Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

Our previous studies implicated the involvement of protein kinase-A in the inhibitory effects of isoproterenol and relaxin on oxytocin-stimulated phosphoinositide turnover in rat myometrium. To understand the possible mechanisms involved, the properties and regulation of phospholipase-C (PLC) in purified myometrial plasma membranes from estrogen-primed rats were studied. The PLC activity measured with exogenous [3H]phosphatidylinositol 4,5-bisphosphate as substrate was Ca2+ dependent. The nonhydrolyzable GTP analog guanosine 5'-(3-O-thio)triphosphate stimulated PLC activity with a ED50 of 1.6 microM and shifted the calcium dependence curve to the left. Guanosine 5'-(3-O-thio)triphosphate-stimulated phosphatidylinositol 4,5-bisphosphate hydrolysis was inhibited by activation of endogenous and exogenous cAMP-dependent protein kinase (PKA). The effects of endogenous and exogenous PKA were significantly reversed by IP20, a potent synthetic peptide inhibitor of PKA. In the presence of [gamma-32Pi]ATP and exogenous PKA, 32Pi was incorporated in an IP20-sensitive manner into major bands at approximately 17,000, 20,000-24,000, 33,000, 38,000, 40,000-44,000, and other higher mol wt. These data indicate that one or more GTP-binding proteins mediate activation of membrane-bound PLC in rat myometrium. Phosphorylation of one or more membrane-associated proteins by PKA may regulate myometrial PLC activity and play a role in the inhibitory effects of isoproterenol and relaxin.
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PMID:Protein kinase-A inhibits phospholipase-C activity and alters protein phosphorylation in rat myometrial plasma membranes. 132 60

The insulin-like growth factor-binding protein IGF-BP1 is a major secretory protein of human endometrial stromal cells decidualized in culture. Anion exchange chromatography and nondenaturing gel electrophoresis showed IGF-BP1 to exist in five electrophoretically and chromatographically distinct isoforms. IGF-BP1 variants migrated as a quintet on nondenaturing polyacrylamide gels and as a single band (28 kDa) on sodium dodecyl sulfate-polyacrylamide gels. Alkaline phosphatase treatment reduced the IGF-BP1 variants to a single band. Cells incubated with [32P]orthophosphate for 12 h secreted four 32P-labeled IGF-BP1 phosphovariants, and their migration coincided with those bands that were eliminated by alkaline phosphatase treatment. In cells treated with medroxyprogesterone acetate and relaxin, the concentration of phosphorylated IGF-BP1 was increased dramatically as compared with controls. All the phosphovariants were confirmed to be IGF-BP1 by their ability to be supershifted on nondenaturing polyacrylamide gels after binding a monoclonal antibody to IGF-BP1. Thin layer electrophoresis of IGF-BP1 acid hydrolysates showed IGF-BP1 to be phosphorylated exclusively on serine. Non-phosphorylated IGF-BP1 was phosphorylated by the catalytic subunit of the cAMP-dependent protein kinase and casein kinase II in vitro. This suggests that IGF-BP1 may be a substrate of multiple protein kinases in vivo.
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PMID:Insulin-like growth factor-binding protein-1 is phosphorylated by cultured human endometrial stromal cells and multiple protein kinases in vitro. 165 36

Preincubation of Fura 2-loaded rat myometrial cells with H-8, an inhibitor of protein kinase A, for 1 h reversed the inhibitory effects of 8-(4-chlorophenylthio)-cAMP (CPTcAMP) on the oxytocin-stimulated increase in (Ca2+)i (intracellular free calcium), with an EC50 of 47 microM. H-8 also prevented the inhibition by relaxin and isoproterenol of the oxytocin-induced increase in (Ca2+)i. The EC50 of H-8 in reversing the relaxin effect was 42 microM. H-8 reversal of the effect of relaxin on (Ca2+)i was evident both in the absence of extracellular calcium and in cells pretreated with pertussis toxin. H-8 also reversed the inhibitory effects of relaxin and CPTcAMP on the oxytocin-induced increase in [3H]inositol phosphate formation and [3H]phosphoinositide hydrolysis. Preincubation of myometrial cells for 1 h with H-7, another protein kinase inhibitor, only partially attenuated the inhibition by relaxin and CPTcAMP of the oxytocin-induced increase in (Ca2+)i and [3H]inositol phosphate formation at concentrations 4-5 times greater than those of H-8. Acute (15-min) exposure to phorbol myristate acetate (1.0 microM) did not affect basal (Ca2+)i or the oxytocin-stimulated increases in (Ca2+)i or inositol phosphate formation. These results imply a regulatory role for protein kinase A in the inhibition of the oxytocin-induced increase in (Ca2+)i and inositol phosphate formation by relaxants.
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PMID:Involvement of protein kinase A in the regulation of intracellular free calcium and phosphoinositide turnover in rat myometrium. 196 19

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

The effect of the obligatory precursor of prostaglandin biosynthesis, arachidonic acid, on the release of relaxin by porcine luteal cells was examined by use of a reverse hemolytic plaque assay. In this assay, luteal cells were cocultured in monolayers with protein-A-coupled ovine erythrocytes. In the presence of porcine relaxin antiserum and complement, a zone of hemolysis, a plaque, developed around relaxin-releasing luteal cells, identified as large luteal cells (LLCs). The rate of development of plaques in time-course studies and the area of plaques were then used as an index of the rate of relaxin release and cumulative amount of hormone released, respectively. Incubation of collagenase-dispersed luteal cells derived from early pregnant pigs with 0.1-100 microM arachidonic acid (AA) resulted in dose-dependent increases in the rate of plaque formation. Despite AA stimulation, however, only 55-65% of all LLCs formed plaques during the experimental incubation period (up to 12 h). Minimally and maximally effective doses were about 1 and 10 microM, respectively. In the presence of 10 microM AA, maximal plaque formation occurred significantly faster (1-2 h) than in controls (4-8 h; P less than 0.05). The percentage of plaque-forming cells (plaque-forming LLCs) was, likewise, significantly greater in 10 microM AA-treated monolayers than in controls during the first 3-4 h of incubation. Similarly, agents that liberate endogenous AA (phospholipase A2 and melittin) also stimulated relaxin release. The stimulatory effect of AA (10 microM) on relaxin release was almost wholly blocked by a cyclooxygenase inhibitor (ibuprofen; 20 microM); but not by a lipooxygenase inhibitor (nordihydroguaretic acid; 20 microM). However, the same dose of ibuprofen (20 microM) failed to modulate the stimulatory effect of prostaglandin E2 (1 microM) or phorbol diester (4 beta-phorbol 12 beta-myristate 13 alpha-acetate; 50 nM) on the rate of relaxin release. These results indicate that a product(s) of the cyclooxygenase pathway of AA metabolism participates in the control of relaxin release, but that this metabolite(s) is not essential to the biological action of at least one stimulatory secretagogue. Moreover, this metabolite failed to influence a subpopulation of nonresponsive LLCs. These data taken in association with our previous demonstration that the pathways of both calcium mobilization and protein kinase-C activation are implicated in the regulation of relaxin release, are consistent with the view that AA liberation may amplify the actions of other signalling mechanisms.
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PMID:Analysis of relaxin release by cultured porcine luteal cells using a reverse hemolytic plaque assay: effects of arachidonic acid, cyclo- and lipooxygenase blockers, phospholipase A2, and melittin. 250 67

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

Prorenin (Pro) is synthesized in a number of human utero-placental tissues, including chorion, decidua, villous placenta and probably mesenchymal cells. The release of Pro from these extra-renal tissues follows new protein synthesis and appears to utilize the constitutive secretory pathway. Unlike processing in the kidney, very little of the Pro is subsequently cleaved to the smaller product (active renin). Primary signals which regulate Pro include protein hormones and peptides (relaxin, endothelin, hCG), amines (epinephrine, norepinephrine, and related beta adrenergic agents), and eicosanoids. These agents increase the mRNA for prorenin at a time before peak secretory effects are noted. Other extracellular signals have negative regulatory effects. These include angiotensin, endotoxin and cytokines (TNF-alpha and interleukin-1 B). There is also evidence that glucocorticoid receptor activation has an inhibitory effects on Pro release in placenta. Second messengers involved in the regulation of Pro include cyclic AMP and protein kinase A (PKA), protein kinase C (PKC), and calcium. The possible biological effect(s) of the extracellular Pro are unknown but may be due to direct generation of angiotensin I. Since angiotensin-peptides have a number of trophic effect on both vascular and non-vascular tissues, regulation of utero-placental Pro by autocrine, paracrine or endocrine signalling may be critical in normal fetal and/or placental development.
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PMID:Regulation of utero-placental prorenin. 748 44

Relaxin, a hormone that is elevated during pregnancy, can suppress myometrial contractile activity. Ca(2+)-activated K+ channels (KCa) play a role in the modulation of uterine contractions and myometrial Ca2+ homeostasis and have been implicated in the control of smooth muscle excitability. We now show that relaxin stimulates KCa channels in cell-attached patches in a cell line derived from term pregnant human myometrium. This effect was prevented by the protein kinase A (PKA) antagonist, the Rp diastereomer of adenosine 3',5'-cyclic monophosphothioate (Rp-cAMPS). After patch excision, the channel was activated by PKA and inhibited by alkaline phosphatase. These data suggest that relaxin may promote myometrial quiescence in part by stimulation of KCa channels via a PKA-mediated mechanism.
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PMID:Relaxin stimulates myometrial calcium-activated potassium channel activity via protein kinase A. 765 12

Oxytocin increases myometrial intracellular free calcium by promotion of calcium entry and release of calcium from intracellular stores. Calcium release from intracellular stores is secondary to an increase in phosphoinositide (PI) turnover and generation of IP3. We have explored the biochemical basis for the coupling of oxytocin (OT) to phospholipase C (PLC). Rat myometrial membranes contain PLC beta, gamma, and delta isoforms as well as the GTP-binding proteins G alpha(q) and G alpha(11). Oxytocin stimulates both GTPase and PLC activity in rat and human myometrial membranes. These data and available structural information suggest that the oxytocin receptor couples to PLC through a GTP-binding protein. In support of this hypothesis, an antibody generated against the specific C-terminal region of G alpha(q) and G alpha(11) inhibits both the oxytocin-stimulated GTPase and PLC activities. This inhibition is reversed by neutralization of the antibody with the antigenic peptide. The data indicate that the oxytocin receptor couples to PLC, presumably of the beta subclass, via interaction with proteins of the G alpha(q/11) subclass. In the nonpregnant, estrogen-primed rat, the stimulation of PI turnover by oxytocin is inhibited by the hormone relaxin and by pertussis toxin. The effects of both of these agents are mediated by the action of cAMP-dependent protein kinase. In plasma membranes, GTP-stimulated PLC activity can also be inhibited by treatment with protein kinase A. These data suggest that cAMP-dependent phosphorylation at a step involving GTP-binding protein/PLC coupling can exert a negative effect on the stimulation of IP3 formation by oxytocin and thereby affect contraction/relaxation in the myometrium.
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PMID:Mechanisms regulating oxytocin receptor coupling to phospholipase C in rat and human myometrium. 871 99

Relaxin, a reproductive hormone of the insulin-like growth factor family, increases heart rate in experimental animals but its other actions on cardiac function and cellular mechanisms responsible for the positive chronotrophic effect remain unknown. We have studied the actions of human recombinant gene-2 relaxin on the release of atrial natriuretic peptide (ANP) and cardiac function (heart rate, contractile force, perfusion pressure) as well as the underlying signal transduction mechanisms by using the isolated perfused spontaneously beating rat heart preparation. The administration of relaxin into the perfusion fluid at concentrations of 1.5, 3 or 10 nM for 30 min caused a dose-dependent sustained increase in heart rate, while contractile force and perfusion pressure remained unchanged. In addition, infusion of relaxin at a concentration of 10 nM into the perfusate produced a gradual 1.5-fold increase in immunoreactive ANP (IR-ANP) secretion (from 456 +/- 76 to 701 +/- 124 pg/ml, F = 4.5, P < 0.001). The ANP secretory and chronotrophic effects of relaxin appear to involve the activation of protein kinase C, since administration of a protein kinase C inhibitor staurosporine at a concentration of 30 nM completely blocked the effect of relaxin (10 nM) on IR-ANP secretion (P < 0.001) and heart rate (P < 0.001). A cAMP-dependent protein kinase inhibitor, H-89 (100 nM), also substantially reduced the ANP secretory effect of relaxin and attenuated the increase in heart rate during the sustained phase of the relaxin infusion (P < 0.001). KN-62 (3 microM), a Ca2+/calmodulin-dependent protein kinase inhibitor, decreased the positive chronotrophic effect of relaxin (P < 0.001) but did not influence significantly the effect of relaxin on IR-ANP release in isolated perfused rat heart preparation. These results provide the first evidence that relaxin stimulates the secretion of ANP from isolated perfused rat hearts. Our results also suggest that relaxin modulates ANP secretion by activation of protein kinase C and cAMP-dependent protein kinase pathways.
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PMID:Relaxin stimulates atrial natriuretic peptide secretion in perfused rat heart. 888 68


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