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)

Insulin stimulates glucose transport by promoting translocation of GLUT4 proteins from the perinuclear compartment to the cell surface. It has been previously suggested that the microtubule-associated motor protein kinesin, which transports cargo toward the plus end of microtubules, plays a role in translocating GLUT4 vesicles to the cell surface. In this study, we investigated the role of Rab4, a small GTPase-binding protein, and the motor protein KIF3 (kinesin II in mice) in insulin-induced GLUT4 exocytosis in 3T3-L1 adipocytes. Photoaffinity labeling of Rab4 with [gamma-(32)P]GTP-azidoanilide showed that insulin stimulated Rab4 GTP loading and that this insulin effect was inhibited by pretreatment with the phosphatidylinositol 3-kinase (PI3-kinase) inhibitor LY294002 or expression of dominant-negative protein kinase C-lambda (PKC-lambda). Consistent with previous reports, expression of dominant-negative Rab4 (N121I) decreased insulin-induced GLUT4 translocation by 45%. Microinjection of an anti-KIF3 antibody into 3T3-L1 adipocytes decreased insulin-induced GLUT4 exocytosis by 65% but had no effect on endocytosis. Coimmunoprecipitation experiments showed that Rab4, but not Rab5, physically associated with KIF3, and this was confirmed by showing in vitro association using glutathione S-transferase-Rab4. A microtubule capture assay demonstrated that insulin stimulation increased the activity for the binding of KIF3 to microtubules and that this activation was inhibited by pretreatment with the PI3-kinase inhibitor LY294002 or expression of dominant-negative PKC-lambda. Taken together, these data indicate that (i) insulin signaling stimulates Rab4 activity, the association of Rab4 with kinesin, and the interaction of KIF3 with microtubules and (ii) this process is mediated by insulin-induced PI3-kinase-dependent PKC-lambda activation and participates in GLUT4 exocytosis in 3T3-L1 adipocytes.
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PMID:Insulin-induced GLUT4 translocation involves protein kinase C-lambda-mediated functional coupling between Rab4 and the motor protein kinesin. 1283 75

Non-esterified fatty acid (free fatty acid)-induced activation of the novel PKC (protein kinase C) isoenzymes PKCdelta and PKCtheta correlates with insulin resistance, including decreased insulin-stimulated IRS-1 (insulin receptor substrate-1) tyrosine phosphorylation and phosphoinositide 3-kinase activation, although the mechanism(s) for this resistance is not known. In the present study, we have explored the possibility of a novel PKC, PKCdelta, to modulate directly the ability of the insulin receptor kinase to tyrosine-phosphorylate IRS-1. We have found that expression of either constitutively active PKCdelta or wild-type PKCdelta followed by phorbol ester activation both inhibit insulin-stimulated IRS-1 tyrosine phosphorylation in vivo. Activated PKCdelta was also found to inhibit the IRS-1 tyrosine phosphorylation in vitro by purified insulin receptor using recombinant full-length human IRS-1 and a partial IRS-1-glutathione S-transferase-fusion protein as substrates. This inhibition in vitro was not observed with a non-IRS-1 substrate, indicating that it was not the result of a general decrease in the intrinsic kinase activity of the receptor. Consistent with the hypothesis that PKCdelta acts directly on IRS-1, we show that IRS-1 can be phosphorylated by PKCdelta on at least 18 sites. The importance of three of the PKCdelta phosphorylation sites in IRS-1 was shown in vitro by a 75-80% decrease in the incorporation of phosphate into an IRS-1 triple mutant in which Ser-307, Ser-323 and Ser-574 were replaced by Ala. More importantly, the mutation of these three sites completely abrogated the inhibitory effect of PKCdelta on IRS-1 tyrosine phosphorylation in vitro. These results indicate that PKCdelta modulates the ability of the insulin receptor to tyrosine-phosphorylate IRS-1 by direct phosphorylation of the IRS-1 molecule.
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PMID:Modulation of human insulin receptor substrate-1 tyrosine phosphorylation by protein kinase Cdelta. 1458 92

Although protein kinase D (PKD), like protein kinase C (PKC), possesses a C1 domain that binds phorbol esters and diacylglycerol, the structural differences from PKC within this and other domains of PKD imply differential regulation by lipids and ligands. We characterized the phorbol ester and phospholipid binding properties of a glutathione S-transferase-tagged full-length PKD and compared them with those of PKC-alpha and -delta. We found that PKD is a high-affinity phorbol ester receptor for a range of structurally and functionally divergent phorbol esters and analogs and showed both similarities and differences in structure-activity relations compared with the PKCs examined. In particular, PKD had lower affinity than PKC for certain diacylglycerol analogs, which might be caused by a lysine residue at the 22 position of the PKD-C1b domain in place of the tryptophan residue at this position conserved in the PKCs. The membrane-targeting domains in PKD are largely different from those in PKC; among these differences, PKD contains a pleckstrin homology (PH) domain that is absent in PKC. However, phosphatidylinositol-4,5-bisphosphate PIP2, a lipid ligand for some PH domains, reconstitutes phorbol 12,13-dibutyrate (PDBu) binding to PKD similarly as it does to PKC-alpha and -delta, implying that the PH domain in PKD may not preferentially interact with PIP2. Overall, the requirement of anionic phospholipids for the reconstitution of [3H]PDBu binding to PKD was intermediate between those of PKC-alpha and -delta. We conclude that PKD is a high-affinity phorbol ester receptor; its lipid requirements for ligand binding are approximately comparable with those of PKC but may be differentially regulated in cells through the binding of diacylglycerol to the C1 domain.
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PMID:Ligand structure-activity requirements and phospholipid dependence for the binding of phorbol esters to protein kinase D. 1464 64

A cDNA clone encoding a rac-like small GTP binding protein was isolated from a cDNA library of Chinese cabbage (Brassica campestris L. ssp. pekinensis) flower buds and named Brac1. The Brac1 cDNA contains an open reading frame encoding 198 amino acid residues with an estimated molecular mass of 21,690 Da and this coding region has conserved residues and motifs unique to the Rho subfamily of proteins. The deduced amino acid sequence of the Brac1 protein is closely related to that of Arabidopsis thaliana Arac3 (91%), but it shares relatively little homology with other members of the Ras superfamily (about 30% identity). To further characterize Brac1, a pGBrac1 expression vector consisting of PCR-amplified Brac1 cDNA plus glutathione S-transferase (GST) and pBKS(+)II was used to purify the protein. Using a PEI-cellulose/TLC plate, GTPase activity of this protein was confirmed and competition binding studies, using the guanine nucleotides, ATP, UTP and CTP, revealed that the di- and triphosphate forms of guanine nucleotides strongly bind Brac1. Membrane-bound PLD activity was synergistically enhanced by Brac1 in the presence of protein kinase C, but not in the presence of ARF (ADP-ribosylation factor). Genomic analysis indicated that Brac1 belongs to a multigene family. Brac1 transcripts were expressed in all the organs of Brassica, but were especially prevalent in flower buds.
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PMID:A rac-like small G-protein from Brassica campestris activates a PKC-dependent phospholipase D. 1469 72

Calponin has been implicated in the regulation of smooth muscle contraction through its interaction with F-actin and inhibition of the actin-activated MgATPase activity of phosphorylated myosin. Calponin has also been shown to interact with PKC. We have studied the interaction of calponin with PKC-alpha and with the low molecular weight heat-shock protein (HSP)27 in contraction of colonic smooth muscle cells. Particulate fractions from isolated smooth muscle cells were immunoprecipitated with antibodies to calponin and Western blot analyzed with antibodies to HSP27 and to PKC-alpha. Acetylcholine induced a sustained increase in the immunocomplexing of calponin with HSP27 and of calponin with PKC-alpha in the particulate fraction, indicating an association of the translocated proteins in the membrane. To examine whether the observed interaction in vivo is due to a direct interaction of calponin with PKC-alpha, a cDNA of 1.3 kb of human calponin gene was PCR amplified. PCR product encoding 622 nt of calponin cDNA (nt 351-972 corresponding to amino acids 92-229) was expressed as fusion glutathione S-transferase (GST) protein in the vector pGEX-KT. We have studied the direct association of GST-calponin fusion protein with recombinant PKC-alpha in vitro. Western blot analysis of the fractions collected after elution with reduced glutathione buffer (pH 8.0) show a coelution of GST-calponin with PKC-alpha, indicating a direct association of GST-calponin with PKC-alpha. These data suggest that there is a direct association of translocated calponin and PKC-alpha in the membrane and a role for the complex calponin-PKC-alpha-HSP27, in contraction of colonic smooth muscle cells.
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PMID:Direct association and translocation of PKC-alpha with calponin. 1472 9

Scleroderma, a disease involving excessive collagen deposition, can be studied using fibroblasts cultured from affected tissues. We find that curcumin, the active component of the spice turmeric, causes apoptosis in scleroderma lung fibroblasts (SLF), but not in normal lung fibroblasts (NLF). This effect is likely to be linked to the fact that although curcumin induces the expression of the phase 2 detoxification enzymes heme oxygenase 1 and glutathione S-transferase P1 (GST P1) in NLF, SLF are deficient in these enzymes, particularly after curcumin treatment. The sensitivity of cells to curcumin-induced apoptosis and the expression of GST P1 (but not heme oxygenase 1) are regulated by the epsilon isoform of protein kinase C (PKCepsilon). SLF, which contain less PKCepsilon and less GST P1 than NLF, become less sensitive to curcumin-induced apoptosis and express higher levels of GST P1 when transfected with wild-type PKCepsilon, but not with dominant-negative PKCepsilon. Conversely, NLF become sensitive to curcumin-induced apoptosis and express lower levels of GST P1 when PKCepsilon expression or function is inhibited. The subcellular distribution of PKCepsilon also differs in NLF and SLF. PKCepsilon is predominantly nuclear or perinuclear in NLF but is associated with stress fibers in SLF. Just as PKCepsilon levels are lower in SLF than in NLF in vitro, PKCepsilon expression is decreased in fibrotic lung tissue in vivo. In summary, our results suggest that a signaling pathway involving PKCepsilon and phase 2 detoxification enzymes provides protection against curcumin-induced apoptosis in NLF and is defective in SLF. These observations suggest that curcumin may have therapeutic value in treating scleroderma, just as it has already been shown to protect rats from lung fibrosis induced by a variety of agents.
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PMID:Curcumin-induced apoptosis in scleroderma lung fibroblasts: role of protein kinase cepsilon. 1474 95

In past studies, we demonstrated regulation of CFTR Cl channel function by protein kinase C (PKC)-epsilon through the binding of PKC-epsilon to RACK1 (a receptor for activated C-kinase) and of RACK1 to human Na(+)/H(+) exchanger regulatory factor (NHERF1). In this study, we investigated the site of RACK1 binding on NHERF1 using solid-phase and solution binding assays and pulldown, immunoprecipitation, and (36)Cl efflux experiments. Recombinant RACK1 binding to glutathione S-transferase (GST)-tagged PDZ1 domain of NHERF1 was 10-fold higher than its binding to GST-tagged PDZ2 domain of NHERF1. PDZ1 binds to RACK1 in a dose-dependent manner and vice versa, with similar binding constants of 1.67 and 1.26 microg, respectively. Interaction of the PDZ1 domain with RACK1 was not blocked by binding of activated PKC-epsilon to RACK1. A GST-tagged PDZ1 domain pulled down endogenous RACK1 from Calu-3 cell lysate. An internal 11-amino acid motif embedding the GYGF carboxylate binding loop of PDZ1 binds to RACK1, inhibits binding of recombinant NHERF1 and RACK1, pulls down endogenous RACK1 from Calu-3 cell lysate, and blocks coimmunoprecipitation of endogenous RACK1 with endogenous NHERF1 but does not affect cAMP-dependent activation of CFTR. A similar amino acid sequence in the PDZ2 domain did not bind RACK1. Our results indicate binding of Calu-3 RACK1 predominantly to the PDZ1 domain of NHERF1 at a site encompassing the GYGF loop of the PDZ1 domain and a site on RACK1 distinct from a PKC-epsilon binding site. CFTR activation by cAMP-generating agent is not affected by loss of RACK1-NHERF1 interaction.
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PMID:Role of a PDZ1 domain of NHERF1 in the binding of airway epithelial RACK1 to NHERF1. 1507 2

4-Hydroxy-2-trans-nonenal (4-HNE), one of the major end products of lipid peroxidation, has been shown to induce apoptosis in a variety of cell lines. It appears to modulate signaling processes in more than one way because it has been suggested to have a role in signaling for differentiation and proliferation. We show for the first time that incorporation of 4-HNE-metabolizing glutathione S-transferase (GST) isozyme, hGSTA4-4, into adherent cell lines HLE B-3 and CCL-75, by either cDNA transfection or microinjection of active enzyme, leads to their transformation. The dramatic phenotypic changes due to the incorporation of hGSTA4-4 include rounding of cells and anchorage-independent rapid proliferation of immortalized, rounded, and smaller cells. Incorporation of the inactive mutant of hGSTA4-4 (Y212F) in cells by either microinjection or transfection does not cause transformation, suggesting that the activity of hGSTA4-4 toward 4-HNE is required for transformation. This is further confirmed by the fact that mouse and Drosophila GST isozymes (mGSTA4-4 and DmGSTD1-1), which have high activity toward 4-HNE and subsequent depletion of 4-HNE, cause transformation whereas human GST isozymes hGSTP1-1 and hGSTA1-1, with minimal activity toward 4-HNE, do not cause transformation. In cells overexpressing active hGSTA4-4, expression of transforming growth factor beta1, cyclin-dependent kinase 2, protein kinase C betaII and extracellular signal regulated kinase is upregulated, whereas expression of p53 is downregulated. These studies suggest that alterations in 4-HNE homeostasis can profoundly affect cell-cycle signaling events.
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PMID:Transfection with 4-hydroxynonenal-metabolizing glutathione S-transferase isozymes leads to phenotypic transformation and immortalization of adherent cells. 1509 8

We report that albumin is translocated to the nucleus in response to oxidative stress. Prior measurements have demonstrated that in concert with known transcription factors albumin binds to an antioxidant response element, which controls the expression of glutathione S-transferase and other antioxidant enzymes that function to mediate adaptive cellular responses [Holderman, M. T., Miller, K. P., Dangott, L. J., and Ramos, K. S. (2002) Mol. Pharmacol. 61, 1174-1183]. To investigate the mechanisms underlying this adaptive cell response, we have identified linkages between calcium signaling and the nuclear translocation of albumin in JB6 epithelial cells. Under resting conditions, albumin and the calcium regulatory protein calmodulin (CaM) co-immunoprecipitate using antibodies against either protein, indicating a tight association. Calcium activation of CaM disrupts the association between CaM and albumin, suggesting that transient increases in cytosolic calcium levels function to mobilize intracellular albumin to facilitate its translocation into the nucleus. Likewise, nuclear translocation of albumin is induced by exposure of cells to hydrogen peroxide or a phorbol ester, indicating a functional linkage between reactive oxygen species, calcium, and PKC-signaling pathways. Inclusion of an antioxidant enzyme (i.e., superoxide dismutase) blocks nuclear translocation, suggesting that the oxidation of sensitive proteins functions to coordinate the adaptive cellular response. These results suggest that elevated calcium transients and associated increases in reactive oxygen species contribute to adaptive cellular responses through the mobilization and nuclear translocation of cellular albumin.
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PMID:Calmodulin involvement in stress-activated nuclear localization of albumin in JB6 epithelial cells. 1518 87

Exocytic insertion of H(+)-ATPase into the apical membrane of inner medullary collecting duct (IMCD) cells is dependent on a soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein target receptor (SNARE) complex. In this study we determined the role of Munc-18 in regulation of IMCD cell exocytosis of H(+)-ATPase. We compared the effect of acute cell acidification (the stimulus for IMCD exocytosis) on the interaction of syntaxin 1A with Munc-18-2 and the 31-kDa subunit of H(+)-ATPase. Immunoprecipitation revealed that cell acidification decreased green fluorescent protein (GFP)-syntaxin 1A and Munc-18-2 interaction by 49 +/- 7% and increased the interaction between GFP-syntaxin 1A and H(+)-ATPase by 170 +/- 23%. Apical membrane Munc-18-2 decreased by 27.5 +/- 4.6% and H(+)-ATPase increased by 246 +/- 22%, whereas GP-135, an apical membrane marker, did not increase. Pretreatment of IMCD cells with a PKC inhibitor (GO-6983) diminished the previously described changes in Munc-18-2-syntaxin 1A interaction and redistribution of H(+)-ATPase. In a pull-down assay of H(+)-ATPase by glutathione S-transferase (GST)-syntaxin 1A bound to beads, preincubation of beads with an approximately twofold excess of His-Munc-18-2 decreased H(+)-ATPase pulled down by 64 +/- 16%. IMCD cells that overexpress Munc-18-2 had a reduced rate of proton transport compared with control cells. We conclude that Munc-18-2 must dissociate from the syntaxin 1A protein for the exocytosis of H(+)-ATPase to occur. This dissociation leads to a conformational change in syntaxin 1A, allowing it to interact with H(+)-ATPase, synaptosome-associated protein (SNAP)-23, and vesicle-associated membrane protein (VAMP), forming the SNARE complex that leads to the docking and fusion of H(+)-ATPase vesicles.
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PMID:Munc-18-2 regulates exocytosis of H(+)-ATPase in rat inner medullary collecting duct cells. 1524 Mar 46


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