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)

Atrial natriuretic peptide (ANP) binds to a transmembrane receptor having intrinsic guanylyl cyclase activity; this receptor has been designated GC-A. Binding of ANP to GC-A stimulates its catalytic activity, resulting in increased production of the second messenger, cyclic GMP. Here we show that GC-A can be expressed in insect cells using a recombinant baculovirus and that the expressed protein retained its abilities to bind ANP and to function as an ANP-activated guanylyl cyclase. In addition, GC-A produced in insect cells was absolutely dependent on the presence of adenine nucleotides for activation by ANP. Millimolar concentrations of ATP were required for optimal activation. The relative potencies of various nucleotides for activation was adenosine 5'-O-(thiotriphosphate) greater than ATP greater than ADP, adenosine 5'-(beta, gamma-imino)triphosphate greater than ADP beta S. AMP had no effect. These studies suggest that binding of an adenine nucleotide, most likely to the protein kinase-like domain of GC-A, is absolutely required for ANP activation. Regulation of guanylyl cyclase activation by adenine nucleotides represents a novel mechanism for the modulation of signal transduction, possibly analogous in some respects to the role of guanine nucleotides and G proteins in the regulation of adenylyl cyclase activity.
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PMID:Adenine nucleotides are required for activation of rat atrial natriuretic peptide receptor/guanylyl cyclase expressed in a baculovirus system. 167 58

We investigated the involvement of second messenger systems in the control by pituitary cytotropic factor (CTF) of tyrosine hydroxylase (TH) expression in primary cultures of hypothalamic cells. Forskolin, an activator of adenylyl cyclase, as well as Sp-cAMP[S] [(Sp)-cyclic adenosine 3',5'-monophosphothioate], a cAMP agonist, and theophylline, an inhibitor of phosphodiesterase activity, stimulate the secretion of dihydroxyphenylalanine (DOPA) and dopamine (DA), suggesting a role for cAMP-dependent protein kinase in the secretion of catecholamines by hypothalamic dopaminergic cells. When cells were cultured with either CTF or forskolin for 14 days, a progressive increase in the secretion of DOPA and DA was observed throughout the period of incubation. At the end of the 2-week culture period, the amount of TH in the cells, determined by immunoblot analysis, was appreciably increased compared to controls. When the cells were analyzed immunocytochemically for TH, the TH-positive cells that had been incubated with CTF or forskolin for 2 weeks were found to have neurites that appeared larger than those of TH-positive cells in the controls. The diameters of the perikarya of TH-positive cells in cultures incubated with CTF also appeared larger than the controls. After incubation of hypothalamic cells with CTF for 96 h, the amount of TH mRNA in the cultures was significantly increased. When membranes isolated from PC12 cells were incubated for 10 min with 50 microM forskolin, the specific activity of adenylyl cyclase was increased 20-fold; CTF had no effect on adenylyl cyclase activity of PC12 cell membranes. Yet, CTF significantly (P less than 0.001) stimulated the secretion of DOPA and DA by PC12 cells. When hypothalamic cells were incubated with both forskolin and CTF, using doses of each that stimulated maximal secretion, the secretion of DOPA and DA was equal to sum of the secretions with each stimulant alone. These additive actions of forskolin and CTF and the failure of CTF to activate adenylyl cyclase in membranes of PC12 cells suggest that forskolin and CTF stimulate catecholamine secretion by hypothalamic dopaminergic cells through different mechanisms, perhaps through different protein kinases. When hypothalamic cells were incubated with CTF and W-7 [N-(6-aminohexyl)5-chloro-1-naphthalenesulfonamide], an inhibitor of calmodulin, the secretion of DOPA was significantly (P less than 0.001) less than that in cultures that were not incubated with W-7. The findings of this study suggest that TH expression in hypothalamic dopaminergic cells is controlled by redundant protein kinases, including cAMP-dependent protein kinase and Ca2+/calmodulin-dependent protein kinase.
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PMID:Tyrosine hydroxylase expression in hypothalamic cells: analysis of the roles of adenosine 3',5'-monophosphate- and Ca2+/calmodulin-dependent protein kinases in the action of pituitary cytotropic factor. 168 36

Although the existence of plasminogen activator (PA) activity and the factors that regulate it in ovarian granulosa cells of both mammalian and avian species have been extensively documented, very little information has been generated concerning the control of PA activity in the adjacent thecal layer. This study was conducted to evaluate the effects of several physiological and pharmacological agents on PA activity in dispersed cells from the thecal layer of the largest preovulatory follicle in the hen ovary 17-16 h before ovulation. LH (50 and 100 ng) in the presence of 3-isobutyl-1-methylxanthine (0.01 mM) stimulated an approximate 25% increase in cell-associated PA activity, possibly via elevated levels of cAMP. Prostaglandin E1 and E2 (PGE1 and PGE2; 0.1 and 1 microM), but not PGI2 or PGF2 alpha (1 microM), enhanced PA activity and cAMP formation, effects that were potentiated by 0.01 mM 3-isobutyl-1-methylxanthine. Activation of Gs with cholera toxin (0.01-10 ng/tube) or adenylyl cyclase with forskolin (0.01-10 microM) stimulated cAMP formation and PA activity in a dose-dependent manner. Exposure of cells to the cAMP analog 8-bromo-cAMP (0.1-5 mM) caused similar increases in thecal cell PA activity. Incubation of cells with phorbol 12-myristate 13-acetate (PMA; 3.2-162 nM), an agonist known to activate protein kinase-C, resulted in a dose-dependent increase in PA activity. However, an equimolar concentration of phorbol 13-monoacetate (162 nM), an inactive analog of PMA that does not activate protein kinase-C, was without effect. Coincubation of cells with forskolin (1 microM) and PMA (32 nM) resulted in a synergistic stimulation of secreted PA activity, apparently via an enhancement of adenylyl cyclase activity. Treatment of cells with the calcium ionophore A23187 (0.01-1 microM) suppressed basal PA activity. However, PA activity stimulated by PMA (32 nM) was synergistically increased after coincubation with a 0.05-microM concentration of A23187, but was inhibited at doses of 0.5 and 1 microM. Taken collectively, the data indicate that PA activity is present in the thecal layer of the largest preovulatory follicle in the ovary of the domestic hen. Furthermore, several endocrine factors (i.e. LH and PGs) were found to stimulate PA activity, possibly via both the adenylyl cyclase-cAMP-protein kinase-A and phosphoinositide-protein kinase-C pathways. In light of these findings, we propose that the preovulatory increase in PGs and LH activates PA in the thecal layer of the largest preovulatory follicle, resulting in proteolytic degradation of the follicular connective tissue and, ultimately, ovulation.
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PMID:Control of plasminogen activator activity in the thecal layer of the largest preovulatory follicle in the hen ovary. 169 Jun 37

G-proteins couple hormonal activation of receptors to the regulation of specific enzymes and ion channels. Gs and Gi are G-proteins which regulate the stimulation and inhibition, respectively, of adenylyl cyclase. We have constructed two chimeric cDNAs in which different lengths of the alpha subunit of Gs (alpha s) have been replaced with the corresponding sequence of the Gi alpha subunit (alpha i2). One chimera, referred to as alpha i(54)/s' replaces the NH2-terminal 61 amino acids of alpha s with the first 54 residues of alpha i. Within this sequence there are 7 residues unique to alpha s, and 16 of the remaining 54 amino acids are nonhomologous between alpha i and alpha s. The second chimera, referred to as alpha i/s(Bam), replaces the first 234 amino acids of alpha s with the corresponding 212 residues of alpha i. Transient expression of alpha i(54)/s in COS-1 cells resulted in an 18- to 20-fold increase in cyclic AMP (cAMP) levels, whereas expression of either alpha i/s(Bam) or the wild-type alpha s polypeptide resulted in only a 5- to 6-fold increase in cellular cAMP levels. COS-1 cells transfected with alpha i showed a small decrease in cAMP levels. Stable expression of the chimeric alpha i(54)/s polypeptide in Chinese hamster ovary (CHO) cells constitutively increased both cAMP synthesis and cAMP-dependent protein kinase activity. CHO clones expressing transfected alpha i/s(Bam) or the wild-type alpha s and alpha i cDNAs exhibited cAMP levels and cAMP-dependent protein kinase activities similar to those in control CHO cells. Therefore, the alpha i(54)/s chimera behaves as a constitutively active alpha s polypeptide, whereas the alpha i/s(Bam) polypeptide is regulated similarly to wild-type alpha s. Expression in cyc-S49 cells, which lack expression of wild-type alpha s, confirmed that the alpha i(54)/s polypeptide is a highly active alpha s molecule whose robust activity is independent of any change in intrinsic GTPase activity. The difference in phenotypes observed upon expression of alpha i(54)/s or alpha i/s(Bam) indicates that the NH2-terminal moieties of alpha s and alpha i function as attenuators of the effector enzyme activator domain which is within the COOH-terminal half of the alpha subunit. Mutation at the NH2 terminus of alpha s relieves the attenuator control of the Gs protein and results in a dominant active G-protein mutant.
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PMID:Mutation of the Gs protein alpha subunit NH2 terminus relieves an attenuator function, resulting in constitutive adenylyl cyclase stimulation. 169 62

Somatostatin (SRIF) reduces growth hormone releasing hormone (GRF)-stimulated growth hormone (GH) release from avian and mammalian adenohypophyseal cells. The present studies examined the intracellular mechanisms mediating SRIF inhibition of GRF-stimulated GH release from chicken pituitary cells. Increases (P less than 0.05) in GH release were observed in the presence of (1) GRF; (2) the adenylyl cyclase stimulator, forskolin; (3) a cAMP analog, 8-bromo-cAMP; (4) the phosphodiesterase inhibitor 3-isobutyl-l-methyl-xanthine (IBMX) combined with GRF; (5) a tumor-promoting phorbol ester and protein kinase C activator, phorbol 12-myristate, 13-acetate (PMA); (6) a diacylglycerol analog, 1,2-dioctanoyl-glycerol (DiC8); and (7) a calcium ionophore, A23187, alone and in combination with PMA. Somatostatin (10 ng/ml) reduced the release of GH stimulated by GRF, forskolin, and 8-bromo cAMP and the GRF-provoked release of GH in the presence of IBMX (P less than 0.05). Somatostatin, however, did not influence GH release in the presence of the protein kinase C activators, PMA or DiC8, or the calcium ionophore A23187. These data suggest that SRIF inhibits GRF-provoked GH release by reducing the ability of the cAMP-protein kinase A but not of the calcium or protein kinase C intracellular message pathways to stimulate GH release.
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PMID:Possible involvement of adenylyl cyclase-cAMP-protein kinase a pathway in somatostatin inhibition of growth hormone release from chicken pituitary cells. 170 26

1. Octimibate, 8-[(1,4,5-triphenyl-1H-imidazol-2-yl)oxy]octanoic acid, is reported to have antithrombotic properties. This is in addition to its antihyperlipidaemic effects which are due to inhibition of acylCoA:cholesterol acyltransferase (ACAT). The aim of this study was to investigate the mechanism of the antithrombotic effect of octimibate, and to determine whether the effects of octimibate are mediated through prostacyclin receptors. 2. In suspensions of washed (plasma-free) human platelets, octimibate is a potent inhibitor of aggregation; its IC50 is approx. 10 nM for inhibition of aggregation stimulated by several different agonists, including U46619 and ADP. The inhibitory effects of octimibate on aggregation are not competitive with the stimulatory agonist; the maximal response is suppressed but there is no obvious shift in potency of the agonist. In platelet-rich plasma, octimibate inhibits agonist-stimulated aggregation with an IC50 of approx. 200 nM. 3. Octimibate also inhibits agonist-stimulated rises in the cytosolic free calcium concentration, [Ca2+]i, in platelets. Both Ca2+ influx and release from intracellular stores are inhibited. The effects of octimibate on aggregation and [Ca2+]i are typical of agents that act via elevation of adenosine 3':5'-cyclic monophosphate (cyclic AMP). Similar effects are seen with forskolin, prostacyclin (PGl2) and iloprost (a stable PGl2 mimetic). 4. Octimibate increases cyclic AMP concentrations in platelets and increases the cyclic AMP-dependent protein kinase activity ratio. Octimibate stimulates adenylyl cyclase activity in human platelet membranes, with an EC50 of 200 nM. The maximal achievable activation of adenylyl cyclase by octimibate is 60% of that obtainable with iloprost. Octimibate has no effect on the cyclic GMP-inhibited phosphodiesterase (phosphodiesterase-ITI), which is the major cyclic AMP-degrading enzyme in human platelets.5. Octimibate inhibits, apparently competitively, the binding of [3H]-iloprost (a stable PGl2 mimetic) to platelet membranes; the estimated Ki is 150 nm. 6. The platelets of different species show considerable differences in the apparent potency of their inhibition of aggregation by octimibate; platelets from cynomolgus monkeys are 3 fold more sensitive than those from humans, while rat, cat and cow platelets are 50, 100, and 250 fold less sensitive than human platelets. The sensitivity of these different species to iloprost, however, varies over a range of only 10 fold with no obvious difference between primates and non-primates. 7. Octimibate appears to be a potent agonist (aggregation), or partial agonist (adenylyl cyclase), at prostacyclin receptors and is the first non-prostanoid agent of this type to be identified. The species differences in relative potency of octimibate and iloprost may reflect the existence of receptor subtypes.
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PMID:Octimibate, a potent non-prostanoid inhibitor of platelet aggregation, acts via the prostacyclin receptor. 171 May 26

These studies examined the cellular basis for the inhibitory effects of triiodothyronine (T3) on growth hormone-releasing factor (GRF)-evoked growth hormone (GH) release from chicken anterior pituitary cells in vitro. A primary monolayer culture of anterior pituitaries from 4- to 8-week-old White Leghorn cockerels was performed as previously described by this laboratory. Following a 72-hr preincubation period, cells were washed and incubated (2 hr) with either secretagogues or media alone (control). T3 (20 ng/ml) or vehicle was added to cells during both the preincubation (48-72 hr) and incubation (2 hr period. Triiodothyronine reduced (P less than 0.05) GH release (ng/ml) in response to (1) GRF; (2) the adenylyl cyclase stimulator, forskolin; (3) the cAMP analog and protein kinase A activator, 8-bromo cAMP; and (4) the phorbol ester and protein kinase C activator, phorbol 12-myristate 13-acetate. Triiodothyronine reduced (P less than 0.05) the intracellular content of GH and total GH (released GH and intracellular GH) irrespectively of whether secretagogues were also present. When GH release was expressed as a percentage of total GH [released GH/(intracellular GH + released GH)], percentage GH released in response to GRF, or the protein kinase A, protein kinase C, or calcium pathway activators was not as great in T3-treated versus non-T3-treated cells. These data indicate that T3 inhibits GRF-evoked GH release by reducing the availability of intracellular stores of GH and by also inhibiting second messenger-stimulated GH release pathways.
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PMID:Triiodothyronine (T3) inhibition of growth hormone secretion by chicken pituitary cells in vitro. 172 15

1. The effects of the protein kinase inhibitors H-7 and H-8 were investigated on diastolic depolarization of the action potential with microelectrodes and on the pacemaker current if with the two-microelectrode voltage clamp in canine cardiac Purkinje fibres. 2. Both 200 microM-H-7 and 100 microM-H-8 had no significant effect on the slope of diastolic depolarization but eliminated the actions of isoprenaline (1 microM). 3. We examined the actions of H-7 and H-8 on if in the presence and absence of isoprenaline. H-7 (200 microM) shifted the pacemaker current if in the negative direction on the voltage axis, whereas 100 microM-H-8 had no significant effect by itself. Both 200 microM-H-7 and 100 microM-H-8 can reverse or prevent the actions of isoprenaline (1-5 microM) on if. 4. We applied activators of the cyclic AMP cascade down-stream to the beta-receptor, to further evaluate where H-7 and H-8 might be exerting their effects. When exposing Purkinje fibres to an adenylyl cyclase activator (forskolin, 10-50 microM), a phosphodiesterase inhibitor (IBMX, 100 microM) and a permeable cyclic AMP analogue (8-chlorophenylthio-cyclic AMP, 200 microM-1 mM), the amplitude of if was increased. H-7 and H-8 at 100-200 microM eliminated each of these actions. 5. These results suggest that a phosphorylation process is involved in the modulation of the pacemaker current, if, in Purkinje fibres. The different actions of H-7 and H-8 on basal if suggest the hypothesis that other protein kinases, possibly protein kinase C, might also be involved in regulating basal phosphorylation of if in Purkinje fibres.
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PMID:Effects of protein kinase inhibitors on canine Purkinje fibre pacemaker depolarization and the pacemaker current i(f). 180 68

Plasma membrane receptors that couple to guanine nucleotide-binding regulatory proteins (G proteins) undergo a variety of rapid (minutes) and longer term (hours) regulatory processes induced by ligands. For the beta 2-adrenergic receptor (beta 2AR), the rapid processes include functional desensitization, mediated by phosphorylation of the receptor by the cAMP-dependent protein kinase and the beta-adrenergic receptor kinase, as well as a loss of hydrophilic ligand binding proposed to represent sequestration of receptors into a cellular compartment distinct from the plasma membrane. The slower processes include beta 2AR down-regulation (i.e., a decrease in the total cellular complement of receptors). It is not yet known whether beta 2AR phosphorylation and/or sequestration are prerequisites for down-regulation of the receptor. Like other G protein-coupled receptors, the beta 2AR molecule spans the plasma membrane seven times, and the cytoplasmic carboxyl-terminal domain has been proposed to contain molecular determinants for each of these regulatory processes. We replaced four serine and threonine residues located within a 10-amino acid segment of this domain of beta 2AR and thereby prevented agonist-promoted phosphorylation, sequestration, and rapid desensitization of the adenylyl cyclase response. In contrast, these mutations did not affect functional coupling to the stimulatory G protein Gs or long-term down-regulation. These findings thus define a small, hitherto unappreciated region of the receptor molecule that may selectively subserve its rapid regulation. In addition, with the demonstration that beta 2AR does not have to be phosphorylated or sequestered in order to enter the down-regulation pathway, the results suggest that the classical receptor endocytosis model may not be appropriate for beta 2AR regulation.
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PMID:A small region of the beta-adrenergic receptor is selectively involved in its rapid regulation. 184 41

Human SK-N-MC neurotumor cells express beta 1- but not beta 2-adrenergic receptors. Following exposure of the cells to isoproterenol, there was no reduction in the maximum response of adenylyl cyclase to the agonist but a 3-fold shift to less sensitivity in the concentration response. This desensitization was very rapid and dose dependent; half-maximal effects occurred at 10 nM isoproterenol. A similar shift was observed when membranes from control cells were incubated with ATP and the catalytic subunit of cyclic AMP-dependent protein kinase (PKA). No shift, however, was observed in intact cells exposed to either dibutyryl cyclic AMP or dopamine, which stimulates adenylyl cyclase in these cells through D1 dopamine receptors. To pursue the role of protein kinases in the desensitization process, cells were made permeable, loaded with a PKA inhibitor or with heparin, an inhibitor of the beta-adrenergic receptor kinase (beta ARK), and exposed to isoproterenol. The PKA inhibitor but not heparin blocked the agonist-mediated desensitization. In contrast, desensitized human tumor cells (HeLa and A431), which express beta 2-adrenergic receptors, exhibited both a shift in concentration response and a reduction in maximum response; the former was blocked by the PKA inhibitor and the latter by heparin. Our results indicated that whereas both human beta 1- and beta 2-adrenergic receptors are susceptible to PKA, only the beta 2 receptors are susceptible to beta ARK. These differences in desensitization may be due to differences in receptor structure as the human beta 1 receptor has fewer potential phosphorylation sites for beta ARK in the carboxyl terminus than the human beta 2 receptor.
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PMID:Desensitization of the human beta 1-adrenergic receptor. Involvement of the cyclic AMP-dependent but not a receptor-specific protein kinase. 185 Apr 9


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