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)

An 80-kDa protein labeled with [3H]myristic acid in C6 glioma and N1E-115 neuroblastoma cells has been identified as the myristoylated alanine-rich C kinase substrate (MARCKS protein) on the basis of its calmodulin-binding, acidic nature, heat stability, and immunochemical properties. When C6 cells preincubated with [3H]myristate were treated with 200 nM 4 beta-12-O-tetradecanoylphorbol 13-acetate (beta-TPA), labeled MARCKS was rapidly increased in the soluble digitonin fraction (maximal, fivefold at 10 min) with a concomitant decrease in the Triton X-100-soluble membrane fraction. However, phosphorylation of this protein was increased in the presence of beta-TPA to a similar extent in both fractions (maximal, fourfold at 30 min). In contrast, beta-TPA-stimulated phosphorylation of MARCKS in N1E-115 cells was confined to the membrane fraction only and no change in the distribution of the myristoylated protein was noted relative to alpha-TPA controls. These results indicate that although phosphorylation of MARCKS by protein kinase C occurs in both cell lines, it is not directly associated with translocation from membrane to cytosol, which occurs in C6 cells only. The cell-specific translocation of MARCKS appears to correlate with previously demonstrated differential effects of phorbol esters on stimulation of phosphatidylcholine turnover in these two cell lines.
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PMID:Dissociation of phosphorylation and translocation of a myristoylated protein kinase C substrate (MARCKS protein) in C6 glioma and N1E-115 neuroblastoma cells. 845 32

Infection is a frequent complication and the major cause of death among end-stage renal patients. Polymorphonuclear phagocytes (PMNL) are important in host defense mainly because of bacterial destruction by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-related free radical production following phagocytosis. In this study, hexose monophosphate pathway glycolytic activity, delivering energy to NADPH oxidase, is evaluated in vivo and in vitro, in healthy controls and in dialyzed renal failure patients. Our results show a marked parallel and correlated inhibition in the response to three stimuli for phagocytic activity (Staphylococcus aureus, formyl-methionine-leucine-phenylalanine, phorbol myristic acid) in predialysis samples. These data point to a main suppression of metabolic pathways, possibly beyond protein kinase C. This response is further suppressed at the 15th minute of cuprophane dialysis, for all stimuli studied (-40 to -94%; p < 0.001) except PMA. PMNL response remains intact during dialysis with non-complement-activating dialyzers. In vitro experiments confirm decreased PMNL glycolytic activity after the suspension of cuprophane fragments in normal whole blood. We conclude that polymorphonuclear cell energy delivery to NADPH oxidase is impaired in patients with end-stage renal failure. The impaired response against various stimuli is different in predialysis blood samples compared to samples collected during cuprophane dialysis, and may be related to two different conditions. These events probably contribute to the acquired immune suppression of uremia and the high incidence of infection among dialysis patients.
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PMID:Depressed phagocytosis in hemodialyzed patients: in vivo and in vitro mechanisms. 845 76

Subchronic exposure of rats to allylamine (AAM) modulates aortic smooth muscle cells (SMCs) from a quiescent to a proliferative phenotype. This response is associated with alterations in phospholipid metabolism and protein kinase C (PKC) activity. The present studies were conducted to evaluate the effects of long-term exposure to phorbol 12-myristate 13-acetate (PMA) on phospholipid metabolism in SMCs derived from control and AAM-treated animals, cells of varying proliferative potential. Measurements of 32P/[3H]myristic acid incorporation into parent phospholipids and phosphatidic acid (PA) and the extent of PKC-mediated histone phosphorylation were conducted following exposure of pre- and postconfluent subcultures of SMCs to PMA for 3 h. Increased 32P incorporation into phosphatidylcholine (PC) was observed in both pre- and postconfluent cultures of control and AAM cells treated with PMA relative to vehicle. This response was attenuated in pre- and postconfluent AAM cells relative to control counterparts. PMA enhanced 32P incorporation into phosphatidylinositol (PI) in preconfluent cultures of control cells, but decreased 32P incorporation in cultures of AAM cells relative to vehicle. A similar relationship was observed in the PI profile of postconfluent cultures. The alterations in primary phospholipid profiles induced by PMA correlated with the loss of PKC-mediated histone phosphorylation in the cytosolic and particulate fractions of both cell types. The pattern of 32P incorporation into PA, a phospholipid metabolite, paralleled that of PC in cultures of both cell types. In the presence of ethanol, vehicle-treated control and AAM cells exhibited a modest increase in phosphatidylethanol (PEt) formation, as measured by [3H]myristic acid incorporation. PMA enhanced PEt formation in control and AAM cultures, but selectively decreased [3H]myristic acid incorporation into PA in AAM cells. These data demonstrate that long-term PMA treatment differentially modulates phospholipid metabolism in aortic SMCs of varying proliferative potential. These alterations are associated with modulation of PLD-mediated hydrolysis of membrane phospholipids.
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PMID:Differential phospholipid metabolism in rat aortic smooth muscle cells of varying proliferative potential upon long term exposure to phorbol 12-myristate 13-acetate. 848 85

The acute retrovirus AKT8, isolated from an AKR mouse T-cell lymphoma, transforms mink lung cells in culture and is oncogenic when inoculated into newborn mice. The oncogene carried by this virus, v-akt, arose by recombination between Gag and the 5' untranslated region of the cellular gene c-akt. v-akt encodes a 105 kilodalton (kd) Gag-Akt fusion protein which is phosphorylated on serine and threonine residues. c-akt encodes a 55 kd serine-threonine protein-kinase, which is related to members of the protein kinase C (PKC) family and contains an SH2-like domain. The SH2-like and catalytic domains of Akt were expressed in E. coli as fusions to the carboxy-terminus of the Maltose binding protein (MBP). Antibodies against these proteins were raised in rabbits and they were used to determine the potential myristylation and subcellular localization of the v-akt and c-akt protein products. Immunoprecipitation of v-akt and c-akt from lysates of [35S]methionine and [3H]myristic acid labeled AKT8 transformed mink lung cells revealed that only v-akt was myristylated. Fractionation of Dounce-homogenized cellular extracts from uninfected and v-akt-transformed mink lung and PA317 cells and from uninfected PC12 cells by differential centrifugation showed that while the c-akt protein was localized primarily in the cytosol (90%), the v-akt protein was dispersed among the cellular compartments with approximately 40% on the plasma membranes, approximately 30% in the nucleus and approximately 30% in the cytosol. To determine whether the differences in post-translational modification and subcellular distribution between c-akt and v-akt translated into oncogenicity differences between the two proteins, we used retrovirus based constructs to express them both in the nontumorigenic rat T cell lymphoma line 5675. Intraperitoneal (IP) inoculation of the parental and c-akt expressing 5675 cells in nude Balb/c mice revealed that neither was oncogenic. In sharp contrast to these results, v-akt expressing 5675 cells inoculated in nude Balb/c mice were found to be highly oncogenic.
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PMID:The proteins encoded by c-akt and v-akt differ in post-translational modification, subcellular localization and oncogenic potential. 851 Sep 38

Platelet-activating factor (PAF) has been shown to stimulate phospholipase D (PLD) activity in human endometrium. The effect of 17 beta-oestradiol on PAF- and 12-O-tetradecanoylphorbol 13-acetate (TPA)-evoked PLD activity assayed as an accumulation of [3H]phosphatidylbutanol was examined in [3H]myristic acid labelled in a human endometrial epithelial cell line HEC-1B. TPA stimulated PLD activity in a dose-dependent manner whereas PAF had no significant effect on PLD activity. Following 48 h pretreatment with 100 nM 17 beta-oestradiol, PAF evoked PLD activity while leaving inositol trisphosphate accumulation in myo-[2-3H] inositol-labelled HEC-1B cells unaffected. In the 17 beta-oestradiol-treated cells, TPA-stimulated PLD activity was significantly elevated at 100 nM TPA (P < 0.05) and 1 microM TPA (P < 0.05) compared to responses in the untreated cells, suggesting that 17 beta-oestradiol may upregulate PKC activity. Interestingly, following a 30 min pretreatment of HEC-1B cells with a range of 17 beta-oestradiol concentrations. TPA (10 nM) and PAF (100 nM) stimulated PLD activity. However, TPA-stimulated PLD activity levels fell 10-fold while PAF-mediated PLD activity remained elevated at 10 nM and 100 nM concentrations of 17 beta-oestradiol suggesting a different mechanism of activation. These results indicate that 17 beta-oestradiol can upregulate PAF-induced PLD activity in HEC-1B cells.
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PMID:Oestradiol-17 beta modulates PAF-evoked phospholipase D activity but not inositide-lipid hydrolysis in human endometrial cell line, HEC-1B. 852 9

Low density lipoproteins (LDL) are risk factors in atherosclerosis and oxidative modification of LDL to oxidized LDL (OX-LDL) increases its atherogenicity. Development of atherosclerosis likely involves OX-LDL-mediated smooth muscle cell (SMC) proliferation. However, the mechanism(s) of SMC proliferation by OX-LDL is unknown. We hypothesized that OX-LDL may mediate SMC proliferation by activation of phospholipase D (PLD) through the generation of the second-messenger, phosphatidic acid (PA). To test this hypothesis, activation of PLD by OX-LDL was investigated in [3H]myristic acid- or [32P]orthophosphate-labeled rabbit femoral artery smooth muscle cells (RFASMC) in the presence of 0.5% ethanol or 0.05% butanol. Phospholipase D activation, as measured by labeled phosphatidylethanol (PEt) or phosphatidylbutanol (PBt) formation, was enhanced (3- to 5-fold) by OX-LDL. This activation of PLD was specific for OX-LDL, as native LDL or acetylated LDL had no effect. Further, OX-LDL-mediated [32P]PEt formation was dose- and time-dependent. To determine the mechanism(s) of OX-LDL-induced PLD activation, the role of protein kinase C (PKC) and Ca2+ was investigated. Pretreatment of [32P]orthophosphate-labeled RFASMC with known inhibitors of PKC such as staurosporine, calphostin-C, or H-7, had no effect on OX-LDL-induced PLD activation. Also, down-regulation of PKC by 12-O-tetradecanoylphorbol 13-acetate (TPA) (100 nM, 18 h) did not alter the OX-LDL-mediated [32P]PEt formation. However, pretreatment of RFASMC with genistein, a putative inhibitor of tyrosine kinases, attenuated the OX-LDL-mediated [32P]PEt formation. In addition, exposure of RFASMC to sodium orthovanadate, an inhibitor of phosphatases, enhanced the OX-LDL-mediated PLD activation. The effects of genistein and vanadate on PLD activation were specific for OX-LDL as these agents did not alter the TPA-induced [32P]PEt formation. Treatment of quiescent RFASMC with OX-LDL increased [3H]thymidine incorporation into DNA. This enhanced incorporation of [3H]thymidine into DNA was also mimicked by exogenously added phosphatidic acid (PA) or lysophosphatidic acid (LPA). These findings suggest that OX-LDL is a potent activator of the PLD pathway in SMC. The activation of PLD by OX-LDL generates second-messengers like PA and/or LPA which modulate mitogenesis. Thus, these results indicate that OX-LDL, in atherosclerotic lesions, may enhance SMC proliferation through the modulation of signal transduction pathways including activation of PLD.
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PMID:Oxidized low density lipoprotein-mediated activation of phospholipase D in smooth muscle cells: a possible role in cell proliferation and atherogenesis. 855 88

The partially purified myosin-bound phosphatase had an associated protein kinase that phosphorylated the holoenzyme, primarily on the large (130-kDa) subunit. Phosphorylation of the 130-kDa subunit resulted in inhibition of phosphatase activity. The major site of phosphorylation was threonine 654 of the 130-kDa subunit or threonine 695 of the 133-kDa isoform. Phosphorylation of the large subunit did not dissociate the holoenzyme. Dephosphorylation of the large subunit was achieved by the holoenzyme, and addition of the catalytic subunit of the type 2A enzyme did not increase the rate of dephosphorylation. The associated kinase was inhibited by chelerythrine, with half-maximal inhibition at approximately 5 microM (in 150 microM ATP). The associated kinase phosphorylated two synthetic peptides, one corresponding to the sequence flanking the phosphorylated threonine, i.e. 648-661 of the 130-kDa subunit, and the other to a known protein kinase C substrate, i.e. a modified sequence from the autoinhibitory region of epsilon protein kinase C. The associated kinase was activated by arachidonic and oleic acid and to a lesser extent by myristic acid. The protein kinase that phosphorylated the 130-kDa subunit and resulted in inhibition of myosin phosphatase activity was not identified.
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PMID:Phosphorylation of the large subunit of myosin phosphatase and inhibition of phosphatase activity. 861 39

MARCKS is a protein kinase C (PKC) substrate which binds calcium/calmodulin and actin, and which has been implicated in cell motility, phagocytosis, membrane traffic, and mitogenesis. MARCKS cycles on and off the membrane via a myristoyl electrostatic switch (McLaughlin, S., and Aderem, A.(1995) Trends Biochem. Sci. 20, 272-276). Here we define the molecular determinants of the myristoyl-electrostatic switch. Mutation of the N-terminal glycine results in a nonmyristoylated form of MARCKS which does not bind membranes and is poorly phosphorylated. This indicates that myristic acid targets MARCKS to the membrane, where it is efficiently phosphorylated by PKC. A chimeric protein in which the N terminus of MARCKS is replaced by a sequence, which is doubly palmitoylated, is phosphorylated by PKC but not released from the membrane. Thus two palmitic acid moieties confer sufficient membrane binding energy to render the second, electrostatic membrane binding site superfluous. Mutation of the PKC phosphorylation sites results in a mutant which does not translocate from the membrane to the cytosol. A mutant in which the intervening sequence between the myristoyl moiety and the basic effector domain is deleted, is not displaced from the membrane by PKC dependent phosphorylation, fulfilling a theoretical prediction of the model. In addition to the nonspecific membrane binding interactions conferred by the myristoyl-electrostatic switch, indirect immunofluorescence microscopy demonstrates that specific protein-protein interactions also specify the intracellular localization of MARCKS.
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PMID:Molecular determinants of the myristoyl-electrostatic switch of MARCKS. 870 37

1. External application of the unsaturated fatty acid arachidonic acid (AA) to frog ventricular cells caused a large inhibition (approximately 85%) of the L-type calcium current (ICa,L) previously stimulated by the beta-adrenergic agonist isoprenaline (Iso). The concentration producing half-maximal inhibition (K1/2) was 1.52 microM. The inhibitory effect did not affect the peak current-voltage relationship but produced a negative shift in the inactivation curve. 2. The inhibitory effect of AA also occurred in cells internally perfused with cAMP and non-hydrolysable analogues of cAMP. These data suggest that AA is acting by a mechanism located beyond adenylyl cyclase and does not involve changes in intracellular cAMP levels. 3. AA also inhibited the calcium current stimulated by internal perfusion with the catalytic subunit of protein kinase A (PKA), suggesting that AA acts downstream of channel phosphorylation. 4. The inhibitory effect of AA on the isoprenaline- or cAMP-stimulated ICa,L is largely reduced in cells internally perfused with the thiophosphate donor analogue of ATP, ATP gamma S, or protein phosphatase 1 and 2A inhibitors like microcystin (MC) or okadaic acid (OA). External application of the phosphatase inhibitor calyculin (Caly) also reduced the AA effect. These data suggested that the AA effect on ICa,L involves activation of protein phosphatase activity. 5. The effect of AA on ICa,L was not affected by staurosporine, an inhibitor of protein kinases. It was also unaffected in cells internally perfused with GTP gamma S. These results suggest that neither a PKC- nor a G-protein-mediated mechanism are likely to be involved in the effect of AA on ICa,L. 6. A saturated fatty acid, myristic acid (MA), had no inhibitory effect on the isoprenaline-stimulated Ca2+ current, whereas, in the same cells arachidonic acid produced approximately 85% inhibition of ICa,L. 7. The inhibitory effect of AA was not affected by exposing the cells to indomethacin (Indo), an inhibitor of the metabolism of AA by cyclo-oxygenase, nor nordihydroguaiaretic acid (NDGA), an inhibitor of the lipoxygenase pathway. However, the non-metabolizable analogue of AA, 5,8,11,14-eicosatetraynoic acid (ETYA), was without effect on the isoprenaline-stimulated ICa,L. 8. These results suggest that AA inhibits ICa,L via a mechanism which involves, in part, stimulation of protein phosphatase activity. This process could provide a new mechanism in the modulation of calcium channel activity.
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PMID:Effect of arachidonic acid on the L-type calcium current in frog cardiac myocytes. 873 95

The present study examines whether nitrogen dioxide (NO2)-induced activation of protein kinase C (PKC) is associated with increased expression of specific PKC isoforms and/or with enhanced generation of phosphatidylcholine(PC)-derived diacylglycerol (DAG) in pulmonary artery endothelial cells (PAEC). Western blot analysis revealed that exposure to 5 ppm NO2 resulted in increased expression of PKC alpha and epsilon isoforms in both cytosol and membrane fractions in a time-dependent fashion compared with controls. A time-dependent elevated expression of PKC isoform beta was observed in the cytosol fraction only of N02-exposed cells. PKC isoform gamma was not detectable in either the cytosolic or membrane fractions from control or N02-exposed cells. Scatchard analysis of [3h]phorbol 12,13-dibutyrate (PDBu) binding showed that exposure to N02 for 24 h increased the maximal number of binding sites (Bmax) from 15.2 +/- 2.3 pmol/mg (control) to 42.3 +/- 5.3 pmol/mg (p < 0.01, n = 4) (NO2-exposed). Exposure to NO2 significantly increased PC specific-phospholipase C and phospholipase D activities in the plasma membrane of PAEC (p < 0.05 and p < 0.001, respectively). When [3H]-myristic acid-labeled cells were exposed to NO2, significantly increased radioactivity was associated with cellular DAG. These results show for the first time that exposure of PAEC to NO2 results in elevated expression of specific PKC isoforms and in enhanced generation of cellular DAG, and the latter appears to arise largely from the hydrolysis of plasma membrane PC.
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PMID:NO2-induced expression of specific protein kinase C isoforms and generation of phosphatidylcholine-derived diacylglycerol in cultured pulmonary artery endothelial cells. 876 15


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