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)

Relatively little is known about the substrate specificity of individual protein kinase C (PKC) isozymes, particularly with respect to physiologically relevant substrates. One class of prominent cellular substrates for PKC is represented by the myristoylated alanine-rich C kinase substrate, or MARCKS, protein. In the present study, we have used a baculovirus expression system to coexpress human MARCKS with eight different isozymes of PKC, to determine which isozymes are capable of phosphorylating MARCKS in intact cells. In Sf9 cells, coexpression of MARCKS with individual PKC isozymes led to the following increases in MARCKS phosphorylation: alpha, 3.6-fold; beta iota, 4.6-fold; beta mu, 2.7-fold; gamma, 4.8-fold; delta, 3.0-fold; epsilon, 4.3-fold; and eta, 4.9-fold. In most cases, stimulation of cells with a phorbol ester led to a slight increase (20-30%) in MARCKS phosphorylation. PKC zeta did not phosphorylate MARCKS to any appreciable extent above control. In addition, in vitro kinetic analysis of PKC zeta showed that it has a 1000-fold lower affinity for a synthetic peptide comprising the MARCKS phosphorylation site domain compared to mixed conventional PKC isozymes from rat brain. These data indicate that MARCKS is a substrate in intact cells for at least seven isozymes of PKC: alpha; beta iota; beta mu; gamma; delta; epsilon; and eta. The isozyme PKC zeta does not appear to phosphorylate MARCKS in vivo or with significant affinity in vitro. Thus, PKC zeta, which is not activated by phorbol esters or diacylglycerol, also appears to behave differently with respect to this class of important cellular PKC substrates.
 ...
PMID:MARCKS phosphorylation by individual protein kinase C isozymes in insect Sf9 cells. 883 63

The myristoylated alanine-rich C kinase substrate, or MARCKS protein, is a widely expressed, prominent substrate for protein kinase C. Although the exact function of MARCKS has not been elucidated, targeted disruption of the MARCKS gene (Macs) in mice has shown that MARCKS plays a crucial role in the development of the central nervous system. Mice deficient in MARCKS exhibited universal perinatal death with defects in neurulation, fusion of the cerebral hemispheres, formation of the great forebrain commissures, and retinal and cortical lamination (Stumpo et al., Proc. Natl. Acad. Sci. USA 92, 944-948, 1995). In the present studies, a transgene consisting of approximately 3.4 kb of promoter from the human MARCKS gene (MACS), with an epitope tag sequence inserted at the carboxyl terminus of the MARCKS coding region, was able to complement completely MARCKS deficiency in mice. Thus, the human transgene contained all of the elements necessary for normal developmental expression of MARCKS. To test the importance of MARCKS myristoylation to its developmental role, an otherwise identical transgene was constructed in which the glycine at the amino terminus of MARCKS was mutated to an alanine. This mutation, which resulted in the expression of nonmyristoylated MARCKS, was successful in partially rescuing the Macs null phenotype. Specifically, about 25% of these mice survived the perinatal period; these survivors appeared to develop normally except for slightly decreased body size. In both the survivors and the nonsurvivors, all of the known anatomical defects associated with MARCKS deficiency were corrected by expression of the nonmyristoylated human protein. These results indicate that myristoylation of MARCKS is not required for the protein to correct many of the developmental abnormalities characteristic of its deficiency.
 ...
PMID:Nonmyristoylated MARCKS complements some but not all of the developmental defects associated with MARCKS deficiency in mice. 887 59

Neuroblastoma and glioma cells differentially express isoforms of protein kinase C (PKC) and myristoylated PKC substrates (e.g. MARCKS). Correlation with metabolism of membrane phospholipids suggests that PKC-alpha and MARCKS may be required to mediate phosphatidylcholine turnover stimulated by phorbol ester (beta-TPA). To evaluate relationships to neural cell differentiation, SK-N-SH human neuroblastoma cells were treated with 20 nM beta-TPA. In beta-TPA-treated cells, growth arrest and differentiation occurred (neurite extension; 40-60% decrease in cell number, total protein and RNA). By day 4, mRNA for PKC-alpha and MARCKS increased and, after an initial decrease, PKC-alpha protein also increased. At day 4, phosphatidylcholine synthesis was 3-5 fold greater than in control cells. In contrast, C6 glioma cells treated with beta-TPA showed no growth arrest, decreased PKC-alpha protein (< 20%) and lower phosphatidylcholine synthesis. Thus, induced differentiation of human neuroblastoma cells involved increased expression of PKC-alpha and MARCKS and synthesis of phosphatidylcholine, consistent with involvement of PKC-alpha and MARCKS in regulation of phosphatidylcholine turnover during neurite growth.
 ...
PMID:Protein kinase C isoforms and growth, differentiation and phosphatidylcholine turnover in human neuroblastoma cells. 890 63

The lateral membrane organization of phosphatidylserine, diacylglycerol, substrate, and Ca(2+)-dependent protein kinase C in large unilamellar vesicles was investigated by using fluorescence digital imaging microscopy. The formation of phosphatidylserine domains could be induced by either Ca2+, the MARCKS peptide, or protein kinase C. However, only Ca2+ could induce diacylglycerol to partition into the phosphatidylserine domains. In the complete protein kinase C assay mixture, two separate triple-labeling experiments demonstrated the colocalization of phosphatidylserine, protein kinase C, diacylglycerol, and the MARCKS peptide in domains. The amounts of all the labeled components in whole vesicles and in domains were measured at various concentrations of either phosphatidylserine, Ca2+, diacylglycerol, or the MARCKS peptide or with the addition of polylysine. The role of each component in forming membrane domains and in mediating the enzyme activity was analyzed. The results indicated that the inclusion of the MARCKS peptide in the domains, not just the binding of the substrate to vesicles, was especially important for PKC activity. The formation of PKC domains required the presence of DAG and Ca2+ at physiological ionic strength. The PKC activity was proportional to the amounts of PKC and substrate in the domains. The results also showed that the MARCKS peptide left the domains after being phosphorylated. A model for the activation of protein kinase C involving sequestering of the reaction components into membrane domains is proposed. The efficiency of the reaction was greatly increased by concentrating the activators, the enzyme, and the substrate into domains.
 ...
PMID:Formation of membrane domains during the activation of protein kinase C. 890 94

We previously identified a novel src- and ras-suppressed gene, 322, encoding a mitogenic regulatory function (Lin, X., Nelson, P. J., Frankfort, B., Tombler, E., Johnson, R., and Gelman, I. H. (1995) Mol. Cell. Biol. 15, 2754-2762). Here, we characterize the 322 gene product as an in vivo and in vitro substrate of protein kinase C (PKC). Hence, we named this product SSeCKS (pronounced essex) for Src Suppressed C Kinase Substrate. Rabbit polyclonal sera raised against glutathione S-transferase (GST)-SSeCKS recognized a myristylated 280/290-kDa doublet in Rat-6 fibroblasts. SSeCKS levels in src- and ras-transformed Rat-6 cells were 15- and 8-fold less, respectively, than those in untransformed cells. Short-term addition of phorbol ester resulted in a 5-fold increase in SSeCKS phosphorylation which was inhibited by bis-indolylmaleimide. In vitro phosphorylation of GST-SSeCKS by purified rabbit brain PKC-alpha was enhanced by phosphatidylserine and blocked by excess PKC pseudosubstrate inhibitor peptide. GST-SSeCKS bound purified PKC-alpha or PKC from Rat-6 lysates in a phosphatidylserine-dependent manner. Four SSeCKS domains containing Lys/Arg-rich motifs similar to the PKC phosphorylation site in MARCKS were phosphorylated in vitro by PKC. Immunofluorescence analysis showed SSeCKS present throughout the cytoplasm with enrichment in podosomes and at the cell edge. Short-term addition of phorbol esters caused the movement of SSeCKS from plasma membrane sites to the perinucleus coincident with a loss of actin stress fibers. These data suggest a role for SSeCKS in the control of cellular cytoskeletal architecture.
 ...
PMID:A novel src- and ras-suppressed protein kinase C substrate associated with cytoskeletal architecture. 891 Apr 68

Bovine alveolar macrophages (BAM) were stimulated with quartz dusts, metal oxide-coated silica particles, and zymosan. To investigate the role of protein kinase C (PKC) in the mechanism of agonist-induced activation 12-O-tetradecanoyl phorbol 13-acetate (TPA), staurosporine, and the PKC specific inhibitor GF 109203X were applied. PKC activity was determined by means of a continuous fluorescence assay [1]. The assay is based on the measurement of fluorescence decrease caused by phosphorylation of an acrylodan-labelled MARCKS peptide, a specific substrate of PKC. The PKC fluorescence assay was verified with the purified enzyme, but it could not be adapted to cytosolic and membrane homogenates of BAM, as it is sensitive to the activity of proteases. PKC-mediated protein phosphorylation in intact BAM was achieved by mapping the [32P]phosphoproteins with an optimized horizontal 2D electrophoresis technique with subsequent autoradiography and image analysis. Agonist- and time-dependent changes of phosphoprotein patterns in BAM were detected and analysed.
 ...
PMID:Protein kinase C activity and phosphoprotein pattern in stimulated alveolar macrophages. 892 Jul 28

The roles of protein kinase C and its substrates in development are poorly understood. Recently, we disrupted the mouse gene for a major cellular substrate for protein kinase C, the MARCKS protein (Proc. Natl. Acad. Sci. USA, 92, 944-948, 1995). The resulting phenotype consisted of universal perinatal lethality, agenesis of the corpus callosum and other forebrain commissures, and neuronal ectopia and other cortical and retinal lamination disturbances. These mice also had high frequencies of exencephaly (25% overall, 35% in females). In the present study, we have examined the normal expression of MARCKS and the various isozymes of protein kinase C at the time of cranial neural tube closure, in an attempt to correlate MARCKS expression in time and anatomical location with the exencephaly characteristic of MARCKS deficiency. Failure of neural tube closure occurred at various sites in the cranial neural tube, suggesting a cellular functional defect that was not limited to a specific location. Non-exencephalic MARCKS-deficient embryos appeared to be anatomically normal on embryonic day (E) 8.5-9.5. MARCKS and PKC alpha were expressed at the plasma membrane of the neuroepithelial cells comprising the future neural tube, as well as in the surface ectoderm and underlying mesenchyme. Endogenous protein kinase C species, comprising either or both alpha and delta, were capable of phosphorylating MARCKS in intact E8.5 embryos. Thus, MARCKS is expressed at the plasma membranes of the specific cell types involved in cranial neurulation; its deficiency presumably results in a still-to-be-elucidated functional defect in these cells that leads to exencephaly in a high proportion of cases.
 ...
PMID:Developmental expression of MARCKS and protein kinase C in mice in relation to the exencephaly resulting from MARCKS deficiency. 892 69

Phosphorylation of myristoylated alanine-rich protein kinase C substrate (MARCKS) in intact cells has been employed as an indicator for activation of protein kinase C (PKC). Specific PKC isoenzymes responsible for MARCKS phosphorylation under physiological conditions, however, remained to be identified. In our present study using stably transfected NIH 3T3 cell clones we demonstrate that expression of constitutively active mutants of either conventional cPKC-alpha or novel nPKC-epsilon increased phosphorylation of endogenous MARCKS in the absence of phorbol 12,13-dibutyrate in intact mouse fibroblasts, implicating that each of these PKC isoforms itself is sufficient to induce enhanced MARCKS phosphorylation. Similarly, ectopic expression of a constitutively active mutant of PKC-theta significantly increased MARCKS phosphorylation compared to vector controls, identifying PKC-theta as a MARCKS kinase. The PKC-specific inhibitor GF 109203X (bisindolylmaleimide I) reduced MARCKS phosphorylation in intact cells at a similar dose-response as enzymatic activity of recombinant isoenzymes cPKC-alpha, nPKC-epsilon, and nPKC-theta in vitro. Consistently, phorbol 12,13-dibutyrate-dependent MARCKS phosphorylation was significantly reduced in cell lines expressing dominant negative mutants of either PKC-alpha K368R or (dominant negative) PKC-epsilon K436R. The fact, that the constitutively active PKC-lambda A119E mutant did not alter the MARCKS phosphorylation underscores the assumption that atypical PKC isoforms are not involved in this process. We conclude that under physiological conditions, conventional cPKC-alpha and novel nPKC-epsilon, but not atypical aPKC-lambda are responsible for MARCKS phosphorylation in intact NIH 3T3 fibroblasts.
 ...
PMID:Conventional PKC-alpha, novel PKC-epsilon and PKC-theta, but not atypical PKC-lambda are MARCKS kinases in intact NIH 3T3 fibroblasts. 902 Jan 16

The snake venom phospholipase A2 neurotoxin, beta-bungarotoxin, acts presynaptically to alter acetylcholine release in both the peripheral and central nervous systems. In investigating the mechanism of this action, we found that beta-bungarotoxin inhibited phosphorylation of synapsin I, GAP-43 and MARCKS in rat brain synaptosomes. This inhibition was not due to the inhibition of ATP synthesis, action of arachidonic acid metabolites, or stimulation of phosphatase activities. Furthermore, the activities of Ca2+/calmodulin-kinase II, cAMP-kinase and protein kinase C were not altered by beta-bungarotoxin in either synaptic plasma membranes or cytosol. When synaptic plasma membranes were treated with beta-bungarotoxin, MARCKS phosphorylation was inhibited, and this inhibition was overcome by the addition of exogenous protein kinase C. These results suggest that the interaction between MARCKS and endogenous protein kinase C is altered by beta-bungarotoxin. In contrast, Naja naja atra phospholipase A2, a typical phospholipase A2 enzyme, had effects on phosphorylation which were different from those of beta-bungarotoxin: (1) inhibition of phosphorylation of synapsin I in intact synaptosomes was less potent than that by beta-bungarotoxin; (2) it stimulated basal phosphorylation of GAP-43 and MARCKS; and (3) it increased the activity of protein kinase C. The inhibition of synapsin I phosphorylation by N. n. atra phospholipase A2 in intact synaptosomes may be due to the inhibition of ATP synthesis. The stimulation of GAP-43 and MARCKS by N. n. atra phospholipase A2 can be explained by the production of arachidonic acid, which stimulated protein kinase C activity to a similar extent as that caused by N. n. atra phospholipase A2. Thus, the mechanism of action of beta-bungarotoxin appears to be quite different from that of a phospholipase A2 enzyme, suggesting that phospholipase A2 activity of beta-bungarotoxin may not be essential for its action. beta-Bungarotoxin may be a useful tool to study the physiological role of phosphorylation of synaptosomal proteins in neurotransmitter release.
 ...
PMID:Mechanism of action of beta-bungarotoxin, a presynaptically acting phospholipase A2 neurotoxin: its effect on protein phosphorylation in rat brain synaptosomes. 902 77

The role of hydration in the catalytic activity and membrane binding of rat brain protein kinase C (PKC) was investigated by modulating the activity of water with polyethylene glycols with molecular weights of 1000-20000 and dextran with a molecular weight of 20000. These polymers create an osmotic stress due to their exclusion from hydration shells and crevices on proteins, causing dehydration. Polymers larger than 1000 caused an activation of the PKC-catalyzed phosphorylation of histone, while PEG 1000 had no significant effect. The extent of activation by PEG and dextran 20000 was larger than that of PEG 6000 or 8000 when vesicles were composed of 1:1 POPS/POPC, suggesting the presence of at least two distinct regions of exclusion on PKC: one inaccessible to PEGs larger than 1000 and the other inaccessible only to PEGs of > 10000. The extent of activation was dependent on the composition of the vesicles used. If basal activity (without PEG) was low (e.g. with low PS content in membranes), then the extent of activation was similar for all polymers larger than 1000. Binding of PKC to membranes containing 50 mol % PS was unaffected by PEG 6000 but was inhibited by PEG 20000. At a low PS content of 10%, both PEG 6000 and 20000 inhibited binding. This suggests that PKC becomes hydrated upon binding to membranes. Under conditions in which all of the enzyme is membrane-bound, both Km and Vmax for the phosphorylation of histone increased linearly with osmotic stress induced by PEG 6000. Thus, PKC becomes hydrated with 2311 +/- 476 water molecules upon binding of histone and is dehydrated by 1349 +/- 882 water molecules in going to the transition state. Km and Vmax for phosphorylation of the MARCKS peptide also increase with osmotic stress induced by PEG 6000. When protamine sulfate was used as a substrate (cofactor-independent), Vmax for the reaction was unaffected, but Km decreased with osmotic pressure (with PEG 6000), suggesting that PKC becomes dehydrated upon binding protamine. Similar results were found with a peptide substrate derived from the pseudosubstrate site of PKC epsilon. Since dextran, a polymer unrelated in structure to PEG, could cause a similar activation of PKC, the effects seen are likely due to osmotic stress and not to specific binding of PEG to PKC. Also, results obtained with PE-linked PEG were opposite to those with free PEG. PE-linked PEGs of 2000 and 5000 caused an inhibition of PKC-catalyzed phosphorylation of histone when present in membranes. If a specific interaction occurred with PEG, this would be expected to occur even with PE-PEG. The effects observed with free PEG are also independent of ionic strength. Free PEG had no effect on the bilayer to hexagonal phase transition temperature of DEPE membranes, suggesting that the effects on PKC activity are not a consequence of changes in membrane properties at the osmotic pressures used.
 ...
PMID:Role of water in protein kinase C catalysis and its binding to membranes. 904 27


<< Previous 1 2 3 4 5 6 7 8 9 10