EC:2.7.11.13 - FACTA Search

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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)

1-beta-D-Arabinofuranosylcytosine (ara-C) is an effective antileukemic agent that misincorporates into DNA. Recent studies have demonstrated that ara-C treatment is associated with transient induction of the c-jun early response gene. The present studies have examined the effects of ara-C on c-jun expression in a phorbol ester-resistant variant of the HL-60 myeloid leukemia cell line, designated HL-525, that is deficient in protein kinase C (PKC)-mediated signal transduction and fails to respond to 12-O-tetradecanoylphorbol-13-acetate with induction of c-jun transcripts. The results demonstrate that treatment of HL-525 cells with ara-C is associated with transcriptional activation of the c-jun gene. We also demonstrate that ara-C treatment is associated with activation of a PKC-like activity. Partial purification of this Ca(2+)-independent activity has demonstrated phosphorylation of synthetic peptides derived from (a) amino acids 4-14 of myelin basic protein and (b) the pseudosubstrate region of PKC (amino acids 19-31), with substitution of Ala25 with serine. The finding that the ara-C-induced activity is inhibited by the pseudosubstrate PKC(19-36) supports the activation of a PKC-like enzyme. Because PKC can act upstream of the mitogen-activated protein (MAP) kinases, we studied the effects of ara-C treatment on MAP kinase activity. The results demonstrate that MAP kinase is activated in ara-C-treated cells and that the kinetics of this activation are similar to those of the PKC-like activity. Because 12-O-tetradecanoylphorbol-13-acetate has little, if any, effect on the PKC-like and MAP kinase activities in HL-525 cells, these findings suggest that ara-C activates a distinct signaling cascade that may contribute to induction of the c-jun gene.
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PMID:1-beta-D-arabinofuranosylcytosine activates serine/threonine protein kinases and c-jun gene expression in phorbol ester-resistant myeloid leukemia cells. 805 58

The repetitive passages of a Schwann cell culture results in the appearance of immortalized cells. In order to investigate the direct effects of cyclic AMP (cAMP) on Schwann cell proliferation, we used the immortalized Schwann cells because the responses of a short-term Schwann cell culture to agents increasing the intracellular cAMP are more complicated and it does not seem that all of them are due to the direct effects of cAMP. By adding up to 200 microM of forskolin, an adenylate cyclase activator, to the culture medium, Schwann cell proliferation was inhibited and the intracellular 1,2-diacylglycerol (DG) level was decreased in a dose-dependent manner to 44 and 53% of the control values, respectively. The protein phosphorylation activity in the cytosol from the cell treated with 100 microM forskolin, assayed with myelin basic protein as the acceptor, decreased to 78% and this inhibition was then reversed by the addition of 1-oleoyl-2-acetyl-sn-glycerol (OAG), a membrane-permeable DG, to the assay mixture. The cell proliferation inhibited by forskolin was also restored by the addition of OAG. These data suggest that cAMP inhibits both the activity of protein kinase C (PKC) and consequently cell proliferation through suppression of intracellular DG level, an activator of PKC. Since the inositol 1,4,5-triphosphate level and the hydrolysis of phosphatidylcholine to DG and phosphorylcholine were not affected, forskolin therefore appears to suppress the de novo synthesis of DG.
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PMID:Inhibition of the proliferation of cultured immortalized Schwann cells by forskolin with a decreased basal level of diacylglycerol. 806 31

PHAS-I is a heat- and acid-stable protein that is phosphorylated on Ser/Thr residues in response to insulin and growth factors. To investigate the phosphorylation of PHAS-I, the protein was expressed in bacteria and purified for use as substrate in protein kinase reactions in vitro. Recombinant PHAS-I was rapidly and stoichiometrically phosphorylated by mitogen-activated protein (MAP) kinase. At saturating MgATP, the Km and Vmax observed with PHAS-I were almost identical to those obtained with myelin basic protein, one of the best MAP kinase substrates. PHAS-I was also phosphorylated at a significant rate by casein kinase II and protein kinase C. To investigate sites of phosphorylation, PHAS-I was digested with collagenase and phosphopeptides were resolved by reverse phase high performance liquid chromatography. Almost all of the phosphate introduced by MAP kinase was recovered in the peptide, Leu-Met-Glu-Cys-Arg-Asn-Ser-Pro-Val-Ala-Lys-Thr. 32P was released in the seventh cycle of Edman degradation, identifying the Ser (Ser64) as the phosphorylated residue. Ser64 was also phosphorylated in response to insulin in rat adipocytes. We conclude that PHAS-I is a substrate for MAP kinase both in vivo and in vitro. As PHAS-I is one of the most prominent insulin-stimulated phosphoproteins in adipocytes, it may qualify as the major MAP kinase substrate in these cells.
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PMID:Phosphorylation of PHAS-I by mitogen-activated protein (MAP) kinase. Identification of a site phosphorylated by MAP kinase in vitro and in response to insulin in rat adipocytes. 808 23

Incubation of the human renal carcinoma cell line CaKi-1 with interferon-gamma (IFN-gamma) or the phorbol ester, phorbol-12-myristate 13-acetate (PMA) strongly stimulated the immunocytochemical expression of the intercellular adhesion molecule-1 (ICAM-1) in a dose-dependent manner. Since PMA is capable of activating the Ca2+/phospholipid-dependent protein kinase C (PKC), we investigated the role of this kinase during IFN-gamma signal transduction. Calcium ionophore A23187 significantly enhanced IFN-gamma- and PMA-induced ICAM-1 staining. While staurosporine, H7 and sphingosine, three known PKC inhibitors, blocked the PMA effect, only staurosporine abrogated the action of IFN-gamma. Finally, 24 h of PMA pretreatment with subsequent IFN-gamma stimulation enhanced ICAM-1 staining above values from cultures where IFN-gamma was omitted. This occurred despite the fact that 24 h of PMA pretreatment abolished the effect of IFN-gamma on PKC activation, as determined by acetylated myelin basic protein 4-14 phosphorylation. In conclusion, these results suggest that additional events other than PKC activation are required for complete regulation of ICAM-1 antigen by IFN-gamma in the whole cell population. Hence, other Ca(2+)-dependent signalling pathway(s) mediated by IFN-gamma receptors must act. Further studies are needed to elucidate these specific pathway(s) activated during IFN-gamma stimulation in our model.
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PMID:Role of protein kinase C during interferon-gamma- and phorbol ester-stimulated immunocytochemical expression of ICAM-1 in human renal carcinoma cells. 810 44

The c-Raf-1 protein kinase plays a central role in the mitogenic response of cells to growth factors, cytokines, and many oncogenes. Despite the critical importance of this enzyme, very little is known of its biochemical properties or mechanisms of regulation. In these experiments, we used the only candidate physiologic substrate identified as yet for c-Raf-1, mitogen-activated protein kinase kinase (MAPKK), to examine enzymatic characteristics and candidate modulators of c-Raf-1, c-Raf-1 was purified from Sf9 cells infected with recombinant baculovirus encoding a histidine-tagged c-Raf-1. The Km values of c-Raf-1 for ATP and MAPKK were 11.6 microM and 0.8 microM, respectively, and the stoichiometry of phosphorylation of MAPKK by c-Raf-1 was 1.67 mol of phosphate per mol of MAPKK. In contrast to prior reports, Mg2+ was the preferred cation at Mg2+ and Mn2+ concentrations > 5 mM. c-Raf-1 substrate specificity was extremely restricted, consistent with the identification of only one candidate physiologic substrate to date and highlighting the necessity of using MAPKK rather than artificial substrates in c-Raf-1 activity assays. Of multiple potential substrates tested, the only one phosphorylated to > 20% of the level of MAPKK phosphorylation was myelin basic protein (22%). Heat-denatured MAPKK was phosphorylated at only 2% the level of native MAPKK, indicating that the restricted substrate specificity may be due to tertiary-structural requirements. We also examined whether c-Raf-1 activity is modulated by lipid binding to the cysteine finger region in its regulatory domain. Of multiple mitogen-stimulated or cell-membrane lipids tested, only phosphatidylserine and diacylglycerol in the presence of Ca2+ (2.5 mM) increased c-Raf-1 kinase activity significantly (1.5-fold). The increase is probably not of physiologic significance because it was about two orders of magnitude less than the stimulation of protein kinase C by these lipids. On gel-filtration chromatography, the peak of c-Raf-1 kinase activity and immunoreactivity eluted at a predicted molecular mass of > 150 kDa, suggesting that active c-Raf-1 (but not inactive c-Raf-1) exists as a multimeric complex. This complex may not include p21ras, however, because immunoreactive p21ras was not identified in the active fractions.
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PMID:Enzymatic characteristics of the c-Raf-1 protein kinase. 810

The Saccharomyces cerevisiae PKC1 gene encodes a homolog of mammalian protein kinase C (Levin, D. E., Fields, F.O., Kunisawa, R., Bishop, J.M., and Thorner, J. (1990) Cell 62, 213-224). A protein of 150 kDa is recognized by a polyclonal antiserum raised against a trpE-Pkc1 fusion protein. In subcellular fractionations, Pkc1p associates with the 100,000 x g particulate fraction. This association is resistant to extraction with high salt concentrations, alkali buffer, or nonionic detergents, suggesting that Pkc1p may be associated with a large protein complex. Pkc1p modified at its COOH terminus with two repeats of the Staphylococcus aureus protein A IgG-binding fragment (ZZ sequence tag) was able to fully restore the growth defects of a pkc1ts strain at restrictive temperature. ZZ-tagged Pkc1p was partially purified by chromatography on DEAE-Sepharose, followed by IgG-Sepharose. In vitro, Pkc1p phosphorylates the pseudosubstrate peptide and myelin basic protein, but not histones. Replacing an isoleucine with an arginine 2 amino acids COOH-terminal of the acceptor serine in the substrate peptide resulted in a 10-fold decrease of Km. Pkc1p activity was independent of cofactors such as phospholipids, diacylglycerol, and Ca2+, known to activate several mammalian protein kinase C isoenzymes, making it a rather distantly related member of the protein kinase C superfamily.
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PMID:Protein kinase C in yeast. Characteristics of the Saccharomyces cerevisiae PKC1 gene product. 820 4

The protein kinase C (PKC) family of serine-threonine kinases comprises at least eight members. These are differentially expressed, show varying affinities for activators such as Ca2+ and lipid species, and are therefore thought to play wide-ranging roles in the regulation of such cellular processes as differentiation, growth, and secretion. The aim of this study was to identify new PKC isoforms in the insulin-secreting cell line RINm5F that might be activated by the alterations in lipid metabolism that accompany nutrient-stimulated insulin release. Fragments of cDNA, derived from RINm5F cell mRNA, were amplified by the polymerase chain reaction using degenerate oligonucleotide primers corresponding to highly conserved regions in the catalytic domains of all known PKCs. A novel sequence generated by this approach was subsequently used to screen cDNA libraries. The entire 587-amino acid coding region of a new PKC isoform, PKC iota, was deduced from two overlapping clones isolated from a human kidney cDNA library. The amino acid sequence of PKC iota showed greatest homology to PKC zeta, with 72% identity overall rising to 84% in the catalytic domain. In contrast, the homology of PKC iota to the other isoforms was less pronounced, with < 53% identity even in the highly conserved catalytic region. Further similarities between PKC zeta and PKC iota included a highly conserved pseudosubstrate sequence, the absence of an apparent Ca(2+)-binding region, and the presence of only one cysteine-rich, zinc finger-like domain. Northern blot analysis, using the full-length PKC iota clone as a probe, revealed a single 4.6-kilobase transcript present predominantly in lung and brain, but also expressed at lower levels in many tissues including pancreatic islets. In CHO-K1 cells stably expressing the PKC iota cDNA under the human beta-actin promoter, the protein was detected as a 65-kDa band by Western blotting using an antibody to the COOH terminus of PKC zeta (conserved in PKC iota). Extracts of transfected CHO-K1 cells also displayed a significantly increased kinase activity using myelin basic protein as a substrate. The results suggest that PKC iota should be included in the atypical subgroup of PKCs whose definitive member is PKC zeta. As such, PKC iota is unlikely to be activated by the diacylglycerol that is derived from phosphoinositide hydrolysis, but might be a target for novel lipid activators that are elevated during nutrient-stimulated insulin secretion.
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PMID:Molecular cloning and characterization of PKC iota, an atypical isoform of protein kinase C derived from insulin-secreting cells. 822 78

Platelet-derived growth factor (PDGF) is a potent mitogen for a variety of cells. The calcium/phospholipid-dependent protein kinase C (PKC) represents a major signal transduction pathway for many growth stimuli including PDGF. Various isoforms of PKC are differentially expressed in the same or in different cells and tissues, and diverse stimuli may selectively activate one or more PKC isoforms. We studied the effect of PDGF on DNA synthesis and on the activity of PKC in human mesangial cells and vascular pericytes in the glomerular microvascular bed. PKC activity was measured as the amount of phosphorylated myelin basic protein-derived peptide substrate in the absence and presence of an inhibitor, a peptide spanning the pseudosubstrate region of PKC. PDGF (15 ng/ml) stimulated PKC activity within 5 min, and the effect was sustained for 60 min. Pretreatment of mesangial cells with 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), an inhibitor of PKC, abolished the stimulation of PKC and DNA synthesis in response to PDGF. This effect of H-7 was specific, because H-7 did not inhibit the tyrosine phosphorylation of the PDGF receptor in vivo when added to the cells or the in vitro kinase activity in the PDGF beta-receptor immunoprecipitates. Utilizing isotype-specific antibodies against PKC-alpha, -beta, or -gamma for immunoprecipitation of PDGF-treated mesangial cell extracts, followed by assay of PKC activity, we demonstrated the activation of PKC-alpha only. Northern blot analysis of mRNA prepared from mesangial cells also revealed two transcripts, 3.7 kb and 1.8 kb, that hybridized with cDNA specific for PKC-alpha.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Involvement of PKC-alpha in PDGF-mediated mitogenic signaling in human mesangial cells. 823 43

Treatment of human myeloid leukemia cells with 12-O-tetradecanoylphorbol-13-acetate (TPA), an activator of protein kinase C (PKC), is associated with induction of monocytic differentiation. Since PKC can act immediately upstream to the cytoplasmic Raf-1 serine/threonine protein kinase, we studied activation of Raf-1 during induction of the differentiated monocytic phenotype. The results demonstrate that Raf-1 is activated during TPA-induced monocytic differentiation of HL-60 cells. In contrast, there was little effect of TPA on this kinase in an HL-60 variant, designated HL-525, which is resistant to TPA-induced differentiation. Treatment of both HL-60 and HL-525 cells with okadaic acid, an inhibitor of serine/threonine protein phosphatases 1 and 2A, was associated with Raf-1 activation and induction of the monocytic phenotype. Since Raf-1 can activate the mitogen-activated protein (MAP) kinases, we also studied the relationship between MAP kinase activation and monocytic differentiation. Treatment of HL-60, but not HL-525, cells with TPA was associated with increased MAP kinase activity as determined by phosphorylation of myelin basic protein and the c-Jun Y peptide. Okadaic acid-induced differentiation of both HL-60 and HL-525 cells was similarly accompanied by increases in MAP kinase activity. These findings indicated that activation of Raf-1/MAP kinase signaling is associated with induction of a differentiated monocytic phenotype and that okadaic acid bypasses a defect in this cascade in TPA-treated HL-525 cells. While recent studies have shown that HL-525 cells are deficient in PKC beta, the present results demonstrate that PKC beta expression is up-regulated in the HL-525 variant by treatment with retinoic acid. The results also demonstrate that retinoic acid-treated HL-525 cells respond to TPA with activation of Raf-1 and MAP kinase, as well as induction of monocytic differentiation. Taken together, the results indicate that activation of Raf-1/MAP kinase signaling is associated with monocytic differentiation and that stimulation of serine/threonine protein phosphorylation by TPA or okadaic acid is sufficient for reversal of the leukemic HL-60 phenotype.
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PMID:Activation of Raf-1 and mitogen-activated protein kinases during monocytic differentiation of human myeloid leukemia cells. 828 41

Recent investigations identified a new signal transduction pathway, termed the sphingomyelin pathway, which may mediate the action of tumor necrosis factor (TNF) alpha and interleukin-1 beta (Mathias, S., Younes, A., Kan, C., Orlow, I., Joseph, C., and Kolesnick, R. N. (1993) Science 259, 519-522). This pathway is initiated by hydrolysis of sphingomyelin to ceramide by a neutral sphingomyelinase and stimulation of a ceramide-activated Ser/Thr protein kinase. Recent investigations demonstrated that kinase activity is proline-directed, recognizing substrates in which the phosphoacceptor site is followed by a proline residue. Until now, the kinase has been defined only as a membrane-bound activity capable of phosphorylating a peptide derived from the sequence surrounding Thr669 of the epidermal growth factor receptor. In the present studies, the kinase was quantitatively extracted from membrane with detergent and separated from protein kinase C by anion-exchange chromatography and isoelectric focusing. Ceramide-activated protein kinase was resolved as an exclusively membrane-bound, 97-kDa protein with a pI of 7.05. Kinase activity toward the epidermal growth factor receptor peptide co-purified with activity toward a generic proline-directed substrate, myelin basic protein. Kinase activity was reconstituted by a denaturation-renaturation procedure and demonstrated activity toward self (autophosphorylation) and exogenous substrate (myelin basic protein). Autophosphorylation occurred exclusively on serine residues. These activities were enhanced to 7-fold of control by ceramide and TNF alpha. These investigations provide additional evidence for a role for ceramide-activated protein kinase in signal transduction for TNF alpha.
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PMID:Renaturation and tumor necrosis factor-alpha stimulation of a 97-kDa ceramide-activated protein kinase. 830 Jun 38

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