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 phosphorylation sites in the myristoylated alanine-rich C kinase substrate or MARCKS protein consist of four serines contained within a conserved, basic region of 25 amino acids, termed the phosphorylation site domain. A synthetic peptide comprising this domain was phosphorylated by both protein kinase C and its catalytic fragment with high affinity and apparent positive cooperativity. Tryptic phosphopeptides derived from the peptide appeared similar to phosphopeptides derived from the phosphorylated intact protein. The peptide was phosphorylated by cAMP- and cGMP-dependent protein kinases with markedly lower affinities. In peptides containing only one of the four serines, with the other three serines replaced by alanine, the affinities for protein kinase C ranged from 25 to 60 nM with Hill constants between 1.8 and 3.0. The potential pseudosubstrate peptide, in which all four serines were replaced by alanines, inhibited protein kinase C phosphorylation of histone or a peptide substrate with an IC50 of 100-200 nM with apparently non-competitive kinetics; it also inhibited the catalytic fragment of protein kinase C with a Ki of 20 nM, with kinetics of the mixed type. The peptide did not significantly inhibit the cAMP- and cGMP-dependent protein kinases. It inhibited Ca2+/calmodulin-dependent protein kinases I, II, and III by competing with the kinases for calmodulin. In addition, the peptide inhibited the Ca2+/calmodulin-independent activity of a proteolytic fragment of Ca2+/calmodulin protein kinase II, with an IC50 approximately 5 microM. Thus, the phosphorylation site domain peptide of the MARCKS protein is a high affinity substrate for protein kinase C in vitro; the cognate peptide containing no serines is a potent but not completely specific inhibitor of both protein kinase C and its catalytic fragment.
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PMID:Protein kinase C substrate and inhibitor characteristics of peptides derived from the myristoylated alanine-rich C kinase substrate (MARCKS) protein phosphorylation site domain. 165 Mar 59

The growth cone, the motile tip of developing neuronal processes, is considered responsible for the exact guidance of axons and synaptogenesis. High activity of tyrosine kinases in growth cones may contribute to the functions of growth cones. Our previous work revealed that vinculin is one of the endogenous substrates for intrinsic tyrosine kinases in the growth cone particle (GCP) fraction isolated from fetal rat brain. In the present study, we examined tyrosine phosphorylation and immunoblot analysis of vinculin in various fractions from fetal rat brains and adult synaptosomal fraction. Tyrosine phosphorylation of vinculin in the GCP fraction was more prominent than in any other fraction from fetal brain or synaptosomes from adult. Compared to other fractions, however, the enrichment of vinculin in the GCP fraction was not observed. Tyrosine phosphorylation of vinculin in the fraction was inhibited by genistein, a specific tyrosine kinase inhibitor. Although vinculin was also phosphorylated by protein kinase C in the GCP fraction, it incorporated a much smaller amount of 32P than MARCKS protein or GAP-43. The cytoskeletal subfraction from the GCP fraction contained a considerable amount of vinculin and it was one of the major substrates for tyrosine kinases in the GCP cytoskeleton. The membrane skeleton from the GCP fraction contained a low amount of vinculin but showed high kinase activity that phosphorylated vinculin. Taken together, our results suggest that tyrosine phosphorylation of vinculin contributes to the cytoskeletal organization of growth cones.
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PMID:Tyrosine phosphorylation and immunodetection of vinculin in growth cone particle (GCP) fraction and in GCP-cytoskeletal subfractions. 172 70

The cell-permeant heavy metal chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine(TPEN) was found to counteract phorbol ester-induced actin reorganization in PTK2 and Swiss 3T3 cells. By using fluorescence and the higher resolution technique of photoelectron microscopy to monitor actin patterns, 15-min pretreatment with 25-50 microM TPEN was found to dramatically reduce actin alterations resulting from subsequent phorbol ester treatment in PTK2 cells. Similar results were obtained with Swiss 3T3 cells using 50 microM TPEN for 1.5 h. Phorbol ester-induced actin alterations are thought to depend on activation of protein kinase C (PKC). In contrast to the phorbol ester effect, the PKC-independent actin cytoskeletal disruption caused by staurosporine and cytochalasin B was unaffected by TPEN pretreatment. TPEN did not block phorbol ester-induced activation of PKC in Swiss 3T3 cells, as observed by the phosphorylation of the 80K PKC substrate protein (MARCKS protein). TPEN also did not inhibit partially purified PKC from Swiss 3T3 cells in an in vitro PKC-specific commercial assay. To establish that the effect of TPEN is the removal of metal ions and not some other nonspecific effect of TPEN, a series of transition metal ions was added at the end of the TPEN pretreatment. The results indicate that the transient but dramatic phorbol ester-induced reorganization of the actin cytoskeleton in cultured cells depends on an interaction of PKC with a heavy metal, probably zinc.
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PMID:Phorbol ester-induced actin cytoskeletal reorganization requires a heavy metal ion. 180 24

The MARCKS (myristylated alanine-rich C-kinase substrate) protein is an abundant calmodulin-binding protein that is a major and specific endogenous substrate of protein kinase C (PKC). Stimulation of cells with phorbol esters or other activators of PKC has been shown previously to result in rapid phosphorylation of MARCKS proteins and redistribution of these myristylated C-kinase substrates from membrane to cytosol. Here we show that NIH3T3 murine fibroblasts transformed by p21-HA-C-RAS or pp60-V-SRC oncoproteins have markedly reduced levels of p68-MARCKS and that most of the remaining MARCKS protein is found in the cytosol. 3T3 cells containing a nontransforming oncoprotein p26-BCL2, in contrast, exhibited normal levels and distribution of p68-MARCKS. When taken together with recent evidence that MARCKS proteins are involved in regulating organization of the membrane cytoskeleton, our findings suggest that oncoprotein-mediated alterations in MARCKS protein levels and subcellular distribution may contribute to the development or maintenance of the transformed phenotype.
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PMID:Transformed 3T3 cells have reduced levels and altered subcellular distribution of the major PKC substrate protein MARCKS. 183 87

Adducin is a membrane-skeletal protein which is a candidate to promote assembly of a spectrin-actin network in erythrocytes and at sites of cell-cell contact in epithelial tissues. The complete sequence of both subunits of human adducin, alpha (737 amino acids), and beta (726 amino acids) has been deduced by analysis of the cDNAs. The two subunits have strikingly conserved amino acid sequences with 49% identity and 66% similarity, suggesting evolution by gene duplication. Each adducin subunit has three distinct domains: a 39-kD NH2-terminal globular protease-resistant domain, connected by a 9-kD domain to a 33-kD COOH-terminal protease-sensitive tail comprised almost entirely of hydrophilic amino acids. The tail is responsible for the high frictional ratio of adducin noted previously, and was visualized by EM. The head domains of both adducin subunits exhibit a limited sequence similarity with the NH2-terminal actin-binding motif present in members of the spectrin superfamily and actin gelation proteins. The COOH-termini of both subunits contain an identical, highly basic stretch of 22 amino acids with sequence similarity to the MARCKS protein. Predicted sites of phosphorylation by protein kinase C include the COOH-terminus and sites at the junction of the head and tail. Northern blot analysis of mRNA from rat tissues, K562 erythroleukemia cells and reticulocytes has shown that alpha adducin is expressed in all the tissues tested as a single message size of 4 kb. In contrast, beta adducin shows tissue specific variability in size of mRNA and level of expression. A striking divergence between alpha and beta mRNAs was noted in reticulocytes, where alpha adducin mRNA is present in at least 20-fold higher levels than that of beta adducin. The beta subunit thus is a candidate to perform a limiting role in assembly of functional adducin molecules.
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PMID:Primary structure and domain organization of human alpha and beta adducin. 184 Jun 3

In Swiss 3T3 fibroblasts a peptide mitogen bombesin, which acts through the phospholipase C-protein kinase C signaling pathway, stimulates DNA synthesis in a manner strictly dependent on the medium calcium concentration: [3H]thymidine incorporation into DNA in the presence of a saturating concentration of bombesin (10(-8) M) is 4-fold greater at 3.0 mM extracellular calcium as compared with a value obtained at 0.03 mM calcium. In the present study we attempted to identify the site and the mechanism of action of Ca2+ influx along the bombesin-induced mitogenic signaling pathway, by comparing bombesin effects at 0.03 and 3.0 mM of medium calcium. Bombesin induces the same extent of increases in [3H]inositol phosphates after 1 min, and comparable sustained increases in the cellular content of 1,2-diacylglycerol for up to 4 h, at either 0.03 or 3.0 mM calcium. Bombesin induces the same extent of phosphorylation of MARCKS protein, the major cellular substrate for protein kinase C, irrespective of the medium calcium concentration for at least 4 h. Moreover, diverse cellular responses elicited by bombesin, including c-fos expression, activation of microtubule-associated protein 2 kinase and S6 kinase, glucose uptake, and protein synthesis but not the release of arachidonic acid and its metabolites, are induced similarly at either 0.03 or 3.0 mM calcium. Down-regulation of cellular protein kinase C nearly completely abolishes bombesin effects on c-fos expression, S6 kinase activation, glucose uptake, and DNA synthesis. These results suggest that the target of Ca2+ influx in bombesin-induced mitogenic signaling pathway is not located along the phospholipase C-protein kinase C signal transduction system including cellular events in early G1 phase that exist downstream to protein kinase C action.
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PMID:Role of Ca2+ influx in bombesin-induced mitogenesis in Swiss 3T3 fibroblasts. 184 53

The expression of a major cellular substrate for protein kinase C, the MARCKS protein, is regulated in a cell-, tissue-, and developmental stage-specific fashion; in addition, this expression can be stimulated acutely by various cytokines in certain cell types. We have begun to characterize the human gene in order to elucidate the genetic elements responsible for this highly regulated expression. We first cloned a human MARCKS cDNA, which encoded a predicted protein of 332 amino acids (Mr 31,600) that was approximately 89, 74, and 59% identical to the bovine, mouse, and chicken proteins, respectively. Regions conserved at the amino acid level included the amino-terminal myristoylation consensus sequence, the site of intron splicing, and the phosphorylation site domain. The human cDNA was used to demonstrate that tumor necrosis factor-alpha could rapidly stimulate MARCKS gene transcription in the human promyelocytic leukemia cell line HL60. Genomic clones were then isolated; sequence analysis identified a putative promoter region that had no TATA box and contained multiple transcription initiation sites in a region spanning 57 base pairs (bp). This was followed by a 5'-untranslated region of approximately 400 bp, which displayed a complex predicted secondary structure with a delta G of -73.4 kcal/mol. Plasmid constructions containing between 52 and 1453 bp of the human MARCKS promoter linked to the human growth hormone gene were then used in transient expression experiments. Constructions containing 52 and 110 bp of the MARCKS promoter did not exhibit promoter function while the larger constructions all exhibited promotor function; the 248-bp fragment of the MARCKS promoter was 80% as effective as the human ferritin promoter in stimulating expression of human growth hormone in intact cells. Using an insert from the human genomic clone as a probe, we identified human chromosome 6, q21-qter, as the location of the MARCKS gene; this has been assigned the gene symbol MACS.
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PMID:The human myristoylated alanine-rich C kinase substrate (MARCKS) gene (MACS). Analysis of its gene product, promoter, and chromosomal localization. 186 Aug 46

A 25-amino acid peptide, containing the four protein kinase C (PKC) phosphorylation sites and the calmodulin (CaM) binding domain of the myristoylated alanine-rich C kinase substrate (MARCKS) protein, has been synthesized and used to determine the effects of phosphorylation on its binding and regulation of CaM. PKC phosphorylation of this peptide (3.0 mol of Pi/mol of peptide) produced a 200-fold decrease in its affinity for CaM. PKC phosphorylation of the peptide resulted in its dissociation from CaM over a time course that paralleled the phosphorylation of 1 mol of serine/mol of peptide. The peptide inhibited CaM's binding to myosin light chain kinase and CaM's stimulation of phosphodiesterase and calcineurin. PKC phosphorylation of the peptide resulted in a rapid release of bound CaM, allowing its subsequent binding to myosin light chain kinase (t1/2 = 1.6 min), stimulation of phosphodiesterase (t1/2 = 1.2 min) and calcineurin (t1/2 = 1.7 min). Partially purified MARCKS protein produced a similar inhibition of CaM-phosphodiesterase which was reversed by PKC phosphorylation. PKC phosphorylation of the peptide occurred primarily at serine 8 and serine 12, and phosphorylation of serine 12 regulated peptide affinity for CaM. Thus, PKC phosphorylation of the peptide and the MARCKS protein results in the rapid release of CaM and the subsequent activation of CaM-dependent enzymes. This process might allow for interplay between PKC and CaM-dependent signal transduction pathways.
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PMID:Phosphorylation-dependent binding of a synthetic MARCKS peptide to calmodulin. 200 42

Although insulin is known to activate several protein serine/threonine protein kinases, its ability to activate protein kinase C remains controversial. We reinvestigated this question, taking advantage of several technical advances such as the development of fibroblast cell lines that overexpress normal human insulin receptors, and the development of antibodies to and expression vectors for the myristoylated, alanine-rich C kinase substrate (MARCKS) protein, a major cellular substrate for protein kinase C. In HIR 3.5 cells, a mouse 3T3 cell derivative that expresses about 6 x 10(6) human insulin receptors/cell, insulin (70 nM for 10 min) stimulated phosphorylation of the MARCKS protein by approximately 2-fold (p less than 0.005). This phosphorylation was not further increased by different times of insulin exposure, different insulin concentrations, or longer periods of serum deprivation. The insulin stimulation represented about 14% of the response to phorbol 12-myristate 13-acetate and about 17% of the response to 10% fetal calf serum. No significant stimulation of MARCKS protein phosphorylation was seen in four other insulin-sensitive cell lines, in which insulin is known to activate other protein serine/threonine kinases: HIRC-B, BC3H-1, 3T3-L1 adipocytes, and H35 rat hepatoma cells made to stably express the MARCKS protein. In these four cell lines, serum and/or phorbol 12-myristate 13-acetate exerted a large stimulatory effect on MARCKS protein phosphorylation. We conclude that insulin may activate protein kinase C to a minor extent in certain cell types that vastly overexpress insulin receptors; however, we believe that this effect of insulin is unlikely to be of physiological importance.
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PMID:Insulin activation of protein kinase C: a reassessment. 204 Jun 11

A protein of 80 kDa apparent molecular mass was found to be specifically myristolylated in rat brain cytosols derived from either whole brain or synaptosomes. The attachment of the fatty acid took place in the absence of protein synthesis, since the cytosols did not incorporate [14C]lysine into protein, nor did cycloheximide affect the incorporation of the myristic acid into the protein. The fatty acid was incorporated into the protein via an acid-labile/alkali-resistant band, and Pronase digestion of the labelled protein showed that the lipid was covalently linked to a glycine residue. Together, these data suggested that the myristic acid was amide-linked to the N-terminal residue of the protein. The protein was identified as one of the major protein kinase C substrates, the MARCKS (myristoylated alanine-rich C kinase substrate) protein, by showing that Ca2+ stimulated its phosphorylation, by its heat stability and by immune precipitation (using an antiserum to the MARCKS protein). Incorporation of myristic acid into intact protein continued for up to 12 h, despite the fact that over this period some degradation of the protein could be demonstrated. In pulse-chase experiments, the pattern of loss of the incorporated fatty acid was similar to that of the protein itself, and therefore the loss of radioactivity probably reflects protein degradation rather than specific de-acylation of the protein. Together, these results suggest that there is a pool of unacylated MARCKS protein in the rat brain.
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PMID:Evidence for a non-myristoylated pool of the 80 kDa protein kinase C substrate of rat brain. 224 73


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