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)

NGA is a human melanoma-associated antigen recognized by a panel of murine monoclonal antibodies developed in this laboratory. NGA consists of a 23.5 kDa core protein which is glycosylated in vivo to give a family of glycoproteins (30-60 kDa). Treatment of human melanoma G361 cells with the phorbol ester PMA resulted in apparent partial inhibition of NGA glycosylation. After PMA treatment, NGA appeared as 3 different bands of 24, 29 and 34 kDa on SDS-PAGE. The 29 kDa band is similar to the one obtained by treatment with the ionophore monensin, which inhibits NGA O-glycosylation. PMA can modulate plasma membrane ion exchange, most likely by activating protein kinase C. In G361 cells PMA may produce the same net effect as monensin, by impairing transport in the Golgi complex and consequently inhibiting protein O-glycosylation through an ionophore-like effect. Treatment of G361 cells with both PMA and protein kinase C inhibitors re-established the usual NGA glycosylation pattern. Thus the observed effect of PMA on NGA glycosylation is reversible and appears to be mediated by protein kinase C activation.
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PMID:Inhibition of neuroglandular antigen (NGA) glycosylation by phorbol ester in human melanoma cells. 264 70

The tumor promoter PMA has been shown to induce the expression of a 28-kDa/32-kDa early activation Ag, termed EA 1, on resting T cells. Under nonreducing conditions, EA 1 was detected by SDS-PAGE as a diffuse band in the 60-kDa region. In this study, this diffuse band was resolved into 56-kDa and 60-kDa bands. Endoglycosidase F treatment of EA 1 resulted in the appearance of a single band with a Mr of 48 kDa. Upon reduction, the 48-kDa band was shown to be composed of 24-kDa peptides. Diagonal gel electrophoresis showed that the major band of EA 1 was composed of a series of disulfide-linked homodimers with subunits of the same 24-kDa core protein that were differentially glycosylated. This analysis also revealed in a minor population of the EA 1 molecules, the presence of proteins of different Mr associated with the core protein. The signal requirements for the induction of EA 1 were investigated. The putative cellular action of PMA is the activation of protein kinase C (PKC). To further investigate the role of PKC activation in the expression of EA 1, the synthetic diacylglycerol, 1,2-sn-dioctanoylglycerol (diOG) was examined for its ability to substitute for PMA. DiOG induced EA 1 expression in a dose dependent manner. H-7, a relatively selective inhibitor of PKC, blocked diOG and PMA induced EA 1 expression. HA1004, a selective inhibitor of cAMP- and cGMP-dependent protein kinases, had no effect. In kinetic studies, EA 1 expression was seen as early as 1 h in diOG- and PMA-activated T cells. However, diOG did not completely mimic PMA-induced EA 1 expression. By 18 h, diOG-induced EA 1 expression was markedly reduced, whereas PMA-induced EA 1 expression was persistent. The role of calcium in EA 1 expression was investigated. mAb against CD3 potentiated diOG-induced EA 1 expression. This potentiation appeared to correlate with the ability of the anti-CD3 mAb to induce rises in intracellular calcium. Addition of EGTA to the media blocked the potentiation of diOG induced EA 1 expression by these mAb. The role of calcium in EA 1 expression was further demonstrated by the ability of ionomycin to potentiate EA 1 expression. These results demonstrate that PKC activation is the primary pathway for the induction of EA 1. However, calcium-dependent pathways appear to have a secondary role.
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PMID:The 28-kDa/32-kDa activation antigen EA 1. Further characterization and signal requirements for its expression. 326 2

The mechanisms by which a stimulatory monoclonal antibody (mAb), called mAb F11, induces granular secretion and aggregation in human platelets have been characterized. Fab fragments of mAb F11, as well as an mAb directed against the platelet Fc gamma RII receptor (mAb IV.3) were found to inhibit mAb F11-induced platelet secretion and aggregation, indicating that the mAb F11 IgG molecule interacts with the Fc gamma RII receptor through its Fc domain and with its own antigen through its Fab domain. The mAb F11 recognized two platelet proteins of 32 and 35 kDa on the platelet membrane surface, as identified by Western blot analysis. We purified both proteins from human platelet membranes using DEAE-Sepharose chromatography followed by mAb F11 affinity chromatography. When added to platelet-rich plasma, the purified proteins dose-dependently inhibited mAb F11-induced platelet aggregation. The purified protein preparation also competitively inhibited the binding of 125I-labelled mAb F11 to intact platelets. The N-terminal 26 amino acid sequences of both the 32 and 35 kDa proteins were identical and contained a single unblocked serine in the N-terminal position. When digested with N-glycanase, the 32 and 35 kDa proteins were converted into a single approximately 29 kDa protein, indicating that these two proteins are derived from the same core protein but differ in their degree of glycosylation. Internal amino acid sequence analysis of the F11 antigen provided information concerning 68 amino acids and suggested two consensus phosphorylation sites for protein kinase C (PKC). The phosphorylation by PKC of the isolated F11 antigen was observed following stimulation by phorbol 12-myristate 13-acetate. Databank analysis of the N-terminal and internal amino acid sequences of the F11 antigen indicated that the N-terminal sequence exhibited the highest degree of similarity to the variable region of the alpha-chain of human T-cell receptors (TCR). In contrast, the F11 internal sequences did not exhibit any similarity to the TCR. Our results demonstrate that the F11 antigen is a novel platelet membrane surface glycoprotein which becomes cross-linked with the Fc gamma RII receptor when platelets are activated by the stimulatory mAb F11. These mechanisms may be relevant to the production of immune thrombocytopenia by platelet-activating antibodies.
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PMID:Mechanisms of platelet activation by a stimulatory antibody: cross-linking of a novel platelet receptor for monoclonal antibody F11 with the Fc gamma RII receptor. 764 39

We previously demonstrated that the core protein of hepatitis C virus (HCV) can suppress gene expression and replication of hepatitis B virus (HBV) in a human hepatoma cell line (HuH-7). In this study, we have characterized the phosphorylation property of HCV core protein and examined the effect of phosphorylation on its suppressive activity of HBV. Our results indicated that both the full-length HCV core protein (22 kDa) and its processed or degraded forms (14 to 18 kDa) were phosphorylated in insect cells. As demonstrated by using the glutathione S-transferase fusion protein expression system and in vitro transcription and translation system, the phosphorylation of HCV core protein was carried out by protein kinase A (PKA) and protein kinase C (PKC) in vitro. In both kinase reactions, it was determined that the phosphorylated amino acid was a serine residue. The potential phosphorylated sites in core protein were identified as residues Ser-53 and Ser-116 for PKA and Ser-53 and Ser-99 for PKC. Comparison of the phosphorylation intensities of the wild type and Ser mutants suggested that Ser-99 and Ser-116 were the major phosphorylation sites for PKC and PKA, respectively. The phosphorylation of Ser-99 and Ser-116, but not Ser-53, in HCV core protein was essential for the suppressive activity of HCV core protein on HBV gene expression and replication in HuH-7 cells. Mutation of the former two serine residues to alanine or aspartate residues led to a drastic loss of the inhibitory effects of HCV core protein on HBV gene expression (both transcription and antigen production) and pregenomic RNA encapsidation, as well as the release of HBV virus particles. In contrast, the Ser-53 mutant conferred the same level of suppressive activity as the wild type did. This property is in accordance with the observation that Ser-99 and Ser-116 are the predominant phosphorylation sites in the HCV core construct. All serine mutants (including those with mutations in PKA, PKC, and both kinase recognition sites) of HCV core protein retained the ability to translocate into the nucleus. Furthermore, wild-type HCV core protein diminished its suppressive activity when cells were treated with PKA or PKC inhibitor. In conclusion, HCV core protein is a phospho-protein and in HuH-7 cells, its trans suppression of HBV gene expression and replication is positively regulated by PKA and PKC. The role of phosphorylation in the control of trans-suppressive activity cannot be reproduced by introducing an acidic residue.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Modulation of the trans-suppression activity of hepatitis C virus core protein by phosphorylation. 781 94

Phosphorylation of core particles derived either from hepatitis B viruses or from livers of hepatitis B-infected individuals has been long recognized, but the nature and function of the phosphorylating enzyme remained unknown. By immunoblotting with a monoclonal antibody, we have now detected protein kinase C within the liver-derived core particles. To study the significance of the encapsidated protein kinase C for the viral life cycle, we established an in vitro assembly system consisting of Escherichia coli-expressed core protein, protein kinase C, and in vitro-synthesized hepatitis B virus RNA. Phosphorylation of the core protein led to a reduced RNA encapsidation capacity of the core particles. Furthermore, RNA and protein kinase C competed for their target sequence, which is the carboxy-terminal arginine-rich domain of the core protein. This finding implies that phosphorylation of the nucleic acid binding site in the core protein occurs within the particles after encapsidation of protein kinase C, pregenomic RNA, and viral polymerase at a later step during viral genome maturation.
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PMID:Effect of core protein phosphorylation by protein kinase C on encapsidation of RNA within core particles of hepatitis B virus. 796 89

Focal adhesion formation in fibroblasts results from complex transmembrane signaling processes initiated by extracellular matrix molecules. Although a role for integrins with attendant tyrosine kinases has been established, there is evidence that cell surface heparan sulfate proteoglycans (HSPGs) are also involved with an associated role of protein kinase C. The identity of the proteoglycan has remained elusive, but we now report that syndecan 4 (ryudocan/amphiglycan) is present in focal adhesions of a number of cell types. Affinity-purified antibodies raised against a unique portion of the cytoplasmic domain of syndecan 4 core protein recognized an HSPG of similar characteristics to those of syndecan 4. These antibodies stained focal adhesions only after cell permeabilization and recognized differing mammalian species. Syndecan 4 was associated with focal adhesions that contained either beta 1 or beta 3 integrin subunits and those that formed on substrates of fibronectin, laminin, vitronectin, or type I collagen. No focal adhesions were found that were vinculin-containing but lacked syndecan 4. In contrast, syndecan 2, whose cytoplasmic domain is closely homologous to syndecan 4, does not appear to be a focal adhesion component. Thus, syndecan 4 represents a new transmembrane focal adhesion component, probably involved in their assembly.
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PMID:Syndecan 4 heparan sulfate proteoglycan is a selectively enriched and widespread focal adhesion component. 801 4

A protein extract of mouse seminal-vesicle secretion was used to immunize mature mice (Balb/c) of both sexes. Results of Western-blot analyses for these secretory proteins indicated that only one minor protein component could be recognized by the autoantisera prepared from either autoimmunization of male mice or isoimmunization of female mice. The autoantigen was purified from seminal-vesicle secretion. The purified autoantigen retained the ability to induce autoantibody formation. The autoantigen has glycoprotein characteristics: the majority of the carbohydrate is N-linked and the remainder is O-linked. Rabbit antibodies to the autoantigen were used to isolate the corresponding cDNA from a mouse seminal-vesicle cDNA library. The primary structure deduced from the cDNA sequence was confirmed by direct amino acid sequence determination. The results indicate that the core protein consists of 131 amino acid residues. Analysis of the primary structure indicates that the autoantigen has two potential acceptor sites for the N-linked carbohydrate at Asn-12 and Asn-122, three potential phosphorylation sites for casein kinase II at Thr-55, Ser-68 and Thr-76, and three potential phosphorylation sites for protein kinase C at Thr-28, Thr-40 and Thr-124. The core protein and the carbohydrate portion together have a molecular mass of 19 kDa. Results from Western- and Northern-blot analyses for various tissues indicate that the seminal vesicle is the sole organ producing this autoantigen. Expression of this autoantigen gene was stimulated by testosterone.
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PMID:Primary structure and characterization of an androgen-stimulated autoantigen purified from mouse seminal-vesicle secretion. 828 54

NG2 is a chondroitin sulfate proteoglycan that is expressed on dividing progenitor cells of several lineages including glia, muscle, and cartilage. It is an integral membrane proteoglycan with a core glycoprotein of 300 kDa. In the present study we have characterized three molecular forms of the NG2 core protein expressed by different cell lines. Many cell lines that express the full length 300-kDa NG2 core protein also release a 290-kDa form into the medium. This species lacks the cytoplasmic domain but contains almost the entire ectodomain. Two core protein species, the intact 300-kDa form and a truncated 275-kDa form, are expressed at the surface of an NG2-transfected cell line U251NG52. The 275-kDa species lacks the cytoplasmic domain and at least 64 amino acids of the ectodomain. Mild trypsinization of B49 cells also generates the 275-kDa species, suggesting that this component is produced by proteolysis of the 300-kDa form. Conversion of the 300-kDa species to the 275-kDa form in U251NG52 cells is stimulated by reagents such as phorbol esters, which activate protein kinase C. Phorbol esters are also known to induce expression of metalloproteinases such as collagenase and stromelysin, which could be responsible for cleavage of the 300-kDa core protein. Although B49 cells do not spontaneously produce the truncated 275-kDa species, use of monoclonal antibodies against NG2 to block the interaction between NG2 and type VI collagen results in the appearance of the 275-kDa component in these cells. Thus the interaction between NG2 and type VI collagen, which contains a Kunitz-type proteinase inhibitor sequence in the alpha 3 chain, may protect the proteoglycan against proteolysis. This is consistent with the observed deficiency of U251NG52 cells in anchoring type VI collagen at the surface.
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PMID:Generation of truncated forms of the NG2 proteoglycan by cell surface proteolysis. 859 Aug 8

PGs play an important role in regulating articular chondrocyte function in both normal and pathological states. However, the mechanisms of the effects of PG on chondrocyte function remain undefined. We, therefore, examined the effects of PGE1, PGE2, and PGE2 alpha on second messenger generation in relation to DNA and aggrecan synthesis in the nontransformed rat RCJ 3.1C5.18 (RCJ) chondrocyte cell line. RCJ cells were grown under minimal attachment conditions on a composite collagen-agarose (0.15%/0.8%) gel to maintain a differentiated phenotype. PGE1 and PGE2 (0.001-100 microM) produced a similar dose-related increase in cAMP accumulation, with a maximal 8-fold increase over basal values, whereas PGF2 alpha produced a minimal 1.3-fold increase in cAMP levels only at 100 microM. On the other hand, both PGE2 and PGE2 alpha raised the intracellular free calcium ([Ca2+]i) concentration, derived primarily from extracellular sources, whereas PGE1 was without effect on [Ca2+]i. These three PGs also had divergent effects on DNA synthesis, as measured by [3H]thymidine ([3H]TdR) incorporation. PGF2 alpha (0.001-5 microM) produced a dose-related increase in [3H]TdR incorporation, with a maximal 1.6-fold increase over baseline values at 5 microM and a slight decline to below maximal levels at 10 microM. PGE2 exhibited a contrasting inverse biphasic response, with an initial small suppressive effect that was maximal at 0.1 microM and a secondary stimulatory phase producing a small increase over control values at 5 microM. PGE1 had a uniformly suppressive effect, producing a 30% decrease at 10 microM. Despite the divergent effects of PGE1, PGE2, and PGE2 alpha on second messenger generation and DNA synthesis, all three PGs produced a dose-related stimulation of aggrecan synthesis. PGF2 alpha was the most potent, producing significant stimulation at 0.001 microM and a maximal 104% increase at 5 microM. PGE1 and PGE2 were approximately equipotent and approximately 60% as effective as PGF2 alpha in stimulating aggrecan synthesis. Northern analysis demonstrated that the effects of PG on aggrecan synthesis were not accompanied by changes in aggrecan core protein steady state messenger RNA levels. Thus, the effects of PG on aggrecan production in RCJ cells appear to be regulated at the posttranscriptional level. Forskolin and (Bu)2cAMP mimicked the suppressive effects of PGE1 on [3H]TdR incorporation, as well as the stimulatory effect of PGE1 on aggrecan synthesis. In addition, the phorbol ester 12-O-tetradecanoyl phorbol acetate mimicked PGF2 alpha stimulation of [3H]TdR incorporation and aggrecan synthesis, and the effects of PGE2 alpha on these processes were blocked by protein kinase C inhibitors. Therefore, it appears that in mammalian chondrocytes, PGE1 primarily activates the cAMP-protein kinase A second messenger system, PGE2 alpha affects primarily the Ca2(+)-protein kinase C system, and PGE2 activates both pathways. Moreover, PG posttranscriptional regulation of aggrecan synthesis in chondrocytes involves both the cAMP-protein kinase A and Ca2(+)-protein kinase C second messenger systems.
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PMID:Effects of prostaglandins on deoxyribonucleic acid and aggrecan synthesis in the RCJ 3.1C5.18 chondrocyte cell line: role of second messengers. 864 Nov 67

Platelet-derived growth factor (PDGF) stimulates not only the proliferation and migration of arterial smooth muscle cells (ASMCs), but also the transcription, translation, and posttranslational processing of versican, a large chondroitin sulfate proteoglycan present in the extracellular matrix of blood vessels. PDGF receptor tyrosine kinase activity is required for signaling events associated with mitogenic and motogenic stimulation of cells by PDGF. Therefore, we have asked if inhibiton of tyrosine kinase activity by genistein also blocks the stimulation of both versican core protein synthesis and glycosaminoglycan (GAG) chain modifications induced by PDGF in ASMCs. The tyrosine kinase inhibitor, genistein, in a dose-dependent manner, reversibly inhibits PDGF-stimulated ASMC cell proliferation and RNA and core protein expression of versican, without affecting the expression of decorin and biglycan. In contrast, genistein does not affect the increase in GAG chain elongation that is induced by PDGF. This suggests that different aspects of the biosynthesis of versican are differentially regulated. To determine if such differential regulation involves downstream activation of protein kinase C, ASMCs were treated with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) to directly activate this kinase. In comparison to PDGF stimulation, TPA has little effect on expression of versican mRNA expression, nor does TPA stimulate ASMC cell proliferation. However, like PDGF, TPA increases [35S]sulfate incorporation into proteoglycans and GAG chain elongation. These results indicate that PDGF-induced GAG chain elongation, which is not inhibited by genistein treatment and is stimulated by protein kinase C activation, involves signaling pathways different from those that regulate PDGF-stimulated versican mRNA and protein expression.
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PMID:Genistein selectively inhibits platelet-derived growth factor-stimulated versican biosynthesis in monkey arterial smooth muscle cells. 905 68


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