共查询到20条相似文献,搜索用时 15 毫秒
1.
M Gschwendt W Kittstein F Horn H Leibersperger F Marks 《Journal of cellular biochemistry》1989,40(3):295-307
The phosphorylation of an Mr 82,000 protein (p82) in the Triton X-100 extract of the particulate fraction of mouse epidermis is dependent on the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) or diacylglycerol and phospholipid and, contrary to protein kinase C (PKC)-catalyzed phosphorylation, cannot be activated by calcium plus phospholipid. The novel p82 kinase differs also from PKC in many other respects, such as substrate specificity, turnover rate, and sensitivity to inhibitors. The p82 kinase can be separated from PKC by chromatography on phenyl sepharose and does not react with a polyclonal PKC antiserum. Like PKC, the novel kinase phosphorylates its substrate on threonine and serine, but not on tyrosine. Similar to PKC, the epidermal p82-kinase system is down-modulated after TPA treatment of mouse skin, with a half-life of around 5 h. Down-modulation is also accomplished by the phorbol ester RPA, but not by the Ca2+ ionophore A23187, and it is inhibited by the immunosuppressive agent cyclosporin A. In addition to down-modulation, TPA treatment of the animals activates a phosphatase that dephosphorylates phosphorylated p82 in the extract of the particulate fraction. 相似文献
2.
Brief treatment of intact thymocytes with TPA and other tumor promoters causes a reduction in protein kinase C activity from the cytosol and an increase in kinase activity in the particulate fraction. In contrast to the activity in the cytosol, which is absolutely dependent on the addition of Ca2+, phosphatidylserine and diolein, the activity in the particulate fraction is independent of these agents. Analysis of target specificity of the particulate kinase activity using exogenous and endogenous substrates suggests that the increased phosphorylation in the particulate fraction is catalysed by protein kinase C with altered catalytic properties. Although interleukin-1 and TPA are both co-mitogens for murine thymocytes, interleukin-1 does not share with TPA its property to alter protein kinase activity in the cytosolic and particulate fractions. 相似文献
3.
Protein kinase C (PKC) from bovine neutrophils was purified 1420-fold. Subcellular fractionation analysis of bovine neutrophil homogenate in the presence of EGTA indicated that more than 95% of the PKC activity was present in the soluble fraction. The purification procedure from cytosol involved sequential chromatographic steps on DE-52 cellulose, Mono Q, and phenyl-Sepharose. Whereas bovine brain PKC could be resolved into four isoenzymatic forms by chromatography on a hydroxylapatite column, bovine neutrophil PKC was eluted in a single peak, suggesting that it corresponded to a single isoform. The apparent molecular weight of bovine neutrophil PKC was 82,000, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. By filtration on Sephadex G-150, a molecular weight of 85,000 was calculated, indicating that bovine neutrophil PKC in solution is monomeric. Its isoelectric point was 5.9 +/- 0.1. Bovine neutrophil PKC was autophosphorylated in the presence of [gamma-32P]ATP, provided that the medium was supplemented with Mg2+, Ca2+, phosphatidylserine, and diacylglycerol; phorbol myristate acetate could substitute for diacylglycerol. Autophosphorylated PKC could be cleaved by trypsin to generate two radiolabeled peptides of Mr 48,000 and 39,000. The labeled amino acids were serine and threonine. During the course of the purification procedure of bovine neutrophil PKC, a protein of Mr 23,000, which was abundant in the cytosolic fraction of the homogenate, was found to exhibit a strong propensity to PKC-dependent phosphorylation in the presence of [gamma-32P]ATP, Mg2+, Ca2+, phosphatidylserine, and diacylglycerol. This protein was recovered together with PKC in one of the two active peaks eluted from the Mono Q column at the second step of PKC purification.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
4.
In mammalian systems, Ca2+/diacylglycerol-activated phospholipid-dependent protein kinase (C-kinase) appears to play an important role in regulating physiological responses that outlast the transient rise in cytosolic Ca2+. Electrophysiological experiments in neurons of the nudibranch mollusc, Hermissenda crassicornis, have suggested a role for C-kinase in the long-lasting reductions in early and late K+ currents that have been observed following associative learning. Accordingly, we have investigated the catalytic properties of C-kinase in Hermissenda CNS. Following homogenization in Ca2+-free buffer, C-kinase can be separated from Ca2+/calmodulin-dependent protein kinase by centrifugation; C-kinase activity is found in the supernatant whereas essentially all of the Ca2+/calmodulin-dependent protein kinase is found in the membrane fraction. Addition of Ca2+, phosphatidylserine, and diacylglycerol to the cytosol results in phosphorylation of at least eight endogenous proteins. The Hermissenda CNS C-kinase can also phosphorylate lysine-rich histone, a substrate for mammalian C-kinase. The molluscan enzyme exhibits phospholipid specificity in that phosphatidylserine is much more effective than phosphatidylethanolamine, phosphatidylcholine, phosphatidylinositol, and phosphatidic acid. Addition of diacylglycerol, in the presence of Ca2+ and phosphatidylserine, increases the activity of the C-kinase. The percentage of activation by diacylglycerol is larger at lower Ca2+ concentrations. Enzyme activity is inhibited by trifluoperazine and polymixin B sulfate. These studies indicate that the Hermissenda C-kinase is catalytically similar to mammalian C-kinase. 相似文献
5.
Pancreatic islet homogenates display protein kinase C activity. Although the rate of histone phosphorylation by islet homogenates is not enhanced by Ca2+ alone, the Ca2+ ion markedly augments reaction velocity in the presence of phosphatidylserine and at low concentrations (20 nM--0.2 microM) of the tumor-promoting agent 12-0-tetradecanoylphorbol-13-acetate (TPA). At a higher concentration (2.0 microM), TPA stimulates histone phosphorylation even in the absence of Ca2+. Ca-calmodulin also stimulates protein phosphorylation but the latter effect is apparently mediated by a Ca-calmodulin-responsive protein kinase distinct from the protein kinase C. In the presence of phosphatidylserine, retinoic acid (0.1 microM) fails to cause any obvious change in protein kinase C activity. However, in the 0.1-100.0 microM range, retinoic acid confers a limited responsiveness to TPA in the absence of phosphatidylserine. These findings support the view that Ca2+ may regulate protein phosphorylation in the pancreatic B-cell through several distinct pathways. 相似文献
6.
In Vivo Translocation and Down-Regulation of Protein Kinase C Following Intraventricular Administration of Tetanus Toxin 总被引:5,自引:5,他引:0
A single intraventricular injection into adult rats of 100 mouse lethal doses of tetanus toxin (TeTox) produces a marked intracellular redistribution of Ca2+/phosphatidylserine (PtdSer)-dependent protein kinase C (PKC) activity. Changes are particularly pronounced in hypothalamus, hippocampus, and spinal cord structures. Translocation of PKC from the inactive cytosolic compartment to a membrane-bound active form is followed by a time-dependent reduction in both total activity and enzyme protein. The down-regulation of PKC activity in the hypothalamus is accompanied by a marked increase in a Ca2+/PtdSer-independent kinase activity, predominantly in the cytosolic fraction. Our data identify PKC as a possible indirect target for TeTox and suggest that down-regulation of the enzyme may provide a clue for tetanus neurotoxicity. 相似文献
7.
Paul T. Kelly Rick K. Yip Steven M. Shields Michael Hay 《Journal of neurochemistry》1985,45(5):1620-1634
Synaptic junctions (SJs) from rat forebrain were examined for Ca2+/calmodulin (CaM)-dependent kinase activity and compared to synaptic plasma membrane (SPM) and postsynaptic density (PSD) fractions. The kinase activity in synaptic fractions was examined for its capacity to phosphorylate endogenous proteins or exogenous synapsin I, in the presence or absence of Ca2+ plus CaM. When assayed for endogenous protein phosphorylation, SJs contained approximately 25-fold greater amounts of Ca2+/CAM-dependent kinase activity than SPMs, and fivefold more activity than PSDs. When kinase activities were measured by phosphorylation of exogenous synapsin I, SJs contained fourfold more activity than SPMs, and 10-fold more than PSDs. The phosphorylation of SJ proteins of 60- and 50-kilodalton (major PSD protein) polypeptides were greatly stimulated by Ca2+/CaM; levels of phosphorylation for these proteins were 23- and 17-fold greater than basal levels, respectively. Six additional proteins whose phosphorylation was stimulated 6-15-fold by Ca2+/CAM were identified in SJs. These proteins include synapsin I, and proteins of 240, 207, 170, 140, and 54 kilodaltons. The 54-kilodalton protein is a highly phosphorylated form of the major PSD protein and the 170-kilodalton component is a cell-surface glycoprotein of the postsynaptic membrane that binds concanavalin A. The CaM-dependent kinase in SJ fractions phosphorylated endogenous phosphoproteins at serine and/or threonine residues. Ca2+-dependent phosphorylation in SJ fractions was strictly dependent on exogenous CaM, even though SJs contained substantial amounts of endogenous CaM (15 micrograms CaM/mg SJ protein). Exogenous CaM, after being functionally incorporated into SJs, was rapidly removed by sequential washings. These observations suggest that the SJ-associated CaM involved in regulating Ca2+-dependent protein phosphorylation may be in dynamic equilibrium with the cytoplasm. These findings indicate that a brain CaM-dependent kinase(s) and substrate proteins are concentrated at SJs and that CaM-dependent protein phosphorylation may play an important role in mechanisms that underlie synaptic communication. 相似文献
8.
Phorbol Esters Induce Neurotransmitter Release in Cholinergic Synaptosomes from Torpedo Electric Organ 总被引:2,自引:2,他引:0
The effect of phorbol esters and so the involvement of Ca2+/phospholipid-dependent protein kinase (protein kinase C;PKC) in the release of acetylcholine (ACh) was studied using Torpedo electric organ synaptosomes. 12-O-Tetradecanoylphorbol 13-acetate (TPA), a known activator of PKC, induced neurotransmitter release in a concentration-dependent manner and increased the potassium-evoked release of ACh. The effect of TPA was shown to be independent of the extrasynaptosomal calcium concentration. TPA-induced ACh release was reversed by H-7, an inhibitor of PKC activity. This drug showed no effect on potassium-evoked ACh release. Botulinum toxin, a strong blocker of potassium-induced ACh release in that synaptosomal preparation, showed no inhibitory effect on the TPA-induced ACh release. Our results suggest that activation of PKC potentiates the release of an ACh pool that is not releasable by potassium depolarization, independently of the extracellular calcium concentration. 相似文献
9.
We examined whether protein kinase C activation plays a modulatory or an obligatory role in exocytosis of catecholamines from chromaffin cells by using PKC(19-31) (a protein kinase C pseudosubstrate inhibitory peptide), Ca/CaM kinase II(291-317) (a calmodulin-binding peptide), and staurosporine. In permeabilized cells, PKC (19-31) inhibited the phorbol ester-mediated enhancement of Ca2(+)-dependent secretion as much as 90% but had no effect on Ca2(+)-dependent secretion in the absence of phorbol ester. The inhibition of the phorbol ester-induced enhancement of secretion by PKC (19-31) was correlated closely with the ability of the peptide to inhibit in situ phorbol ester-stimulated protein kinase C activity. PKC(19-31) also blocked 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced phosphorylation of numerous endogenous proteins in permeabilized cells but had no effect on Ca2(+)-stimulated phosphorylation of tyrosine hydroxylase. Ca/CaM kinase II(291-317), derived from the calmodulin binding region of Ca/calmodulin kinase II, had no effect on Ca2(+)-dependent secretion in the presence or absence of phorbol ester. The peptide completely blocked the Ca2(+)-dependent increase in tyrosine hydroxylase phosphorylation but had no effect on TPA-induced phosphorylation of endogenous proteins in permeabilized cells. To determine whether a long-lived protein kinase C substrate might be required for secretion, the lipophilic protein kinase inhibitor, staurosporine, was added to intact cells for 30 min before permeabilizing and measuring secretion. Staurosporine strongly inhibited the phorbol ester-mediated enhancement of Ca2(+)-dependent secretion. It caused a small inhibition of Ca2(+)-dependent secretion in the absence of phorbol ester which could not be readily attributed to inhibition of protein kinase C. Staurosporine also inhibited the phorbol ester-mediated enhancement of elevated K(+)-induced secretion from intact cells while it enhanced 45Ca2+ uptake. Staurosporine inhibited to a small extent secretion stimulated by elevated K+ in the absence of TPA. The data indicate that activation of protein kinase C is modulatory but not obligatory in the exocytotoxic pathway. 相似文献
10.
Monoclonal antibodies (8/1, 10/10, and 25/3) against rat brain type II protein kinase C were used for the immunochemical characterization of this kinase. These antibodies immunoprecipitated the type II protein kinase C in a dose-dependent manner but did neither to the type I nor III isozyme. Immunoblot analysis of the tryptic fragments from protein kinase C revealed that all three antibodies recognized the 27-38-kDa fragments, the phospholipid/phorbol ester-binding domain, but not the 45-48-kDa fragments, the kinase catalytic domain. The immune complexes of the kinase and the antibodies retained 70-80% of the kinase activity which was dependent on Ca2+ and phosphatidylserine and further activated by diacylglycerol or tumor-promoting phorbol ester. With antibody 8/1, the kinetic parameters with respect to Km for ATP and histone and K alpha for phosphatidylserine and phorbol 12,13-dibutyrate were not significantly influenced. However, the antibody causes variable effects on the K alpha for Ca2+ under different assay conditions. When determined in the presence of phosphatidylserine, the K alpha for Ca2+ was reduced by an order of magnitude (37 +/- 8 to 2.0 +/- 1.8 microM); in the presence of phosphatidylserine and phorbol 12,13-dibutyrate, the K alpha for Ca2+ was not significantly altered; and in the presence of phosphatidylserine and dioleoylglycerol, the kinase became an apparently Ca2+-independent enzyme. The effects of antibody 8/1 on the kinetic parameters of the enzyme for phorbol ester binding were different from those for kinase activity. This antibody causes a 20-30% reduction in phorbol ester binding and a 2-fold increase (1.9 +/- 0.2 to 3.9 +/- 0.3 micrograms/ml) in the concentration of phosphatidylserine required for half-maximal binding, but is without significant influence on those parameters for Ca2+ and phorbol 12,13-dibutyrate. The differential effects of antibody 8/1 on kinase activity and phorbol ester binding with respect to the kinetic parameter of phosphatidylserine suggest that the roles of this phospholipid in supporting phorbol ester binding and kinase activation are different. In the presence of the antibody, the autophosphorylations of the phospholipid/phorbol ester-binding domain and the kinase domain were reduced; the reduction was more pronounced for the former than for the latter. These results suggest that the epitope for antibody 8/1 is localized within the phospholipid/phorbol ester-binding domain at the region adjacent to the kinase domain so that the autophosphorylations of both domains are affected. 相似文献
11.
The Ca2+/phosphatidylserine-stimulated protein kinase C (PKC) appears to exist as interconvertible inactive, soluble and active, membrane-bound forms. Changes in the bimodal distribution of PKC induced by diacylglycerol or tumor-promoting phorbol esters have been proposed to regulate the activity of this kinase [Nishizuka, Y. (1984) Nature (London) 308, 693-698]. A rapid microassay for assessment of protein kinase C translocation between cytosol and membranes was developed. This procedure, which relied on the selective digitonin-mediated release of cytoplasmic proteins, eliminated potential homogenization and fractionation artifacts. PKC activity toward histone H1 was determined after limited trypsinolysis, which abolished the Ca2+/phospholipid requirement of the enzyme and prevented interference by inhibitory proteins. Complete translocation of PKC to the membrane fraction and subsequent down-regulation of the kinase in response to 12-O-tetradecanoylphorbol-13-acetate treatment of Swiss 3T3 cells could be demonstrated by this method. Platelet-derived growth factor, insulin-like growth factor 1, vasopressin, and prostaglandin F2 alpha facilitated partial conversions of PKC to the membrane-bound form in quiescent 3T3 cells. 相似文献
12.
Two forms of protein kinase C (PKC) activity in cytosol of cultured rat mesangial cells have been characterized in vitro by using histone H1 or endogenous proteins as substrates. Histones H1-phosphorylation was significantly increased only when calcium, phosphatidylserine (PS) and 1,2-diacylglycerol (DAG) or phorbol myristate acetate (PMA) were present together in the incubation medium. EGTA, a calcium chelator, completely inhibited this activity. Upon hydroxyapatite chromatography (HPLC), the PKC activity was eluted as a main peak at 150 mM potassium phosphate with a shoulder at 180 mM. Both peaks corresponded to the type III PKC from rat brain and were identified as PKC alpha isoform by immunoblot analysis. In contrast with what was observed using histone H1, the increased phosphorylation of endogenous proteins in the presence of a mixture of Ca2+/PS, plus either DAG or PMA, was only partly reduced by EGTA. Moreover, the level of the PKC activity detected in the presence of EGTA was comparable to the level of kinase activity, measured in the presence of PS alone or associated with DAG or PMA. This suggests that mesangial cells contain PKC activity which does not absolutely require calcium. Polyacrylamide gel electrophoresis revealed that patterns of phosphorylated mesangial cell proteins are different depending on whether calcium was added or not. In the presence of calcium, PKC strongly phosphorylated the proteins of 53,000 molecular weight, a doublet of 37,000-39,000, the 24,000 and the triplet of 17,000-20,000-22,000 molecular weight. The addition of EGTA to the assays suppressed completely the labelling of most proteins; only the 20,000 molecular weight protein remained strongly labelled, while the 39,000 molecular weight band was only faintly visible. The same patterns of phosphorylations were obtained after omission of calcium in the assays containing only PS and DAG (or PMA). So, the main substrates of calcium-dependent PKC are proteins of 53,000, 39,000, 37,000, 22,000, 24,000 and 17,000 molecular weight while the protein of 20,000 molecular weight appears to be the main substrate of calcium-independent PKC. The existence in mesangial cells of at least two forms of PKC, which phosphorylate specific endogenous proteins, emphasizes the complexity of the phospholipid-dependent regulatory cascade and raises the possibility that actions of different regulators may be transduced through distinct PKC isozymes. 相似文献
13.
Characterization of calcium-independent forms of protein kinase C-beta in phorbol ester-treated rabbit platelets 总被引:1,自引:0,他引:1
S L Pelech M Samiei D L Charest S L Howard H Salari 《The Journal of biological chemistry》1991,266(14):8696-8705
The subcellular distribution, size, and activation state of protein kinase C (PKC) were studied after short term exposure of rabbit platelets to a saturating dose of 12-O-tetradecanoylphorbol 13-acetate (TPA). Cytosolic and Nonidet P-40-solubilized particulate extracts prepared from TPA-treated platelets were subjected to analytical column chromatography on Mono Q, hydroxylapatite, and Superose 6/12. PKC activity was assayed according to the ability of the enzyme to phosphorylate (i) histone H1 in the presence of the activators calcium, diacylglycerol, and phosphatidylserine; (ii) histone H1 after proteolytic activation of PKC with trypsin; and (iii) protamine in the absence of calcium and lipid. Within 1 min of TPA treatment of platelets, greater than 95% of the PKC activity was particulate associated, as assessed by all three methods. The particulate PKC activity from 1-min TPA-treated cells eluted from Mono Q with approximately 0.35 M NaCl (peak I), and it was highly dependent upon Ca2+ and lipid for optimal histone H1 phosphorylation. With longer exposure times of platelets to TPA, the disappearance of the Mono Q peak I form of PKC was correlated with the production of new PKC species that were released from Mono Q with approximately 0.4 M NaCl (peak II), approximately 0.5 M NaCl (peak III), and approximately 0.6 M NaCl (peak IV). These last forms of PKC were still lipid activated but exhibited little Ca2+ dependence. The Mono Q peak III form displayed a particularly high level of histone H1 phosphorylating activity in the absence of lipid and Ca2+. All of these forms behaved as approximately 65-kDa proteins on Superose 6/12, but on sodium dodecyl sulfate-polyacrylamide gels, Western blotting with anti-PKC-beta antibodies revealed immunoreactive polypeptides of approximately 79 kDa (Mono Q peaks I, II, and IV) and approximately 100-kDa (Mono Q peak III). Hydroxylapatite column chromatography permitted partial resolution of the Mono Q peaks I and II forms, which were eluted within a concentration range of potassium phosphate (100-150 mM) which was typical of the beta isozyme of PKC. Treatment of the Mono Q peak III and IV PKC forms with alkaline phosphatase resulted in the production of the peak I form, which implicated protein phosphorylation in the interconversion of the various PKC forms. 相似文献
14.
S Hoshina M Ueffing M Morotomi I B Weinstein 《Biochemical and biophysical research communications》1991,176(1):505-510
Protein kinase C (PKC) is a Ca2(+)- and phospholipid-dependent serine and threonine protein kinase which binds and is activated by tumor promoters such as the phorbol ester 12-0-tetradecanoylphorbol-13-acetate (TPA). PKC can be activated in vitro by phosphatidylserine (PS) plus Ca2+. We report here that the compound fecapentaene-12 can replace the requirement for PS in the activation of PKC by Ca2+. In addition, at low concentrations fecapentaene-12 can enhance the activation of PKC by Ca2+ and PS. It can also either enhance or inhibit activation of PKC by the tumor promoter teleocidin, depending on the assay conditions. These results are of interest since fecapentaene is known to be a potent mutagen that is produced by Bacteroides species present in the lumen of the human colon. The present studies raise the possibility that this compound might also play a role in colon cancer by altering the activity of PKC. 相似文献
15.
Protein kinase C from small intestine epithelial cells 总被引:1,自引:0,他引:1
G Velasco C F Iglesias P Domínguez F Barros S Gascón P S Lazo 《Biochemical and biophysical research communications》1986,139(3):875-882
Protein kinase C activity has been identified in cytosolic and membrane fractions from rat and rabbit small intestine epithelial cells. The cytosolic fraction comprised about the 75% of total activity. Protein kinase C activity was resolved from other protein kinase activities by ion exchange chromatography. Phosphatidylserine or phosphatidylinositol were required for protein kinase C to be active. In addition, the activity was enhanced by the presence of a diacylglycerol. Diolein and dimyristin were the most effective (13-14 fold activation). In the presence of phosphatidylserine and diolein, the Ka for activation by Ca2+ was 10(-7)M. The phorbol ester TPA substituted for diacylglycerol in activating protein kinase C. Brush border and basolateral membranes contained protein kinase C activity, although the specific activity of the basal lateral membranes was four-fold higher than the specific activity of the brush border membranes. The presence of PKC in small intestine epithelial cells might have important implications in the Ca2+ mediated control of ionic transport in this tissue. 相似文献
16.
We have examined endogenous cyclic AMP-stimulated phosphorylation of subcellular fractions of rat brain enriched in synaptic plasma membranes (SPM), purified synaptic junctions (SJ), and postsynaptic densities (PSD). The analyses of these fractions are essential to provide direct evidence for cyclic AMP-dependent endogenous phosphorylation at discrete synaptic junctional loci. Protein kinase activity was measured in subcellular fractions using both endogenous and exogenous (histones) proteins as substrates. The SJ fraction possessed the highest kinase activity toward endogenous protein substrates, 5-fold greater than SPM and approximately 120-fold greater than PSD fractions. Although the kinase activity as measured with histones as substrates was only slightly higher in SJ than SPM fractions, there was a marked preference of kinase activity toward endogenous compared to exogenous substrates in SJ fractions but in SPM fractions. Although overall phosphorylation in SJ fractions was increased only 36% by 5 micron cyclic AMP, there were discrete proteins of Mr = 85,000, 82,000, 78,000, and 55,000 which incorporated 2- to 3-fold more radioactive phosphate in the presence of cyclic AMP. Most, if not all, of the cyclic AMP-independent kinase activity is probably catalyzed by catalytic subunit derived from cyclic AMP-dependent kinase, since the phosphorylation of both exogenous and endogenous proteins was greatly decreased in the presence of a heat-stable inhibitor protein prepared from the soluble fraction of rat brain. The specific retention of SJ protein kinase(s) activity during purification and their resistance to detergent solubilization was achieved by chemical treatments which produce interprotein cross-linking via disulfide bridges. Two SJ polypeptides of Mr = 55,000 and 49,000 were photoaffinity-labeled with [32P]8-N3-cyclic AMP and probably represent the regulatory subunits of the type I and II cyclic AMP-dependent protein kinases. The protein of Mr = 55,000 was phosphorylated in a cyclic AMP-stimulated manner suggesting autophosphorylation as previously observed in other systems. 相似文献
17.
Protein Kinase C in Primary Astrocyte Cultures: Cytoplasmic Localization and Translocation by a Phorbol Ester 总被引:7,自引:7,他引:0
The distribution of calcium-activated, phospholipid-dependent protein kinase (protein kinase C) in supernatant and particulate fractions of primary cultures of rat astrocytes and its translocation by a phorbol ester were studied. We observed that 91% of protein kinase C activity in astrocytes was in the supernatant fraction, as measured by lysine-rich histone phosphorylation assay. Attempts to uncover latent activity in the particulate fraction were unsuccessful. Approximately 75% of the supernatant protein kinase C activity could be translocated to the particulate fraction by prior treatment (30-60 min) of the cultures with 100 nM 12-O-tetradecanoyl-phorbol 13-acetate (TPA), but not with 4 alpha-phorbol, an inactive phorbol ester. Investigation of endogenous substrates for protein kinase C showed that TPA treatment brought about an increase in phosphorylation in membrane proteins and a decrease in phosphorylation of supernatant proteins. These findings indicate that the distribution of protein kinase C in astrocytes differs substantially from that in whole brain tissue, where approximately two-thirds of the protein kinase C activity is associated with the particulate fraction. Because protein kinase C is concentrated in the cytosol of astrocytes and most of this activity can be translocated to membranes, astrocytes may be particularly well-suited to respond to signals that activate phosphoinositide-linked receptors in brain. 相似文献
18.
P J Robinson 《The Journal of biological chemistry》1992,267(30):21637-21644
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. 相似文献
19.
Phorbol ester stimulation of insulin release and secretory-granule protein phosphorylation in a transplantable rat insulinoma. 总被引:5,自引:2,他引:3
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The effects of tumour-promoting phorbol esters on protein-phosphorylation reactions and secretion in rat insulinoma tissue were investigated with the objective of assessing the possible role of Ca2+- and phospholipid-dependent protein kinases (protein kinase C) in insulin release. 4 beta-Phorbol 12-myristate 13-acetate (TPA) was a potent secretagogue at concentrations above 0.1 microM. TPA-induced release was inhibited by adrenaline or omission of Ca2+ from the extracellular medium and was augmented by theophylline. These findings suggested that TPA activated an exocytotic process. TPA enhanced the Ca2+- and phospholipid-dependent phosphorylation of histone III-S by a soluble protein fraction of the tissue. Endogenous phosphorylation reactions involving soluble and secretory-granule membrane proteins were also stimulated by TPA in tissue homogenates and reconstituted subcellular fractions. Histone phosphorylation and the granule-protein phosphorylation reactions showed similar concentration-dependencies for activation by both Ca2+ and TPA, thus indicating that the same enzyme was involved. It is concluded that the phosphorylation of cytosolic and membrane protein substrates by protein kinase C may be important in the stimulus-secretion coupling mechanism of insulin release. 相似文献
20.
Characterization of the membrane-bound protein kinase C and its substrate proteins in canine cardiac sarcolemma 总被引:1,自引:0,他引:1
Cardiac sarcolemma was purified from canine ventricles. Enrichment of the sarcolemmal membranes was demonstrated by the high (Na+ + K+)-ATPase activity of 28.0 +/- 1.5 mumol Pi/mg protein per h and the high concentration of muscarinic receptors with the Bmax of 8.2 +/- 2.5 pmol/mg protein as determined by [3H]QNB binding. The purified sarcolemma also contains significant levels of a membrane-bound Ca2+ and phospholipid-dependent protein kinase (protein kinase C). To elucidate the protein kinase C activity in sarcolemma, a prior incubation of the membranes with EGTA and Triton X-100 was necessary. The specific activity of protein kinase C was found to be 131.4 pmol Pi/mg per min, in the presence of 6.25 micrograms phosphatidylserine and 0.5 mM CaCl2. Treatment of sarcolemma with 12-O-tetradecanoylphorbol 13-acetate (TPA) and phorbol 12,13-dibutyrate (PBu2) resulted in a concentration-dependent activation of protein kinase C activity. The effect of TPA and PBu2 on protein kinase C in sarcolemma was independent of exogenous Ca2+ and phosphatidylserine. Polymyxin B inhibited phorbol-ester-induced activation of protein kinase C activity. The distribution of protein kinase C in the cytosolic fraction was also examined. The specific activity of the kinase in the cytosolic fraction was 59.7 pmol Pi/mg per min. However, the total protein kinase C activity in the cytosol was 213500 pmol Pi/min, compared to that of 1025 pmol Pi/min in the sarcolemma isolated from approx. 100 g of canine ventricular muscle. Several endogenous proteins in cardiac sarcolemma were phosphorylated in the presence of Ca2+ and phosphatidylserine. The major substrates for protein kinase C were proteins of Mr 94 000, 87 000, 78 000, 51 000, 46 000, 11 500 and 10 000. Most of these substrate proteins have not been identified before. Other proteins of Mr 38 000, 31 000 and 15 000 were markedly phosphorylated in the presence of Ca2+ only. Phosphorylation of phospholamban (Mr 27 000 and 11 000) was also stimulated in the presence of Ca2+ and phosphatidylserine, but the low Mr form of phospholamban was distinct from two other low Mr substrate proteins for protein kinase C. Polymyxin B was more selective in inhibiting the protein kinase C dependent phosphorylation. On the other hand, trifluoperazine selectively inhibited the phosphorylation of phospholamban and Mr 15 000 protein. Although the exact function of this kinase is unknown, based on these observations, we believe that protein kinase C in the cardiac sarcolemma may play an important role in the cell-surface-signal regulated cardiac function. 相似文献