammalian cells.
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PMID:Mammalian cell lines engineered to identify inhibitors of specific signal transduction pathways. 171 Nov 89

The hematoxylin-stainable protein (HSP) in keratohyalin granules of the newborn rat epidermis was found to have the same amino acid composition and the same inhibitory and immunological properties as cystatin alpha. However, only its pI value (4.7) differed from that of cystatin alpha (5.3). Alkaline phosphatase treatment of HSP changed its pI value from 4.7 to 5.3. This pI change was inhibited by EDTA, an inhibitor of alkaline phosphatase. Furthermore, 32P from [gamma-32P]ATP was incorporated into recombinant cystatin alpha by a protein kinase C (PKC) preparation in the presence of phosphatidyl serine and Ca2+ ions as co-factors. The incorporation increased dose-dependently with the added cystatin alpha and was inhibited significantly by H-7, a specific inhibitor of PKC. SDS-PAGE autoradiography of the 32P-labeled proteins showed that 32P was incorporated into the cystatin alpha. This incorporation was not observed by the action of cAMP-dependent protein kinase. Therefore, it is highly possible that the HSP is a phosphorylated cystatin alpha and that the phosphorylation is catalyzed specifically by PKC.
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PMID:Identification of hematoxylin-stainable protein in epidermal keratohyalin granules as phosphorylated cystatin alpha by protein kinase C. 171 84

We have shown previously that cytoplasmic extracts from actively dividing lymphoid cells are capable of inducing DNA synthesis in isolated nuclei. One of the factors involved in this activity, ADR, appears to be a greater than 90 kDa heat-labile protease. Cytoplasmic extracts prepared from nonproliferating lymphocytes express little to no ADR activity. However, ADR activity can be generated in these extracts by brief exposure to a membrane-enriched fraction of spontaneously proliferating, leukemic human T lymphoblastoid (MOLT-4) cells. This suggests that ADR activity is present in the resting cytoplasm in an inactive or precursor form. This in vitro generation of ADR activity can be inhibited in a dose-dependent manner by the isoquinolinesulfonamide derivative, H-7 (1-(5-isoquinoline-sulfonyl)-2-methylpiperazine dihydrochloride), an inhibitor of both cyclic adenosine monophosphate (cAMP)-dependent protein kinases and protein kinase C (PKC). However, more specific inhibitors of cAMP-dependent protein kinases, including N-[( 2-methylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H8) and N-(2-gua-nidinoethyl)-5-isoquinolinesulfonamide (HA-1004), had little to no effect on the in vitro generation of ADR activity. Furthermore, membranes from MOLT-4 cells depleted of PKC by long-term exposure (24 h) to phorbol esters and calcium ionophores were unable to induce ADR activity in resting peripheral blood lymphocytes extracts. The results of these studies suggest 1) ADR activity is present in resting cell cytoplasm in an inactive or precursor form; and 2) ADR activity can be induced in this resting cytoplasm through a mechanism involving a membrane-associated protein kinase, possibly PKC. The ability of alkaline phosphatase to deplete the activity of preformed ADR suggests the possibility that ADR itself is phosphoprotein.
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PMID:Induction of a cytoplasmic activator of DNA synthesis in lymphocytes is mediated through a membrane-associated protein kinase. 172 28

Alkaline phosphatase from calf intestinal mucosa dephosphorylated histone H1 and fibrinogen that had been phosphorylated with protein kinase C. The reaction velocity was dependent on the ionic strength of the buffer; decreasing with increasing concentration. The pH optimum was around 7, which is lower than pH-optima described for other kinds of substrates. (32P) phosphorylated fibrinogen was dephosphorylated about 20 times faster than (32P)phosphohistone on a weight basis and the reaction continued linearily with time for the longest time tested (3 hs) even at 37 degrees C. As alkaline phosphatase is present in the blood the possible physiological significance of the dephosphorylation of phosphofibrinogen is discussed.
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PMID:Dephosporylation with alkaline phosphatase of histone and fibrinogen phosphorylated with protein kinase C in vitro. 177 20

We applied Southwestern and Western blotting and gel retardation techniques to investigate the changes that occur in the cyclic adenosine monophosphate (cAMP)-responsive element (CRE) binding (CREB) proteins in rapidly growing, chemically induced 5123tc and 5123D Morris hepatomas. Using the CRE sequences from the c-fos, E2A, and somatostatin gene promoters, we identified in the nuclear proteins from normal unstimulated or proliferating rat liver cells six different protein factors of Mr 34,000, 36,000, 40,000, 47,000, 56,000, and 72,000 capable of binding to the element. The Mr 47,000 protein had the highest specificity for the core CRE, suggesting its importance in cAMP-mediated gene expression. We could not find the Mr 47,000 CREB protein in the 5123tc and 5123D hepatomas. Our efforts to detect this protein in the tumors by (a) using the CRE sequence from different gene promoters, (b) altering the protocol for extracting nuclear proteins, or (c) attempting to restore its DNA-binding property by phosphorylation [with endogenous protein kinase(s), a catalytic subunit of cAMP-dependent protein kinase, and protein kinase C/dephosphorylation (with alkaline phosphatase)] were unsuccessful. The loss of tje Mr 47,000 CREB protein from solid tumors of the Morris hepatoma is likely to be related to the neoplastic properties of the tumor cell rather than to cell growth because the level of this protein remained unchanged during a 6-day period of liver regeneration. The nuclear extract from the Morris hepatoma that did not have the Mr 47,000 CRE-binding factor contained proteins immunologically related to the CREB, c-Jun, and c-Fos proteins. We conclude that the Mr 47,000 factor represents a distinct member of the CRE-binding protein family and that its absence from the hepatomas may lead to aberrant expression of cAMP-inducible genes.
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PMID:Changes in cyclic adenosine monophosphate-responsive element binding proteins in rat hepatomas. 182 83


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