Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.13 (protein kinase C)
 49,245 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The cholecystokinin (CCK) receptor on the rat pancreatic acinar cell is a G protein-coupled receptor that is phosphorylated in response to homologous and heterologous agonist stimulation. In this work we have studied the stoichiometry of receptor phosphorylation and have utilized one-dimensional phosphopeptide mapping after cyanogen bromide cleavage to demonstrate that the third intracellular loop is the predominant domain of phosphorylation of this receptor in response to these treatments. Of the average 5 mol of phosphate/mol of receptor, greater than 95% was on the third loop, with the remainder residing on the carboxyl-terminal tail. Serine residues were the site of greater than 95% of phosphorylation, with threonine representing the remainder, and no phosphotyrosine was detected. Further, we have utilized two-dimensional phosphopeptide mapping after subtilisin cleavage to identify differing sites of CCK receptor phosphorylation which are dependent on the agonist utilized to stimulate this cell. Both qualitative and quantitative differences in phosphorylation sites were observed after acinar cell stimulation with different protein kinase C agonists. Further, distinct phosphopeptides on the map were identified as representing substrate(s) of a staurosporine-insensitive kinase activity stimulated only by receptor occupation with native CCK and were felt to represent site(s) of action of a member of the G protein-coupled receptor kinase family. This represents a sensitive and powerful approach that is applicable to sparse receptors residing in their native cellular environment to assess possible differences in patterns of phosphorylation which may be important in agonist-specific receptor regulation.
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PMID:Phosphopeptide mapping of cholecystokinin receptors on agonist-stimulated native pancreatic acinar cells. 753 8

Albeit anthracyclines are widely used in the treatment of solid tumors and leukemias, their mechanism of action has not been elucidated. The present study gives relevant information about the role of nonlamellar membrane structures in signaling pathways, which could explain how anthracyclines can exert their cytocidal action without entering the cell [Tritton, T. R. & Yee, G. (1982) Science 217, 248-250]. The anthracycline daunomycin reduced the formation of the nonlamellar hexagonal (HII) phase (i.e., the hexagonal phase propensity), stabilizing the bilayer structure of the plasma membrane by a direct interaction with membrane phospholipids. As a consequence, various cellular events involved in signal transduction, such as membrane fusion and membrane association of peripheral proteins [e.g., guanine nucleotide-binding regulatory proteins (G proteins and protein kinase C-alpha beta)], where nonlamellar structures (negative intrinsic monolayer curvature strain) are required, were altered by the presence of daunomycin. Functionally, daunomycin also impaired the expression of the high-affinity state of a G protein-coupled receptor (ternary complex for the alpha 2-adrenergic receptor) due to G-protein dissociation from the plasma membrane. In vivo, daunomycin also decreased the levels of membrane-associated G proteins and protein kinase C-alpha beta in the heart. The occurrence of such nonlamellar structures favors the association of these peripheral proteins with the plasma membrane and prevents daunomycin-induced dissociation. These results reveal an important role of the lipid component of the cell membrane in signal transduction and its alteration by anthracyclines.
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PMID:Disruption of cellular signaling pathways by daunomycin through destabilization of nonlamellar membrane structures. 763 36

Parathyroid hormone (PTH) and parathyroid hormone-related peptide (PTHRP) interact with a common G protein-coupled receptor and stimulate production of diverse second messengers (i.e. cAMP, diacylglycerol, and inositol 1,4,5-trisphosphate) that varies depending on the target cell. In renal proximal tubule OK cells, PTH inhibits the activity of the apical membrane Na+/H+ exchanger, although it is unclear whether the signal is transmitted through protein kinase A (PKA) and/or protein kinase C (PKC). To delineate the signaling circuitry, a series of synthetic PTH and PTHRP fragments were used that stimulate the adenylate cyclase-cAMP-PKA and/or phospholipase C-diacylglycerol-PKC pathways. Human PTH-(1-34) and PTHRP-(1-34) stimulated adenylate cyclase and PKC activity, whereas the PTH analogues, PTH-(3-34), PTH-(28-42), and PTH-(28-48), selectively enhanced only PKC activity. However, each peptide fragment inhibited Na+/H+ exchanger activity by 40-50%, suggesting that PKC and possibly PKA were capable of transducing the PTH/PTHRP signal to the transporter. This was corroborated when forskolin and phorbol 12-myristate 13-acetate (PMA), direct agonists of adenylate cyclase and PKC, respectively, both inhibited the Na+/H+ exchanger. The specific PKA antagonist, H-89, abolished the forskolin-mediated suppression of Na+/H+ exchanger activity, but did not prevent the inhibitory effects of PTH-(1-34) or PMA. In comparison, the potent PKC inhibitor, chelerythrine chloride, prevented the inhibition of Na+/H+ exchanger activity mediated by PTH-(28-48) and PMA but did not avert the negative regulation caused by PTH-(1-34) or forskolin. However, inhibition of both PKA and PKC prevented PTH-(1-34)-mediated suppression of Na+/H+ exchanger activity, indicating that PTH-(1-34) acted through both signaling pathways. In addition, Northern blot analysis revealed the presence of only the NHE-3 isoform of the Na+/H+ exchanger in OK cells. In summary, these results demonstrated that NHE-3 is expressed in OK cells and that activation of the PTH receptor can stimulate both the PKA and PKC pathways, each of which can independently lead to inhibition of NHE-3 activity.
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PMID:Parathyroid hormone and parathyroid hormone-related peptide inhibit the apical Na+/H+ exchanger NHE-3 isoform in renal cells (OK) via a dual signaling cascade involving protein kinase A and C. 765 18

With chronic opiate use, opioid receptor desensitization may be one of the important mechanisms underlying the development of opiate tolerance and addiction. Opioid receptors belong to the G protein-coupled receptor superfamily. In this study, the mouse delta-opioid receptor (delta OR) was used in a model system to investigate the role of opioid receptor phosphorylation in receptor desensitization. When expressed in 293 cells and exposed to agonist, the delta OR underwent receptor-specific desensitization within 10 min. This agonist-induced desensitization corresponded temporally to a 3-fold increase in receptor phosphorylation. Phorbol ester, but not forskolin, also stimulated phosphorylation of the delta OR in 293 cells. Although down-regulation of protein kinase C failed to affect agonist-induced receptor phosphorylation, it abolished phorbol ester-induced receptor phosphorylation. Agonist-induced delta OR phosphorylation must therefore involve kinases other than protein kinase C. Whereas overexpression of a dominant negative mutant (K220R) of beta-adrenergic receptor kinase-1 (beta ARK1) in 293 cells significantly reduced agonist-dependent phosphorylation of the delta OR, overexpression of beta ARK1 or G protein-coupled receptor kinase-5 significantly enhanced this phosphorylation. Concordantly, beta ARK1-K220R overexpression reduced agonist-dependent delta OR desensitization, whereas beta ARK1 overexpression enhanced this densensitization. We conclude that short term desensitization of the delta OR involves phosphorylation of the receptor by one or more G protein-coupled receptor kinases.
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PMID:Agonist-dependent phosphorylation of the mouse delta-opioid receptor: involvement of G protein-coupled receptor kinases but not protein kinase C. 765 49

The objectives of the present study were to determine whether the G protein-coupled receptor for PTH and PTH-related protein (PTHrP) is subject to agonist-specific phosphorylation and to characterize the relevant kinase(s). The opossum kidney PTH/PTHrP receptor stably expressed in human embryonic kidney 293 cells was coupled to adenylyl cyclase, with half-maximal activation occurring in the presence of 0.1 nM bovine (b) PTH-(1-34). Immunoprecipitation of extracts of 32P-labeled cells using a monoclonal antibody to the PTH/PTHrP receptor revealed the presence of a major 32P-labeled protein of approximately 85 kilodaltons that was not evident in untransfected 293 cells. bPTH-(1-34) treatment produced a rapid dose-dependent increase in phosphorylation of the 85-kilodalton receptor, with a maximal effect that was 3.5 +/- 0.7-fold (n = 4) over basal. Half-maximal phosphorylation occurred with 10 nM bPTH-(1-34), similar to the hormone concentration required for 50% receptor occupancy. Activation of protein kinase A or protein kinase C with forskolin or phorbol 12-myristate 13-acetate also increased PTH/PTHrP receptor phosphorylation, but to a lesser degree than PTH. Neither of these kinases mediated the effect of PTH, as blockade of the protein kinase A pathway (with H-89) or the protein kinase C pathway (with the bisindolylmaleimide GF 109203X) did not inhibit bPTH-(1-34)-induced PTH/PTHrP receptor phosphorylation. These results suggest that agonist-stimulated PTH/PTHrP receptor phosphorylation may involve a nonsecond messenger-activated kinase, such as a member of the G protein-coupled receptor kinase family.
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PMID:Agonist-stimulated phosphorylation of the G protein-coupled receptor for parathyroid hormone (PTH) and PTH-related protein. 766 44

Cholecystokinin (CCK) is a gastrointestinal hormone that acts through a G protein-coupled receptor to stimulate pancreatic enzyme secretion. In this work, we demonstrate that CCK stimulation of dispersed pancreatic acini results in increased tyrosine phosphorylation of several cellular proteins. This is mediated via a calcium-dependent pathway, also activated by a phenethyl ester analogue of CCK and calcium ionophores, and by a protein kinase C-dependent cascade, also activated by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate. All demonstrable stimulated tyrosine phosphorylation events were inhibited by genistein, with different subsets of proteins affected by staurosporine and H-7. The importance of tyrosine phosphorylation events in agonist-stimulated amylase secretion was studied using genistein and staurosporine as protein kinase inhibitors. Genistein inhibited the secretory response to CCK, its phenethyl ester analogue, and calcium ionophores, all known to stimulate secretion in a calcium-dependent fashion. In contrast, genistein had no effect on the secretory response to 12-O-tetradecanoylphorbol-13-acetate, suggesting that the protein kinase C-dependent tyrosine phosphorylation events were not involved in the secretory mechanism. Furthermore, CCK-induced secretion was not affected by relatively specific protein kinase C inhibition by H-7, but was decreased by staurosporine, an inhibitor of both protein kinase C and tyrosine kinase activities in these cells. These results provide evidence that acinar cell tyrosine phosphorylation is stimulated by agonists acting via calcium-dependent and protein kinase C-dependent pathways, with only the calcium-dependent tyrosine phosphorylation cascade involved in triggering hormone-induced amylase release.
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PMID:A role for cholecystokinin-stimulated protein tyrosine phosphorylation in regulated secretion by the pancreatic acinar cell. 768 71

Beta-adrenergic receptor kinase (beta ARK) is a serine-threonine kinase involved in the process of homologous desensitization of G-coupled receptors. beta ARK is a member of a multigene family, consisting of six known subtypes, also named G protein-coupled receptor kinases (GRK 1-6). In this study we investigated the expression of GRKs during the process of T cell activation, which is of fundamental importance in regulating immune responses. T cell activation was induced by exposing mononuclear leukocytes (MNL) to PHA and confirmed by tritiated thymidine incorporation measurement. A substantial increase of GRK activity (as measured by in vitro phosphorylation of rhodopsin) was found after 48 h (331 +/- 80% of controls) and 72 h (347 +/- 86% of controls) of exposure to PHA. A threefold increase of beta ARK1 immunoreactivity was found in MNL exposed to PHA for 72 h. Persistent activation of protein kinase C (PKC) by 10 nM 12-O-tetradecanoylphorbol-13-acetate (TPA) was able to increase beta ARK activity to the same extent as PHA, suggesting a PKC-mediated mechanism. The kinetic of beta-adrenergic-stimulated cAMP production was substantially modified in TPA and PHA-activated cells, indicating that the increased GRK activity resulted in an increased beta-adrenergic homologous desensitization. A three- to fourfold increase in GRK activity was also observed in a population of T cell blasts (> 97% CD3+) exposed to PHA for 48-72 h. A significant increase in beta ARK1 and beta ARK2 mRNA expression was observed 48 h after mitogen stimulation, while mRNA expression of GRK5 and GRK6 was not changed. In conclusion our data show that the expression of GRK subtypes is actively and selectively modulated according to the functional state of T lymphocytes.
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PMID:Regulation of G protein-coupled receptor kinase subtypes in activated T lymphocytes. Selective increase of beta-adrenergic receptor kinase 1 and 2. 781 17

Acetylcholine (ACh) binding to atrial muscarinic receptors activates an inwardly rectifying K+ current (IK[ACh]) via a pertussis toxin-sensitive GTP-binding protein (GK). The muscarinic K+ channel (termed GIRK1) has been cloned, and the nucleotide sequence contains nine consensus sites for protein kinase C (PKC) phosphorylation (16). Dephosphorylation of the muscarinic K+ channel has been implicated in rapid IK[ACh] desensitization in the presence of agonist (13). Staurosporine is a widely used membrane-permeant inhibitor of PKC and other protein kinases (7), including G protein-coupled receptor kinases. We investigated the role of phosphorylation in the regulation of IK[ACh] by examining the effect of a variety of protein kinase inhibitors. Staurosporine produced a rapid and reversible dose-dependent decrease in IK[ACh], activated by either GTP or guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S). Other PKC inhibitors, including calphostin C and K-252b, were without effect on GTP gamma S-activated IK[ACh]. In excised patches of atrial membrane under nonphosphorylating conditions (0 ATP, 1 mM 5'-adenylylimidodiphosphate), staurosporine reversibly reduced muscarinic K+ channel activity without altering single-channel current amplitude. These results suggest that staurosporine inhibits IK[ACh] by a mechanism independent of intracellular protein kinases.
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PMID:Protein kinase-independent inhibition of muscarinic K+ channels by staurosporine. 817 60

alpha 1-Adrenergic receptors (ARs) are members of the G protein-coupled receptor superfamily. alpha 1-AR subtypes mediate the effects of the sympathetic nervous system, especially those involved in cardiac homeostasis. To investigate signal transduction by a novel subtype (alpha 1D), which we recently cloned, and to compare it with that by the previously characterized alpha 1B-AR, we assessed the ability of each subtype to activate polyphosphoinositide (PI) metabolism, cAMP accumulation, and arachidonic acid release in Chinese hamster ovary (CHO) and COS-1 cells expressing these subtypes after stable or transient transfection, respectively. In COS-1 and CHO cells, both the alpha 1D- and alpha 1B-AR were found to couple to PI hydrolysis through a pertussis toxin-insensitive G protein. Both alpha 1-AR subtypes also increased intracellular cAMP by an indirect mechanism, although this effect was observed only in COS-1 cells and not in CHO cells. Interestingly, alpha 1-AR-stimulated arachidonic acid release was also demonstrated for both subtypes in COS-1 cells. This release was mediated through phospholipase A2 activation and involved a pertussis toxin-sensitive G protein. alpha 1-AR-stimulated arachidonic acid release was dependent upon extracellular calcium and was inhibited by 1 microM nifedipine. Inhibitors of protein kinase C, phospholipase C, and diacylglycerol lipase did not alter alpha 1-AR-stimulated release of arachidonic acid. These findings indicate that in COS-1 cells alpha 1-AR-stimulated arachidonic acid release is most likely coupled to dihydropyridine-sensitive L-type calcium channels via a pertussis toxin-sensitive G protein. The influx of extracellular calcium then stimulates phospholipase A2 to release arachidonic acid. alpha 1-AR-stimulated arachidonic acid release could also be demonstrated in CHO cells and was pertussis toxin sensitive but nifedipine insensitive. These cells were also unresponsive to Bay K8644, indicating a lack of voltage-sensitive calcium channels in CHO cells. Nevertheless, alpha 1-AR activation increased intracellular Ca2+ levels, as assessed by fura-2 fluorescence studies. Neomycin blocked both alpha 1-AR-stimulated PI hydrolysis and increases in intracellular Ca2+ levels but did not inhibit the increase in arachidonic acid release. Taken together, these data indicate that in CHO cells alpha 1-ARs can couple directly to phospholipase A2 activation via a pertussis toxin-sensitive pathway. Thus, in these model systems we demonstrate for the first time that a single alpha 1-AR subtype can activate multiple distinct signal transduction pathways, in which receptor-effector coupling is modulated by distinct G proteins.
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PMID:Coupling of expressed alpha 1B- and alpha 1D-adrenergic receptor to multiple signaling pathways is both G protein and cell type specific. 823 29

The cholecystokinin (CCK) receptor on the rat pancreatic acinar cell is a guanine nucleotide-binding protein (G protein)-coupled receptor, which was recently demonstrated to be phosphorylated in response to agonist stimulation (Klueppelberg et al., J. Biol. Chem. 266: 17744-17746, 1991). In this work, we establish that this receptor is phosphorylated in response to a variety of homologous and heterologous secretagogues and that these phosphorylation events represent action by more than one protein kinase. One subgroup of kinases includes one or more isotype of protein kinase C (PKC), and is capable of playing a role in homologous and heterologous desensitization. A second subgroup of kinases that acts on the CCK receptor was defined by its resistance to 10 microM staurosporine, which was shown to inhibit all PKC in these cells. The activity of the second group of kinases was observed only in response to occupation of the CCK receptor by high concentrations of native hormone, raising the possibility of a "receptor-specific kinase." Similar to the prototypical kinase, beta-adrenergic receptor kinase (beta-ARK), this activity was inhibited in permeabilized cells by heparin. Furthermore, like this enzyme activity, beta-ARK was shown to be resistant to staurosporine. Based on its action on a G protein-coupled receptor, its activation at high concentrations of native agonist, and its pattern of inhibition, we believe that the staurosporine-insensitive CCK receptor kinase activity represents either beta-ARK or a closely related member of the receptor-specific kinase enzyme family.
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PMID:Multiple kinases phosphorylate the pancreatic cholecystokinin receptor in an agonist-dependent manner. 849 11


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