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)

Rat pheochromocytoma PC12 cells differentiate to sympathetic neuron-like cells upon treatment with nerve growth factor (NGF). The ras and src transforming proteins also induce PC12 neuronal differentiation and are likely to involve the protein kinase C signal transduction pathway. Using a number of ras mutants, we have established that the domains of oncogenic ras protein responsible for PC12 differentiation overlap those required for cellular transformation. All of the ras mutants that induced neuronal differentiation also activated c-fos transcription through the dyad symmetry element (DSE). Transforming ras protein activated an intracellular signal pathway, which led to the induction of 12-O-tetradecanoyl phorbol-13-acetate-responsive elements; activation was enhanced by coexpression of the proto-oncogene jun (encoding AP-1) and was further augmented by fos. Nuclear extracts from ras-infected PC12 cells showed an increased AP-1 DNA-binding activity. Transcriptional activation by ras was independent of the cyclic AMP-dependent pathway of signal transduction. We propose a possible involvement of fos and jun in ras-induced differentiation.
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PMID:ras-induced neuronal differentiation of PC12 cells: possible involvement of fos and jun. 250 2

Treatment of PC-12 pheochromocytoma cells with nerve growth factor (NGF) results in the differentiation of these cells into a sympathetic neuron-like phenotype. Although the initial intracellular signals elicited by NGF remain unknown, some of the cellular effects of NGF are similar to those of other growth factors, such as insulin. We have investigated the involvement of a newly identified inositol-containing glycolipid in signal transduction for the actions of NGF. NGF stimulates the rapid generation of a species of diacylglycerol that is labeled with [3H]myristate but not with [3H]arachidonate. NGF stimulates [3H]myristate- or [32P]phosphate-labeled phosphatidic acid production over the same time course. Although NGF alone has no effect on the turnover of inositol phospholipids, it does stimulate the hydrolysis of glycosylphosphatidylinositol. The NGF-dependent cleavage of this lipid is accompanied by an increase in the accumulation of its polar head group, an inositol phosphate glycan, which is generated within 30-60 sec of NGF treatment. In an unresponsive PC-12 mutant cell line, neither the diacylglycerol nor inositol phosphate glycan response is detected. A possible role for the NGF-stimulated diacylglycerol is suggested by the inhibition of NGF-dependent c-fos induction by staurosporin, a potent inhibitor of protein kinase C. These results suggest that, like insulin, some of the cellular effects of NGF may be mediated by the phospholipase C-catalyzed hydrolysis of glycosylphosphatidylinositol.
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PMID:Nerve growth factor stimulates the hydrolysis of glycosylphosphatidylinositol in PC-12 cells: a mechanism of protein kinase C regulation. 253 12

In order to study the role of protein kinase C in the regulation of tyrosine hydroxylase phosphorylation in PC12 cells, the effects of various agonists on diacylglycerol accumulation in PC12 cells were measured and the ability of these agonists to increase the phosphorylation tyrosine hydroxylase in protein kinase C-deficient cells was evaluated. Bradykinin (10 microM) and elevated extracellular K+ (55 mM) increased the accumulation of [3H]diacylglycerol in PC12 cells that had been prelabeled with [3H]arachidonic acid, and so might be expected to activate protein kinase C in these cells; in contrast, nerve growth factor did not increase diacylglycerol accumulation in PC12 cells. Protein kinase C-deficient PC12 cells were prepared by incubating the cells for 24 h with 1 microM phorbol dibutyrate. This treatment resulted in the loss of approximately 90% of the protein kinase C activity in the cells. Control and protein kinase C-deficient cells were incubated with 32Pi for 90 min and then stimulated with various agonists. 32P-labeled tyrosine hydroxylase was isolated from the cells by polyacrylamide gel electrophoresis and subjected to tryptic hydrolysis. 32P-containing phosphopeptides were separated by two-dimensional thin-layer electrophoresis and chromatography, visualized by autoradiography, and quantitated by scintillation counting Treatment of control cells with phorbol dibutyrate increased the incorporation of 32P into one tryptic phosphopeptide (referred to as T3) in tyrosine hydroxylase. Phorbol dibutyrate did not increase the phosphorylation of this peptide in protein kinase C-deficient cells. Bradykinin or 55 mM K+ increased the incorporation of 32P into four tyrosine hydroxylase phosphopeptides, including peptide T3.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Phosphorylation of tyrosine hydroxylase in protein kinase C-deficient PC12 cells. 257 Mar 73

Synthetic peptide substrates specific for cAMP-dependent protein kinase, protein kinase C, ribosomal S6 kinase, and Ca2+/calmodulin-dependent protein kinases were used to monitor regulation of these protein kinases in digitonin-permeabilized PC12 cells following treatment with nerve growth factor (NGF) and epidermal growth factor (EGF). cAMP-dependent protein kinase was not activated by NGF and EGF. In addition, neither the Ca2+/calmodulin-dependent nor -independent activity of a protein kinase similar to Ca2+/calmodulin kinase II was affected by growth factor treatment. However, protein kinase C was rapidly and transiently activated and ribosomal S6 kinase activity was persistently elevated. Maximal protein kinase C activity was observed after 2 to 5 min of treatment and, subsequently, returned to control levels within 30 to 40 min. In contrast, S6 kinase activity was maximal within 15 min of NGF and EGF addition and was stably maintained for at least 24 hr. In addition to protein kinase C and S6 kinase, NGF and EGF regulated a protein kinase that was maximally elevated after 15 to 30 min and returned to control levels within 3 to 5 hr. This kinase (approximately 100 kDa) failed to bind to a calmodulin affinity column and eluted from a cation exchange column as a single major species that was distinct from S6 kinase activity, which eluted as multiple peaks. The findings indicate that at least three protein kinases are rapidly activated in PC12 cells following treatment with NGF and EGF. The distinct durations of activation of each kinase implicates significantly different roles for each in growth factor signalling in PC12 cells.
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PMID:Detection of nerve growth factor and epidermal growth factor-regulated protein kinases in PC12 cells with synthetic peptide substrates. 278 35

We have used a permeabilized cell assay and a synthetic peptide substrate (KRTLRR) to specifically monitor the activity of protein kinase C in PC12 cells preincubated with nerve growth factor (NGF), epidermal growth factor (EGF), or phorbol esters. Pretreatment of PC12 cells with 1 microM 12-O-tetradecanoylphorbol 13-acetate or 1 microM phorbol dibutyrate stimulated the rate of KRTLRR peptide phosphorylation 4.8- and 2.6-fold, respectively. Furthermore, pretreatment of cells with NGF or EGF transiently increased the KRTLRR peptide kinase activity. Peak stimulations of KRTLRR peptide kinase (1.3-2-fold) were observed after 1-5 min of growth factor treatment and returned to control levels within 15-20 min. The KRTLRR peptide kinase activity fulfilled two criteria of protein kinase C. A synthetic peptide inhibitor of protein kinase C inhibited both growth factor- and phorbol ester-stimulated KRTLRR peptide kinase activity. In addition, growth factors and phorbol esters failed to stimulate KRTLRR peptide kinase activity in cells rendered protein kinase C-deficient by long-term treatment with 1 microM 12-O-tetradecanoylphorbol 13-acetate. In contrast to the transient activation of protein kinase C, ribosomal S6 kinase, assayed with the synthetic peptide RRLSSLRA, was persistently activated by NGF and EGF. The findings indicate that protein kinase C serves an early and transient role in the molecular actions of NGF and EGF in PC12 cells.
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PMID:Regulation of protein kinase C by nerve growth factor, epidermal growth factor, and phorbol esters in PC12 pheochromocytoma cells. 278 93

The effects of phorbol esters were investigated on the survival of chick sympathetic neurons in a serum-free culture medium. The protein kinase C activator phorbol 12,13-dibutyrate (PDB) supported about 40% of the plated sympathetic neurons. This number was comparable to that supported by nerve growth factor (NGF). A combination of phorbol ester and NGF did not significantly increase the number of surviving neurons. Phorbol ester-supported sympathetic neurons possessed desipramine-sensitive [3H]-norepinephrine uptake mechanism, and therefore were noradrenegic in character. Two days after the start of cultures, if NGF was replaced by phorbol ester, or phorbol ester was replaced by NGF, the number of surviving sympathetic neurons was essentially the same in both groups, and the uptake of [3H]norepinephrine was also comparable when examined 2 days after the switchover. Interchangeability between phorbol ester and NGF in the survival of sympathetic neurons suggests that both agents act on the same subpopulation of neurons of the chick sympathetic ganglia. The protein kinase C activity of cytosol and particulate fractions of NGF-supported neurons was 0.14 and 0.09 pmol/min/mg protein, respectively. In phorbol ester-supported neurons the activity in the particulate fraction increased by about fivefold. Removal of the phorbol ester after 2 days resulted in restoration of the enzyme activity in less than 1 h, and readdition of the phorbol ester again increased the activity by fivefold. When NGF was added to these neurons (1 microgram for 15 min), there was no change in the enzyme activity. Phorbol 13-acetate was ineffective in supporting sympathetic neurons in culture, as well as in enhancing protein kinase C activity. We also compared the protein kinase C activity of sympathetic neurons supported in culture by NGF and excess potassium (35 mM K+) Neurons supported in culture by 35 mM K+ for 2 days had almost eightfold more protein kinase C activity in their particulate fraction than in cytosol fraction. In NGF-supported neurons were acutely treated with excess K+, the protein kinase C activity was increased in the particulate fraction by about sevenfold in a concentration- and time-dependent manner. Excess K+ plus phorbol ester did not produce an additive effect on protein kinase C activity. PDB and excess K+ had no effect on cyclic AMP content of sympathetic neurons. In summary, the present data suggest that the neurotrophic action of PDB and excess K+ is probably mediated through protein kinase C.
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PMID:Excess K+ and phorbol ester activate protein kinase C and support the survival of chick sympathetic neurons in culture. 284 60

The role of various intracellular signals and of their possible interactions in the control of neurotransmitter release was investigated in PC12 cells. To this purpose, agents that affect primarily the cytosolic concentration of Ca2+, [Ca2+]i (ionomycin, high K+), agents that affect cyclic AMP concentrations (forskolin; the adenosine analogue phenylisopropyladenosine; clonidine) and activators of protein kinase C (phorbol esters) were applied alone or in combination to either growing chromaffin-like PC12-cells, or to neuron-like PC12+ cells differentiated by treatment with NGF (nerve growth factor). In addition, the release effects of muscarinic-receptor stimulation (which causes increase in [Ca2+]i, activation of protein kinase C and decrease in cyclic AMP) were investigated. Two techniques were employed to measure catecholamine release: static incubation of [3H]dopamine-loaded cells, and perfusion incubation of unlabelled cells coupled to highly sensitive electrochemical detection of released catecholamines. The results obtained demonstrate that: (1) release from PC12 cells can be elicited by both raising [Ca2+]i and activating protein kinases (protein kinase C and, although to a much smaller extent, cyclic AMP-dependent protein kinase); and (2) these various control pathways interact extensively. Activation of muscarinic receptors by carbachol induced appreciable release responses, which appeared to be due to a synergistic interplay between [Ca2+]i and protein kinase C activation. The muscarinic-induced release responses tended to become inactivated rapidly, possibly by feedback desensitization of the receptor mediated by protein kinase C. Muscarinic inactivation was prevented (or reversed) by agents that increase, and accelerated by agents that decrease, cyclic AMP. Agents that stimulate release primarily through the Ca2+ pathway (ionomycin and high K+) were found to be equipotent in both PC12- and PC12+ cells, whereas the protein kinase C activator 12-O-tetradecanoyl-phorbol 13-acetate was approx. 10-fold less potent in PC12+ cells, when administered either alone or in combination with ionomycin. In contrast, the cell binding of phorbol esters was not greatly modified by NGF treatment. Thus control of neurotransmitter release from PC12 cells is changed by differentiation, with a diminished role of the mechanism mediated by protein kinase C.
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PMID:Second-messenger control of catecholamine release from PC12 cells. Role of muscarinic receptors and nerve-growth-factor-induced cell differentiation. 285 Jul 96

The addition of nerve growth factor (NGF) to PC12 cells induces an approximate doubling in the cell surface expression of the Thy-1 glycoprotein and the neural cell adhesion molecule (N-CAM) after 24 h of culture. Although both responses are measured at the same time point, their sensitivity to NGF differed with half-maximal induction of Thy-1 apparent at NGF concentrations (approximately 0.1 ng/ml NGF) that had little effect on N-CAM expression. Phorbol ester derivatives capable of activating Ca2+/phospholipid-dependent protein kinase (protein kinase C) and the calcium ionophore A23187 were found to mimic the NGF induction of Thy-1, but not N-CAM. Similar results were observed when a synthetic diacylglycerol was added to PC12 cell cultures. Increased expression of Thy-1 consequent to phorbol ester, calcium ionophore, or NGF treatment was associated with an increase in the expression of the mRNA species that encodes Thy-1. Increased expression of Thy-1 consequent to all three treatments was also reduced by treatment with the transcription inhibitor cordycepin. Treatment of PC12 cells with high concentrations of phorbol esters was found to inhibit the NGF induction of Thy-1, but not N-CAM. Whereas the above results are consistent with activation of protein kinase C underlying the NGF induction of Thy-1, the same data are not consistent with this pathway being important in the N-CAM response.
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PMID:Comparison of the effects of NGF, activators of protein kinase C, and a calcium ionophore on the expression of Thy-1 and N-CAM in PC12 cell cultures. 289 83

In previous studies, we observed that the stimulatory effect of (Bu)2cAMP on aromatase activity of human adipose stromal cells was markedly attenuated when fetal calf serum was present in the culture medium. To determine whether growth factors may be the inhibitors of (Bu)2cAMP-stimulated aromatase activity in serum, the effects of growth factors and phorbol esters on aromatase activity of human adipose stromal cells in monolayer culture were investigated. Epidermal growth factor (EGF), fibroblast growth factor (FGF), and platelet-derived growth factor (PDGF) were all without effect on aromatase activity when added by themselves, but markedly inhibited aromatase activity stimulated by (Bu)2cAMP. On the other hand, nerve growth factor, multiplication-stimulating activity, relaxin, and insulin had no effect on aromatase activity, either by themselves or in the presence of (Bu)2cAMP. Thus, EGF, PDGF, and FGF can mimic the inhibitory action of fetal calf serum on (Bu)2cAMP-stimulated aromatase activity of these cells. By contrast, none of these substances was capable of mimicking the effect of serum to facilitate the stimulatory action of dexamethasone on aromatase activity of these cells. The phorbol esters phorbol-12-myristate-13-acetate, phorbol-12,13-didecanoate, and phorbol-12,13-diacetate were also capable of facilitating the action of (Bu)2cAMP to stimulate aromatase activity, but had little or no action on dexamethasone-stimulated aromatase activity or when added by themselves. It is concluded that aromatase is under multifactorial regulation in human adipose stromal cells. The activity is induced by glucocorticoids and by agents that stimulate cAMP-dependent protein kinase; the latter effect is potentiated by factors that stimulate protein kinase C, but is suppressed by growth factors such as EGF, FGF, and PDGF, whose actions are believed to be mediated by receptor-linked tyrosine kinase activity.
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PMID:Growth factors suppress and phorbol esters potentiate the action of dibutyryl adenosine 3',5'-monophosphate to stimulate aromatase activity of human adipose stromal cells. 300 2

We have examined phosphorylation of nerve growth factor (NGF) receptor in cultured sympathetic neurons and PC12 cells. Dissociated rat superior cervical ganglion neurons or PC12 cells were incubated with 32Pi to label cellular phosphoproteins. Membrane proteins were solubilized, and NGF receptor proteins were immunoprecipitated with the monoclonal antibody 192-IgG. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography showed that NGF receptor components of Mr = 80,000 and Mr = 210,000 were phosphorylated. Phosphorylation of neither species was affected by treating the cells with NGF or phorbol 12-myristate 13-acetate. When the 80,000-Da protein was subjected to complete trypsin proteolysis and then analyzed by reverse phase liquid chromatography, two 32P-labeled peptides were resolved. The more hydrophobic peptide accounted for most of the 32P and contained only phosphoserine; the other peptide contained phosphoserine and phosphothreonine. No phosphotyrosine was detected in the receptor proteins. When receptor molecules from nonlabeled PC12 cells were immunoprecipitated and then incubated in vitro with [gamma-32P]ATP and the cAMP-independent protein kinase FA/GSK-3, phosphorylation occurred predominantly on serine and to a lesser extent on threonine. However, the immunoprecipitated receptor proteins neither autophosphorylated nor were they detectably phosphorylated by cAMP-dependent protein kinase, casein kinase II, or protein kinase C (the Ca2+/phospholipid-dependent enzyme). We conclude that binding units of the NGF receptor are phosphorylated constitutively in at least two sites in intact cells and that they can be phosphorylated by FA/GSK-3 in vitro.
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PMID:Phosphorylation of nerve growth factor receptor proteins in sympathetic neurons and PC12 cells. In vitro phosphorylation by the cAMP-independent protein kinase FA/GSK-3. 302 30


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