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)

In the A20 cell line, we examined the mechanisms that modulate the Fas-mediated apoptotic pathway through the B cell receptor. As in other systems, Fas signaling activates cysteine proteases, leading to specific proteolysis of poly(ADP-ribose) polymerase (PARP) and protein kinase C (PKC) delta. We describe that PKC-epsilon and PKC-zeta proteins are two new IL-1 beta-converting enzyme (ICE) substrates; we found that ICE activation and its proteolytic effects are inhibited by surface IgG (sIgG) cross-linking. Apoptosis induced by Fas ligation is consequently abrogated after sIgG engagement, and sIgG signaling therefore interferes with the apoptotic signal upstream of ICE protease activation. Since the PKC inhibitor bisindolylmaleimide I completely abolishes the protective effect of the sIgG signal, a member of the PKC family is probably responsible for the prevention of ICE cascade activation. Direct activation of PKC by PMA partially mimics the protective effect of sIgG cross-linking against Fas-mediated death in A20 cells. Nevertheless, PMA inhibits neither ICE activation nor the subsequent proteolysis of ICE substrates, suggesting that the PKC responsible for ICE inactivation is a non-PMA-sensitive PKC. In this system, Fas ligation also triggers Bcl-2/Bcl-x down-regulation, an effect inhibited by sIgG cross-linking, the cysteine protease inhibitor acetyl-Tyr-Val-Ala-Asp-chloromethyl ketone, and PMA treatment. In A20 cells, Fas signaling may thus trigger both ICE activation and Bcl-x and Bcl-2 down-regulation. These results indicate that sIgG signaling gives rise to two pathways after PKC activation, one presumably promoted by non-PMA-sensitive PKC, which inactivates the ICE cascade, and another produced by PMA-sensitive PKC, which maintains normal Bcl-2/Bcl-x levels.
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PMID:B cell receptor cross-linking prevents Fas-induced cell death by inactivating the IL-1 beta-converting enzyme protease and regulating Bcl-2/Bcl-x expression. 931 14

Mechanical perturbation has been shown to modulate a wide variety of changes in second message signals and patterns of gene expression in osteoblasts. Embryonic chick osteoblasts were subjected to a dynamic spatially uniform biaxial strain (1.3% applied strain) at 0.25 Hz for a single 2-h period, and osteopontin (OPN), an Arg-Gly-Asp (RGD)-containing protein, was shown to be a mechanoresponsive gene. Expression of opn mRNA reached a maximal 4-fold increase 9 h after the end of the mechanical perturbation that was not inhibited by cycloheximide, thus demonstrating that mechanoinduction of opn expression is a primary response through the activation of pre-existing transcriptional factors. The signal transduction pathways, which mediated the increased expression of opn in response to mechanical stimuli, were shown to be dependent on the activation of a tyrosine kinase(s) and protein kinase A (PKA) or a PKA-like kinase. Selective inhibition of protein kinase C (PKC) had no effect on the mechanoinduction of osteopontin even though opn has been demonstrated to be an early response gene to phorbol 12-myristate 13-acetate (PMA) stimulation. Mechanotransduction was dependent on microfilament integrity since cytochalasin-D blocked the up-regulation of the opn expression; however, microfilament disruption had no effect on the PMA induction of the gene. The microtubule component of the cytoskeleton was not related to the mechanism of signal transduction involved in controlling opn expression in response to mechanical stimulation since colchicine did not block opn expression. Mechanical stimulus was shown to activate focal adhesion kinase (FAK), which specifically became associated with the cytoskeleton after mechanical perturbation, and its association with the cytoskeleton was dependent on tyrosine kinase activity. In conclusion, these results demonstrate that the signal transduction pathway for mechanical activation of opn is uniquely dependent on the structural integrity of the microfilament component of the cytoskeleton. In contrast, the PKC pathway, which also activates this gene in osteoblasts, acts independently of the cytoskeleton in the transduction of its activity.
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PMID:Signal transduction of mechanical stimuli is dependent on microfilament integrity: identification of osteopontin as a mechanically induced gene in osteoblasts. 933 23

The caspase family of proteases plays a critical role in the execution of apoptosis. However, efforts to decipher the molecular mechanisms by which caspases induce cell death have been greatly hindered by the lack of systematic and broadly applicable strategies to identify their substrates. Here we describe a novel expression cloning strategy to rapidly isolate cDNAs encoding caspase substrates that are cleaved during apoptosis. Small cDNA pools (approximately 100 clones each) are transcribed/translated in vitro in the presence of [35S]methionine; these labeled protein pools are then incubated with cytosolic extracts from control and apoptotic cells. cDNA pools encoding proteins that are specifically cleaved by the apoptotic extract and whose cleavage is prevented by the caspase inhibitor acetyl-Tyr-Val-Ala-Asp chloromethylketone are subdivided and retested until a single cDNA is isolated. Using this approach, we isolated a partial cDNA encoding protein kinase C-related kinase 2 (PRK2), a serine-threonine kinase, and demonstrate that full-length human PRK2 is proteolyzed by caspase-3 at Asp117 and Asp700 in vitro. In addition, PRK2 is cleaved rapidly during Fas- and staurosporine-induced apoptosis in vivo by caspase-3 or a closely related caspase. Both of the major apoptotic cleavage sites of PRK2 in vivo lie within its regulatory domain, suggesting that its activity may be deregulated by proteolysis.
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PMID:Specific proteolysis of the kinase protein kinase C-related kinase 2 by caspase-3 during apoptosis. Identification by a novel, small pool expression cloning strategy. 936 3

We previously demonstrated that the anticancer agent and protein kinase C (PKC) inhibitor 7-hydroxystaurosporine (UCN-01) induces apoptosis independently of p53 and protein synthesis in HL60 cells. We now report the associated changes of PKC isoforms. PKCalpha, betaI, betaII, delta, and zeta activities were measured after immunoprecipitation of cytosols from UCN-01-treated HL60 cells. UCN-01 had no effect on PKCzeta and inhibited kinase activity of PKCbetaI, betaII, and delta. PKCalpha activity was initially inhibited at 1 h, and subsequently increased as cells underwent apoptosis 3 h after the beginning of UCN-01 treatment. Camptothecin (CPT) and etoposide (VP-16) also markedly enhanced PKCalpha activity during apoptosis in HL60 cells. However, CPT did not affect PKCbetaI, betaII and zeta, and activated PKCdelta. PKCalpha activation was not due to increased protein levels or proteolytic cleavage but was associated with PKCalpha autophosphorylation in vitro and increased phosphorylation in vivo. We also found that not only PKC delta but also PKC betaI was proteolytically activated in HL60 cells during apoptosis. The PKCalpha activation and hyperphosphorylation were abrogated by N-benzyloxycarbonyl-Val-Ala-Asp(O-methyl)-fluoromethylketone (z-VAD-fmk) under conditions that abrogated apoptosis. z-VAD-fmk also prevented PKCdelta and betaI proteolytic activation. Together these findings suggest that caspases regulate PKC activity during apoptosis in HL60 cells. At least two modes of activation were observed: hyperphosphorylation for PKCalpha and proteolytic activation for PKC delta and betaI.
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PMID:Activation of PKCalpha downstream from caspases during apoptosis induced by 7-hydroxystaurosporine or the topoisomerase inhibitors, camptothecin and etoposide, in human myeloid leukemia HL60 cells. 939 60

We sought to determine the importance of integrins for recovery after acute tubular injury and to investigate the signal transduction pathways for integrin effects on cell cycle regulation involving proliferation and apoptosis. Primary cultures of rat renal proximal tubule epithelial cells were exposed to a superoxide-generating system to induce injury in the absence of overt necrosis. Integrin function was antagonized by the integrin recognition sequence tetrapeptide Gly-Arg-Gly-Asp (GRGD) or monoclonal antibody to beta 1-integrin. Injured cells had reduced thymidine uptake compared with normal cells. The presence of GRGD during recovery from injury caused a further 44% reduction in DNA synthesis but did not affect DNA synthesis in normal cells. Injured cells had an increased proportion of apoptosis that was further accentuated by exposed to GRGD during recovery. Integrin antagonism also stimulated apoptosis in uninjured cells. To investigate signal transduction mechanisms for this effect of integrins, inhibitors and activators of protein tyrosine kinase (PTK) and protein kinase C (PKC) were evaluated. Activation of PKC stimulated cellular proliferation, whereas inhibitors of PKC and PTK had no significant effect. Genistein, a PTK inhibitor, induced apoptosis in normal cells, mimicking the effect of integrin inhibition. On the other hand, PMA, an activator of PKC, prevented cells from becoming apoptotic when exposed to injury plus GRGD. The phosphorylation status of intracellular proteins was evaluated by immunoblotting with antiphosphotyrosine antibody. A similar pattern of decreased phosphorylation was observed after either integrin inhibition, injury, both, or PTK inhibition. These findings suggest that kinase cascades are involved in the effects of integrins on renal epithelial cell proliferation and apoptosis. After injury, an interaction between cells and the extracellular matrix is required for cells to proliferate and contribute to repair rather than to enter an apoptotic pathway.
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PMID:Effects of integrins on proliferation and apoptosis of renal epithelial cells after acute injury. 940 96

We have previously shown that the rat Na(+)-K(+)-ATPase alpha 1-isoform is phosphorylated at Ser-943 by protein kinase A (PKA) and at Ser-23 by protein kinase C (PKC), which in both cases results in inhibition of enzyme activity. We now present evidence that suggests that the phosphorylation of Ser-943 by PKA modulates the response of Na(+)-K(+)-ATPase to PKC. Rat Na(+)-K(+)-ATPase alpha 1 or a mutant in which Ser-943 was changed to Ala-943 was stably expressed in COS cells. The inhibition of enzyme activity measured in response to treatment with the phorbol ester, phorbol 12,13-dibutyrate (PDBu; 10(-6) M), was significantly reduced in the cells expressing the Ala-943 mutant compared with that observed in cells expressing wild-type enzyme. In contrast, for cells expressing Na(+)-K(+)-ATPase alpha 1 in which Ser-943 was mutated to Asp-943, the effect of PDBu was slightly enhanced. The PDBu-induced inhibition was not mediated by activation of the adenosine 3',5'-cyclic monophosphate/PKA system and was not achieved via direct phosphorylation of Ser-943. Sp-5,6-DCI-cBIMPS, a specific PKA activator, increased the phosphorylation of Ser-943, and this was associated with an enhanced response to PDBu. Thus the effect of PKC on rat Na(+)-K(+)-ATPase alpha 1 is determined not only by the activity of PKC but also by the state of phosphorylation of Ser-943.
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PMID:Regulation of rat Na(+)-K(+)-ATPase activity by PKC is modulated by state of phosphorylation of Ser-943 by PKA. 943 4

The integrin beta subunit cytoplasmic domains are important for activation-dependent cell adhesion and adhesion-dependent signaling events. We report an interaction between integrin beta subunit cytoplasmic domain and Rack1, a Trp-Asp (WD) repeat protein that has been shown to bind activated protein kinase C. The Rack1-binding site on integrin beta 2 subunit resides within a conserved, membrane-proximal region. In the yeast two-hybrid assay, WD repeats five to seven of Rack1 (Rack1-WD5/7) interact with integrin beta 1, beta 2, and beta 5 cytoplasmic domain. In eukaryotic cells, Rack1 co-immunoprecipitates with at least two different beta integrins, beta 1 integrins in 293T cells and beta 2 integrins in JY lymphoblastoid cells. Whereas Rack1-WD5/7 binds integrins constitutively, the association of full-length Rack1 to integrins in vivo requires a treatment with phorbol esters, which promotes cell spreading and adhesion. These findings suggest that Rack1 may link protein kinase C directly to integrins and participate in the regulation of integrin functions.
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PMID:Rack1, a receptor for activated protein kinase C, interacts with integrin beta subunit. 944 85

The purpose of this study was to examine which intracellular signalling systems influence the attachment of normal uveal melanocytes and uveal melanoma cells to extracellular matrix (ECM) proteins in vitro. Uveal melanocytes were found to attach strongly to fibronectin in preference to plastic, collagens type I, III or IV, or laminin. In contrast, uveal melanoma cells attached equally well to fibronectin and collagens I, III and IV in preference to plastic or laminin. Manipulation of intracellular cyclic AMP or protein kinase C had little, if any, effect on the attachment of either cell to fibronectin. In contrast, inhibition of calmodulin significantly inhibited the attachment of both normal and transformed cells, as did manipulating intracellular free calcium. We noted that the intracellular free calcium in melanoma cells was less than half that seen in melanocytes. Fibronectin, laminin and Arg-Gly-Asp (RGD) were all capable of acutely increasing the intracellular free calcium in both cells. ECM-induced increases in calcium were more apparent in low density than high density cells and appeared more sustained in melanocytes than in melanoma cells. We conclude that both normal and neoplastic uveal melanocytes require an intracellular signal or signals which involves calcium and calmodulin in the few minutes following cell binding to ECM proteins in order for successful cell attachment to occur. While the transformed cell does not differ significantly from the normal cell in this respect, this dependency on calcium and calmodulin may nevertheless offer an approach for pharmacological intervention in the prevention or arrest of metastatic spread and merits further investigation.
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PMID:Attachment of human uveal melanocytes and melanoma cells to extracellular matrix proteins involves intracellular calcium and calmodulin. 946 15

The extracellular matrix influences the cellular spreading of vascular smooth muscle cells (VSMCs) via integrin receptors. However, the intracellular signaling mechanisms are still incompletely understood. We investigated the hypothesis that VSMCs binding to fibronectin activates the protein kinase C (PKC) pathway, causes differential intracellular PKC isoform translocation, and mediates cell spreading. VSMCs binding to poly-L-lysine or preincubated with Arg-Gly-Asp (RGD) peptides were used as controls. Diacylglycerol (DAG) and phospholipase D (PLD) activity were measured by thin-layer chromatography. Intracellular distribution of PKC isoforms was assessed by confocal microscopy. VSMCs binding to fibronectin induced focal adhesions and cell spreading within 30 minutes. Fibronectin induced a rapid increase in DAG content, peaking at 10 minutes with a sustained response for <1 hour. In contrast, PLD activity was not influenced by specific binding to fibronectin. PKC isoforms alpha, delta, epsilon, and zeta were assessed by confocal microscopy. Fibronectin induced a PKC isoform translocation to the cell nucleus and to focal adhesions within minutes. The nuclear PKCalpha immunoreactivity was transiently increased. PKC isoforms a and epsilon were both translocated to focal adhesions. The intracellular distributions of other PKC isoforms were not influenced by fibronectin. The effects of fibronectin on DAG generation, the translocation of PKCalpha and PKCepsilon, and cell spreading were all abolished by the incubation with RGD peptides. Downregulation of PKC isoforms alpha and epsilon with specific antisense oligodinucleotides resulted in a significant inhibition of cell spreading. Our results show that integrins induce intracellular signaling in VSMCs via DAG and PKC. PKC isoform a is translocated to the nucleus, whereas PKC isoforms alpha and epsilon are translocated to focal adhesions. Both isoforms seem to play a role in inside-out integrin signaling and cell spreading.
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PMID:Integrin-induced protein kinase Calpha and Cepsilon translocation to focal adhesions mediates vascular smooth muscle cell spreading. 946 86

The neuronal protein GAP-43 is concentrated at the growth cone membrane, where it is thought to amplify the signal transduction process. As a model for its neuronal effects, GAP-43 protein injection into Xenopus laevis oocytes strongly augments the calcium-sensitive chloride current evoked by the G protein-coupled receptor stimulation. We have now examined a series of GAP-43 mutants in this system and determined those regions of GAP-43 required for this increase in current flux. As expected, palmitoylation inhibits signal amplification in oocytes by blocking G protein activation. Unexpectedly, a second domain of GAP-43 (residues 35-50) containing a protein kinase C phosphorylation site at residue 41 is also necessary for augmentation of G protein-coupled signals in oocytes. This region is not required for activation of isolated Go but is necessary for GAP-43 binding to isolated calmodulin and to isolated protein kinase C. Substitution of Asp for Ser41 inactivates GAP-43 as a signal facilitator in oocytes. This mutation blocks GAP-43 binding to both protein kinase C and calmodulin. Thus, GAP-43 regulates an oocyte signaling cascade via coordinated, simultaneous G protein activation and interaction with either calmodulin or protein kinase C.
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PMID:GAP-43 augmentation of G protein-mediated signal transduction is regulated by both phosphorylation and palmitoylation. 948 17


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