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)

Raf-1 is a serine/threonine kinase which is essential in cell growth and differentiation. Tyrosine kinase oncogenes and receptors and p21ras can activate Raf-1, and recent studies have suggested that Raf-1 functions upstream of MEK (MAP/ERK kinase), which phosphorylates and activates ERK. To determine whether or not Raf-1 directly activates MEK, we developed an in vitro assay with purified recombinant proteins. Epitope-tagged versions of Raf-1 and MEK and kinase-inactive mutants of each protein were expressed in Sf9 cells, and ERK1 was purified as a glutathione S-transferase fusion protein from bacteria. Raf-1 purified from Sf9 cells which had been coinfected with v-src or v-ras was able to phosphorylate kinase-active and kinase-inactive MEK. A kinase-inactive version of Raf-1 purified from cells that had been coinfected with v-src or v-ras was not able to phosphorylate MEK. Raf-1 phosphorylation of MEK activated it, as judged by its ability to stimulate the phosphorylation of myelin basic protein by glutathione S-transferase-ERK1. We conclude that MEK is a direct substrate of Raf-1 and that the activation of MEK by Raf-1 is due to phosphorylation by Raf-1, which is sufficient for MEK activation. We also tested the ability of protein kinase C to activate Raf-1 and found that, although protein kinase C phosphorylation of Raf-1 was able to stimulate its autokinase activity, it did not stimulate its ability to phosphorylate MEK.
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PMID:Reconstitution of the Raf-1-MEK-ERK signal transduction pathway in vitro. 841 57

Tyrosine kinases are involved in cell signalling of growth factors such as insulin and insulin-like growth factor (IGF-I) and others. Insulin and IGF-I receptors which possibly feedback on insulin release are established in insulin-secreting cells. The role of tyrosine kinase in insulin secretion is controversial. Both the tyrosine kinase inhibitors tyrphostin 25 (TYR) and genistein (GEN), but not its structurally similar albeit biologically inactive analogue daidzein, increase insulin release at 16.7 mM glucose in INS-1 cells, an insulin secreting cell line. Tyrosine kinase activity is inhibited by GEN, but not diadzein. The inhibitory effects of either insulin or IGF-I on insulin release are abolished by 10(-4) M GEN but not by daidzein indicating an involvement of tyrosine kinase in the inhibitory effect of both insulin and IGF-I on insulin release. Since GEN was argued not to be specific for tyrosine kinase, several second messengers were investigated. cAMP is not influenced. The insulinotropic effect of acutely administered TPA is not influenced by GEN while in protein kinase C (PKC)-downregulated cells the insulinotropic effect of GEN is preserved: both indicate no involvement of PKC in GEN effect. Since pertussis toxin (PT) pretreatment has no effect on the inhibitory effects of IGF-I on insulin release, a PT-sensitive G-protein is not likely to be involved. The data indicate that tyrosine kinase is involved in the inhibitory effects of insulin and IGF on insulin release in INS-1 cells, possibly mediating the negative feedback effect.
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PMID:Role of tyrosine kinase in insulin release in an insulin secreting cell line (INS-1). 856 11

Angiotensin II (Ang II) is a potent regulator of proximal tubule functions, including transport, metabolism, and cell proliferation. The opossum kidney (OK) cell line is a useful model of renal proximal tubule. Mitogen-activated protein (MAP) kinases are rapidly phosphorylated and activated in response to various agonists. We investigated Ang II effects on serine/threonine kinase cascades in OK cells. The major findings of the present study are that Ang II stimulated MAP kinase kinase (MAPKK), MAP kinase (MAPK), and S6 kinase activities, and that it increased phosphorylation of Raf-1 kinase and p42 MAP kinase in OK cells. These stimulations of kinases were dose-dependent (from 10(-6) to 10(-11) M). The time course of activation was sequential; the peak stimulation was reached at 5 to 10 minutes for Raf-1 kinase, MAPKK and MAPK, and at 20 minutes for S6 kinase. The activation of MAPK was inhibited by approximately 70% with prolonged 24-hour PMA pretreatment or in the presence of calphostin C or H-7. Tyrosine kinase inhibitors (genistein and herbimycin) did not inhibit AngII-induced MAPK activity. This activation of MAPK was also inhibited via AT1 receptor antagonist, Dup753 and pertussis toxin. This evidence suggests that the activation of serine/threonine cascades by Ang II is largely dependent on PMA-sensitive PKC, and is not dependent on tyrosine kinase and pertussis toxin.
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PMID:Sequential activation of MAP kinase cascade by angiotensin II in opossum kidney cells. 858 39

Prolonged stimulation of gonadotropin receptors in granulosa cells leads to desensitization of the cellular response to gonadotropic hormones which is evident by decrease in cAMP formation. In order to explore the mechanism of desensitization and to examine whether protein phosphorylation may play a role in this phenomenon, we have studied the effect of various stimulators and inhibitors of protein phosphorylation on FSH-induced cAMP formation in the FSH-responsive cell line, GFSHR-17, recently established in our laboratory. Both ovine and human FSH activated the hormone sensitive adenylate cyclase in a dose-dependent manner with an ED50 of 0.5 nM. This stimulation was followed by a sharp decrease in cAMP formation after 30 min incubation of the cell with the hormone. When cells were preincubated for 60 min with staurosporine, cAMP accumulation during 20 min of FSH stimulation was elevated about 500%, compared to cells stimulated by FSH alone. Staurosporine alone showed a negligible effect on cAMP accumulation in these cells. In cells stimulated with forskolin, a non-specific activator of adenylate cyclase, or with cholera toxin (CT), an inhibitor of GTPase activity associated with Gs of adenylate cyclase, preincubation with staurosporine increased cAMP formation in these cells by only 50-70 or 80-120%, respectively. Preincubation of cells with the protein kinase C (PKC) inhibitors chelerythrine and GF109203X increased FSH-stimulated accumulation of cAMP by 50 and 30%, respectively. These drugs exhibit a similar effect on forskolin-stimulated cells. Preincubation of cells for 60 min with a PKC stimulator, TPA, suppressed FSH-mediated cAMP response in these cells by 40%. Tyrosine kinase inhibitors such as AG18, AG33 and genistein exhibit a modest inhibitory effect of up to 20% on FSH-stimulated cAMP accumulation. All the above results were obtained both in the presence and absence of IBMX, a potent inhibitor of the cellular phosphodiesterases. Upon prolonged incubation with FSH (3 h) cells pretreated with staurosporine exhibited a much slower rate of decline in intracellular cAMP levels. Moreover, in desensitized cells, following 1 or 2 h of continuous stimulation with FSH, staurosporine could markedly enhance cAMP formation in the presence of FSH. Our data suggest that staurosporine-sensitive phosphorylation of serine or threonine in the FSH receptor-cyclase system may be responsible for desensitization of the FSH coupled activation of cAMP formation, while reactivation of the system can be achieved by protein dephosphorylation at these specific sites. Because specific inhibition of PKC could not mimic the staurosporine effect on FSH-stimulated cAMP formation, nor could activation of kinase C antagonize it, it is suggested that a specific staurosporine-sensitive receptor kinase may be responsible for modulation of the coupling between the gonadotropin receptor and the adenylate cyclase system.
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PMID:Activation of FSH-responsive adenylate cyclase by staurosporine: role for protein phosphorylation in gonadotropin receptor desensitization. 882 63

Peripheral nerve regeneration comprises the formation of axonal sprouts, their outgrowth as regenerating axons and the reinnervation of original targets. This review focuses on the morphological features of axonal sprouts at the node of Ranvier and their subsequent outgrowth guided by Schwann cells or by Schwann cell basal laminae. Adhesion molecules such as N-CAM, L1 and N-cadherin are involved in the axon-to-axon and axon-to-Schwann cell attachment, and it is suggested that integrins such as alpha 1 beta 1 and alpha 6 beta 1 mediate the attachment between axons and Schwann cell basal laminae. The presence of synaptic vesicle-associated proteins such as synaptophysin, synaptotagmin and synapsin I in the growth cones of regenerating axons indicates the possibility that exocytotic fusion of vesicles with the surface axolemma supplies the membranous components for the extension of regenerating axons. Almost all the subtypes of protein kinase C have been localized in growth cones both in vivo and in vitro. Protein kinase C and GAP-43 are implicated to be involved in at least some part of the adhesion of growth cones to the substrate and their growth activity. The significance of tyrosine kinase in growth cones is emphasized. Tyrosine kinase plays an important role in intracellular signal transduction of the growth of regenerating axons mediated by both nerve trophic factors and adhesion molecules. Growth factors such as NGF, BDNF, CNTF and bFGF are also discussed mainly in terms of the influence of Schwann cells on regenerating axons.
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PMID:Peripheral nerve regeneration. 882 47

Several serine/threonine and tyrosine kinase signal transduction pathways have been recently linked to prolactin (PRL) action in lymphoid cells. Utilizing the lactogen-dependent, rat pre-T lymphoma cell line, Nb2-11, and the autonomous subline, Nb2-SFJCD1, studies were conducted to determine whether PRL- or interleukin-2 (IL-2)-stimulated Nb2 cell proliferation is coupled to the activation of p21ras and mitogen-activated protein (MAP) kinase. Stimulation of Nb2-11 cells, growth-arrested in the early G1 phase of the cell cycle, with PRL or IL-2 rapidly (5-10 min) provoked GTP binding to Ras, enhanced tyrosyl phosphorylation of MAP kinase, significantly increased its enzymatic activity, and caused its nuclear translocation. The phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), which directly activates protein kinase C, similarly activated Ras and MAP kinase but failed to cause its nuclear translocation. Tyrosine kinase antagonism with genistein inhibited PRL-stimulated Ras and MAP kinase activation. In other experiments, Ras and MAP kinase were each found to be constitutively active in the Nb2-SFJCD1 line. The addition of PRL to these cultures enhanced the activity of these signaling proteins. Finally, the effects of PRL, IL-2, TPA, and phosphatase inhibition on Nb2-11 cell population density and [3H]thymidine uptake were compared. The addition of PRL, IL-2, and TPA significantly stimulated[3H] thymidine incorporation, while only the polypeptide growth factors augmented cell density. Phosphatase inhibition had no effect on either parameter. These results indicate that Nb2 cell proliferation is associated with the early activation of Ras and MAP kinase. Moreover, tyrosyl phosphorylation upstream of Ras activation appears to be required for its subsequent stimulation of mediators, which activate MAP kinase. Protein kinase C activation may be coupled to MAP kinase activation but is not sufficient for Nb2 cell proliferation.
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PMID:Rapid activation of mitogen-activated protein kinase and p21ras by prolactin and interleukin 2 in rat Nb2 node lymphoma cells. 884

The mechanism by which human cytomegalovirus (HCMV) enters cells is unknown. We sought evidence that protein phosphorylation plays a role in HCMV infection in two ways: (1) by determining whether the degree of phosphorylation of a constitutively phosphorylated 92.5 kDa putative cell membrane receptor for HCMV gH is changed following exposure to HCMV or monoclonal anti-idiotype antibodies (MAb2) that antigenically mimic HCMV gH, and (2) by studying the effects of protein kinase inhibition on receptor phosphorylation and HCMV adsorption or fusion. Phosphorylation of the 92.5 kDa cell membrane protein was specifically increased within 10 min of incubation with HCMV or MAb2 that had been crosslinked by goat anti-mouse antibodies. In addition, fusion of viral envelope with the cell membrane was inhibited by certain protein kinase inhibitors which also inhibited receptor phosphorylation, while the adsorption of [3H]HCMV to human embryonic lung cells was not affected. Tyrosine kinase inhibitors inhibited virus/cell fusion to a greater extent than protein kinase C (PKC) inhibitors, and an inhibitor which primarily affects cAMP and cGMP kinases had little effect. In addition, fusion was stimulated by preincubating cells with agents that stimulated receptor phosphorylation including a phorbol ester, tyrosine phosphatase inhibitor and serine/threonine phosphatase inhibitor. These data indicate that increased phosphorylation of a 92.5 kDa putative cell membrane protein receptor for gH is an early event in response to HCMV, and that cell protein phosphorylation by tyrosine kinase(s) and PKC may facilitate HCMV/cell membrane fusion.
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PMID:Evidence for the role of cell protein phosphorylation in human cytomegalovirus/host cell fusion. 888 96

Cytokine-stimulated expression of inducible type of nitric oxide synthase (iNOS) seems to be regulated by various signal pathways in a cell-specific manner. In this study, we examined how it was regulated in L6 rat skeletal muscle cells. In L6 cells, the combination of interleukin-1 beta and interferon-gamma induced a marked accumulation of nitrite, a stable metabolite of nitric oxide. In parallel with this reaction, iNOS mRNA expression was achieved at a maximum between 3 and 6 h, and iNOS protein was detectable at 6 h and peaked at 24 h after stimulation. Tyrosine kinase inhibitors, herbimycin A, and genistein suppressed cytokine-induced iNOS expression and nitrite production. Forskolin, an adenosine 3',5'-cyclic monophosphate-dependent protein kinase (PKA) activator, and phorbol 12-myristate 13-acetate, a protein kinase C (PKC)-activating phorbol ester, enhanced these cytokine-induced reactions. These results indicate that iNOS expression by cytokines is mediated via a protein tyrosine kinase-dependent pathway and is positively modulated by both PKA- and PKC-dependent pathways in this cell type.
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PMID:Regulation of inducible nitric oxide synthase expression in L6 rat skeletal muscle cells. 903 8

The purpose of this study was to investigate factors regulating melanogenesis in cultured human uveal melanocytes. The effects of various substances on the melanin content, tyrosinase activity and growth of cultured uveal melanocytes were tested. 12-O-tetradecanoyl-phorbol-13-acetate (a protein kinase C activator) and various cAMP-elevating agents, including isobutylmethylxanthine, cholera, toxin, and dibutyryl-cAMP increased melanin content per culture, tyrosinase activity and cell numbers of uveal melanocytes in a dose dependent manner. Basic fibroblast growth factor (tyrosine kinase activator) stimulated growth but did not affect melanin content per culture of uveal melanocytes in vitro. These results indicate that cAMP-elevating agents and protein kinase C activator stimulate melanogenesis and growth of cultured uveal melanocytes. Tyrosine kinase activator stimulates growth but not melanogenesis of cultured uveal melanocytes.
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PMID:Regulation of melanogenesis by human uveal melanocytes in vitro. 919 91

The induction of atrial and ventricular B-type natriuretic peptide (BNP) gene expression is one of the earliest events occurring during hemodynamic overload. To examine the molecular mechanisms for increased BNP gene expression during cardiac overload, we studied the induction of the BNP gene expression compared with that of atrial natriuretic peptide (ANP) in a modified perfused rat heart preparation. An increase in right atrial pressure of 5 mm Hg resulted in a 1.4-fold (P < .05) and 2.2-fold (P < .01) increase in BNP mRNA levels after 1 and 2 hours, respectively, whereas ANP mRNA levels remained unchanged. Stretching for up to 2 hours also significantly increased right atrial immunoreactive BNP (ir-BNP) levels (from 15.8 +/- 2.2 to 20.1 +/- 1.2 ng/mg, P < .05). Actinomycin D (10 micrograms/mL), a transcriptional inhibitor, completely inhibited the stretch-induced increase in atrial BNP mRNA levels at 1 hour (P < .05) and 2 hours (P < .001), whereas a protein synthesis inhibitor, cycloheximide (90 micrograms/mL), had no effect on basal or direct mechanical stretch-induced increase in right atrial BNP mRNA levels. Furthermore, we examined the role of tyrosine kinase and protein kinase C activities in acute mechanical stretch-induced increase in BNP synthesis. Tyrosine kinase inhibitors lavendustin A (1 mumol/L) and tyrphostin A25 (3 mumol/L) and protein kinase C inhibitors staurosporine (30 nmol/L) and chelerythrine (1 mumol/L) prevented the stretch-induced increase in right atrial ir-BNP concentrations at 2 hours. In addition, chelerythrine inhibited the increase of right atrial BNP mRNA levels stimulated by cardiac overload. These resuls demonstrate that the early increase of BNP mRNA levels by mechanical stretch results from increased transcriptional activation and is independent of protein synthesis. Our results also suggest that protein kinase C and tyrosine kinases activities may be involved in coupling cardiac overload to alterations in atrial BNP synthesis.
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PMID:Involvement of transcriptional and posttranscriptional mechanisms in cardiac overload-induced increase of B-type natriuretic peptide gene expression. 935 43


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