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:2.7.11.11 (AMPK)
12,425 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

32P-labeled acetyl-CoA carboxylase was isolated from 32P-labeled rat epididymal fat pads by avidin-Sepharose affinity chromatography after exposure to epinephrine and insulin. Epinephrine led to an inactivation of the isolated enzyme by a reduction of Vmax, while the insulin stimulation observed in crude extracts did not survive enzyme purification. Both insulin and epinephrine caused only small increases in total 32P content of the enzyme. However, mapping of tryptic 32P-phosphopeptides by high performance liquid chromatography revealed that epinephrine and insulin stimulated the phosphorylation of 32P-peptides specific for each hormone. The major 32P-peptide phosphorylated by epinephrine co-migrated with the major 32P-peptide phosphorylated in vitro by the cAMP-dependent protein kinase, while the 32P-peptide phosphorylated in response to insulin co-migrated with that phosphorylated by casein kinase-I and casein kinase-II. The effects of epinephrine on carboxylase activity and phosphorylation can thus be accounted for by the expected epinephrine-induced activation of the cAMP-dependent protein kinase. While the increase in site-specific phosphorylation caused by insulin cannot be directly linked to insulin-induced activation in crude extracts, these data suggest that casein kinase-I and/or casein kinase-II may mediate the insulin-stimulated phosphorylation of acetyl-CoA carboxylase.
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PMID:Stimulation of site-specific phosphorylation of acetyl coenzyme A carboxylase by insulin and epinephrine. 613 73

Epidermal cells contain 4 separate surface receptors which are linked to adenylate cyclase. Activation of any one of these receptors leads to the accumulation of cAMP within the cell which in turn leads to the activation of cAMP-dependent protein kinase. The levels of cAMP accumulation within the cell caused by the 4 activators are not the same. Epinephrine, histamine, adenosine, and prostaglandins of the "E" series cause easily measurable concentrations of cAMP within 5 min of exposure. Prostaglandin F2 alpha causes only a small nonsignificant increase. Similarly, 2 phosphodiesterase inhibitors, which inhibit the breakdown of cAMP formed within the cell, differ in their ability to accumulate cAMP when cells are exposed to these agents alone. Isobutylmethylxanthine causes a measurable increase in cAMP, while theophylline, a weak inhibitor of phosphodiesterase, gives a nonsignificant increase in cAMP. Recently, experiments have shown that agents that give only slight increases in cAMP by biochemical measurements, that is, prostaglandins F2 alpha and theophylline, are equally able to activate protein kinase within the cell. Since activation of protein kinase is the only mechanism for an increase in cAMP to have a physiologic effect, all of these agents that do activate protein kinase should cause physiologic effects. Using an explant culture system, we show in this paper that this supposition is correct and that all agents that activate protein kinase do result in inhibition of mitotic activity regardless of whether or not they are able to raise cAMP to a level that can be biochemically measured as being significantly different from the baseline value.
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PMID:Agents that activate cyclic AMP-dependent protein kinase inhibit explant culture growth and mitotic activity. 619 22

Studies of rat skeletal glycogen metabolism carried out in a perfused hindlimb system indicated that epinephrine activates phosphorylase via the cascade of phosphorylation reactions classically linked to the beta-adrenergic receptor/adenylate cyclase system. The beta blocker propranolol completely blocked the effects of epinephrine on cAMP, cAMP-dependent protein kinase, phosphorylase, and glucose-6-P, whereas the alpha blocker phentolamine was totally ineffective. Omission of glucose from the perfusion medium did not modify the effects of epinephrine. Glycogen synthase activity in control perfused and nonperfused muscle was largely glucose-6-P-dependent (-glucose-6-P/+glucose-6-P activity ratios of 0.1 and 0.2, respectively). Epinephrine perfusion caused a small decrease in the enzyme's activity ratio (0.1 to 0.05) and a large increase in its Ka for glucose-6-P (0.3 to 1.5 mM). This increase in glucose-6-P dependency correlated in time with protein kinase activation and was totally blocked by propranolol and unaffected by phentolamine. Comparison of the kinetics of glycogen synthase in extracts of control and epinephrine-perfused muscle with the kinetics of purified rat skeletal muscle glycogen synthase a phosphorylated to various degrees by cAMP-dependent protein kinase indicated that the enzyme was already substantially phosphorylated in control muscle and that epinephrine treatment caused further phosphorylation of synthase, presumably via cAMP-dependent protein kinase. These data provide a basis for speculation about in vivo regulation of the enzyme.
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PMID:Epinephrine regulation of skeletal muscle glycogen metabolism. Studies utilizing the perfused rat hindlimb preparation. 624 74

Possible inhibitory effects of insulin on epinephrine-induced changes in the enzymes of glycogen metabolism in skeletal muscle were tested using a perfused rat hindlimb preparation. Epinephrine and/or insulin were infused over a wide range of concentrations. Insulin at 6 X 10(-9) M increased the activity ratio (--Glc-6-P/+Glc-6-P) of glycogen synthase from a basal value of 0.09 +/- 0.01 to 0.13 +/- 0.01 and caused a 23% decrease in the Ka for Glc-6-P. In contrast, epinephrine at 10(-7) M decreased the activity ratio to 0.05 +/- 0.01 and increased the Ka for Glc-6-P 6.3-fold. Insulin was without effect on the concentration of cAMP or the activity ratio (-cAMP/+cAMP) of cAMP-dependent protein kinase and caused a small decrease in the activity ratio (-AMP/+AMP) of phosphorylase, whereas epinephrine caused large increases in all these parameters. Insulin at 6 X 10(-11) to 6 X 10(-8) M had no inhibitory effect on the actions of 10(-8) or 10(-7) M epinephrine on glycogen synthase, phosphorylase or cAMP-dependent protein kinase at 30 min or at earlier times. Insulin (6 X 10(-9) M) also did not alter th concentration of cAMP in the presence of 10(-8) or 10(-7) M epinephrine. These data are not consistent with the view that insulin activates glycogen synthase by producing an inhibitor of cAMP-dependent protein kinase. Nor do they support the hypothesis that insulin acts by decreasing the activity of an inhibitor of a multisubstrate phosphoprotein phosphatase.
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PMID:Studies on the interactions between insulin and epinephrine in the control of skeletal muscle glycogen metabolism. 626 Jul 99

Pig epidermal slices were incubated with various compounds which increased epidermal cAMP (adenosine 3',5'-monophosphate), and the change in cAMP-dependent protein kinase activity ratio was studied by the method of Cherrington et al (J Biol Chem 251:5209-5218, 1976) with modification. Epinephrine (5 x 10(-5) M), histamine (10(-4) M) and adenosine (10(-3) M), potent agonists of epidermal adenyl cyclase, fully activated the protein kinase (PK) during an incubation of 30 to 45 seconds, that was much shorter than that required for maximal cAMP accumulation under the same conditions (5 min). With such a brief stimulus, the epidermal cAMP-PK system did not become refractory and responded to repeated stimuli. Prostaglandin E2 (PGE2) and isobuthylmethylxanthine (IBMX) and ethanol only partially activated the enzyme. Prostaglandin F2 alpha (PGF2 alpha) and theophylline which were much less effective in increasing epidermal cAMP, activated the enzyme to the same extent as PGE2 and IBMX respectively. These results suggest that protein kinase activation takes place in response to a cAMP increase in small locus of the cell. Such an increase in cAMP can be very small or even not measurable when measured as total cAMP in the tissue homogenate. Also, increases above this level may not be physiologic. It is concluded that measurement of cAMP-dependent protein kinase activity ratio is a more direct and more sensitive way to study the effect of compounds which act through cAMP mediated mechanisms.
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PMID:Activation of cAMP-dependent protein kinase in epidermis by the compounds which increase epidermal cAMP. 627 Feb 13

Microsomal cytochrome P450c17 catalyzes both steroid 17 alpha-hydroxylase activity and scission of the C17-C20 steroid bond (17,20-lyase) on the same active site. Adrenal 17 alpha-hydroxylase activity is needed to produce cortisol throughout life, but 17,20-lyase activity appears to be controlled independently in a complex, age-dependent pattern. We show that human P450c17 is phosphorylated on serine and threonine residues by a cAMP-dependent protein kinase. Phosphorylation of P450c17 increases 17,20-lyase activity, while dephosphorylation virtually eliminates this activity. Hormonally regulated serine phosphorylation of human P450c17 suggests a possible mechanism for human adrenarche and may be a unifying etiologic link between the hyperandrogenism and insulin resistance that characterize the polycystic ovary syndrome.
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PMID:Serine phosphorylation of human P450c17 increases 17,20-lyase activity: implications for adrenarche and the polycystic ovary syndrome. 747 52

The involvement of adenosine 3',5'-cyclic monophosphate (cAMP) in the stimulation of ventricular protein synthesis by aortic hypertension or adrenergic agonists in the adult rat heart was investigated. In either the retrogradely or anterogradely perfused heart, aortic hypertension increased protein synthesis rates by up to 19%. However, no changes in cAMP concentrations or in cAMP-dependent protein kinase activity ratios could be detected either at early (< 5 min) or late (90 min) time points. Although isoproterenol, 3-isobutyl-1-methylxanthine, or forskolin raised cAMP concentrations (by up to 4.5-fold) and cAMP-dependent protein kinase ratios (by up to 4-fold), protein synthesis rates were not increased; however, under some perfusion conditions, glucagon did stimulate protein synthesis by 25%. Epinephrine stimulated protein synthesis by up to 32%, an effect that was not prevented by propranolol. Phenylephrine also stimulated protein synthesis, an effect that was prevented by prazosin but was unaffected by yohimbine. These findings implicate the alpha 1-adrenoceptor in the regulation of cardiac protein synthesis. Because changes in adenine nucleotide concentrations were similar in hearts perfused with epinephrine or with the agents that raised cAMP, it is unlikely that adenine nucleotide depletion is responsible for the failure to observe effects of the latter group of agents on protein synthesis. Although isoproterenol or forskolin raised cAMP concentrations in isolated ventricular cardiomyocytes where ATP depletion was minimal, neither stimulated protein synthesis. alpha 1-Adrenergic agonists stimulate phosphoinositide hydrolysis in the heart (Brown, J. H., I. L. Buxton, and L. L. Brunton. Circ. Res. 57:532-537, 1985). Aortic hypertension doubled the rate of phosphoinositide hydrolysis in the perfused heart. We suggest that the phosphoinositide-linked signal transduction pathway is more likely to be involved in stimulation of cardiac protein synthesis by hypertension or adrenergic agonism than the adenylyl cyclase/cAMP-linked pathway.
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PMID:cAMP and protein synthesis in isolated adult rat heart preparations. 769 91

In the present study wild-type and various mutant hamster beta 2-adrenergic receptors (beta ARs) expressed in L cells were used to examine potential molecular mechanisms involved in the desensitization of hormonal stimulation of adenylyl cyclase observed after long term exposure to low concentrations of epinephrine. The mutant beta ARs included deletion mutants, D(259-262)beta AR and D(343-348)beta AR, that lack the consensus sites for cAMP-dependent protein kinase (cAPK) and protein kinase C (PKC) and a truncation mutant, T(354)beta AR, that lacks the putative consensus sites for beta AR kinase. Epinephrine stimulation of adenylyl cyclase was desensitized in all four cell lines after growth for 24 hr in the presence of 3 nM epinephrine, and this desensitization was characterized by a 3-4-fold increase in the EC50 for epinephrine stimulation of adenylyl cyclase. In addition, the Vmax was significantly decreased in the cells with the wild-type beta AR and the D(343-348)beta AR. The desensitization was not masked by high concentrations of magnesium and was accompanied by a 40-70% down-regulation of beta ARs. In the cells treated with 3 nM epinephrine, prostaglandin E1 stimulation of adenylyl cyclase was decreased 11% in cells with the wild-type beta AR and forskolin stimulation was decreased 25-36% with all but the D(259-262)beta AR mutant. These results demonstrated that phosphorylations of the cAPK/PKC consensus sites and the serine- and threonine-rich segment of the carboxyl-terminal tail of the beta AR were not required for the desensitization caused by 3 nM epinephrine, thus further differentiating it from cAPK- or PKC-mediated desensitization and the desensitization attributed to beta AR kinase.
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PMID:Beta 2-adrenergic receptor mutants reveal structural requirements for the desensitization observed with long-term epinephrine treatment. 839 17

Tentative identification of the G protein-coupled receptor kinase 2 and 5 (GRK2 and GRK5) sites of phosphorylation of the beta2-adrenergic receptor (betaAR) was recently reported based on in vitro phosphorylation of recombinant receptor (Fredericks, Z. L., Pitcher, J. A., and Lefkowitz, R. J. (1996) J. Biol. Chem. 271, 13796-13803). Phosphorylated residues identified for GRK2 were threonine 384 and serines 396, 401, and 407. GRK5 phosphorylated these four residues as well as threonine 393 and serine 411. To determine if mutation of these sites altered desensitization, we have constructed betaARs in which the threonines and serines of the putative GRK2 and GRK5 sites were substituted with alanines. These constructs were further modified to eliminate the cAMP-dependent protein kinase (PKA) consensus sites. Mutants betaARs were transfected into HEK 293 cells, and standard kinetic parameters were measured following 10 microM epinephrine treatment of cells. The mutant and wild type (WT) receptors were all desensitized 89-94% after 5 min of 10 microM epinephrine stimulation and 96-98% after a 30-min pretreatment. There were no significant changes observed for any of the mutant betaARs relative to the WT in the extent of 10 microM epinephrine-induced internalization (77-82% after 30 min). Epinephrine treatment for 1 min induced a rapid increase in the phosphorylation of the GRK5 and PKA- mutant betaARs as well as the WT. We conclude that sites other than the GRK2 and GRK5 sites identified by in vitro phosphorylation are involved in mediating the major effects of the in vivo GRK-dependent desensitization of the betaAR.
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PMID:Desensitization of beta2-adrenergic receptors with mutations of the proposed G protein-coupled receptor kinase phosphorylation sites. 951 68

Intramuscular triacylglycerol is an important energy store and is also related to insulin resistance. The mobilization of fatty acids from this pool is probably regulated by hormone-sensitive lipase (HSL), which has recently been shown to exist in muscle and to be activated by both adrenaline and contractions. Adrenaline acts via cAMP-dependent protein kinase (PKA). The signalling mediating the effect of contractions is unknown and was explored in this study. Incubated soleus muscles from 70 g male rats were electrically stimulated to perform repeated tetanic contractions for 5 min. The contraction-induced activation of HSL was abolished by the protein kinase C (PKC) inhibitors bisindolylmaleimide I and calphostin C and reduced 50% by the mitogen-activated protein kinase kinase (MEK) inhibitor U0126, which also completely blocked extracellular signal-regulated kinase (ERK) 1 and 2 phosphorylation. None of the inhibitors reduced adrenaline-induced HSL activation in soleus muscle. Both phorbol-12-myristate-13-acetate (PMA), which activates PKC and, in turn, ERK, and caffeine, which increases intracellular Ca2+ without eliciting contraction, increased HSL activity. Activated ERK increased HSL activity in supernatant from basal but not from electrically stimulated muscle. In conclusion, in muscle, PKC can stimulate HSL through ERK. Contractions and adrenaline enhance muscle HSL activity by different signalling mechanisms. The effect of contractions is mediated by PKC, at least partly via the ERK pathway.
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PMID:Contractions activate hormone-sensitive lipase in rat muscle by protein kinase C and mitogen-activated protein kinase. 1279 77


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