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)

We have examined the cell-free heterologous desensitization of adenylyl cyclase in plasma membrane preparations from S49 wild-type (WT) and kin- cells (which lack cAMP-dependent protein kinase) incubated with purified catalytic subunit of cAMP-dependent protein kinase (cA.PKc). cA.PKc caused a rapid (t1/2 = 40 s) decrease in the hormone responsiveness of adenylyl cyclase in the WT membrane preparations that mimicked the intact cell heterologous desensitization; that is, there was an increase in the Kact for both epinephrine and prostaglandin E1 (PGE1) stimulations of adenylyl cyclase induced at the receptor level because neither forskolin- nor NaF-stimulated activity was affected. The desensitization was independent of agonist occupancy of the receptor, and the effects were blocked both by the active fragment (amino acids 5-22) of the specific inhibitor of cA.PK and by p[NH]ppA. cA.PKc treatment of kin- membranes resulted in a heterologous desensitization that resembled the effects of WT adenylyl cyclase, with the exception that forskolin-stimulated activity was also reproducibly decreased by 24%. cA.PKc had no effect on WT membranes isolated from cells that had previously undergone maximal heterologous desensitization during treatment with 10 microM forskolin. In contrast, cA.PKc-induced heterologous desensitization of kin- membranes was additive with the epinephrine-induced homologous desensitization of intact cells. Cell-free desensitizations were reversed by incubation of membranes with cA.PKc and ADP, conditions that drive the kinase reaction backward. The similarities of our cell-free cA.PKc-mediated heterologous desensitization of adenylyl cyclase with the intact cell desensitization support our hypothesis that heterologous desensitization of the WT lymphoma cells is mediated by cA.PK via a mechanism independent of homologous desensitization.
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PMID:Cell-free heterologous desensitization of adenylyl cyclase in S49 lymphoma cell membranes mediated by cAMP-dependent protein kinase. 254 97

Continuous exposure of cells to neurotransmitter or hormone agonists often results in a rapid desensitization of the cellular response. For example, pretreatment of Chinese hamster fibroblasts (CHW cells) expressing beta 2-adrenergic receptors (beta 2AR) with low (nanomolar) concentrations of isoproterenol, a beta-adrenergic agonist, causes decreases in the sensitivity of the cellular adenylyl cyclase response to the agonist, without changing the maximal responsiveness. In contrast, exposure of CHW cells to high (micromolar) concentrations of isoproterenol results in decreases in both sensitivity and the maximal responsiveness to agonist. To explore the role(s) of receptor phosphorylation in these processes, we expressed in CHW cells three mutant beta 2AR genes encoding receptors lacking putative phosphorylation sites for the cAMP-dependent protein kinase A and/or the cAMP-independent beta 2AR kinase. Using these mutants we found that exposure of cells to low concentrations of agonist appears to preferentially induce phosphorylation at protein kinase A sites. This phosphorylation correlates with the decreased sensitivity to agonist stimulation of the adenylyl cyclase response. At higher agonist concentrations phosphorylation on both the beta 2AR kinase and protein kinase A sites occurs, and only then is the maximal cyclase responsiveness elicited by agonist reduced. We conclude that low or high concentrations of agonist elicit phosphorylation of beta 2AR on distinct domains, with different implications for the functional coupling of the receptors with effector molecules.
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PMID:Phosphorylation sites on two domains of the beta 2-adrenergic receptor are involved in distinct pathways of receptor desensitization. 254 14

The G-protein GS couples hormone-activated receptors with adenylyl cyclase and stimulates increased cyclic AMP synthesis. Transient expression in COS-1 cells of cDNAs coding for the GS alpha-subunit (alpha S) or alpha S cDNAs having single amino acid mutations Gly49----Val or Gly225----Thr elevated cyclic AMP levels, resulting in the activation of cyclic AMP dependent protein kinase. Stable expression in Chinese hamster ovary cells of alpha S Val49 cDNA resulted in a small constitutive elevation of cyclic AMP that was sufficient to persistently activate cyclic AMP dependent protein kinase activity 1.5-2-fold over basal activity. Stable expression of wild-type alpha S or alpha S Thr225 in Chinese hamster ovary cells was less effective in sustaining elevated cyclic AMP synthesis and kinase activation compared to alpha SVal49.
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PMID:Mutation of glycine 49 to valine in the alpha subunit of GS results in the constitutive elevation of cyclic AMP synthesis. 254 81

The molecular basis for heterologous desensitization of the beta-adrenergic receptor (beta AR) was investigated by site-directed mutagenesis of the beta AR protein. Rapid heterologous desensitization of agonist-stimulated adenylyl cyclase activity was observed when L cells expressing the wild-type beta AR were incubated with 50 nM epinephrine. This desensitization response could be mimicked in a cell-free system by incubation with cAMP-dependent protein kinase (cA.PK). Deletion of amino acid residues 259-262 from the beta AR, removing one of the two consensus sequences in the receptor for phosphorylation by cA.PK, abolished the ability of the receptor to undergo rapid heterologous desensitization. In contrast, deletion of the other cA.PK consensus sequence (residues 343-348) or truncation of the Ser/Thr-rich C-terminal tail of the beta AR (deletion of residues 354-418) did not affect this heterologous desensitization process. These results suggest that the action of cA.PK on amino acid residue(s) contained within the sequence 259-262 of the beta AR is required for rapid heterologous desensitization of the receptor in response to agonists.
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PMID:Identification of a specific site required for rapid heterologous desensitization of the beta-adrenergic receptor by cAMP-dependent protein kinase. 255 Jul 73

The exact nature of the interaction(s) between cAMP and calcium-sensitive phospholipid-dependent protein kinase-C effector pathways is not well understood in many tissues, including the ovary. In the present work we have evaluated the ability of protein kinase-C to modulate receptor-and nonreceptor-mediated cAMP generation in acute suspension cultures of swine luteal cells. Cells were exposed to LH (1 micrograms/ml), forskolin (100 microM), cholera toxin (1 microgram/ml), pertussis toxin (100 ng/ml), and/or phorbol ester [12-O-tetradecanoylphorbol-13-acetate (TPA)] for 0-90 min. TPA had no effect on basal cAMP accumulation, but increased (P less than 0.05) LH-, forskolin-, and cholera toxin-activated cAMP formation, with maximal facilitation at 30, 45, and 60 min, respectively. This facilitative effect was robust, as it could be demonstrated in both the presence and absence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (0.5 mM). TPA increased dose-dependent LH (0.1-1 microgram/ml)-, forskolin (3-300 microM-, and cholera toxin (0.3-10 microgram/ml)-stimulated cAMP accumulation. TPA induced a dose-dependent (0.3-30 ng/ml) increase in cAMP accumulation when incubated with the half-maximally effective (ED50) and maximally effective doses of LH (0.8 and 1 microgram/ml, respectively), forskolin (10 and 300 microM), and cholera toxin (0.2 and 3 micrograms/ml). TPA had an ED50 for this functional activation of 6.1 (67% confidence interval, 4.4-9.7) nM. The stimulatory effect of TPA could be mimicked by two synthetic diacylglycerols, 1,2-Dioctanoylglycerol and 1-oleoyl-2-acetylglycerol, but not by inactive phorbol esters. In addition, TPA augmented the stimulatory effect of pertussis toxin when combined with maximally effective doses of LH, forskolin, and cholera toxin. The stimulatory action of TPA on cAMP production was limited to endogenous cellular adenylyl cyclase. Bacterially derived adenylyl cyclase toxin isolated from Bordetella pertussis resulted in a dose-dependent increase in cAMP formation over 60 min, which was not facilitated by phorbol ester. We conclude that stimulatory coupling exists between the calcium-dependent protein kinase-C and cAMP-generating systems in swine luteal cells. This stimulatory coupling is enacted in part at the levels of both the guanine binding and the catalytic subunits of adenylyl cyclase.
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PMID:Facilitative actions of the protein kinase-C effector system on hormonally stimulated adenosine 3',5'-monophosphate production by swine luteal cells. 255 49

As with many other receptor-effector systems, the responsiveness of the beta-adrenergic receptor (beta AR)/adenylyl cyclase system undergoes desensitization upon agonist exposure. Phosphorylations of the receptor by the cAMP-dependent protein kinase (protein kinase A) and the beta AR kinase appear to play roles in such desensitization phenomena, but the functional significance of the receptor phosphorylation in intact cells has not been previously assessed. In this study, we constructed and expressed in a mammalian fibroblast line the normal (wild type) human beta 2 AR and mutant forms of the receptor that lack the putative phosphorylation sites for these two protein kinases. The two consensus sequences for phosphorylation by protein kinase A were altered by changing serines 261, 262 and 345, 346 to alanines. In another mutant, the 11 serines and threonines at the carboxy terminus of the protein that constitute the putative beta AR kinase phosphorylation sites were changed to alanines or glycines. The mutated receptors did not differ from the wild type in their affinities for agonists or antagonists or in their ability to mediate agonist stimulation of adenylyl cyclase. Moreover, their levels of expression in the cultured cells were the same. When stimulated with the beta AR agonist isoproterenol, cells bearing either the wild type or mutant receptors generated cAMP at essentially identical rates for the first 2 min. Cells bearing wild type receptors then showed a rapid desensitization characterized by a markedly diminished rate of cAMP production after the first few minutes of stimulation. However, cells bearing either of the mutated forms of the receptor showed much less desensitization and continued to generate cAMP at a rate 3-4 times greater than that observed in cells expressing the wild type receptor. In contrast, intact cell cAMP levels stimulated by prostaglandin E1 and forskolin were not different between cells bearing wild type or mutant beta AR. These results suggest an important physiological role for phosphorylation of the beta AR in regulating rapid agonist-induced desensitization in intact cells.
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PMID:Altered patterns of agonist-stimulated cAMP accumulation in cells expressing mutant beta 2-adrenergic receptors lacking phosphorylation sites. 255 15

The plasma-membrane ATPase of Saccharomyces cerevisiae is a proton pump whose activity, essential fro proliferation, is subject to regulation by nutritional signals. The previous finding that the CDC25 gene product is required for the glucose-induced H+-ATPase activation suggested that H+-ATPase activity is regulated by cAMP. Analysis of starvation-induced inactivation and glucose-induced activation of the H+-ATPase in mutants affected in activity of the RAS proteins, adenylyl cyclase or cAMP-dependent protein kinase showed that nutritional regulation of H+-ATPase activity does not depend directly on any of these factors. We conclude that adenlyl cyclase does not mediate all nutritional responses. This also indicates that the specific CDC25 requirement for the glucose-induced activation of the H+-ATPase identifies a new function for the CDC25 gene product, a function that appears to be independent of CDC25-mediated modulation of the RAS/adenylyl cyclase/cAMP pathway.
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PMID:cAMP- and RAS-independent nutritional regulation of plasma-membrane H+-ATPase activity in Saccharomyces cerevisiae. 255 50

We report here that, contrary to previously reported findings, treatment of S49 wild-type (WT) lymphoma cells with 0-50 nM epinephrine resulted in a heterologous desensitization of adenylyl cyclase (EC 4.6.1.1)--that is, epinephrine and prostaglandin E1 (PGE1) stimulations of adenylyl cyclase were reduced. Observation of this heterologous desensitization required the assay of adenylyl cyclase with submillimolar concentrations of Mg2+ and low concentrations of epinephrine. Also, whereas previously there had been no evidence for any role of cAMP-dependent protein kinase in the desensitization of the WT beta-adrenergic receptor, our data comparing the characteristics of the desensitization in WT, kin-, and cyc- lymphoma cells [where kin- and cyc- refer to variants of S49 WT cells lacking cAMP-dependent protein kinase activity (kin-) and the alpha subunit of the stimulatory guanine nucleotide-binding regulatory protein (cyc-)] now suggest that cAMP-dependent protein kinase mediates the heterologous desensitization of adenylyl cyclase. Specifically, we found that only the WT cells exhibited epinephrine-induced heterologous desensitization. The kin- and cyc- cells exhibited only homologous desensitization, and much higher concentrations of epinephrine were required to elicit the homologous desensitization in the variants relative to the heterologous desensitization of the WT. Treatment of WT and cyc- cells with dibutyryl cAMP or treatment of WT with forskolin or PGE1 caused the heterologous desensitization of adenylyl cyclase, indicating that neither receptor occupancy nor activation of adenylyl cyclase was necessary for the heterologous desensitization.
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PMID:Activation of cAMP-dependent protein kinase is required for heterologous desensitization of adenylyl cyclase in S49 wild-type lymphoma cells. 283 Jun 19

Skin from hairless mice was incubated with two synthetic retinoids, isotretinoin and etretinate, and the cAMP content as well as the activity of cAMP-dependent protein kinase were determined. A crude plasma membrane preparation was used to measure adenylyl cyclase activity. Neither isotretinoin (10(-6) and 10(-5)M) nor etretinate (10(-6)-10(-4)M) produced any significant changes in adenylyl cyclase activity. Tissue cAMP levels also remained unaltered after treatment with these retinoids. Although the protein kinase activity ratios remained constant over the concentration range of each retinoid, absolute protein kinase activity was stimulated by treatment with etretinate. These data suggest that cAMP may not mediate the action of retinoids in skin, and that the stimulation of protein kinase activity caused by etretinate probably involves an alternative mechanism.
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PMID:Cyclic AMP and cyclic AMP-dependent protein kinase in mouse skin. II. In vitro effects of isotretinoin and etretinate. 300 9

Phosphoprotein patterns in two mutants of Saccharomyces cerevisiae, cdc25-20(ts) and cdc25-20(ts) bcy1, were analysed by two-dimensional polyacrylamide gel electrophoresis. Comparison with the phosphoprotein patterns of the mutants cyr1-2(ts) and bcy1, analysed in a previous study, demonstrated not only that the CDC25 gene product is a positive element in the regulation of adenylyl cyclase activity, as suggested by recent studies, but that it is also a negative element in the phosphorylation of a 31 kDa protein (p31c and p31d), a protein whose phosphorylation is correlated with cell cycle arrest, and dephosphorylation with cell cycle initiation, respectively. Moreover, the phosphorylation phenotype of p31c and p31d suggests that the activity of the CDC25 protein is subject to feedback regulation by cAMP-dependent protein kinase, and that the CDC25 protein is a key element in an ammonium (NH+4) signal-response system.
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PMID:Identification of a 31 kDa protein in Saccharomyces cerevisiae whose phosphorylation is controlled negatively by the CDC25 gene product. 307 84


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