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)

Brief exposure to 12-O-tetradecanoylphorbol 13-acetate (TPA) caused a uniformly flattened population of mouse lung epithelial cells to become more heterogeneous; some cells rounded up, and others detached to overlap with flatter cells. Actin stress fiber organization was disrupted, and F-actin accumulated in lemellipodia. Vinculin dissociated from the focal adhesion plaques to diffuse throughout the cytoplasm. Inhibition of protein kinase C (PKC) activity blocked these effects of TPA. After 8 h of TPA exposure, actin filaments reassembled and vinculin again localized to the cell periphery. Calpain inhibition attenuated the decrease of PKC-alpha protein and PKC activity from the membrane fraction, and prevented the redistribution of cytoskeletal elements. Talin immunostaining was widespread throughout control cells but was localized to the periphery 8 h after treatment with TPA or with inhibitors of PKC and calpain. Both vinculin and talin concentrations increased with prolonged TPA treatment. PKC-zeta and calpain II were not appreciably affected by TPA exposure. Translocation of PKC-alpha to the membrane, followed by its calpain-induced downmodulation, is apparently required for the reversible pattern of cytoskeletal changes caused by TPA.
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PMID:Cytoskeletal architecture in mouse lung epithelial cells is regulated by protein-kinase C-alpha and calpain II. 892 11

Intracellular proteolysis by the calpains, a family of Ca2+ activated cysteine proteases, is a ubiquitous yet poorly understood process. Their action is implicated in an array of cellular and pathologic processes, including long-term potentiation, synaptic remodeling, protein kinase C and steroid receptor activation, ischemic cellular injury, and apoptosis. Unlike most proteases, the calpains display unusually strict substrate specificity, often cleaving only one or two bonds in proteins with hundreds of potential sites. Studies of synthetic peptides have defined sequences that modulate their specificity, but little data exist in the context of a bona fide protein. A prominent substrate for mu-calpain is alpha II spectrin (fodrin, brain spectrin), which is cleaved between Tyr1176 and Gly1177 within spectrin's 11th structural repeat unit. We have cloned and characterized human fetal brain alpha II spectrin (GenBank no. U26396) and identified a new Thr1300 to Ile polymorphism. From this clone, recombinant GST-fusion proteins representing repeat units 8-14 have been prepared and used to systematically explore the in vitro determinants of mu-calpain sensitivity. Twenty different amino acids were substituted by site-directed mutagenesis for wild-type Val1175, the penultimate (P2) residue flanking the major calpain cleavage site in alpha II spectrin. Gly, Pro, and Asp, and to a lesser extent Phe and Glu, substantively inhibited the susceptibility of this site to mu-calpain; other substitutions yielded lesser effects. Dynamic molecular modeling of the 11th structural repeat of human alpha II spectrin incorporating the various mutations suggests that the calpain cleavage site with its flanking calmodulin binding domain interrupts helix C of alpha II spectrin's 11th repetitive unit without significantly disrupting the repeat's triple-helical motif. This model predicts that the critical Tyr1176-Gly1177 bond occurs in a highly exposed loop juxtaposed between helix C and the calmodulin binding domain and that mutations at the P2 position subtly alter the conformation about this site. We conclude that secondary and tertiary conformational features surrounding the cleavage site, rather than the linear sequence itself, dominate the determinants that define alpha II spectrin's mu-calpain susceptibility.
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PMID:Site-directed mutagenesis of alpha II spectrin at codon 1175 modulates its mu-calpain susceptibility. 899 18

A key regulatory event controlling platelet activation is mediated through the phosphorylation of several cellular proteins by protein-tyrosine kinases. The related adhesion focal tyrosine kinase (RAFTK) is a novel cytoplasmic tyrosine kinase and a member of the focal adhesion kinase (FAK) gene family. FAK phosphorylation in platelets is integrin-dependent, occurs in a late stage of platelet activation, and is dependent on platelet aggregation. In this study, we have investigated the involvement of RAFTK phosphorylation during different stages of platelet activation. Treatment of platelets with thrombin induced, in as early as 10 s, a rapid tyrosine phosphorylation of RAFTK in a time- and concentration-dependent manner. Treatment of platelets with thrombin in the absence of stirring or pretreatment of platelets with RGDS peptide prevented platelet aggregation, but not RAFTK phosphorylation. Furthermore, phosphorylation of RAFTK did not require integrin engagement since platelets treated with the 7E3 inhibitory antibodies that block fibrinogen binding to glycoprotein IIb-IIIa did not inhibit RAFTK phosphorylation. Similarly, platelets treated with LIBS6 antibodies, which specifically activate glycoprotein IIb-IIIa, did not induce RAFTK phosphorylation. Stimulation of platelets by several agonists such as collagen, ADP, epinephrine, and calcium ionophore A23187 induced RAFTK phosphorylation. Tyrosine phosphorylation of RAFTK in platelets is regulated by calcium and is mediated through the protein kinase C pathway. Phosphorylation of RAFTK is dependent upon the formation of actin cytoskeleton as disruption of actin polymerization by cytochalasin D significantly inhibited this phosphorylation. The RAFTK protein appears to be proteolytically cleaved by calpain in an aggregation dependent manner upon thrombin stimulation. These results demonstrate that RAFTK is tyrosine-phosphorylated during an early phase of platelet activation by an integrin- independent mechanism and is not dependent on platelet aggregation, suggesting different mechanisms of regulation for FAK and RAFTK phosphorylation during platelet activation.
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PMID:Tyrosine phosphorylation of the novel protein-tyrosine kinase RAFTK during an early phase of platelet activation by an integrin glycoprotein IIb-IIIa-independent mechanism. 909 53

Thrombopoietin has an essential role in megakaryopoiesis and thrombopoiesis. To investigate the signaling processes induced by thrombopoietin, we have employed human platelets and recently demonstrated that thrombopoietin induces rapid tyrosine phosphorylation of Jak-2, Tyk2, Shc, Stat3, Stat5, p120(c-cbl) and other proteins in human platelets. Because the apparent molecular weight of a major tyrosine phosphorylated protein in platelets stimulated by thrombopoietin is approximately 85 to 95 kD, we examined the possibility that this could be Vav, a 95-kD proto-oncogene product. Specific antisera against Vav recognized the same 95 kD protein in lysates of Jurkat cells, which are known to express Vav, and platelets, indicating that platelets have Vav. Thrombopoietin induced rapid tyrosine phosphorylation of Vav in platelets without an elevation in cytosolic free calcium concentration or activation of protein kinase C. Vav was also tyrosine phosphorylated upon treatment of platelets with thrombin, collagen, or U46619, which activate phospholipase C, leading to an increased ionized calcium concentration and activation of protein kinase C. Ionomycin or phorbol 12-myristate 13-acetate (PMA) also induces tyrosine phosphorylation of Vav, suggesting that an increase in ionized calcium concentration or activation of protein kinase C may lead to phosphorylation of Vav. Thrombopoietin also induced tyrosine phosphorylation of Vav in FDCP-2 cells, genetically engineered to express human c-Mpl (FDCP-hMpl5). However, neither ionomycin nor PMA induced an increase in tyrosine phosphorylation of Vav in FDCP-hMpl5 cells, suggesting that the calcium and protein kinase C pathways of Vav phosphorylation may be unique to platelets. Further, Vav became incorporated into the Triton X-100 insoluble 10,000 g sedimentable residue in an aggregation-dependent manner, suggesting that it may have a regulatory role in platelet cytoskeletal processes. Vav was constitutively associated with a 28-kD adapter protein, Grb2, which is also incorporated into the cytoskeleton in an aggregation-dependent fashion. Lastly, we found that Vav is cleaved when there is activation of calpain, a protease that may have a role in postaggregation signaling processes. Our data suggest that thrombopoietin and other agonists may induce tyrosine phosphorylation of Vav by different mechanisms and Vav may also be involved in signaling during platelet aggregation by its redistribution to the cytoskeleton.
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PMID:Thrombopoietin and thrombin induce tyrosine phosphorylation of Vav in human blood platelets. 910 97

In the red blood cell membrane, sodium-proton exchange (NHE-1) exchanges intracellular H(+), Li(+), and Na(+) with extracellular Na(+). In hypertensives (HT), the maximal velocity of translocation (V max)of Na(+)/H(+) and of Na(+)/Li(+) exchange modes are higher, while apparent affinity for external Na(+) of Na(+)/Li(+) exchange and Hill's coefficient for H(+) activation of Na(+)/H(+) exchange are lower than in normotensive subjects (NT). We have therefore examined the effects of protein kinase C (PKC) and insulin on red blood cell membrane phosphorylation and on the kinetic properties of cation heteroexchange. In red cell from NT, PMA-induced activation of PKC reduced K(m) for H(+) of NHE but it did not affect V(max) and K(m) for Na(+). In red cell from HT, PMA-induced a greater PKC stimulation and membrane phosphorylation of band 3,4.1,4.9 than in NT and it did not significantly reduced K(m) for H(i). On the contrary, in HT PKC activation significantly increased Hill's coefficient of NHE. The larger activation of PKC in HT could be due to downregulation secondary to higher membrane calpain activity. Incubation of red cells with insulin decreases K(m) for external Na(+) and increases V(max) of Na(+)/Li(+) exchange. Therefore, we have examined the relationships between Na(+)-activation kinetics of Na(+)/Li(+) exchange and fasting insulin levels. Na(+)-stimulated Li(+) efflux was studied by raising Na(+)up to 300 mM isoosmotically to measure K(m) for Na(+) and V (max). Li(+) efflux saturated at 150 mM external Na(+)in NT but not in HT because in HT it exhibited a two fold higher Na(+) Km. V(max) was higher in HT than in NT. In hyperinsulinemic (fasting insulin > 10 mu U/ml) HT, V(max) and Na(+) Km were higher than in normoinsulinemic HT. In NT, hyperinsulinemia was not associated to abnormal kinetic properties of Na(+)/Li(+)exchange. Stepwise multiple regression analysis confirmed that the main determinants of a high Km were blood pressure and insulin. Our results show that posttranslational effects of PKC and insulin affect the kinetic properties of NHE-1 in red blood cells and suggest that the differences observed between hypertensives and normotensive subjects can be accounted for by PKC activation and insulin exposure.
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PMID:Posttranslational effects of protein kinase C and insulin on red cell membrane phosphorylation and cation heteroexchange in hypertension. 916 39

Calpains, the thiol proteinases of the calcium-dependent proteolytic system, are regulated by a natural inhibitor, calpastatin, which is present in brain tissue in two forms. Although both calpastatins are highly active on human erythrocyte calpain, only one form shows a high inhibitory efficiency with both rat brain calpain isozymes. The second calpastatin form is almost completely inactive against homologous proteinases and can be converted into an active one by exposure to a phosphoprotein phosphatase, also isolated from rat brain. Phosphorylation of the active calpastatin by protein kinase C and protein kinase A promotes a decrease in its inhibitory efficiency. The interconversion between the two inhibitor forms seems involved in the adjustment of the level of intracellular calpastatin activity on specific cell requirements.
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PMID:Modulation of rat brain calpastatin efficiency by post-translational modifications. 927 42

Calpain (calcium-activated neutral protease) has been implicated as playing a role of neuronal injury in cerebral ischemia and excitotoxicity. Here we report that, in addition to extreme excitotoxic conditions [N-methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and kainate challenges], other neurotoxins such as maitotoxin, A23187, and okadaic acid also induce calpain activation, as detected by m-calpain autolytic fragmentation and nonerythroid alpha-spectrin breakdown. Under the same conditions, calmodulin-dependent protein kinase II-alpha (CaMPK-IIalpha) and neuronal nitric oxide synthase (nNOS) are both proteolytically cleaved by calpain. Such fragmentation can be reduced by calpain inhibitors (acetyl-Leu-Leu-Nle-CHO and PD151746). In vitro digestion of protein extract from cortical cultures with purified mu- and m-calpain produced fragmentation patterns for CaMPK-IIalpha and nNOS similar to those produced in situ. Also, several other calpain-sensitive calmodulin-binding proteins (plasma membrane calcium pump, microtubule-associated protein 2, and calcineurin A) and protein kinase C-alpha are also degraded in neurotoxin-treated cultures. Lastly, in a rat pup model of acute excitotoxicity, intrastriatal injection of NMDA resulted in breakdown of CaMPK-IIalpha and nNOS. The degradation of CaMPK-IIalpha, nNOS, and other endogenous calpain substrates may contribute to the neuronal injury associated with various neurotoxins.
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PMID:Neuronal nitric oxide synthase and calmodulin-dependent protein kinase IIalpha undergo neurotoxin-induced proteolysis. 928 22

SH-SY-5Y human neuroblastoma cells were treated with 22 microM of a synthetic peptide corresponding to amino acid residues 25-35 of beta-amyloid (betaA) or 3 microM calcium ionophore A23187 in culture medium containing 1.8 mM extracellular calcium. Both agents increased tau immunoreactivity towards antibodies (PHF-1, ALZ-50) that recognize epitopes common with paired helical filaments (PHFs) and towards an antibody (5E2) that recognized a phosphate-independent tau epitope. However, only ionophore increased immunoreactivity with an additional phosphate-dependent antibody (AT-8) that recognized an epitope of tau when phosphorylated, and induced a corresponding decrease in immunoreactivity towards an additional antibody (Tau-1) that recognizes the same site when that site is not phosphorylated. Moreover, the ionophore-mediated increase in PHF-1 was blocked by EGTA, by the calpain inhibitor calpeptin and by the PKC inhibitor H7, while that evoked by betaA treatment was not inhibited by any of these treatments. Since ionophore-mediated calpain activation induces proteolytic PKC activation, we further examined the influence of PKC inhibition on betaA and ionophore-mediated PHF-1 induction. Antisense oligonucleotide-mediated downregulation of PKCepsilon in a stable transfectant SH-SY-5Y subclone diminished the ionophore-mediated, but not the betaA-mediated, increase in PHF-1 immunoreactivity. These data indicate specific differences in the intracellular cascade of events invoked by betaA and ionophore A23187. Moreover, although betaA invoked calcium influx in these cells, our findings further suggest that the induction of tau hyperphosphorylation by betaA may not be due to calcium influx.
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PMID:Beta-amyloid and ionophore A23187 evoke tau hyperphosphorylation by distinct intracellular pathways: differential involvement of the calpain/protein kinase C system. 933 63

In previous studies of topical application of calphostin C, a specific inhibitor of the regulatory domain of protein kinase C (PKC), and calpeptin, a selective inhibitor of calpain, to spastic canine basilar artery (BA) researchers have suggested that the catalytic fragment of PKC (known as PKM) is probably formed by a limited proteolysis of continuously activated mu-calpain, but there has been no direct evidence for PKM formation in vasospasm. The present immunoblot study with anti-PKCalpha antibody shows a significant decrease in cytosolic 80-kD PKCalpha and a concomitantly significant increase in membrane PKCalpha in the spastic canine BA. In addition, an immunoblot study in which cleavage site-directed antibodies were used demonstrated a significant increase in immunoreactive 45-kD PKM. The changes in membrane PKCalpha and PKM were enhanced with the lapse of time after subarachnoid hemorrhage. The cleavage site-directed antibodies distinguish the proteolyzed from the unproteolyzed forms of PKC for in situ analyses of enzyme regulation mediated by proteolysis. The data indicate that PKCalpha in spastic canine BA is translocated to the cell membrane, where PKCalpha is rapidly cleaved into PKM as a result of proteolysis of the isozyme by mu-calpain but not by m-calpain. The authors hypothesize that mu-calpain is continuously activated in spastic canine BA and produces PKM by limited proteolysis of PKCalpha.
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PMID:Generation of the catalytic fragment of protein kinase C alpha in spastic canine basilar artery. 934 85

One of the responses of activated platelets to certain stimuli is the shedding of microparticles. Many studies have attempted to characterize the role of microparticles under various clinical situations or experimental conditions. Pathological levels of fluid shear stress may occur in diseased small arteries and arterioles partially obstructed by atherosclerosis or vasospasm and such shear stress may induce the activation and aggregation of circulating platelets. We investigated whether high shear stress could cause both platelet aggregation and shedding of microparticles from the platelet plasma membrane. A cone-plate 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 remnant platelets showed procoagulant activity on their surfaces. Investigation of the mechanisms involved in shear-dependent microparticle generation showed that binding of von Willebrand factor to platelet glycoprotein Ib, influx of extracellular calcium, and activation of platelet calpain were required to generate microparticles under high shear stress conditions. Activation of protein kinase C promoted shear-dependent microparticle formation. These findings suggest that local generation of microparticles in atherosclerotic arteries, the site at which pathological levels of shear stress could occur, contributes to arterial thrombosis by providing and expanding a catalytic surface for the coagulation cascade.
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PMID:[Shear stress and platelet-derived microparticles]. 936 69


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