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)

The export of vesicular stomatitis virus glycoprotein (VSV-G) from the endoplasmic reticulum (ER) involves sorting and concentration, and has been proposed to require the function of heterotrimeric G proteins. To begin to identify the basic elements of a potential signaling pathway involved in vesicle assembly, we have examined whether protein kinase C (PKC) is required for ER to Golgi transport. Calphostin C, a specific inhibitor of the highly conserved cysteine-rich C6H2 motif present in the regulatory domain of PKC was found to be a potent inhibitor of export of VSV-G and vesicle budding from the ER in vivo and in vitro (IC50 approximately 60 nM). In contrast, the diacylglycerol analog phorbol 12-myristate 13-acetate, which activates PKC, enhanced the migration of VSV-G from the ER to pre-Golgi intermediates. Neither reagent had detectable effects on the oligomerization of VSV-G prior to export nor perturbed transport of protein between compartments of the Golgi stack. In contrast to the striking effects of calphostin C, reagents that inhibit the function of the catalytic domain of PKC (including the general kinase inhibitor staurosporine, as well as the more specific inhibitors H-7, H-8, pseudosubstrate inhibitor, or chelerythrine) did not inhibit export from the ER. Export was also insensitive to down-regulation of various PKC isoforms. These results suggest that a novel protein containing the conserved C6H2 motif may serve as a potential link in a signaling pathway regulating vesicle budding from the ER.
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PMID:Export of protein from the endoplasmic reticulum is regulated by a diacylglycerol/phorbol ester binding protein. 752 13

Aggregation of the high-affinity receptors for IgE (Fc epsilon RI) on mast cells activates nonreceptor kinases and other enzymes, thereby initiating a complex biochemical cascade. We have employed a chemical cross-linker in order to stabilize the association of Fc epsilon RI with other cellular proteins that are involved in the early events. We reacted the water-soluble, membrane-impermeant chemical cross-linker 3,3'-dithiobis(sulfosuccinimidyl propionate) (DTSSP) with permeabilized rat mucosal mast cells of the RBL line and analyzed immunoprecipitates of the receptor in detergent lysates of cells that had biosynthetically incorporated [35S]cysteine. Gel electrophoresis revealed substantial amounts of nonreceptor components only when the cells had been reacted with DTSSP. Receptors isolated from cells whose receptor-bound IgE had been aggregated with antigen prior to cross-linking yielded a similar pattern of 35S-labeled proteins. However, when the cells had also been exposed to [gamma-32P]ATP, the proteins associated with the cross-linked, aggregated receptors revealed enhanced incorporation of 32P compared to those associated with cross-linked, unaggregated receptors. A variety of antibodies were used to try to identify the associated proteins. Of those tested for, two, the src-like kinase p53/56lyn and the delta isoform of protein kinase C, were associated with the cross-linked Fc epsilon RI in amounts much greater than could be accounted for by nonspecific cross-linking.
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PMID:Chemical cross-linking of IgE-receptor complexes in RBL-2H3 cells. 753 96

The Caenorhabditis elegans Unc-13 protein is a novel member of the phorbol ester receptor family having a single cysteine-rich region with high homology to those present in protein kinase C (PKC) isozymes and the chimaerins. We expressed the cysteine-rich region of Unc-13 in Escherichia coli and quantitatively analyzed its interactions with phorbol esters and related analogs, its phospholipid requirements, and its inhibitor sensitivity. [3H]Phorbol 12,13-dibutyrate [3H]PDBu bound with high affinity to the cysteine-rich region of Unc-13 (Kd = 1.3 +/- 0.2 nM). This affinity is similar to that of other single cysteine-rich regions from PKC isozymes as well as n-chimaerin. As also described for PKC isozymes and n-chimaerin, Unc-13 bound diacylglycerol with an affinity about 2 orders of magnitude weaker than [3H]PDBu. Structure-activity analysis revealed significant but modest differences between recombinant cysteine-rich regions of Unc-13 and PKC delta. In addition, Unc-13 required slightly higher concentrations of phospholipid for reconstitution of [3H]PDBu binding. Calphostin C, a compound described as a selective inhibitor of PKC, was also able to inhibit [3H]PDBu binding to Unc-13, suggesting that this inhibitor is not able to distinguish between different classes of phorbol ester receptors. In conclusion, although our results revealed some differences in ligand and lipid cofactor sensitivities, Unc-13 represents a high affinity cellular target for the phorbol esters as well as for the lipid second messenger diacylglycerol, at least in C. elegans. The use of phorbol esters or some "specific" antagonists of PKC does not distinguish between cellular pathways involving different PKC isozymes or novel phorbol ester receptors such as n-chimaerin or Unc-13.
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PMID:Characterization of the cysteine-rich region of the Caenorhabditis elegans protein Unc-13 as a high affinity phorbol ester receptor. Analysis of ligand-binding interactions, lipid cofactor requirements, and inhibitor sensitivity. 753 38

We have examined the mechanism for the selective down-regulation of protein kinase C epsilon (nPKC epsilon) in rat pituitary GH4C1 cells responding to thyrotropin-releasing hormone (TRH) stimulation. Among various low molecular weight protease inhibitors examined, only a cysteine protease inhibitor (calpain inhibitor I, N-acetyl-Leu-Leu-norleucinal) blocked the down-regulation of nPKC epsilon. Furthermore, the introduction of a synthetic calpastatin peptide, an exclusively specific inhibitor of calpain, into the cells also reduced the down-regulation, suggesting the involvement of calpain among all the intracellular cysteine proteases in this process. In accordance, we observed TRH-induced translocation of m-calpain from the cytosol to the membrane and the concomitant up-regulation of calpastatin isoforms; presumably, the former represents activation of the protease initiating the kinase degradation, while the latter constitutes a negative feedback system protecting the cells from activated calpain. These results suggest that in GH4C1 cells, TRH mobilizes both protease (m-calpain) and inhibitor (calpastatin) as a strictly regulating system for the nPKC epsilon pathway mediating TRH signals.
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PMID:The role of the calpain-calpastatin system in thyrotropin-releasing hormone-induced selective down-regulation of a protein kinase C isozyme, nPKC epsilon, in rat pituitary GH4C1 cells. 755 44

In previous studies we have shown that protein kinase inhibitors and extracellular calcium can affect dramatically the assembly of tight junctions (TJ) and the localization of the TJ protein cingulin at sites of cell-cell contact in renal epithelial (MDCK) cells. To characterize in more detail the relationships between kinase activity and junction organization, we have studied the effects of the protein kinase C agonist phorbol myristate acetate (PMA) on the intracellular localization of cingulin, E-cadherin, desmoplakin and actin microfilaments in confluent MDCK monolayers. To study cingulin phosphorylation, MDCK cells were metabolically labelled with [32P]orthophosphate and immunoprecipitates were prepared with anti-cingulin antiserum. We show here that cingulin is phosphorylated in vivo on serine, and its specific phosphorylation is not significantly changed by treatment of confluent MDCK monolayers with PMA, with the protein kinase inhibitor H-7, or with the calcium chelator EGTA. Metabolic labeling with a pulse of [35S]methionine/cysteine showed that at normal extracellular calcium net cingulin biosynthesis was not affected by PMA or H-7. During junction assembly by calcium switch, H-7 did not change the specific phosphorylation of the immunoprecipitated cingulin, however, it prevented the increase in the amount of cingulin in the immunoprecipitates, suggesting that H-7 may block tight junction assembly by interfering with cellular processes that lead to the accumulation and stabilization of TJ proteins at sites of cell-cell contact.
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PMID:Phosphorylation of the tight junction protein cingulin and the effects of protein kinase inhibitors and activators in MDCK epithelial cells. 759 31

CD6 is a 105/130 kDa monomeric T cell surface glycoprotein that has been shown to play a role in human T cell activation. Recently a partial mouse CD6 cDNA sequence was described. We have isolated full-length cDNA clones including the initiation codon and sequence encoding the full signal peptide, as well as an additional 39 amino acids within the cytoplasmic domain as compared to the previously reported clone. The predicted full-length mouse CD6 protein contains 665 amino acids and has the features of a type I integral membrane protein. The extracellular domain of mouse CD6 is composed of three repeated cysteine-rich domains similar to those in human CD6, mouse and human CD5, and other members of a family of proteins whose prototype is the type I macrophage scavenger receptor. In marked contrast to the previously published human CD6 sequence, the mouse sequence predicts a long cytoplasmic tail that is not closely related to other proteins and possesses two proline-rich motifs containing the SH3-domain binding consensus sequence, three protein kinase C phosphorylation site motifs, nine casein kinase-2 phosphorylation site motifs, and a serine-threonine-rich motif repeated three times. Northern blot analysis revealed that mouse CD6 mRNA is expressed predominantly in thymus, lymph node, and spleen. A polyclonal antiserum was raised against mouse CD6 by gene gun plasmid DNA immunization of rabbits with the mouse CD6 cDNA in an expression vector. In immunofluorescence analysis this polyclonal antiserum positively stained the surface of cells transfected with the mouse CD6 cDNA in an expression vector, as well as most normal mouse thymocytes and peripheral T cells. CD6 protein is expressed on most CD4+CD8+ double-positive and CD4+ or CD8+ single-positive thymocytes, and is expressed at highest levels on mature CD3high thymocytes. The expression of mouse CD6 in thymocytes and peripheral T cells correlates closely with the expression of the related CD5 molecule. The polyclonal rabbit anti-mouse CD6 Abs immunoprecipitated a major polypeptide of 128 kDa from resting and 130 kDa from PMA- and FCS-activated mouse thymocytes and lymph node cells; it is likely that this increase in size upon activation is due to phosphorylation of mouse CD6 as has been described for human CD6. These data demonstrate that mouse thymocytes and T cells express a 130-kDa cell surface protein homologous to human CD6.
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PMID:Identification of a mouse protein homologous to the human CD6 T cell surface protein and sequence of the corresponding cDNA. 759 75

The three-dimensional structure of the second cysteine-rich domain of protein kinase C alpha (residues 95-159) was determined in aqueous solution by two-dimensional proton nuclear magnetic resonance and simulated annealing based calculations. On the basis of 687 distance constraints derived from assigned nuclear Overhauser effect (NOE) connectivities, a total of 10 converged structures were obtained from 40 runs of calculations. The atomic root-mean-square (RMS) difference about the mean coordinate positions (excluding residues 1-7, 16-17, 30-34, and 55-65) is 0.55 A for backbone atoms (N, C alpha, C') and 1.07 A for all non-hydrogen atoms. The molecular scaffold is maintained by triple-stranded and double-stranded twisted beta-sheets packed against an alpha-helix and two independent zincs are coordinated by His8, Cys38, Cys41, Cys57 and Cys21, Cys24, His46, Cys49, respectively. It should be noted that the metal ligands from the two sites are interleaved and this is thought to be a new structural motif of a zinc finger domain. Based on the resultant structure, we propose an interaction site of the cysteine-rich domain of protein kinase C with diacylglycerols and phorbol esters.
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PMID:Solution structure of cysteine-rich domain of protein kinase C alpha. 762 23

In order to identify cDNAs that can induce oncogenic transformation, a retroviral vector was used to transfer a library of cDNAs from the murine 32D hemopoietic cell line into NIH 3T3 fibroblasts. We have identified and recovered a provirus containing a 1.8-kilobase pair cDNA whose expression causes morphological transformation in NIH 3T3 cells. The transforming cDNA contains a complete open reading frame that encodes a protein (designated Lfc) with a region of sequence similarity to the product of the lbc oncogene. This region includes a domain that is characteristic of the CDC24 family of guanine nucleotide exchange factors in tandem with a pleckstrin homology (PH) domain. The Lfc protein is distinguished from Lbc by a 150-amino acid NH2-terminal extension that contains a cysteine- and histidine-rich domain similar to the diacylglycerol-binding site (zinc butterfly) found in protein kinase C. NH2- and COOH-terminal deletion analysis revealed that both the PH and putative guanine nucleotide exchange factor domains are required, but the zinc butterfly is dispensable, for transformation. Although the removal of the PH domain of the Lfc protein completely eliminated its ability to transform NIH 3T3 cells, replacement of this domain with an isoprenylation site restored all of its transforming activity. This suggests that a PH domain-dependent recruitment of the Lfc protein to the cellular membrane is a necessary step for cellular transformation. The lfc gene is expressed in a broad range of tissues as well as in a variety of hemopoietic and non-hemopoietic cell lines. Lfc appears to be a new member of a growing family of proteins that are likely to act as activators of Ras-like proteins in a developmental or cell-lineage specific manner.
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PMID:Expression cloning of lfc, a novel oncogene with structural similarities to guanine nucleotide exchange factors and to the regulatory region of protein kinase C. 762 63

The human colon cancer cell lines HCT 116 (poorly differentiated) and GEO (well differentiated) express the mitogenic peptide transforming growth factor alpha (TGF-alpha). The secretion of TGF-alpha was enhanced by phorbol 12-myristate 13-acetate (PMA), indicating the possible role of protein kinase C (PKC) in the formation of mature TGF-alpha. Cells were metabolically labeled with 35S-cysteine and the formation of the mature 6 kDa TGF-alpha polypeptide from the 17 kDa pro-TGF-alpha precursor was determined. The conversion of pro-TGF-alpha was complete in 2-4 hr with the HCT 116 cells showing faster kinetics of TGF-alpha formation than GEO cells. HCT 116 cells secreted more TGF-alpha than GEO cells and the rate and extent of formation of TGF-alpha was enhanced by PMA in both cell lines. The expression of several PKC isozymes by HCT 116 and GEO cells was examined by immunoblotting. The expression of all isozymes examined was higher in HCT 116 cells compared with GEO cells. Calphostin C, an inhibitor of PKC, reduced the enzyme activity and significantly inhibited the PMA-induced secretion of TGF-alpha by both cell lines. Two agonists of PKC that act on specific PKC isozymes, thymeleatoxin and 12-deoxyphorbol 13-phenylacetate 20-acetate (dPPA), stimulated the release of TGF-alpha into the medium to the same extent as PMA. Since dPPA has been reported to stimulate PKC-beta 1 specifically, our results suggest a potential role for PKC-beta in the processing of pro-TGF-alpha by these 2 human colon carcinoma cell lines.
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PMID:Pro-transforming growth factor-alpha processing in human colon carcinoma cells: role of protein kinase C. 763 77

Intracellular protein phosphorylation by protein kinase C (PKC) plays a major role in the translation of extracellular signals into cellular events. Speculations on the structural basis for PKC activation are based on sequence homology between their cysteine-rich domains (CRD) and the DNA-binding 'zinc-fingers'. We produced a fragment comprising the second CRD (CRD2) of rat PKC-alpha and determined its three-dimensional structure in solution by NMR spectroscopy. This revealed that CRD2 adopts a globular fold allowing two non-consecutive sets of zinc-binding residues to form two separate metal-binding sites. The fold is different to those previously proposed and allows insight into the molecular topology of a family of homologous proteins.
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PMID:Solution structure of a cysteine rich domain of rat protein kinase C. 766 52


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