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:2.7.11.13 (protein kinase C)
49,245 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have investigated coupling between the epidermal growth factor (EGF) receptor and the phospholipase C (PLC)/protein kinase C (PKC) signal-transduction system in normal skin fibroblasts and keratinocytes, for which EGF and transforming growth factor alpha (TGF-alpha) are mitogenic. EGF and TGF-alpha induced a rapid increase in tyrosine phosphorylation of the EGF receptor, in both fibroblasts and keratinocytes, but failed to induce tyrosine phosphorylation of PLC-gamma 1 or detectable phosphoinositide hydrolysis, as measured by two sensitive assays. In fibroblasts, EGF induced phosphatidylcholine (PC) hydrolysis, resulting in increased diacylglycerol (DAG). In contrast, in keratinocytes, there was no detectable PC hydrolysis or elevation of DAG in response to EGF or TGF-alpha. EGF and TGF-alpha activated PKC in fibroblasts, as evidenced by increased phosphorylation of a specific cellular PKC substrate (myristoylated alanine-rich C-kinase substrate, 'MARCKS'). In keratinocytes, TGF-alpha and EGF induced only a modest increase in MARCKS protein phosphorylation. This apparent modest activation of PKC, in the absence of detectable DAG formation, may have been mediated by arachidonic acid, which was released from keratinocytes in response to TGF-alpha, and has been shown to stimulate PKC activity in vitro. These data demonstrate that (1) in dermal fibroblasts and keratinocytes, which express normal levels of EGF receptors, EGF receptor activation is not coupled to tyrosine phosphorylation of PLC-gamma 1 or PtdIns hydrolysis, suggesting that these events are not required for the mitogenic activity of EGF or TGF-alpha in these cells, (2) coupling of EGF receptor to PC hydrolysis is cell-type specific, and (3) in skin fibroblasts, DAG, formed through EGF-induced PC hydrolysis, is capable of activating PKC.
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PMID:Differential induction of phosphatidylcholine hydrolysis, diacylglycerol formation and protein kinase C activation by epidermal growth factor and transforming growth factor-alpha in normal human skin fibroblasts and keratinocytes. 769 May 46

MARCKS is a protein kinase C (PKC) substrate that is phosphorylated during neurosecretion, phagocyte activation and growth factor-dependent mitogenesis. MARCKS binds calcium/calmodulin and crosslinks F-actin, and both these activities are regulated by PKC-dependent phosphorylation. We present evidence here that PKC-dependent phosphorylation also regulates the cycling of MARCKS between the plasma membrane and Lamp-1-positive lysosomes. Immuno-fluorescence and immunoelectron microscopy, and subcellular fractionation, demonstrated that MARCKS was predominantly associated with the plasma membrane of resting fibroblasts. Activation of PKC resulted in MARCKS phosphorylation and its displacement from the plasma membrane to Lamp-1-positive lysosomes. MARCKS phosphorylation is required for its translocation to lysosomes since mutating either the serine residues phosphorylated by PKC (phos-) or the PKC inhibitor staurosporine, prevented MARCKS phosphorylation, its release from the plasma membrane, and its subsequent association with lysosomes. In the presence of lysosomotropic agents or nocodazole, MARCKS accumulated on lysosomes and returned to the plasma membrane upon drug removal, further suggesting that the protein cycles between the plasma membrane and lysosomes. In contrast to wild-type MARCKS, the phos- mutant did not accumulate on lysosomes in cells treated with NH4Cl, suggesting that basal phosphorylation of MARCKS promotes its constitutive cycling between these two compartments.
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PMID:Protein kinase C regulates MARCKS cycling between the plasma membrane and lysosomes in fibroblasts. 772 Jul 2

The inhibitory effects of three novel staurosporine-derived compounds were tested with five different types of protein kinases, including protein kinase C (PKC). IC50 values of two of these compounds were found to be 300 to > 5000 times lower for PKC alpha beta gamma (a mixture of the PKC isoenzymes alpha, beta and gamma) than for any of the other protein kinases. The inhibitory action of the most selective inhibitor was tested also with the Ca(2+)-unresponsive PKC isoenzyme delta and was found to suppress PKC alpha beta gamma and PKC delta differentially. The highly specific PKC inhibitors are active both in cell culture and in vivo. They inhibit the PKC-catalyzed phosphorylation of the specific PKC substrate MARCKS in Swiss-3T3 fibroblasts and the okadaic acid-induced edema of the mouse ear. However, the more complex biological processes triggered by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate in mouse skin, such as inflammation, stimulation of cellular hyperproliferation and tumor promotion, remain largely unaffected upon topical application of these compounds.
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PMID:Lack of an effect of novel inhibitors with high specificity for protein kinase C on the action of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate on mouse skin in vivo. 783 93

Phosphorylation of three physiological substrates of protein kinase C (PKC), MARCKS, neuromodulin (Nm), and neurogranin (Ng), was analyzed to determine their relative efficacy as substrates of PKC alpha, beta, and gamma and sensitivities to inhibition by calmodulin (CaM) and S100. Comparison of the Vmax/Km of the phosphorylation of each individual substrate indicated the order of efficacy as PKC substrate was MARCKS > Nm > Ng. Phosphorylation of these proteins in a mixture by PKC beta and gamma was indistinguishable from that when each individual substrate was phosphorylated by these two isozymes. In contrast, the rates of PKC alpha-catalyzed phosphorylation of Nm and Ng in a mixture also containing MARCKS were significantly reduced as compared to that when Nm or Ng was individually phosphorylated by this isozyme. When these substrates were present in a mixture, both CaM and S100 inhibited the PKC-catalyzed phosphorylation of MARCKS to a higher degree than that of Nm or Ng. Protease-activated catalytic fragment of PKC (PKM) was used to determine the effects of Ca2+ and phospholipid on the CaM and S100-mediated inhibition of PKC substrate phosphorylation. CaM and S100 inhibited the PKM-catalyzed phosphorylation of MARCKS only in the presence of Ca2+ and addition of phosphatidylserine (PS)/dioleoylglycerol (DG) did not influence the inhibitory effect. Phosphorylation of Nm or Ng by PKM was inhibited by CaM to a higher degree in the absence than in the presence of Ca2+. S100 was ineffective in inhibiting the phosphorylation of Nm and Ng without Ca2+ and only poorly effective in the presence of Ca2+. The CaM-mediated inhibition of Nm or Ng phosphorylation by PKM was also not affected by PS/DG either with or without Ca2+. The results presented here demonstrate that MARCKS is a preferred substrate of PKC and its phosphorylation by PKC is most sensitive to inhibition by regulatory proteins such as CaM and S100.
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PMID:Differential responses of protein kinase C substrates (MARCKS, neuromodulin, and neurogranin) phosphorylation to calmodulin and S100. 784 Jun 34

1. We have previously shown that acute exposure to the HIV coat protein gp120 interferes with the beta-adrenergic regulation of astroglial and microglial cells (Levi et al., 1993). In particular, exposure to 100 pM gp120 for 30 min depressed the phosphorylation of vimentin and glial fibrillary acidic protein (GFAP) induced by isoproterenol in rat cortical astrocyte cultures. In the present study we have extended our analysis on the effects of gp120 on astroglial protein phosphorylation. 2. We found that chronic (3-day) treatment of the cells with 100 pM gp120 before exposure to isoproterenol was substantially more effective than acute treatment in depressing the stimulatory effect of the beta-adrenergic agonist on vimentin and GFAP phosphorylation. 3. Even after chronic treatment with gp120, no differences were found in the levels and solubility of these proteins. 4. Besides stimulating the phosphorylation of intermediate filament proteins, isoproterenol inhibited the incorporation of 32P into a soluble acidic protein of 80,000 M(r), which was only minimally present in Triton X-100-insoluble extracts. 5. Treatment of astrocytes with a phorbol ester or exposure to 3H-myristic acid indicated that the acidic 80,000 M(r) protein is a substrate for protein kinase C (PKC) and is myristoylated, thus suggesting that it is related to the MARCKS family of PKC substrates. 6. Acute (30-min) treatment with 100 pM gp120 totally prevented the inhibitory effect of isoproterenol on the phosphorylation of the 80,000 M(r) MARCKS-like protein. 7. Our studies corroborate the hypothesis that viral components may contribute to the neuropathological changes observed in AIDS through the alteration of signal transduction systems in glial cells.
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PMID:Human immunodeficiency virus protein gp120 interferes with beta-adrenergic receptor-mediated protein phosphorylation in cultured rat cortical astrocytes. 784 74

We have recently shown that AVP causes a protein kinase C (PKC)-dependent increase in ACTH release and biosynthesis in ovine anterior pituitary cells. In these cells, AVP also causes the translocation of PKC from the cytosol to the cell membrane which is maximal at 5 min, but the intracellular events distal to protein kinase C activation that underlie ACTH secretion have not been well characterized to date. Since the MARCKS protein has been implicated in neurosecretion and is phosphorylated by PKC in synaptosomes, studies were carried out to determine whether AVP might cause MARCKS phosphorylation in the ovine anterior pituitary, and to determine whether this phenomenon might be temporally correlated with PKC translocation and the release of ACTH. When cytosolic fractions of rat brain, ovine anterior pituitary, and cultured ovine anterior pituitary cells were incubated with purified PKC, several proteins were phosphorylated including those in the region of 83-85 kDa. After precipitation of the proteins with 40% acetic acid, the 83-85 kDa phosphoproteins were selectively recovered in the acid soluble phase. Phosphopeptide maps of either the 83 or 85 kDa proteins were generated with Staphylococcus aureus V8 protease and revealed 13 and 9 kDa phosphopeptides, which are characteristic of the authentic MARCKS protein. An identical phosphopeptide map was also obtained when the MARCKS protein was selectively extracted from intact 32P-labeled anterior pituitary cells. MARCKS phosphorylation was markedly increased when ovine anterior pituitary cells were exposed to 1 microM phorbol 12-myristate 13-acetate (PMA). When the cells were exposed to 1 microM AVP, MARCKS phosphorylation increased at 15 s and reached the maximal plateau value at 30 s. MARCKS phosphorylation then started to diminish at 2 min, and baseline levels were attained by 10 min. In the same cells, AVP stimulated ACTH release in a biphasic manner-during the first 30 s, there resulted a rapid burst of ACTH secretion that was followed by a slower, but sustained rate of secretion. We conclude that: (1) AVP causes a rapid, and reversible, phosphorylation of the MARCKS protein in the ovine anterior pituitary; (2) since the AVP-induced increase in MARCKS phosphorylation occurs much earlier in these cells than does PKC trans-location, MARCKS phosphorylation may provide a more sensitive index of the onset of PKC activation than the translocation assay; (3) the close temporal association between MARCKS phosphorylation and the rapid early release of ACTH suggests that MARCKS phosphorylation may be involved in the initial intracellular events that underly exocytosis of the hormone.
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PMID:Arginine vasopressin (AVP) causes the reversible phosphorylation of the myristoylated alanine-rich C kinase substrate (MARCKS) protein in the ovine anterior pituitary: evidence that MARCKS phosphorylation is associated with adrenocorticotropin (ACTH) secretion. 785 29

The MARCKS protein is a widely distributed cellular substrate for protein kinase C. It is a myristoylprotein that binds calmodulin and actin in a manner reversible by protein kinase C-dependent phosphorylation. It is also highly expressed in nervous tissue, particularly during development. To evaluate a possible developmental role for MARCKS, we disrupted its gene in mice by using the techniques of homologous recombination. Pups homozygous for the disrupted allele lacked detectable MARCKS mRNA and protein. All MARCKS-deficient pups died before or within a few hours of birth. Twenty-five percent had exencephaly and 19% had omphalocele (normal frequencies, < 1%), indicating high frequencies of midline defects, particularly in cranial neurulation. Nonexencephalic MARCKS-deficient pups had agenesis of the corpus callosum and other forebrain commissures, as well as failure of fusion of the cerebral hemispheres. All MARCKS-deficient pups also displayed characteristic lamination abnormalities of the cortex and retina. These studies suggest that MARCKS plays a vital role in the normal developmental processes of neurulation, hemisphere fusion, forebrain commissure formation, and formation of cortical and retinal laminations. We conclude that MARCKS is necessary for normal mouse brain development and postnatal survival.
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PMID:MARCKS deficiency in mice leads to abnormal brain development and perinatal death. 786 70

Neurogranin, neuromodulin, and MARCKS are among the most prominent substrates of protein kinase C (PKC) in the mammalian brain. These phosphoproteins were dephosphorylated by three isoforms of rat brain calcineurin, also known as calmodulin (CaM)-dependent protein phosphatase (CaMPP). The three CaMPP isozymes dephosphorylate neurogranin, the most favorable substrate among the three tested, with subtle differences in their responses to divalent metal ions, Mn2+ and Ni2+. Dephosphorylation of neurogranin by all three CaMPP isozymes, CaMPP-1, -2, and -3, were stimulated to a higher extent by Mn2+ than by Ni2+ in the presence of CaM and Ca2+. The Km values of neurogranin in the presence of Mn2+ were lower than those in the presence of Ni2+ for CaMPP-1 and -2, but that for CaMPP-3 was comparable with either divalent metal ion. The Vmax values were higher in the presence of Mn2+ than those of Ni2+ for all three isozymes. Neurogranin and neuromodulin, both phosphorylated by PKC at a single site, were dephosphorylated completely by CaMPP; however, MARCKS, phosphorylated by PKC at three sites, was partially dephosphorylated by this phosphatase. A higher extent of dephosphorylation of MARCKS could be achieved by the combination of CaMPP and protein phosphatase 2A and a complete dephosphorylation of this protein was observed with protein phosphatase 1. Protein phosphatase 1 and 2A were also effective in a complete dephosphorylation of neurogranin and neuromodulin. Amino acid sequence analysis of the tryptic phosphopeptides derived from MARCKS dephosphorylated by CaMPP and protein phosphatase 2A revealed that the former preferentially dephosphorylated Ser155 and the latter Ser162 of rat brain MARCKS. Both phosphatases dephosphorylated poorly of Ser151. Because of the high concentration of CaMPP in the brain and the colocalization of this phosphatase with major PKC substrates in the various brain regions, it is likely that CaMPP is a phosphatase with potential to reverse the action of PKC.
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PMID:Dephosphorylation of protein kinase C substrates, neurogranin, neuromodulin, and MARCKS, by calcineurin and protein phosphatases 1 and 2A. 786 22

Free radical formation and subsequent lipid peroxidation may participate in the pathogenesis of tissue injury, including the brain injury induced by hypoxia or trauma and cardiac injury arising from ischemia and reperfusion. However, the exact cellular mechanisms by which the initial oxidative insult leads to the ultimate tissue damage are not known. A number of reports have indicated that protein kinase C (PKC) may be activated following oxidative stress and that this enzyme may play an important role in the steps leading to cellular damage. In this work, we have examined in a cell model whether PKC is activated following oxidative exposure. UC11MG cells, a human astrocytoma cell line, were treated with H2O2. Incubation with 0.5 mM H2O2 increased malondialdehyde levels by as early as 15 minutes. To assess the effects of H2O2 treatment on PKC activation, we measured phosphorylation of an endogenous PKC substrate, the MARCKS (myristoylated alanine-rich C kinase substrate) protein. Treatment of cells with 0.2-1.0 mM H2O2 resulted in a rapid increase in MARCKS phosphorylation. Phosphorylation was stimulated approximately 2.5-fold following treatment with 0.5 mM H2O2 for ten minutes. Treatment with phorbol 12-myristate 13-acetate, a PKC activator, increased MARCKS phosphorylation approximately 4-fold. The H2O2-induced MARCKS phosphorylation was inhibited by the addition of the kinase inhibitors H-7 and staurosporine. Furthermore, specific down-regulation of PKC by phorbol ester also inhibited H2O2-induced MARCKS phosphorylation. These results indicate that PKC is rapidly activated in cells following an oxidative exposure and that this cell system may be a good model to further investigate the role of PKC in regulating oxidative damage in the cell.
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PMID:Oxidant-induced activation of protein kinase C in UC11MG cells. 788 45

Protein phosphorylation in response to toxic doses of glutamate has been investigated in cerebellar granule cells. 32P-labelled cells have been stimulated with 100 microM glutamate for up to 20 min and analysed by one and two dimensional gel electrophoresis. A progressive incorporation of label is observed in two molecular species of about 80 and 43 kDa (PP80 and PP43) and acidic isoelectric point. Glutamate-stimulated phosphorylation is greatly reduced by antagonists of NMDA and non-NMDA glutamate receptors. The effect of glutamate is mimicked by phorbol esters and is markedly reduced by inhibitors of protein kinase C (PKC) such as staurosporine and calphostin C. PP80 has been identified by Western blot analysis as the PKC substrate MARCKS (myristoylated alanine-rich C kinase substrate), while antibody to GAP-43 (growth associated protein-43), the nervous tissue-specific substrate of PKC, failed to recognize PP43. Our results suggest that PKC is responsible for the early phosphorylative events induced by toxic doses of glutamate in cerebellar granule cells.
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PMID:Glutamate-induced protein phosphorylation in cerebellar granule cells: role of protein kinase C. 789 41


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