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)

Phosphorylation of the beta-adrenergic receptor (beta AR) is closely associated with homologous desensitization of the beta-adrenergic receptor-coupled adenylate cyclase system. Homologous desensitization and receptor phosphorylation also occur in cell mutants which are deficient in their cAMP-dependent protein kinase (kin- mutant of S49 lymphoma cells). beta AR phosphorylation is mediated by a cAMP-independent protein kinase which phosphorylates the receptor only when it is occupied by a beta-agonist. During the time course of desensitization the beta AR kinase (beta ARK) activity is translocated from a cytoplasmic to a plasma membrane location. beta ARK translocation can also be effected by prostaglandin E1 (PGE1) suggesting that this beta ARK may represent a more general enzyme capable of phosphorylating other adenylate cyclase-coupled receptors. Thus, beta ARK may play a key role in the process of homologous desensitization of adenylate cyclase coupled receptors. Extracellular hormones interact with specific receptors at the outer surface of the plasma membrane and thus initiate a cellular response. One of the best studied transmembrane signalling systems known to be coupled to the occupancy of cell surface receptors is adenylate cyclase. The adenylate cyclase system is composed of various components all of which have been purified to homogeneity (Shorr et al., 1982; Homcy et al., 1983; Benovic et al., 1984; Codina et al., 1984; Northup et al., 1980; Sternweis et al., 1981; Bokoch et al., 1984; Pfeuffer et al., 1985). Initially, agonist binding to the receptor promotes coupling of the occupied receptor to one of the guanine nucleotide binding regulatory proteins. These proteins are members of a family of heterotrimeric proteins consisting of alpha, beta and gamma subunits. Stimulatory receptors like the beta-adrenergic (Cerione et al., 1984) or glucagon (Iyengar et al., 1979) receptors couple to the stimulatory regulatory protein Ns (or Gs) whereas inhibitory receptors like the alpha 2-adrenergic (Jacobs et al., 1976) or M2-muscarinic (Harden et al., 1982) receptors couple to the inhibitory regulatory protein Ni (or Gi). Prolonged exposure to agonist hormones, either stimulatory or inhibitory, results in an attenuation of the response to the hormonal activation, a phenomenon called tachyphylaxis or desensitization (Harden, 1983; Sibley and Lefkowitz, 1985; Sharma et al., 1975). One of the best studied models for desensitization is the beta-adrenergic receptor-coupled adenylate cyclase system. In this system two different forms of desensitization have been characterized.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:The beta-adrenergic receptor kinase: role in homologous desensitization in S49 lymphoma cells. 284 12

Preparations of the catalytic subunit of cAMP-dependent protein kinase from rabbit skeletal muscle, which appear to be homogeneous by SDS-polyacrylamide gel electrophoresis, were often found to contain a hormone-like factor (HLF) which causes an immediate rise, then a decline of intracellular cAMP in a B-lymphoma cell line. Active HLF is released when the fractions that contain it in an inactive form are incubated with cAMP prior to chromatography, or passed through an immobilized cAMP column. HLF seems to be a peptide: it loses its cell-stimulating capability after proteolysis and has an apparent molecular mass of 2.2-2.5 kDa.
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PMID:A cAMP-triggered release of a hormone-like peptide. 284 57

Addition of 12-O-tetradecanoylphorbol-13-acetate (TPA) to S49 lymphoma cells (wild type and a cyclic AMP-dependent protein kinase-lacking clone) has little effect alone but doubles accumulation of cyclic AMP in response to isoproterenol. The effect is immediate and has an apparent affinity and order of potency characteristic of the activation of protein kinase C by phorbol esters. Enhancement does not reflect an altered time course of the beta-adrenergic response, enhanced affinity of the cellular beta-receptor for agonist, or decreased degradation and export of cellular cyclic AMP. Reduction of the beta-adrenergic response by somatostatin does not remove the effect of TPA nor does TPA abolish the effect of somatostatin. Phorbol ester enhances cyclic AMP accumulation in response to cholera toxin in wild type and UNC clones but not in H21a or cyc-. TPA also enhances cAMP accumulation in response to forskolin in wild type cells. The effect of TPA is stable to rapid preparation of membranes. In adenylate cyclase assays on membranes from cells treated with TPA, the activation by guanosine 5'-(beta, gamma-imino)triphosphate is enhanced by 40% with no change in lag time; the effect of beta-agonist plus Gpp(NH)p is similarly enhanced; activation by Mn2+ is unchanged. We conclude that phorbol ester facilitates the productive interaction of the alpha subunit of the transducer protein Gs with the catalytic unit of adenylate cyclase, hypothetically via an action of protein kinase C.
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PMID:Enhancement of adenylate cyclase activity in S49 lymphoma cells by phorbol esters. Putative effect of C kinase on alpha s-GTP-catalytic subunit interaction. 285 14

Agonist-promoted desensitization of adenylate cyclase is intimately associated with phosphorylation of the beta-adrenergic receptor in mammalian, avian, and amphibian cells. However, the nature of the protein kinase(s) involved in receptor phosphorylation remains largely unknown. We report here the identification and partial purification of a protein kinase capable of phosphorylating the agonist-occupied form of the purified beta-adrenergic receptor. The enzyme is prepared from a supernatant fraction from high-speed centrifugation of lysed kin- cells, a mutant of S49 lymphoma cells that lacks a functional cAMP-dependent protein kinase. The beta-agonist isoproterenol induces a 5- to 10-fold increase in receptor phosphorylation by this kinase, which is blocked by the antagonist alprenolol. Fractionation of the kin- supernatant on molecular-sieve HPLC and DEAE-Sephacel results in a 50- to 100-fold purified beta-adrenergic receptor kinase preparation that is largely devoid of other protein kinase activities. The kinase activity is insensitive to cAMP, cGMP, cAMP-dependent kinase inhibitor, Ca2+-calmodulin, Ca2+-phospholipid, and phorbol esters and does not phosphorylate general kinase substrates such as casein and histones. Phosphate appears to be incorporated solely into serine residues. The existence of this novel cAMP-independent kinase, which preferentially phosphorylates the agonist-occupied form of the beta-adrenergic receptor, suggests a mechanism that may explain the homologous or agonist-specific form of adenylate cyclase desensitization. It also suggests a general mechanism for regulation of receptor function in which only the agonist-occupied or "active" form of the receptor is a substrate for enzymes inducing covalent modification.
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PMID:Beta-adrenergic receptor kinase: identification of a novel protein kinase that phosphorylates the agonist-occupied form of the receptor. 287 55

We have used wild-type and variants of the T-lymphoma cell line S49 to explore internalization and down-regulation of adenylate cyclase-linked beta-adrenergic receptors. Internalization was defined by the loss in "surface receptors" detected at 4 degrees C on intact cells by the antagonists [3H]CGP-12177 or [125I]iodocyanopindolol, whereas down-regulation was defined as the loss in total cellular content of receptors [( 125I]iodocyanopindolol binding assayed at 37 degrees C). In wild-type cells, the beta-adrenergic agonist isoproterenol induced a rapid (t 1/2, approximately equal to 1 min) and reversible loss in surface receptors. The surface sites were lost at a rate similar to the rate of desensitization of beta-adrenergic receptor-mediated cyclic AMP generation of S49 cells. A series of S49 variants (cyc-, UNC, H21a) having lesions in NS (the guanine nucleotide binding protein that couples beta-receptors to adenylate cyclase) or with absent cAMP-dependent protein kinase activity (kin-), had a loss in surface sites that was equivalent to that of wild-type cells. By contrast, S49 variant cells having lesions in NS showed variable rates and extents of down-regulation of beta-adrenergic receptors. In wild-type and kin- S49 cells, beta-receptors down-regulated with a t 1/2 of approximately equal to 4 hr. Down-regulation was blunted in the cyc- and UNC variants that have altered coupling of receptors to NS, but it was faster in the H21a variant that retains receptor-NS interaction. Recovery of receptors after down-regulation occurred at a similar rate (t 1/2, approximately equal to 6 hr) in wild-type, UNC, and H21a cells. These results demonstrate that internalization of beta-adrenergic receptors may be necessary, but is not sufficient, to explain agonist-induced receptor down-regulation in S49 cells. The variable expression in the development of down-regulation in S49 variants implies that receptor-NS interaction regulates the fate of receptors linked to the stimulation of adenylate cyclase.
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PMID:Genetic analysis of beta-adrenergic receptor internalization and down-regulation. 298 40

Protein phosphorylation in intact S49 mouse lymphoma cells was studied by using high-resolution two-dimensional gel electrophoresis of proteins labelled with [35S]methionine or [32P]Pi. In wild-type cells substrates for cyclic AMP-stimulatable phosphorylation exhibited high basal phosphorylation; in mutant cells deficient in activities of either cyclic AMP-dependent protein kinase or adenylate cyclase, basal phosphorylation of most of these substrates was negligible. Analysis of tryptic phosphopeptides from proteins labelled with [32P]Pi in wild-type cells suggested that identical sites were phosphorylated under conditions of both basal and hormonally elevated concentrations of cyclic AMP. These results argue that most basal phosphorylation is a consequence of partial activation of cyclic AMP-dependent protein kinase and that this activation is attributable to basal concentrations of cyclic AMP. For the intermediate filament protein vimentin, basal phosphorylation was largely at a site distinct from that stimulated by increased cyclic AMP, and basal phosphorylation was not markedly different in mutant and wild-type cells. Vimentin phosphorylated at both sites was not observed. Cyclic AMP treatment resulted in enhanced phosphorylation at the cyclic AMP-specific site and decreased phosphorylation at the cyclic AMP-independent site.
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PMID:Basal phosphorylation of cyclic AMP-regulated phosphoproteins in intact S49 mouse lymphoma cells. 298 11

High-resolution two-dimensional gel electrophoresis of proteins labeled with either 32Pi or [35S]methionine was used to study interactions between cyclic AMP and tetradecanoyl phorbol acetate (TPA) at the level of intracellular protein phosphorylation. Cultured S49 mouse lymphoma cells were used as a model system, and mutant sublines lacking either the catalytic subunit of cyclic AMP-dependent protein kinase or the guanyl nucleotide-binding "Ns" factor of adenylate cyclase provided tools to probe mechanisms underlying the interactions observed. Three sets of phosphoproteins responded differently to TPA treatment of wild-type and mutant cells: Phosphorylations shown previously to be responsive to activation of intracellular cyclic AMP-dependent protein kinase were stimulated by TPA in wild-type cells but not in mutant cells, a subset of phosphorylations stimulated strongly by TPA in mutant cells was inhibited in wild-type cells, and two novel phosphoprotein species appeared in response to TPA only in wild-type cells. The latter two classes of TPA-mediated responses specific to wild-type cells could be evoked in adenylate cyclase-deficient cells by treating concomitantly with TPA and either forskolin or an analog of cyclic AMP. Three conclusions are drawn from our results: 1) TPA stimulates adenylate cyclase in wild-type cells causing increased phosphorylation of endogenous substrates by cyclic AMP-dependent protein kinase, 2) activated cyclic AMP-dependent protein kinase inhibits phosphorylation (or enhances dephosphorylation) of a specific subset of TPA-dependent phosphoproteins, and 3) cyclic AMP-dependent events facilitate TPA-dependent phosphorylation of some substrate proteins.
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PMID:Interactions between cyclic AMP- and phorbol ester-dependent phosphorylation systems in S49 mouse lymphoma cells. 299 37

From an S49 mouse lymphoma cell subline that carries an electrophoretic marker mutation in one allele for a regulatory (R) subunit of cyclic AMP-dependent protein kinase, 130 cyclic AMP-resistant mutants were isolated and characterized. Of the 77 independent spontaneous and mutagen-induced isolates identified, 74 had kinases with increased apparent activation constants (KaS) for cyclic AMP-dependent activation. The "Ka" phenotype was invariably correlated with an apparent structural lesion in one R subunit allele. "Charge-shift" lesions in 43 independent isolates were mapped to small regions within the R subunit by two-dimensional gel analysis of partial proteolysis peptides. Nine Ka mutations were distinguished by differences in charge or peptide maps of mutant R subunits, and the mutations were clustered in two regions associated with the cyclic AMP-binding sites of the R subunit. The relative frequencies of different mutations differed among spontaneous, ethyl methanesulfonate-induced, and N-methyl-N'-nitro-N-nitrosoguanidine-induced isolates. Mutation frequencies were also markedly different for the two R subunit alleles; this allele preference was strongest for mutagen-induced lesions in the more carboxy terminal cyclic AMP-binding site.
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PMID:Hotspots for spontaneous and mutagen-induced lesions in regulatory subunit of cyclic AMP-dependent protein kinase in S49 mouse lymphoma cells. 300 2

Murine lymphoma cell lines such as WEHI-7 exhibit a cytolytic response to both cAMP and glucocorticoids. We have exploited this behavior to ask if cyclic AMP-dependent protein kinase plays a role in regulating glucocorticoid receptor function. We have found that cAMP-resistant cell lines containing a defective cAMP-dependent protein kinase activity give rise to spontaneous steroid-resistant variants at a high frequency (approximately 10(-7)) relative to wild type cells (less than 10(-10)). Unlike previous results with wild type cells, nearly complete loss of glucocorticoid receptor function was observed in a single selection using unmutagenized cAMPr derivatives of WEHI-7. Thus, the initial selection of the cAMPr phenotype serves as a permissive step toward the acquisition of glucocorticoid resistance in WEHI-7. In addition, cAMP was found to increase the levels of steroid binding in these cell lines, and the dose response was dependent upon the phenotype of the cyclic AMP-dependent protein kinase. The results demonstrate an important role for cAMP in regulating glucocorticoid receptor activity and strongly suggest that this novel two-step selection scheme leads to the isolation of new forms of glucocorticoid resistance.
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PMID:Cyclic AMP-dependent protein kinase promotes glucocorticoid receptor function. 300 79

Virtually all known biological actions stimulated by beta-adrenergic and other adenylate cyclase coupled receptors are mediated by cAMP-dependent protein kinase. Nonetheless, "homologous" or beta-adrenergic agonist-specific desensitization does not require cAMP. Since beta-adrenergic receptor phosphorylation may be involved in desensitization, we studied agonist-promoted receptor phosphorylation during homologous desensitization in wild-type S49 lymphoma cells (WT) and two mutants defective in the cAMP-dependent pathway of beta-agonist-stimulated protein phosphorylation (cyc- cannot generate cAMP in response to beta-adrenergic agonists; kin- lacks cAMP-dependent kinase). All three cell types demonstrate rapid, beta-adrenergic agonist-promoted, stoichiometric phosphorylation of the receptor which is clearly not cAMP mediated. The amino acid residue phosphorylated is solely serine. These data demonstrate, for the first time, that catecholamines can promote phosphorylation of a cellular protein (the beta-adrenergic receptor) via a cAMP-independent pathway. Moreover, the ability of cells with mutations in the adenylate cyclase-cAMP-dependent protein kinase pathway to both homologously desensitize and phosphorylate the beta-adrenergic receptors provides very strong support for the notion that receptor phosphorylation may indeed be central to the molecular mechanism of desensitization.
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PMID:A novel catecholamine-activated adenosine cyclic 3',5'-phosphate independent pathway for beta-adrenergic receptor phosphorylation in wild-type and mutant S49 lymphoma cells: mechanism of homologous desensitization of adenylate cyclase. 300 28


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