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)

The myristoylated alanine-rich C kinase substrate, or MARCKS protein, has been implicated in several cellular processes, yet its physiological function remains unknown. We have studied the molecular basis of its membrane association in a cell-free system in order to help elucidate its regulation and function. First, we showed that the MARCKS protein incorporated [3H]myristate when its mRNA was translated in vitro in reticulocyte lysates. The myristoylated protein bound rapidly to freshly fractionated cell membranes, while a nonmyristoylated mutant associated to a much lesser extent (< 15% of wild type). To determine whether this binding was due to a specific cytoplasmic-face protein "receptor," as is seen with pp60v-src, we pretreated the membranes in several ways. Prior treatment of membranes with heat (100 degrees C for 3 min) or trypsin did not affect subsequent MARCKS binding. Binding was markedly decreased in 50 mM EDTA, 0.5 M NaCl, or 1.0% Triton X-100; it was restored to normal after removal of the NaCl and EDTA but was still decreased after removal of the Triton X-100. These findings argued against the existence of a protein receptor for the MARCKS protein on cellular membranes. Finally, MARCKS protein phosphorylated in vitro with protein kinase C bound to the cell membranes to the same extent as the nonphosphorylated protein; this binding was also unaffected by an excess of a synthetic peptide corresponding to the phosphorylation site domain of the protein. We conclude that, at least in this in vitro system, the membrane association of the MARCKS protein is primarily dependent on the amino-terminal myristate moiety and does not appear to involve a specific cytoplasmic-face protein receptor.
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PMID:Membrane association of the myristoylated alanine-rich C kinase substrate (MARCKS) protein appears to involve myristate-dependent binding in the absence of a myristoyl protein receptor. 133 70

The mechanism by which TGF-b1 affects granulosa cell physiology as well as the modulation of TGF-b1 activity by FSH are not understood. We tested the hypothesis that TGF-b1 exerts its effects on granulosa cells via activation of protein kinase C (PKC). Immunoprecipitation of the MARCKS protein from 32P labeled rat granulosa cells was used to assay PKC activation. 20 minute treatment with TGF-b1 (8 ng/ml), forskolin (30 microM), and TPA (200 nM) all caused an increase in MARCKS phosphorylation as quantified by densitometric scanning. FSH did not increase MARCKS phosphorylation above control levels while exposure of cells to both FSH and TGF-b1 (10 ng/ml) decreased phosphorylation of the MARCKS protein to control levels. These data suggests that (1) TGF-b1 signal transduction in rat granulosa cells may partially involve phosphorylation of the MARCKS protein; and, (2) in granulosa cells FSH can modulate TGF-b1 induced MARCKS phosphorylation.
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PMID:Transforming growth factor-beta (TGF-b) induced phosphorylation of the myristoylated alanine rich C kinase substrate (MARCKS) protein in ovarian granulosa cells is modulated by follicle stimulating hormone (FSH). 133 98

We have compared the properties of a rat aorta-derived protein kinase C substrate (p75) with those of 80 kDa kinase C substrates from rat brain (MARCKS) and rabbit aorta (p80). Rat aortic p75 appeared to be closely related to rat brain MARCKS on the basis of: solubility in perchloric acid and trichloroacetic acid, heat stability, isoelectric point (pI approximately 4.2), overall V8 protease phosphopeptide map, and immunocrossreactivity with an antibody directed against the N-terminal domain of MARCKS. However, p75 could be distinguished from rat brain MARCKS and from the rabbit aorta-derived p80 on the basis of its consistently more rapid electrophoretic mobility in SDS-containing gels, and in terms of a unique proteolytic phosphopeptide found in MARCKS but not in aortic p75. We conclude that p75 probably belongs to the family of protein kinase C substrates represented by MARCKS, and that differences in post-translational processing (glycosylation) or mRNA processing may account for the unique properties of the p75 protein in rat aortic tissue.
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PMID:Comparison of an endogenous protein kinase C substrate in rat aorta with rat brain MARCKS. 133 18

Activation of protein kinase C (PKC) by tumor-promoting phorbol esters leads to the phosphorylation of an 80-kilodalton PKC substrate (known as MARCKS) in murine fibroblasts. In BALB/c 3T3 cells stably transformed by v-Src, phorbol esters were unable to induce phosphorylation of MARCKS. Western blot analysis and in vitro kinase assays showed that both PKC protein levels and kinase activity were unchanged in v-Src-transformed relative to the parental nontransformed BALB/c 3T3 cells. However, MARCKS protein levels were reduced in v-Src-transformed cells relative to nontransformed cells. MARCKS RNA levels were also correspondingly reduced in v-Src-transformed cells. Nuclear "run-on" assays showed decreased transcription of MARCKS in v-Src-transformed cells. Thus, the absence of MARCKS in v-Src-transformed cells could be explained by a down-regulation of MARCKS transcription. Inhibiting the protein tyrosine kinase activity of v-Src with herbimycin A restored MARCKS RNA levels, MARCKS transcription, and MARCKS protein, suggesting that down-regulation of MARCKS in v-Src-transformed BALB/c 3T3 cells is a direct effect of v-Src.
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PMID:MARCKS protein is transcriptionally down-regulated in v-Src-transformed BALB/c 3T3 cells. 137 Apr 66

We have examined the regulation of expression of 80K/MARCKS, a major and specific protein kinase C (PKC) substrate of Swiss 3T3 fibroblasts. Addition of bombesin (10 nM) to confluent quiescent cultures of these cells induced a dramatic and sustained down-regulation of 80-kDa mRNA and protein levels to a minimum of 5% of control within 8 and 48 h, respectively, without depletion of PKC activity. In contrast, the effect of phorbol 12,13-dibutyrate on 80K/MARCKS mRNA levels was transient, and recovery of these transcripts correlated with the loss of PKC activity. The ability of bombesin to down-regulate 80K/MARCKS mRNA levels was dose-dependent (ED50 0.5 nM) and was abolished by both the specific bombesin antagonist [Leu13 psi (CH2NH),Leu14]bombesin and by prior depletion of PKC. Of a range of agents tested, platelet-derived growth factor (PDGF), but not insulin or Ca2+ ionophore, also down-regulated 80K/MARCKS mRNA to 24% of control within 5 h. Prior down-regulation of PKC abolished the effect of PDGF at a concentration of 7 ng/ml. Surprisingly, at higher doses (25 ng/ml), PDGF induced the down-regulation of 80K/MARCKS mRNA in a PKC-independent manner. Furthermore, elevation of cAMP, either through receptor-mediated mechanisms (e.g. prostaglandin E1) or by direct stimulation of adenylate cyclase (e.g. forskolin), also caused a marked dose-dependent depletion of 80K/MARCKS mRNA levels, which were further reduced by co-administration with cAMP-phosphodiesterase inhibitors. The rate of transcription of the 80K/MARCKS gene was unaltered by treatment of cells with either bombesin, PDGF, or forskolin/1-methyl-3-isobutylxanthine. These results indicate a role for both PKC-dependent and -independent pathways in growth factor-induced down-regulation of 80K/MARCKS expression, through a post-transcriptional mechanism.
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PMID:The expression of 80K/MARCKS, a major substrate of protein kinase C (PKC), is down-regulated through both PKC-dependent and -independent pathways. Effects of bombesin, platelet-derived growth factor, and cAMP. 137 35

The cell surface lipophosphoglycan (LPG) from Leishmania donovani promastigotes is a potent inhibitor of purified protein kinase C (PKC) activity in vitro. In this study, we have investigated the effect of LPG on the activation process of PKC in murine bone marrow-derived macrophages. The extent and kinetics of calcium ionophore A23187-induced [3H] phorbol dibutyrate binding to macrophages were not affected by LPG pretreatment or infection with either wild-type or LPG-deficient promastigotes, indicating no effect on the association of calcium-dependent PKC with the plasma membrane. In contrast, LPG inhibited the phosphorylation of both the PKC-specific VRKRTRLLR substrate peptide and MARCKS, and endogenous PKC substrate, in 1-oleoyl-2-acetyl-glycerol-stimulated macrophages. These observations provide direct evidence that LPG effectively inhibits PKC activity in intact macrophages. Finally, depletion of PKC rendered macrophages more permissive for the proliferation of L. donovani, suggesting that inhibition of PKC-dependent events contributes to the survival of this parasite within its host cell.
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PMID:Inhibition of macrophage protein kinase C-mediated protein phosphorylation by Leishmania donovani lipophosphoglycan. 138 36

The myristoylated, alanine-rich C kinase (PKC) substrate (MARCKS) is a major, specific substrate of PKC that is phosphorylated during macrophage and neutrophil activation, growth factor-dependent mitogenesis and neurosecretion. MARCKS is also a calmodulin-binding protein and binding of calmodulin inhibits phosphorylation of the protein by PKC. Several recent observations from our laboratories suggest a role for MARCKS in cellular morphology and motility. First, in macrophages MARCKS is located at points of cellular adherence where actin filaments insert at the plasma membrane and is released to the cytoplasm upon activation of PKC. Second, during neutrophil chemotaxis MARCKS undergoes a cycle of release from, and reassociation with, the plasma membrane. Third, in vitro, MARCKS is an F-actin cross-linking protein whose activity is inhibited by PKC-mediated phosphorylation and by binding to calmodulin. MARCKS therefore appears to be a regulated cross-bridge between actin and the plasma membrane. Regulation of the plasma membrane-binding and actin-binding properties of MARCKS represents a convergence of the PKC and calmodulin signal transduction pathways in the control of actin cytoskeleton-plasma membrane interactions.
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PMID:Calmodulin and protein kinase C cross-talk: the MARCKS protein is an actin filament and plasma membrane cross-linking protein regulated by protein kinase C phosphorylation and by calmodulin. 139 31

Activation of protein kinase C (PKC) by phorbol 12-myristate 13-acetate (PMA) was compared with calcium/phosphatidylserine (Ca/PS). The substrate specificity of PKC was more limited with PS/PMA. Substrates could be divided into three overlapping groups according to their relative level of phosphorylation: C1, relatively preferred substrates with Ca/PS, included dephosphin, histone, and peptide GS1-10. C2, relatively preferred with PS/PMA, included myelin basic protein and MARCKS. C3, substrates independent of activators. PS/PMA altered the Vmax of PKC for substrate, and decreased the Km for Mg2+. Differential substrate phosphorylation by PS/PMA also occurred for PKC isozymes resolved by hydroxylapatite chromatography and was most dramatic for PKC-alpha, which could no longer phosphorylate histone or GS1-12. Differential activities of PKC were also observed in synaptosol and in intact synaptosomes where PMA stimulated phosphorylation of MARCKS, but not dephosphin. It was further shown that dephosphin was indeed a substrate of PKC in the intact synaptosomes by use of a repolarization-dependent dephosphin phosphorylation assay. The differential PKC activities could also be distinguished by inhibitors. H-7 was equipotent, palmitoylcarnitine did not inhibit in vitro C2 phosphorylation, but inhibited dephosphin in intact synaptosomes, and sphingosine did not inhibit C1 substrates and was without effect on dephosphin in intact synaptosomes. Therefore PS/PMA alters or limits the substrate specificity of PKC, leading to a differential substrate phosphorylation in vitro and in intact synaptosomes and differential inhibitor sensitivity. The pattern of protein phosphorylation observed after PKC activation in intact cells will therefore be dependent upon the activator.
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PMID:Differential stimulation of protein kinase C activity by phorbol ester or calcium/phosphatidylserine in vitro and in intact synaptosomes. 140 Apr 74

The aim was to examine systematically the potencies of protein kinase C inhibitors as a function of the kinase activator. Protein kinase C is activated by at least four stimulators: calcium plus phosphatidylserine (Ca/PS), phorbol 12-myristate 13-acetate plus PS (PS/PMA), arachidonic acid plus calcium (Ca/AA) and the synthetic peptide activator PCK530-558. With histone or GS1-12 as substrates, protein kinase C was maximally activated by Ca/PS, or to maxima of 62%, 89% or 82% with PS/PMA, Ca/AA or PKC530-558, respectively. One group of inhibitors, including H-7 and staurosporine, were equipotent, regardless of the activator. All other inhibitors showed variable selectivity, dependent upon the activator. A second group of inhibitors, including sphingosine and lipophosphoglycan, were eight or 200 times more potent for inhibition of PS/PMA-stimulated activity (relative to Ca/PS) and a third group, including retinal and palmitoylcarnitine, were 14 or 262 times more potent towards Ca/PS-stimulated activity. A final group (rhodamine 6G) was nine times more potent when Ca/AA was the activator. Similar results were obtained using the endogenous substrates dephosphin or MARCKS in synaptosol. Phosphorylation of MARCKS was stimulated by PS/PMA or Ca/PS, while phosphorylation of dephosphin was stimulated only by Ca/PS. The phosphorylation of either by Ca/PS-activated kinase was nine times more potently inhibited by palmitoylcarnitine, while phosphorylation of MARCKS by PS/PMA-activated kinase was 10 times more potently inhibited by sphingosine. H-7 inhibited both at similar concentrations. A model encompasses these differences in potency if the inhibitors are divided into four groups (A-D) according to their competitive inhibition with the appropriate activator or at the active site. The non-selective inhibitors interact at the active sites of protein kinase C (group A). The compounds which preferentially inhibit PS/PMA-activated kinase (sphingosine and lipophosphoglycan) are competitive inhibitors of PMA and 1,2-diacylglycerol (group B), those selective for Ca/PS-activated kinase (palmitoylcarnitine and retinal) are competitive with PS (group C) and those selective for Ca-AA activation (rhodamine 6G) are likely to be competitive with fatty acid (group D). Therefore, the effectiveness of protein kinase C inhibitors is dependent upon the activator employed.
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PMID:Potencies of protein kinase C inhibitors are dependent on the activators used to stimulate the enzyme. 141 56

The myristoylated, alanine-rich C-kinase substrate, or MARCKS protein, is a major cellular substrate for protein kinase C that is also a high-affinity calmodulin-binding protein. In addition, it is the prototype of a small family of myristoylated, calmodulin-binding protein kinase C substrate proteins. We isolated a phage clone from a mouse genomic library that spanned the entire coding sequence of the mouse MARCKS protein. The first 612 bp of the putative promoter was 89% identical to a corresponding region of the human promoter, and contained at least 59 potential transcription factor binding sites in analogous locations; both human and mouse promoters lacked TATA boxes. The mouse genomic probe was used to localize the mouse gene to chromosome 10, in the middle of a linkage group that corresponds to a region on human chromosome 6q. These data strongly suggested that the human gene would localize to 6q21. This was confirmed by studies of DNA from a patient with del(6)(q21), in which expression of the human gene encoding MARCKS, MACS, was only about 50% of normal; MARCKS mRNA expression in lymphoblast RNA from this patient was only 22% of normal. These studies confirm that the mouse and human MARCKS proteins are products of the same genes in their respective species; differences in their primary sequence can therefore be attributed to species variation rather than to the existence of related genes.
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PMID:Chromosomal mapping of the human (MACS) and mouse (Macs) genes encoding the MARCKS protein. 142 22


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