Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.1 (protein kinase)
 81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

TSH regulation of insulin and insulin-like growth factor-I (IGF-I) receptor kinases has been studied in FRTL5 cultured thyroid cells. Preincubation of intact cells with TSH increased by 2-fold insulin and IGF-I receptor autophosphorylation and phosphorylation of the p175 endogenous substrate for the receptors. Enhanced phosphorylations reached a maximum within 30 min, were maintained for 30 min more, and vanished after 120 min of TSH incubation. TSH dose-responses exhibited half-maximal and maximal effects at 1 and 10 pM, respectively. In vitro, insulin as well as IGF-I receptors purified from cells treated with 10 pM TSH also exhibited 2-fold enhanced receptor autophosphorylation and kinase activity toward the exogenous substrate poly(Glu,Tyr) (4:1). At variance with TSH, cell incubation with either 8-bromo-cAMP or the protein kinase-C activator 12-O-tetradecanoylphorbol-13-acetate inhibited insulin and IGF-I receptor kinases. In intact cells, TSH stimulation of insulin and IGF-I receptor kinases was accompanied by enhanced turnover of phosphate on autophosphorylated receptors, increased receptor tyrosine phosphorylation, and decreased receptor serine/threonine phosphorylation in response to insulin. Incubation of in vivo labeled insulin and IGF-I receptors with extracts from TSH-treated cells also decreased receptor phosphoserine and phosphothreonine content. Furthermore, preincubation of insulin and IGF-I receptors with extracts from TSH-treated cells enhanced in vitro autophosphorylation. The latter effect was inhibited by the serine/threonine phosphatase inhibitors fluoride and okadaic acid, but not by the tyrosine phosphatase inhibitor vanadate. The data suggest that in FRTL5 cells, TSH induces the activity of a Ser/Thr protein phosphatase, which dephosphorylates insulin and IGF-I receptors and enhances their endogenous kinases.
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PMID:Thyrotropin regulates autophosphorylation and kinase activity in both the insulin and the insulin-like growth factor-I receptors in FRTL5 cells. 131 Dec 44

Insulin-like growth factor-I (IGF-I) stimulates the proliferation of many cell types, including astrocytes. Astrocytes are a population of brain cells highly enriched in IGF-I receptors, which unlike neurons, retain the ability to proliferate in the adult brain. Although astrocyte proliferation in response to IGF-I is well documented, the intracellular mechanisms that mediate this phenomenon are poorly defined. Interestingly, activation of protein kinase-C (PKC) by IGF-I has been observed in several cell types. In this report we first characterized the mitogenic effects of IGF-I on highly purified type I rat astrocyte cultures. Next, we determined whether IGF-I activates PKC in our cultures. Finally, since astrocyte proliferation is stimulated by both IGF-I and the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA), we decided to test the possible involvement of PKC in the mitogenic activity of IGF-I on astrocytes. IGF-I stimulated the DNA synthesis rate in rat astrocytes. Analysis of the time course revealed that IGF-I (10 nM) induces maximal stimulation of [3H]thymidine incorporation (a 4-fold increase) 16-18 h after exposure. TPA also stimulated mitogenesis in our cultures. The dose-response of [3H]thymidine incorporation induced by IGF-I and TPA indicated that 10 nM was the lowest concentration producing a maximal effect for both agents. Analysis of proteins by Western blot revealed that 10 nM IGF-I translocates PKC(alpha), the predominant PKC isoform in astrocyte cultures, from the cytosol to the membrane fraction within 20 min. A similar activation of PKC was achieved with 100 nM TPA. When astrocytes were exposed to IGF-I (10 nM) and TPA (10 nM) in combination, [3H]thymidine uptake was significantly higher than the uptake induced by either IGF-I (10 nM) or TPA (10 nM) alone. However, the effect of IGF-I plus TPA was not fully additive. In a second experiment, the mitogenic effect of IGF-I was partially abolished in cells depleted of PKC by preincubation with high concentrations of TPA (300 nM). Finally, incubation of astrocytes with the PKC inhibitor H-7 at 20 microM, a concentration that completely blocked the mitogenic action of TPA, only reduced the ability of IGF-I to stimulate DNA synthesis by 50%. In summary, our results demonstrate that IGF-I can rapidly activate PKC in astrocytes, and that PKC activation is involved in the mitogenic effect of IGF-I on these cells. However, we conclude that IGF-I also stimulates astrocyte proliferation through PKC-independent pathways.
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PMID:Involvement of protein kinase-C in the mitogenic effect of insulin-like growth factor-I on rat astrocytes. 139 38

We have reported previously that TSH and insulin-like growth factor-I (IGF-I) synergistically stimulate DNA synthesis and elevate the 1,2-diacylglycerol (1,2-DG) content of FRTL-5 thyroid cells and have suggested that protein kinase-C (PKC) may mediate the growth-promoting effects of these hormones. We now present evidence that the effects of TSH on 1,2-DG content are associated with commensurate changes in PKC activity. We measured 1,2-DG content and PKC activity in TSH-deprived growth-arrested cells when TSH was readded. Cells were maintained in medium containing a high dose of insulin (which interacts with IGF-I receptors) and no TSH. When cells incubated in the absence of TSH were reexposed to TSH for 24 h, the 1,2-DG content increased to 234 +/- 22% of the control value, and the ratio of PKC activity in membrane and cytosol fractions of cell homogenates, an index of the state of activation of PKC in situ, increased to 323 +/- 42% of the control value. In cells growing under the influence of TSH in medium containing a high dose of insulin, we found that PKC activity varied during growth. Total cellular PKC activity (3.2 +/- 0.1 nmol/min.micrograms DNA) and the ratio of membrane/cytosol PKC activity (0.24 +/- 0.002) were high during exponential proliferation and fell progressively to 1.1 +/- 0.08 nmol/min.micrograms DNA and 0.12 and fell progressively to 1.1 +/- 0.08 nmol/min.micrograms DNA and 0.12 +/- 0.01, respectively, as cells attained confluence. The specific activity of membrane-associated PKC was 3.0 +/- 0.37 nmol/mg.min in early exponential growth and declined to 0.72 +/- 0.14 nmol/mg.min as cell proliferation ceased. The 1,2-DG content also varied during growth, with a peak occurring during exponential growth, followed by a decline as cells attained a confluent state. These data are consistent with the hypothesis that the growth-promoting effects of TSH in FRTL-5 cells are mediated, at least in part, by 1,2-DG activation of PKC. Since we have demonstrated previously that the effect of TSH to elevate 1,2-DG is, in turn, mediated by cAMP, this represents a special example of the interaction of these two signal transduction systems in regulation of cell proliferation.
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PMID:Protein kinase-C activation during thyrotropin-stimulated proliferation of rat FRTL-5 thyroid cells. 153 8

Fibroblasts represent one of the in vivo sites of insulin-like growth factor-I (IGF-I) production. In this study rat dermal fibroblasts in culture were used as a model system to assess the effect of activation of protein kinase-C on the levels of the mRNAs encoding IGF-I and another growth factor, basic fibroblast growth factor (bFGF). IGF-I and bFGF mRNA levels were determined using a solution hybridization/RNase protection assay. Treatment of cells in serum-free medium containing 0.25% BSA (MEM + BSA) with the tumor-promoting phorbol ester phorbol 12-myristate 13-acetate (PMA) decreased IGF-I and increased bFGF mRNA levels in a time- and dose-dependent fashion. The peak effect of 100 nM PMA on IGF-I mRNA levels occurred at 9 h, whereas the peak effect on bFGF mRNA levels occurred after 3 h of incubation. In dose-response studies, half-maximal inhibition of IGF-I mRNA levels was achieved with approximately 0.08 nM PMA, while half-maximal stimulation of bFGF mRNA levels was achieved with approximately 3 nM PMA. Inhibition of protein synthesis with cycloheximide abrogated the effect of PMA on bFGF mRNA levels, but only partially inhibited the effect of PMA on IGF-I mRNA levels. Studies employing sphingosine or staurosporine to inhibit protein kinase-C or preincubation in high doses of PMA to down-regulate protein kinase-C suggested that the effect of PMA on IGF-I and bFGF mRNA levels was mediated by activation of protein kinase-C, although both staurosporine and sphingosine had independent effects on the levels of these mRNAs and down-regulation of protein kinase-C had a sustained effect on IGF-I mRNA levels. Ligands known to activate protein kinase-C were then tested. Treatment of cells with 100 micrograms/ml of the synthetic diacylglycerol 1-oleoyl-2-acetyl-sn-glycerol decreased IGF-I mRNA levels to 25% and increased bFGF mRNA levels to 520% of the level present in cells maintained in MEM + BSA. Treatment of cells with thrombin or bradykinin also decreased IGF-I mRNA levels and increased bFGF mRNA levels, but whereas the effect of thrombin on IGF-I mRNA levels was marked, the effect of bradykinin was minimal, and whereas the effect of thrombin on bFGF mRNA levels was sustained, the effect of bradykinin was transient.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Activation of protein kinase-C differentially regulates insulin-like growth factor-I and basic fibroblast growth factor messenger RNA levels. 160 84

Recent studies have shown that transforming growth factor-beta (TGF beta) alters DNA synthesis and iodide metabolism in human, porcine, and rat thyroid cells. In the present work we studied the mechanism of TGF beta action in FRTL-5 rat thyroid cells. The cells were treated with TGF beta in the presence of TSH, growth factors, and cellular modulators for various periods of time; then, [3H]thymidine incorporation and DNA content were measured as indicators of DNA synthesis, and [125I]iodide uptake was measured to assess cell function. TGF beta (10 ng/ml) inhibited TSH-induced DNA synthesis and iodide uptake. TGF beta also inhibited DNA synthesis induced by insulin-like growth factor-I, fibroblast growth factor, and endothelial cell growth factor. The protein kinase-A (PKA) activator 8-bromo-cAMP increased both iodide uptake and DNA synthesis; TGF beta inhibited 8-bromo-cAMP-induced [125I]iodide uptake, but not [3H]thymidine incorporation. The protein kinase-C (PKC) activator phorbol 12-myristate 13-acetate increased [3H]thymidine incorporation, and TGF beta inhibited this action of phorbol 12-myristate 13-acetate. The results show that activation of PKA or PKC increases DNA synthesis. TGF beta inhibited PKC-mediated, but not PKA-mediated, DNA synthesis in these cells. The results also show that TGF beta selectively inhibits PKA-mediated iodide uptake, but not PKA-mediated DNA synthesis. These findings suggest that TGF beta is a strong inhibitor of the proliferation and function of thyroid cells.
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PMID:Transforming growth factor-beta blocks protein kinase-A-mediated iodide transport and protein kinase-C-mediated DNA synthesis in FRTL-5 rat thyroid cells. 161 26

Treatment of bovine chromaffin cells with insulin-like growth factor-I (IGF-I) caused the activation of a protein kinase that phosphorylates microtubule-associated protein-2 (MAP-2) in vitro. Activation of MAP-2 kinase by IGF-I varied with the time of treatment (maximal at 10-15 min) and the concentration of IGF-I (maximal at 10 nM). The IGF-I-activated MAP-2 kinase was localized to the soluble fraction of chromaffin cell extracts and required Mg2+ for activity. The IGF-I-activated kinase also phosphorylated myelin basic protein, but had little or no activity toward histones or ribosomal S6 protein. To examine the role of protein tyrosine phosphorylation in the activation of the MAP-2 kinase, we isolated phosphotyrosine (PTyr)-containing proteins from chromaffin cells by immunoaffinity adsorption on anti-PTyr-Sepharose beads. Anti-PTyr-Sepharose eluates from IGF-I-treated cells showed increased MAP-2 kinase activity; thus, the MAP-2 kinase (or a closely associated protein) appears to be a PTyr-containing protein. Treatment of anti-PTyr-Sepharose eluates or crude chromaffin cell extracts with alkaline phosphatase significantly decreased kinase activity toward myelin basic protein, indicating that phosphorylation of the IGF-I-activated kinase is required for its activity.
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PMID:Activation of a microtubule-associated protein-2 kinase by insulin-like growth factor-I in bovine chromaffin cells. 165 24

GH is a major determinant of cytochrome P4502C12 and insulin-like growth factor-I (IGF-I) mRNA expression in rat liver. In the present study, a possible role for protein kinase C (PKC) in the GH-mediated regulation of these two genes was investigated. Addition of bovine GH (bGH) to cultured primary adult rat hepatocytes lead to the formation of diacylglycerol and subsequent induction of P4502C12 and IGF-I mRNA, indicating a PKC-dependent signal transduction. However, stimulation of PKC by phorbol 12-myristate 13-acetate (PMA) or sn-1,2-dioctanoylglycerol treatment, in dose and time-course experiments in the presence or absence of ionomycin, failed to induce either P4502C12 or IGF-I mRNA. On the other hand, down-regulation of PKC by PMA treatment, i.e. 24 h pretreatment, attenuated the bGH induction of both P4502C12 and IGF-I mRNA. One hundred nanomolar PMA reduced the bGH-stimulated expression of both IGF-I mRNA and P4502C12 mRNA (approximately 50%). Treatment with the potent kinase inhibitor staurosporine in combination with bGH caused a dose-dependent decrease of the bGH response with different sensitivities toward the inhibitor for the different mRNA species, IGF-I being less sensitive. These data indicate a permissive role for PKC in the GH-mediated induction of P4502C12 and IGF-I mRNA. When activators of protein kinase A, such as forskolin and 8-Br-cAMP were added to the culture medium opposite effects were observed on the mRNA levels of P4502C12 and IGF-I.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:A role for protein kinases in the growth hormone regulation of cytochrome P4502C12 and insulin-like growth factor-I messenger RNA expression in primary adult rat hepatocytes. 177 Sep 53

Dexamethasone (a synthetic glucocorticoid) inhibited the entry into the S-phase of quiescent chemically transformed mouse fibroblasts (BP-A31) stimulated with 12-O-tetradecanoyl 13-acetate (TPA; a protein kinase-C activator) or with basic fibroblast growth factor. The basal rate of DNA synthesis was also strongly reduced by dexamethasone. In contrast, the mitogenic activity of insulin (acting via the insulin-like growth factor-I receptor) was little or not at all affected by dexamethasone. The antimitogenic activity of dexamethasone was enhanced when the steroid was included in the culture medium 24 h before the addition of mitogens. The effects of dexamethasone were glucocorticoid specific, partially reversed by the antiglucocorticoid RU 486, and prevented by cycloheximide (suggesting the involvement of glucocorticoid-induced protein synthesis in the antimitogenic activity of dexamethasone). Under the conditions of exponential growth in serum-free medium as well as in the presence of TPA, dexamethasone arrested the proliferation of sparsely seeded cells after a delay of 24-48 h. The BP-A31 cells are known to be constitutively competent and express at quiescence certain genes related to the G0/G1 transition in the original nontransformed A31 cell line. Of the transcripts corresponding to these genes, dexamethasone caused a rapid elimination of the JE mRNA, coding for a protein of the family of cytokines. The cell content of c-jun mRNA was also strongly reduced in the cells incubated at quiescence with dexamethasone (in the absence of mitogen). The presence of TPA along with dexamethasone prevented the elimination of c-jun, but not of JE mRNA. Short (30-min; together with the inducers) or long (24-h) treatment of the cells with dexamethasone did not prevent the induction of the c-fos gene expression by either TPA or basic fibroblast growth factor, indicating that dexamethasone does not interfere with mitogenic signal transduction. We conclude that in TPA-stimulated cells, the antiproliferative effect of dexamethasone is not due to interference with the expression of the c-jun gene, but may be related to the decreased level of the JE cytokine mRNA as well as to the synthesis of growth inhibitory protein(s).
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PMID:Antimitogenic effects of dexamethasone in chemically transformed mouse fibroblasts. 190 2

Cultured rat granulosa cells have provided a useful model to examine the hormonal regulation of inhibin secretion. In the present study we have used the cloned rat inhibin alpha- and beta A-subunit cDNAs to characterize the influences of gonadotropins, growth factors, and GnRH on inhibin subunit mRNA levels in granulosa cells obtained from immature estrogen-treated rats. Cells were cultured in medium with or without added hormones. Total RNA from cultured cells was extracted and hybridized with 32P-labeled inhibin alpha- and beta A-subunit cRNA or beta-actin cDNA probes, and inhibin subunit mRNA levels were normalized with beta-actin mRNA levels. Treatment of granulosa cells with FSH increased inhibin alpha- and beta A-subunit mRNA levels in a dose-dependent manner. Similarly, LH, but not PRL, increased alpha- and beta A-subunit mRNA levels in granulosa cells pretreated with FSH to induce functional LH and PRL receptors. The effects of FSH and LH on inhibin subunit mRNA levels were mimicked by forskolin, which increased alpha- and beta A-subunit transcripts in a dose- and time-dependent manner, suggesting involvement of the cAMP-dependent protein kinase-A pathway. Since several growth factors have been shown to influence inhibin secretion, their effects on inhibin subunit mRNA levels were also studied. Treatment of cells with transforming growth factor-beta 1 increased both basal and FSH-stimulated inhibin alpha- and beta A-subunit mRNA content, whereas insulin-like growth factor-I had no significant effect. In contrast, both epidermal growth factor (EGF) and basic fibroblast growth factor (FGF) markedly suppressed both basal and FSH-stimulated inhibin subunit transcript levels. The inhibitory effects of EGF and basic FGF were dose dependent and persisted from 12-72 h of incubation. The regulatory peptide GnRH, which decreases inhibin secretion, was also found to suppress FSH-stimulated inhibin alpha- and beta A-subunit mRNA levels in a dose-dependent manner. Furthermore, the effects of GnRH could be counteracted by coincubation with a GnRH antagonist, suggesting the involvement of specific GnRH-binding sites in GnRH action. These studies indicate that, except for insulin-like growth factor-I, the effects of gonadotropins, growth factors (EGF, basic FGF, and transforming growth factor-beta 1), and GnRH on inhibin secretion are related to their regulation of inhibin alpha- and beta A-subunit mRNA levels.
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PMID:Regulation of inhibin subunit messenger ribonucleic acid levels by gonadotropins, growth factors, and gonadotropin-releasing hormone in cultured rat granulosa cells. 211 34

Relaxin is a member of the insulin family of polypeptide hormones and is known to exert its biological effects on various parts of the mammalian reproductive system. Biologically active human relaxin has been chemically synthesized based on the nucleotide sequence obtained from an ovarian cDNA clone. In the present study synthetic human relaxin was radiolabled by phosphorylation with cAMP-dependent protein kinase and [gamma-32P]ATP to a specific activity of 5000 Ci/mmol. The phosphorylated relaxin was purified on cation exchange high performance liquid chromatography and was shown to co-migrate with relaxin on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Mass spectrometry revealed a single phosphorylated site on the B chain of relaxin. The 32P-relaxin was able to bind to a goat anti-relaxin antibody, and this binding could be displaced by unlabeled relaxin in a concentration-dependent manner. A comparison of the concentration responses of cellular cAMP production stimulated by relaxin and phosphorylated relaxin in a primary human uterine cell line showed that phosphorylation did not affect the in vitro biological efficacy of relaxin. This made it suitable for in situ autoradiographic localization of relaxin binding sites in rat uterine, cervical, and brain tissue sections. Displacement of the binding of 100 pM 32P-relaxin by 100, 10, and 3 nM unlabeled relaxin, but not by 100 nM insulin, insulin-like growth factor-I, and an insulin-like growth factor-I analog, demonstrated the high affinity and specificity of such binding. We conclude that 32P-labeled human relaxin is biologically and immunologically active and that this novel probe binds reversibly and with high affinity to classical (e.g. uterus) and unpredicted (e.g. brain) tissues.
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PMID:Preparation of biologically active 32P-labeled human relaxin. Displaceable binding to rat uterus, cervix, and brain. 216 Sep 76


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