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)

Our studies suggest that protein kinase C is involved in low calcium (Ca2+)-stimulated secretion of parathyroid hormone (PTH) but not directly in high Ca(2+)-stimulated intracellular degradation of PTH to secreted carboxyl-terminal fragments (C-PTH), an important component of Ca(2+)-regulated PTH secretion. The present study was undertaken to determine the presence of calcium-activated proteases, 84 kDa (micro)-calpain and 80 kDa (milli)-calpain, in the bovine parathyroid, and whether they could degrade PTH to C-terminal fragments. Immunocytochemistry of bovine parathyroid tissue using antibodies raised against bovine heart micro- and milli-calpain detected both isoforms of calpain. Western blotting of total bovine parathyroid cell protein prepared from primary cell cultures confirmed the presence of both isoforms of calpain, demonstrated by specific milli- and micro-calpain bands. Purified bovine PTH (bPTH) was incubated in vitro with human erythrocyte micro-calpain and the cleavage products were separated by reverse-phase HPLC. Eluant fractions were assayed with an RIA with equimolar sensitivity to C-PTH and bPTH, and peak areas integrated. Micro-calpain produced a C-PTH peak from bPTH which co-eluted with the major C-PTH secreted by parathyroid cells in culture. C-PTH production by micro-calpain, expressed as per cent area under the curve, increased from 0% in the absence of either micro-calpain or Ca2+, to 71.5% when a 5:1 molar ratio of bPTH to calpain was used. Amino acid sequencing and analysis of the immunoreactive PTH cleavage products indicated the presence of two fragments of bPTH in the C-PTH peak, bPTH47-48 and bPTH69-84. In summary, both isoforms of calpain are present in the bovine parathyroid and calpains may play a role in the Ca(2+)-dependent degradation of PTH to secreted C-terminal fragments.
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PMID:Calcium-activated proteases in the bovine parathyroid gland: potential role in degradation of parathyroid hormone to peptide fragments. 854 14

The protein kinase C inhibitor bisindolylmaleimide GF109203X has a dual effect on the behavior of the neuroblastoma cell line Neuro-2A; when the inhibitor is added in conditions that induce differentiation (absence of serum), neurite outgrowth is potentiated in a dose-dependent manner. However, if the inhibitor is added in growth-promoting conditions (presence of serum), programmed cell death (apoptosis) is induced, as assessed by internucleosomal DNA cleavage and specific immunoassays. This effect is also seen with other specific protein kinase C inhibitors. Bcl2 gene overexpression protects Neuro-2A cells from apoptosis, as has been found in other systems. We also show that calpain I, a neutral Ca(2+)-activated proteinase, participates in this apoptotic pathway. Our results point to a key role of protein kinase C in the regulation of growth and differentiation in Neuro-2A cells.
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PMID:Apoptosis induced by protein kinase C inhibition in a neuroblastoma cell line. 856 75

Calcium influx into SH-SY5Y human neuroblastoma cells after ionophore treatment or transient permeabilization in calcium-containing medium increased ALZ-50 immunoreactivity markedly. This increase was prevented by inhibitors active against calpain or against protein kinase C (PKC), suggesting that both of these enzymes were required to mediate the effect of calcium influx on ALZ-50 immunoreactivity. Treatment with PKC activator TPA increased ALZ-50 immunoreactivity in the absence of calcium influx or after intracellular delivery of the specific calpain inhibitor calpastatin, indicating that the influence of PKC was downstream from that of calpain. Calcium influx also resulted in mu-calpain autolysis (one index of calpain activation) and the transient appearance of PKM (i.e., free PKC catalytic subunits, generated by calpain-mediated cleavage of the regulatory and catalytic PKC domains). Inhibition of calpain within intact cells resulted in a dramatic increase in steady-state levels of total tau (migrating at 46-52 kDa) but resulted in a relatively minor increase in 68-kDa ALZ-50-immunoreactive tau isoforms. Although calcium influx into intact cells resulted in accumulation of ALZ-50 immunoreactivity, total tau levels were, by contrast, rapidly depleted. Incubation of isolated fractions with calpain in the presence of calcium indicated that ALZ-50-immunoreactive tau isoforms were more resistant to calpain-mediated proteolysis than were non-ALZ-50 reactive tau isoforms. These data therefore indicate that calpain may regulate tau levels directly via proteolysis and indirectly through PKC activation. A consequence of the latter action is altered tau phosphorylation, perhaps involving one or more kinase cascades, and the preferential accumulation of ALZ-50-immunoreactive tau isoforms due to their relative resistance to degradation. These findings provide a basis for the possibility that disregulation of calcium homeostasis may contribute to the pathological levels of conversion of tau to A68 by hyperactivation of the calpain/PKC system.
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PMID:Calcium influx into human neuroblastoma cells induces ALZ-50 immunoreactivity: involvement of calpain-mediated hydrolysis of protein kinase C. 862 10

To investigate the intracellular pathways leading to ETOH-induced apoptosis, thymocytes and splenic T and B cells were cultured 16 h with or without ETOH and different stimuli, and apoptotic cell death was determined. At concentrations of 0.4%-2% in culture, ETOH induced apoptosis in all three types of cells, but it had a more profound effect on thymocytes and B cells as compared with its effect on T cells. In thymocytes, ETOH-induced apoptosis was abrogated by chelation of extracellular calcium with EGTA, and inhibition of protein synthesis with CHX, or of PKC with H7 but not of PKA with HA 1004. ETOH potentiated the apoptosis of thymocytes induced with the calcium ionophore A23187 and suboptimal doses of PMA, but it had negligible effect on dAMP- and PGE2-induced apoptosis of thymocytes. In contrast to findings in thymocytes, the ETOH-induced apoptosis of T and B cells was almost completely abrogated by PMA, but not by H7 or CHX. In spleen cells, calcium chelation with EGTA triggered apoptosis. ETOH significantly inhibited EGTA-induced apoptosis of B cells but had little effect on EGTA-induced apoptosis of T cells. IL-4 reduced the ETOH-induced apoptosis of B and T cells, but it was not effective in the prevention of apoptosis of thymocytes. Inhibition of the calcium-dependent neutral protease calpain I did not rescue cells from apoptosis. Moreover, treatment with CI-I potentiated ETOH-induced apoptosis in T cells. These results suggest that both thymocytes and splenic T and B cells have relevant apoptotic pathways that can be induced by ETOH, but the mechanisms of ETOH-induced apoptosis differ in these cells.
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PMID:Different pathways of in vitro ethanol-induced apoptosis in thymocytes and splenic T and B lymphocytes. 865 90

Activated calpain I immunoreactivity (76 kDa band) was detected in membranes prepared from rat brain hippocampal slices using a polyclonal antiserum raised against an N-terminus peptide of the cleaved subunit of calpain I. While basal levels of activated calpain I were stable over incubation times, 1 nM dopamine (DA) produced an initial 32% increase (5 min) in the 76 kDa protein followed by a 53% decrease in this band at 20 min of incubation. The DA-induced changes in activated calpain I immunoreactivity were blocked by the calpain inhibitor peptide, N-acetyl-Leu-Leu-norleucinal(100 microM) or by EGTA. Basal levels of the 76 kDa band were not affected by the calpain inhibitor. These changes in activated calpain I, elicited by DA, are in accord with the DA-induced decreases in the levels of the calpain substrate, gamma PKC (Yurko-Mauro and Friedman; J Cell Biochem [Abstr] 180:80, 1994; J Neurochem 65: 1622-1630, 1995) and suggest that DA activates this Ca(++)-dependent protease in its regulation of neuronal signal transduction.
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PMID:Dopamine-stimulated changes in activated calpain I in rat hippocampal slices. 869 33

Calpains are Ca-activated neutral proteases present in all cells together with an endogenous inhibitor, calpastatin. Proposed substrates are; cytoskeletal proteins like microtubules and actin, protein kinases such as PKC and membrane-bound enzymes like Ca-ATPase and the Ca-channel. In lenses from different species calpains have been detected in decreasing amounts from the epithelium to the cortex to the nucleus. Several substrates for calpain in the lens have been demonstrated: crystallins, vimentin, actin, beaded filaments and MP26 among others. Both studies on animal models and capsulorhexis indicate that calpains are mainly involved in cortical cataract.
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PMID:Calpains in the human lens: relations to membranes and possible role in cataract formation. 872 65

Calponin, a basic smooth-muscle protein capable of binding to F-actin, tropomyosin and calmodulin in vitro, was tested for its expression and subcellular localization in resting and stimulated human platelets. Using immunoblotting techniques calponin was revealed as a single protein band with a molecular weight of 34 kDa. Although calponin has been shown to be proteolytically degraded by calpain, in the presence of the calpain inhibitor E-64 and EGTA a significant hydrolysis of calponin could not be detected. Upon stimulation with 10 microM arachidonic acid calponin became increasingly incorporated into Triton X-100 insoluble cytoskeletal fractions reaching a plateau after 15 s. The accumulation of calponin in the cytoskeletons of stimulated platelets paralleled the polymerization of actin into newly formed microfilaments. Immunofluorescence microscopy revealed a submembranous co-localization of calponin and actin in aggregated platelets. Since isolated calponin is phosphorylated by protein kinase C and Ca2+/calmodulin-dependent protein kinase II thereby losing its inhibitory effect on the actomyosin MgATPase activity, we examined whether changes in cell shape due to platelet stimulation are accompanied by a phosphorylation of calponin. By performing immunoblotting analysis on either resting or stimulated platelets phosphorylation of calponin on tyrosine, serine or threonine residues could not be demonstrated. In line, [32P]radiolabeling experiments were unable to detect phosphate incorporation into calponin. These observations support the hypothesis that calponin plays a physiological role in regulating contraction and secretion of human platelets even in the absence of its phosphorylation.
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PMID:Accumulation of unphosphorylated calponin in the submembranous cytoskeletons of arachidonic acid-stimulated human platelets. 874 89

The alpha isoform of protein kinase C (PKC alpha) is rapidly hydrolyzed by mM Ca(2+)-requiring calpain (calcium-activated neutral proteinase) under cell-free conditions (Shea et al, 1994, FEBS Lett. 350:223). In the present study, we demonstrate that this hydrolysis is inhibited by phosphatidyl serine, diacylglycerol, phosphatidyl choline, phosphatidyl inositol, and phosphatidic acid. With the exception of phosphatidic acid, these phospholipids did not directly inhibit calpain activity as evidenced by degradation of [14C]azocasein, suggesting that the nature of inhibition of calpain-mediated PKC alpha degradation is due to an effect of phospholipids on PKC alpha conformation. These findings suggest that m calpain-mediated PKC alpha hydrolysis may be specifically minimized at the plasma membrane, and leave open the possibility that such a mechanism exists in situ. In addition, the unique inhibition of calpain activity by phosphatidic acid suggests the existence of a specific mechanism by which this phospholipid regulates PKC alpha activity.
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PMID:Phospholipids inhibit proteolysis of protein kinase C alpha by mM calcium-requiring calpain. 878 23

To explore the spatial and temporal localization of PKC isoforms during ischemia, we quantified PKC isoforms in the subcellular fractions in perfused rat heart by immunoblotting using specific antibodies against PKC isoforms. PKCs-alpha and epsilon translocated from the 100000 x g supernatant (S, cytosolic) fraction to the 1000 x g pellet (PI, nucleus-myofibril) and the 1000-100000 x g pellet (P2, membrane) fractions during 5-40 min of ischemia. PKC-delta redistributed from the P2 to the S fraction. A 50-kDa fragment of PKC-alpha appeared during ischemia possibly through calpain action. Immunohistochemical observations showed the different localizations of PKC-alpha, delta, and epsilon in the myocytes. The PKC assay displayed high basal levels of Ca(2+)-independent PKC, the activation of Ca(2+)-dependent PKC in the P1 and P2 fractions, and the activation of Ca(2+)-independent PKC in the P1 fraction after 20 min of ischemia. These observations show that ischemia induces different patterns of translocation of the three PKC isoforms, suggesting differences in their roles.
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PMID:Translocation of protein kinase C-alpha, delta and epsilon isoforms in ischemic rat heart. 887 25

Previous studies have demonstrated that a high level of shear stress can produce platelet aggregation without the addition of any agonist. We investigated whether high shear stress could cause both platelet aggregation and shedding of microparticles from the platelet plasma membrane. A coneplate viscometer was used to apply shear stress and microparticle formation was measured by flow cytometry. It was found that microparticle formation increased as the duration of shear stress increased. Both microparticles and the remnant platelets showed the exposure of procoagulant activity on their surfaces. Investigation of the mechanisms involved in shear-dependent microparticle generation showed that binding of von Willebrand factor (vWF) to platelet glycoprotein lb, influx of extracellular calcium, and activation of platelet calpain were required to generate microparticles under high shear stress conditions. Activation of protein kinase C (PKC) promoted shear-dependent microparticle formation. Epinephrine did not influence microparticle formation, although it enhanced platelet aggregation by high shear stress. These findings suggest the possibility that local generation of microparticles in atherosclerotic arteries, the site that pathologically high shear stress could occur, may contribute to arterial thrombosis by providing and expanding a catalytic surface for the coagulation cascade.
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PMID:High shear stress can initiate both platelet aggregation and shedding of procoagulant containing microparticles. 889 11


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