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)

Occupancy of surface immunoglobulin (sIg) receptor for antigen expressed on resting B cells initiates increased turnover of membrane-associated phosphatidylinositol (PI), which ultimately leads to the enhanced expression of c-myc mRNA. The mechanism which links these initial membrane biochemical changes to subsequent alterations in c-myc transcription is unclear. The present study examines the possible involvement of PKC and its calpain-generated proteolytic fragment, protein kinase M (PKM), in conveying the membrane-associated signal to the nucleus. Utilizing an in vitro phosphorylation assay, we have shown that a calcium-dependent protease, similar to calpain, is involved in the downregulation of membrane-associated PKC induced by anti-immunoglobulin or phorbol 12-myristate 13-acetate (PMA) and ionomycin stimulation of resting B cells. In addition, we have confirmed previous studies showing that PMA and ionomycin are both required for optimal expression of c-myc mRNA. The enhanced expression of c-myc mRNA is sensitive to inhibitors of PKC, such as H-7 and sangavimycin, providing evidence for a prominent role of PKC and/or PKM in the receptor-mediated up-regulation of c-myc message expression. Finally, a calpain inhibitor interferes with the transmission of the membrane-associated signal which induces the increased expression of c-myc mRNA. Our results are consistent with the hypothesis that the calpain-mediated proteolysis of membrane-associated PKC is involved in the sIg-mediated signal transduction pathway.
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PMID:The proteolysis of membrane-associated protein kinase C as a possible component of the signalling pathway leading to c-myc induction in B lymphocytes. 176 Feb 54

Limited proteolysis of protein kinase C (PKC) subspecies with Ca2(+)-dependent neutral protease II (calpain II) was remarkably stimulated by basic polypeptides, such as H1 histone and poly-L-lysine. This stimulatory effect was observed for proteolysis of the active form of PKC, which was associated with phospholipid and diacylglycerol. The inactive form of PKC was far less susceptible to proteolysis, both in the presence and absence of the basic polypeptides. The basic polypeptides did not appear to interact with calpain II, but made the PKC molecule more susceptible to proteolysis. The relative rates of cleavage of type I (gamma), II (beta), and III (alpha) PKC were 2:2:1. The available evidence suggests that, like calpain I, calpain II may also contribute to the down-regulation or depletion of PKC.
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PMID:H1 histone stimulates limited proteolysis of protein kinase C subspecies by calpain II. 176 64

A calpain 1-protein kinase C (PKC) complex was isolated from rabbit skeletal muscle by hydrophobic interaction chromatography on phenyl-sepharose and by strong anion exchange chromatography on Q-Sepharose. Calpain 1 and kinase activities were then dissociated on a phenyl-Sepharose matrix using gradients of decreasing ionic strength. The purified PKC obtained corresponded to conventional PKC and was recognized by a monoclonal antibody specific for alpha and beta isotypes. Leupeptin, calpain inhibitor II, and the more selective calpain inhibitors calpeptin and MDL 28170 did not block the activation of the purified PKC by Ca2+ and phosphatidylserine.
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PMID:Calpain 1-protein kinase C complex: effect of calpain inhibitors after dissociation. 179 35

Purified Ca(2+)-stimulated, Mg(2+)-dependent ATPase (Ca(2+)-ATPase) from human erythrocytes was phosphorylated with a stoichiometry of about 1 mol of phosphate/mol of ATPase at both threonine and serine residues by purified rat brain type III protein kinase C. In the presence of calmodulin, the phosphorylation was markedly reduced. Labeled phosphate from [gamma-32P]ATP was retained on an 86-kDa calmodulin-binding tryptic fragment of Ca(2+)-ATPase but not on 82- and 77-kDa non-calmodulin-binding fragments. Similarly, fragmentation of the phosphorylated Ca(2+)-ATPase by calpain I revealed that calmodulin-binding fragments (127 and 125 kDa) retained phosphate label whereas a non-calmodulin-binding fragment (124 kDa) did not. The calmodulin-binding domain, located about 12 kDa from the carboxyl terminus of the Ca(2+)-ATPase, was thus located as a site of protein kinase C phosphorylation. A synthetic peptide corresponding to a segment of the calmodulin-binding domain (H2 N-R-G-L-N-R-I-Q-T-Q-I-K-V-V-N-COOH) was indeed phosphorylated at the single threonine residue within this sequence. The additional serine phosphorylation site was carboxyl terminal to the calmodulin domain. Phosphorylation by purified type III protein kinase C (canine heart) antagonized the calmodulin activation of the Ca(2+)-ATPase, particularly at lower Ca2+ concentrations (0.2-1.0 microM). By contrast, a purified but unresolved protein kinase C isoenzyme mixture from rat brain stimulated the activity of Ca(2+)-ATPase prepared in asolectin, but not glycerol, by more than 2-fold in the presence of the ionophore A23187, without increasing its Ca2+ sensitivity. The results clearly indicate that human erythrocyte Ca(2+)-ATPase is a substrate of protein kinase C, but the effect of phosphorylation on the activity of the enzyme depends on the isoenzyme form of protein kinase C used and on the lipid associated with the Ca(2+)-ATPase.
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PMID:Protein kinase C phosphorylates the carboxyl terminus of the plasma membrane Ca(2+)-ATPase from human erythrocytes. 182 43

Treatment of the solubilized and purified Ca(2+)-translocating ATPase (Ca(2+)-ATPase) (136 kDa) from human erythrocyte plasma membranes with endoproteinase Glu-C from Staphylococcus aureus strain V8 (V8 protease) yielded transient fragments of 96 kDa and 76 kDa and more stable fragments of 60 kDa and 37/36 kDa (doublet). The presence of calmodulin did not alter the fragmentation pattern. The 60 kDa fragment contains the protein kinase C (bovine brain) phosphorylation site(s), which we previously localized in the C-terminal region [Wang, Wright, Machan, Allen, Conigrave & Roufogalis (1991) J. Biol. Chem. 266, 9078-9085]. On the other hand, the 37/36 kDa fragments possess the ability to form an acyl-phosphate intermediate. Furthermore, the presence of the 60 kDa and 37/36 kDa fragments together results in expression of calmodulin-sensitive Ca(2+)-ATPase activity. However, further degradation of the 60 kDa fragment was coupled with the appearance of calmodulin-independent activity, whereas the 37/36 kDa fragment doublet remained stable. It was concluded that the 60 kDa and the 37/36 kDa fragments: (a) together represent the C-terminal two-thirds of the enzyme, which is functional as an Ca(2+)-ATPase, (b) were produced by a single cleavage near the C-terminal side of the cytosolic catalytic domain, and (c) probably remain physically and functionally associated even after cleavage has occurred. At the C-terminus, the basic calmodulin-binding domain is flanked by two highly acidic regions (domains A and B). Our results indicate that domains A and B, despite containing many Asp and Glu residues, were not readily cleaved by V8 protease, which is known to cleave selectively peptide bonds at the C-terminal side of Asp and Glu. However, if the Ca(2+)-ATPase were pre-digested with calpain I from human erythrocytes, which removed its calmodulin-binding domain (along with domain B), multiple cleavages by V8 protease in domain A were then readily observed. We propose that the calmodulin-binding domain is closely associated with the acidic domains A and B and that these acidic domains might help to co-ordinate the stimulation of the enzyme by calmodulin.
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PMID:Structure--function relationship of the human erythrocyte plasma membrane Ca(2+)-ATPase revealed by V8 protease treatment. 183 79

A small fraction (approximately 5%) of protein kinase C (PKC) in the adult rat brain synaptosomes is tightly associated with Triton X-100-insoluble components (most likely membrane-skeleton elements), and is solubilized only after denaturation with sodium dodecyl sulfate. The kinase domain of this PKC can be released as a soluble form after limited proteolysis with calpain, whereas the regulatory domain which binds phorbol ester remains insoluble. The PKC in this fraction was identified as the beta II-subspecies or its related molecule. Presumably, this enzyme subspecies is responsible for the phosphorylation of a major PKC substrate protein, growth-associated protein-43, which is located in nerve endings as well as in growth cones in association with the membrane-skeleton elements.
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PMID:Protein kinase C in rat brain synaptosomes. Beta II-subspecies as a major isoform associated with membrane-skeleton elements. 183 69

Recent evidence from our laboratory has demonstrated that NK/LAK cell activation of human lymphocytes is protein kinase C (PKC)-dependent. Here, we have investigated the translocation of PKC in human NK cells exposed to sensitive targets or to PMA, a phorbol ester. In NK cells exposed to K562 for 6 hr, we observed a weak translocation of PKC whereas in NK cells exposed to PMA more than 90% of cytosolic PKC was translocated to the membrane in less than 5 min. Stimulation of NK cells with an NK-resistant target, however, did not translocate PKC even after 6 hr. Translocation of PKC to the membrane was followed by the appearance of PKM, the cytosolic calcium/phospholipid (Ca2+/PL)-independent form of PKC. The conversion of PKC to PKM was mediated by calpain, an intracellular calcium-dependent thiol proteinase. When we used two inhibitors of calpain, calpain inhibitor I (CI-I) and calpain inhibitor II (CI-II), both caused a dose-related enhancement of NK-CMC when the inhibitors were present throughout the 3-hr chromium release assay. This enhancement could be circumvented by PMA or by the PKC inhibitor H-7. CI-I and CI-II added together caused a greater increase in NK-CMC than when each was added alone. CI-I and CI-II also enhanced antibody-dependent cell-mediated cytotoxicity (ADCC), substantiating further our previous contention that the activation of both NK-CMC and ADCC may involve a common lytic pathway. Activation of NK cells with IL-2 for 18 hr at 37 degrees C was inhibited in the presence of CI-I. To investigate a possible feedback inhibition mechanism due to the buildup of PKC, we examined phosphatidylinositol (PI) metabolism in NK cells activated by IL-2 in either the presence or the absence of CI-I. We observed a significant decrease in PI turnover when NK cells, activated in the presence of IL-2 and CI-I, were stimulated with K562 as compared to NK cells activated by IL-2 alone, then stimulated with K562.
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PMID:Inhibition of the calpain-mediated proteolysis of protein kinase C enhances lytic activity in human NK cells. 191 39

Enteropathogenic Escherichia coli (EPEC) are a class of diarrheagenic organisms that induce a characteristic attaching and effacing lesion in enterocytes and various cultured cell lines. Infection of cultured HEp-2 cells by EPEC isolates 2036-80 (serotype O119) and E2348-69 (serotype O127) resulted in significant elevation of intracellular free calcium levels, determined quantitatively with the fluorescent calcium indicator dye 2-([2-bis(carboxymethyl)amino-5-methylphenoxy]methyl)-6-methoxy-8- bis(carboxymethyl)aminoquinoline. This effect, which was not observed on infection with non-lesion-forming E. coli strains, was inhibited by dantrolene, a drug that prevents calcium mobilization from intracellular stores. Moreover, activated protein kinase C in infected cells was dissociated from cell membranes by a process that was inhibited by cyclosporin A, suggesting involvement of the calcium-dependent protease calpain. A qualitative method for observing intracellular calcium fluxes by fluorescence microscopy with the recently described fluorescein-based indicator fluo-3 was used to screen a collection of well-characterized E. coli isolates from patients with infantile enteritis. Increased localized calcium-dependent fluo-3 fluorescence was observed only in HEp-2 cells infected with known lesion-forming EPEC strains. We propose that enhancement of intracellular free calcium levels in enterocytes infected with EPEC would result in formation of the characteristic lesion by calcium-dependent activation of actin-depolymerizing proteins, with eventual loss of absorptive capacity.
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PMID:Elevation of intracellular free calcium levels in HEp-2 cells infected with enteropathogenic Escherichia coli. 201 31

Excessive Ca2+ influx through NMDA receptor-coupled channels has been linked to neuronal cell death. Using an in vitro model of transient brain ischemia, we investigated possible protective effects of NMDA receptor antagonists ketamine or MK-801 and of calmidazolium, an inhibitor of intracellular Ca2(+)-activated proteins. Brain ischemia/recovery was simulated in isolated hippocampal slices and injury monitored by measurement of ATP levels. Omission of both glucose and oxygen (but not oxygen alone) for 20 min led to persistent ATP deficits after 4 h recovery. Addition of ketamine or MK-801 at 1 microM permitted ATP to recover within 1 h, as did addition of calmidazolium at 10 microM. Our findings are consistent with other reports that NMDA receptor antagonists can protect neuronal tissue from ischemic damage. The role of inappropriately activated Ca2(+)-mediated signaling processes in the mechanism(s) of such injury is suggested by the protection also seen with calmidazolium, an inhibitor of calmodulin and other structurally related proteins such as calpain(s) and protein kinase C. The inhibition of intracellular Ca2+ target proteins may be an alternative for protection of the brain against injury due to insults that activate NMDA receptors.
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PMID:Ischemic brain injury in vitro: protective effects of NMDA receptor antagonists and calmidazolium. 214 19

Calpain is a Ca2(+)-dependent cysteine endopeptidase and calpastatin is a calpain-specific endogenous inhibitor protein. Both calpain and calpastatin are very widely distributed in various animal tissues and cells. Low (microM) Ca2(+)-requiring calpain I and high (mM) Ca2(+)-requiring calpain II are known to exist. Calpain consists of one heavy (80 kDa) and one light (30 kDa) subunit. The heavy subunits of calpains I and II are different genetic products, whereas the light subunits are the same for both calpains I and II. Molecular cloning as well as protein sequencing revealed that the heavy subunit has four domains, while the light subunit has two domains. The carboxyl terminal domain of each subunit is a calmodulin-like domain, whereas the catalytic site is located in domain 2 of the heavy subunit. Calpastatin has four internally repetitive inhibitory domains. A single domain, or even a truncated 27-mer fragment thereof, possesses inhibitory activity against calpains. Calpain shows a rather broad substrate specificity. It can cleave various enzymes, and cytoskeletal, membrane and receptor proteins. Calpain-catalyzed activation of protein kinase C and transglutaminase may represent a few of the physiological functions of calpain, but a great many other functions can be assigned as well to calpain. Immunohistochemical studies revealed very wide but quite diverse distribution of calpains I and II and calpastatin among various tissues and cells. The expression of the genes for calpain and calpastatin is found to be modulated by retrovirus (HTLV-I) infection to T-lymphocytes. The physiological significance of the calpain and calpastatin system is yet to be elucidated, and accumulating information definitely suggested the role of calpain/calpastatin in health and disease.
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PMID:[Calpain and calpastatin]. 219 87


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