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)

Annexin I (AnxI) contains phosphorylation sites in its "hinge region" that have been implicated in the regulation of cell growth and/or differentiation. A pigeon (Columba livia) isoform of this protein, annexin Icp35 (cp35), has a very similar amino acid sequence overall but an unrelated sequence that lacks phosphorylation sites in the hinge region. We now report the identification and characterization of annexin Icp37 (cp37) from pigeon. Genomic cloning and Southern blot analysis demonstrated that cp37 and cp35 were encoded by separated genes. Prolactin induced the expression of cp35 mRNA but not cp37. The amino acid sequence of cp37 was deduced from a cDNA clone and found to share 93 and 75% sequence identity with cp35 and human AnxI, respectively. The amino acid sequence of cp37 bore similarities to both AnxI and cp35 in the critical hinge region. Like AnxI, cp37 contained consensus phosphorylation sites in its amino acid sequence and was phosphorylated on tyrosine by the EGF receptor/kinase and on serine by protein kinase C in vitro. Despite the functional similarities between cp37 and AnxI, the nucleotide sequence that encoded the hinge region of cp37 was very similar to the analogous region of cp35, but different from that of AnxI. We propose that certain features shared by cp37 and AnxI are the products of convergent evolution. The fact that evolution independently selected for two annexin I-like genes (cp37 and anxI) encoding analogous phosphorylation sites is strong evidence that phosphorylation is important for the regulation of the biological activity of these proteins.
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PMID:Identification and characterization of columbid annexin Icp37. Insights into the evolution of annexin I phosphorylation sites. 138 65

Recent evidence has implicated protein kinase C (PKC) in the etiology of hyperproliferative diseases such as psoriasis and non-melanoma skin cancer. In this study, PKC activity, immunoreactive protein, and phorbol ester-binding kinetics were examined in primary cultures of normal human epidermal keratinocytes (NHEK) in order to elucidate the relationship between PKC and NHEK proliferation and differentiation. NHEK were maintained in a proliferative phase in serum-free low-calcium (0.15 mM) medium, and then were exposed to high calcium (1.6 mM) in order to stimulate growth arrest and differentiation. Staurosporine was inhibitory to Ca(++)-induced differentiation. Scatchard analysis of phorbol binding indicated that exposure to high calcium for 24 h increased the number of binding sites (Bmax) by fivefold. In correlation with the ligand-binding results, PKC activity was extremely low in proliferating (low-calcium) NHEK compared to differentiating cells (high calcium). When assayed after 24, 48, and 72 h, high calcium induced tenfold or greater increases in Ca++/phospholipid-dependent phosphotransferase activity. Immunoblot analysis of NHEK PKC using antibodies directed against the hinge region of PKC alpha/beta also indicated that exposure to high calcium resulted in higher levels of immunoreactive protein. Therefore, PKC in NHEK appears to be upregulated under conditions of Ca(++)-induced growth arrest and differentiation. In addition, NHEK and other human skin cell particulate fractions contain a protein of approximately 116 kDa that is highly immunoreactive to an antibody to PKC alpha/beta, which coelutes from DEAE-sephacel under the same buffer conditions as the 80-kDa PKC.
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PMID:Protein kinase C in normal human epidermal keratinocytes during proliferation and calcium-induced differentiation. 143 Dec 18

The PKC1 gene of Saccharomyces cerevisiae encodes a homolog of mammalian protein kinase C that is required for yeast cell growth and division. To identify additional components of the pathway in which PKC1 functions, we isolated extragenic suppressors of a pkc1 deletion mutant. All of the suppressor mutations were dominant for suppressor function and defined a single locus, which was designated BCK1 (for bypass of C kinase). A molecular clone of one suppressor allele, BCK1-20, was isolated on a centromere-containing plasmid through its ability to rescue a conditional pkc1 mutant. The BCK1 gene possesses a 4.4-kb uninterrupted open reading frame predicted to encode a 163-kDa protein kinase. The BCK1 gene product is not closely related to any known protein kinase, sharing only 45% amino acid identity with its closest known relative (the STE11-encoded protein kinase) through a region restricted to its putative C-terminal catalytic domain. Deletion of BCK1 resulted in a temperature-sensitive cell lysis defect, which was suppressed by osmotic stabilizing agents. Because pkc1 mutants also display a cell lysis defect, we suggest that PKC1 and BCK1 may normally function within the same pathway. Suppressor alleles of BCK1 differed from the wild-type gene in a region surrounding a potential PKC phosphorylation site immediately upstream of the predicted catalytic domain. This region may serve as a hinge between domains whose interaction is regulated by PKC1.
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PMID:Dominant mutations in a gene encoding a putative protein kinase (BCK1) bypass the requirement for a Saccharomyces cerevisiae protein kinase C homolog. 172 97

Members of the protein kinase C (PKC) family are characterized by an NH2-terminal regulatory domain containing binding sites for calcium, phosphatidylserine, and diacylglycerol (or tumor-promoting phorbol esters), a small central hinge region and a COOH-terminal catalytic domain. We have constructed fusion proteins in which the regulatory domain of PKC alpha was removed and replaced by a 19-amino acid leader sequence containing a myristoylation consensus or by the same sequence in which the amino-terminal glycine was changed to alanine to prevent myristoylation. The goal was to generate constitutively active mutants of PKC that were either membrane bound, due to their myristoylation, or cytoplasmic. Western blotting of fractions from COS cells transfected with plasmids encoding wild-type and mutant proteins revealed that PKC alpha resided entirely in a Triton X-100 soluble (TS) fraction, whereas both the myristoylated and nonmyristoylated mutants were associated primarily with the nuclear envelope fraction. A similar mutant that lacked the 19 amino acid leader sequence was also found almost entirely in the nuclear envelope, as was a truncation mutant containing only the regulatory domain, hinge region, and a small portion of the catalytic domain. However, an additional truncation mutant consisting of only the regulatory domain plus the first one-third of the hinge region was almost entirely in the TS fraction. A nonmyristoylated fusion protein containing only the catalytic domain was also found in the nuclear envelope. Immunostaining of cells transfected with these constructs revealed that both the myristoylated and nonmyristoylated mutants were localized in nuclei, whereas wild-type PKC alpha was primarily cytoplasmic and perinuclear. Phorbol dibutyrate treatment of PKC alpha-transfected cells resulted in increased perinuclear and nuclear staining. The results are consistent with a model in which activation of PKC, by phorbol esters or by deletion of the regulatory domain, exposes regions in the hinge and catalytic domains that interact with a PKC "receptor" present in the nuclear envelope, and may explain the ability of wild-type PKC to be translocated to the nucleus under certain conditions.
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PMID:Deletion of the regulatory domain of protein kinase C alpha exposes regions in the hinge and catalytic domains that mediate nuclear targeting. 173 20

The major autophosphorylation sites of the rat beta II isozyme of protein kinase C were identified. The modified threonine and serine residues were found in the amino-terminal peptide, the carboxyl-terminal tail, and the hinge region between the regulatory lipid-binding domain and the catalytic kinase domain. Because this autophosphorylation follows an intrapeptide mechanism, extraordinary flexibility of the protein is necessary to phosphorylate the three regions. Comparison of the sequences surrounding the modified residues showed no obvious recognition motif nor any similarity to substrate phosphorylation sites, suggesting that proximity to the active site may be the primary criterion for their phosphorylation.
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PMID:Autophosphorylation of protein kinase C at three separated regions of its primary sequence. 237 95

The activation of protein kinase C (PKC) is irreversibly regulated by limited proteolysis catalyzed by a calcium-activated neutral cysteine protease, calpain. Calpain cleaves PKC alpha at specific sites in the hinge region between the catalytic and the regulatory domains of this kinase. Here we show a novel method for production of antibodies that bind specifically to the catalytic fragment of PKC alpha but not to the unproteolyzed protein. To detect proteolyzed PKC alpha 'cleavage site-directed antibodies,' which recognize amino-terminal regions in the nascent catalytic fragments and do not cross-react with the unproteolyzed enzymes, were raised using synthetic peptides corresponding to the amino-terminal sequences. The synthetic peptides used in this study were the sequences of human PKC alpha at the cleavage sites by m- and mu-types of calpains (LGPAGNKV and VISPSEDRKQPSNNLDRVKLT, respectively) and they are designated as CF alpha 2, CF alpha 4, in this order. Each synthetic peptide was injected into rabbit after conjugation with a carrier protein. The antibodies thus obtained (anti-CF alpha 2 or -CF alpha 4) specifically reacted with either the 46- or 45-kDa catalytic fragment of PKC alpha, respectively, whereas they did not cross-react with other fragments. Furthermore, the antibodies did not bind to the unproteolyzed enzyme nor fragments of PKC alpha obtained by treatment with other proteinases unless the fragment carried the same amino-terminal sequence. When human platelets were treated with calcium ionophore, the catalytic fragments of PKC alpha (45- and 46-kDa) were detected in the cytosol by immunoblotting with the antibodies. However, these antibodies did not bind unproteolyzed 80-kDa PKC alpha, although this form was dominant in the cytosol of the calcium ionophore-treated human platelets. In addition, the 45-kDa catalytic fragment of PKC alpha was detected in an apoptotic human fibroblast TIG-3 cells cultured in serum-free medium. Our method is applicable for analysis of proteolysis in various cellular states.
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PMID:Antibodies specific for proteolyzed forms of protein kinase C alpha. 749 78

Protein kinase C (PCK) epsilon has been found to have unique properties among the PCK isozymes in terms of its membrane association, oncogenic potential, and substrate specificity. Recently we have demonstrated that PKC epsilon localizes to the Golgi network via its zinc finger domain and that both the holoenzyme and its zinc finger region modulate Golgi function. To further characterize the relationship between the domain organization and the subcellular localization of PKC epsilon, a series of NIH 3T3 cell lines were created, each overexpressing a different truncated version of PKC epsilon. The overexpressed proteins each were designed to contain an epsilon-epitope tag peptide at the COOH terminus to allow ready detection with an antibody specific for the tag. The subcellular localization of the recombinant proteins was analyzed by in vivo phorbol ester binding, immunocytochemistry, and cell fractionation followed by immunoblotting. Results revealed several regions of PKC epsilon that contain putative subcellular localization signals. The presence either of the hinge region or of a 33-amino-acid region including the pseudosubstrate sequence in the recombinant proteins resulted in association with the plasma membrane and cytoskeletal components. The catalytic domain was found predominantly in the cytosolic fraction. The accessibility and thus the dominance of these localization signals is likely to be affected by the overall conformation of the recombinant proteins. Regions with putative proteolytic degradation sites also were identified. The susceptibility of the overexpressed proteins to proteolytic degradation was dependent on the protein conformation. Based on these observations, a model depicting the interaction and hierarchy of the suspected localization signals and proteolytic degradation sites is presented.
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PMID:Protein kinase C epsilon subcellular localization domains and proteolytic degradation sites. A model for protein kinase C conformational changes. 764 54

The autophosphorylation sites of the beta 2 isozyme of protein kinase C (PKC) were recently identified as Ser-16/Thr-17 near the NH2 terminus, Thr-314/Thr-324 in the hinge region, and Thr-634/Thr-641 near the COOH terminus [Flint, A.J., Paladini, R.D. & Koshland, D.E. (1990) Science 294, 408-411]. To define the role of autophosphorylation we constructed three site-directed mutants of PKC beta 1 isozyme in which each pair of phosphorylatable residues is changed to alanine. Wild-type PKC beta 1 and the mutant proteins were transiently overexpressed in COS cells, resulting in at least a 20-fold increase in [3H]phorbol 12,13-dibutyrate binding compared with control transfectants. Enzyme assays of PKC partially purified from transfected cells indicated at least a 5-fold increase in PKC activity upon expression of the wild-type protein or the NH2-terminal and hinge mutants. In contrast, no increased activity was detected upon expression of the COOH-terminal mutant. Immunoblot analysis using a beta isoform-specific antibody showed that wild-type, NH2-terminal mutant, and hinge mutant proteins are similarly distributed between the Triton-soluble and insoluble fractions. In contrast, the COOH-terminal mutant protein is largely Triton-insoluble. Immunoblot analysis also indicated that this mutant is resistant to down-regulation upon chronic exposure of cells to phorbol ester. Moreover, RNA blot analysis showed that overexpression of wild-type PKC but not of the COOH-terminal mutant enhances phorbol ester induction of c-FOS and c-JUN mRNA. Our results indicate that (i) alteration in the NH2-terminal and hinge autophosphorylation sites has no effect on PKC function by the criteria examined and (ii) the COOH-terminal autophosphorylation sites are critical for PKC function and possibly subcellular localization in COS cells.
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PMID:Characterization of site-specific mutants altered at protein kinase C beta 1 isozyme autophosphorylation sites. 832 93

The protein kinase C-alpha (PKC-alpha)-catalyzed phosphorylation of the peptide [Arg]4-Tyr-Gly-Ser-[Arg]5-Tyr is independent of Ca2+ and phospholipid. The binding of this peptide to PKC-alpha induces a conformational change in the enzyme that results in the exposure of hydrophobic groups that subsequently insert into a membrane. Induction of a conformational change in the enzyme by this peptide is demonstrated by susceptibility to trypsin cleavage. Additionally, exposure of hydrophobic sites on the enzyme is shown by the binding of the fluorescent probes PRODAN and bis-ANS and by the partitioning of the enzyme into a Triton X-114-enriched phase. In the presence of a phospholipid bilayer containing phosphatidylserine, this peptide promotes the translocation of PKC-alpha to the membrane in the absence of Ca2+ as observed by increased resonance energy transfer between Trp on the enzyme and dansyl-groups attached to the lipid, as well as by changes in the intrinsic tryptophan fluorescence of the enzyme. Also, once bound to the membrane the peptide.PKC-alpha complex undergoes further conformational change which is evident by an increased sensitivity to trypsin cleavage at the hinge region. These results demonstrate that substrate binding can also induce translocation of PKC to the membrane and suggest that the removal of the pseudosubstrate domain is coupled to a conformational change in the enzyme that results in the exposure of hydrophobic groups.
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PMID:Substrate-induced translocation of PKC-alpha to the membrane. 855 12

Subcellular redistribution (translocation) was initiated by treatment of NIH 3T3 cells overexpressing different epitope-tagged fragments of PKC epsilon with PMA, and was analyzed by immunocytochemistry. The PMA-induced translocation of holo PKC epsilon, as well as fragments epsilon 2 (zinc finger domain + pseudosubstrate domain) and epsilon 7 (zinc finger domain + hinge region) from the Golgi to the plasma membrane was rapid (<10 min), while translocation of fragment epsilon 3 (zinc finger domain) was much slower (30-60 min). These results, combined with results of studies carried out at 20 degrees C to inhibit exocytotic vesicle traffic, indicated that PMA-induced translocation from the Golgi to the plasma membrane may proceed by two distinct mechanisms: a rapid, vesicle independent process noted with holo PKC epsilon (which requires the presence of the pseudosubstrate and/or hinge regions), and a slow, vesicle-dependent pathway observed with the zinc finger fragment.
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PMID:Influence of various domains of protein kinase C epsilon on its PMA-induced translocation from the Golgi to the plasma membrane. 866 Mar 86


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