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)

Using homologous probes for the cloning of related genes within the family of guanine nucleotide-binding protein-coupled receptors, we have cloned the gene for the rhesus macaque D1 dopamine receptor. By using the rat D1 receptor coding sequence as a probe under high stringency conditions, the rhesus D1 receptor gene was isolated from a lambda EMBL3 rhesus genomic DNA library. The rhesus D1 dopamine receptor gene is intronless and encodes a 446-amino acid protein that contains two consensus sites for asparagine-linked glycosylation (Asn-5 and Asn-176) and two consensus sites for cAMP-dependent protein kinase phosphorylation (Thr-136 and Thr-268). The primary amino acid sequence of the rhesus D1 dopamine receptor shows an extremely high degree of similarity (99.6%) to the human D1 receptor. Genomic DNA analyses conducted with high and reduced stringency hybridizations indicate that the rhesus macaque D1 receptor is a member of a large multigene family. Like the human D1 receptor mRNA, the rhesus D1 receptor mRNA is approximately 4 kilobases in size and is localized predominantly in the caudate, with lesser amounts in the hippocampus and cortex. The rhesus D1 receptor coding region was inserted into the cytomegalovirus promoter-driven expression vector pcDNA-1, and the recombinant (pcDNA-D1) was cotransfected with the selectable marker pRSVneo, conferring G418 resistance, into D1 receptor-deficient C6 glioma cells. Analyses of the selected transfectant demonstrate the expression of a high affinity, functional D1 dopamine receptor. The D1 receptor radioligand [3H]SCH 23390 bound transfectant membranes with an affinity (Kd), of 0.3 nM; the D2-selective ligand spiperone, the dopamine receptor ligand clozapine, and the serotonin receptor antagonist ketanserin bound with considerably lower affinities (102, 80, and 95 nM, respectively). Both dopamine and the D1-selective agonist SKF 38393 inhibited the binding of [3H]SCH 23390 to transfectant cell membranes; the binding of these agonists was sensitive to GTP. Dopamine potently stimulated the accumulation of cAMP in transfected C6 cells, whereas SKF 38393 was a partial agonist in these cells. Also, the density of recombinant D1 receptors on the transfectant cells was decreased 40% upon treatment with 10 microM dopamine, indicating that occupation of recombinant D1 receptors by agonists alters surface expression of the receptors.
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PMID:Molecular cloning and expression of the rhesus macaque D1 dopamine receptor gene. 153 68

STE4 encodes the beta-subunit of a heterotrimeric guanine nucleotide-binding protein (G protein) that is an early and essential component of the pheromone signal transduction pathway. From a ste4 deletion strain we have isolated both dominant and recessive suppressors that show increased transcription of pheromone responsive genes and have regained the ability to mate, albeit at a low level. Each of these suppressor mutations suppresses ste4 and ste5 deletions but not deletions in STE7, STE11, or STE12. Among the dominant mutations, we have identified two alleles of STE11, a gene that encodes a protein kinase activity essential for mating. One allele contains an alteration in the putative regulatory domain of the protein kinase; the second allele has an alteration in the catalytic site. In strains carrying these mutations, a second protein kinase required for mating, STE7, becomes hyperphosphorylated, just as it does in wild-type cells treated with pheromone. Thus, a protein kinase cascade appears to be an essential feature of the response pathway and probably connects the receptor/G protein to an identified transcription factor, STE12.
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PMID:Constitutive mutants of the protein kinase STE11 activate the yeast pheromone response pathway in the absence of the G protein. 162 32

Three separate processes may contribute to rapid beta-adrenergic receptor desensitization: functional uncoupling from the stimulatory guanine nucleotide-binding protein Gs, mediated by phosphorylation of the receptors by two distinct kinases, the specific beta-adrenergic receptor kinase (beta ARK) and the cyclic AMP-dependent protein kinase A (PKA), as well as a spatial uncoupling via sequestration of the receptors away from the cell surface. To evaluate the relative importance and potential role of the various processes in different physiological situations, a kinetic analysis of these three mechanisms was performed in permeabilized A431 epidermoid carcinoma cells. To allow a separate analysis of each mechanism, inhibitors of the various desensitization mechanisms were used: heparin to inhibit beta ARK, the PKA inhibitor peptide PKI to inhibit PKA, and concanavalin A treatment to prevent sequestration. Isoproterenol-induced phosphorylation of beta 2 receptors in these cells by beta ARK occurred with a t1/2 of less than 20 sec, whereas phosphorylation by PKA had a t1/2 of about 2 min. Similarly, beta ARK-mediated desensitization of the receptors proceeded with a t1/2 of less than 15 sec, and PKA-mediated desensitization with a t1/2 of about 3.5 min. Maximal desensitization mediated by the two kinases corresponded to a reduction of the signal-transduction capacity of the receptor/adenylyl cyclase system by about 60% in the case of beta ARK and by about 40% in the case of PKA. Receptor sequestration was much slower (t1/2 of about 10 min) and involved no more than 30% of the cell surface receptors. It is concluded that beta ARK-mediated phosphorylation is the most rapid and quantitatively most important factor contributing to the rapid desensitization. This rapidity of the beta ARK-mediated mechanism makes it particularly well suited to regulate beta-adrenergic receptor function in rapidly changing environments such as the synaptic cleft.
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PMID:Comparative rates of desensitization of beta-adrenergic receptors by the beta-adrenergic receptor kinase and the cyclic AMP-dependent protein kinase. 164 31

Light-dependent deactivation of rhodopsin as well as homologous desensitization of beta-adrenergic receptors involves receptor phosphorylation that is mediated by the highly specific protein kinases rhodopsin kinase (RK) and beta-adrenergic receptor kinase (beta ARK), respectively. We report here the cloning of a complementary DNA for RK. The deduced amino acid sequence shows a high degree of homology to beta ARK. In a phylogenetic tree constructed by comparing the catalytic domains of several protein kinases, RK and beta ARK are located on a branch close to, but separate from the cyclic nucleotide-dependent protein kinase and protein kinase C subfamilies. From the common structural features we conclude that both RK and beta ARK are members of a newly delineated gene family of guanine nucleotide-binding protein (G protein)-coupled receptor kinases that may function in diverse pathways to regulate the function of such receptors.
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PMID:The receptor kinase family: primary structure of rhodopsin kinase reveals similarities to the beta-adrenergic receptor kinase. 165 54

Pretreatment of 1321N1 human astrocytoma cells with phorbol 12-myristate-13-acetate or other activators of protein kinase C led to 2.5- to 5-fold increases (sensitization) in subsequent stimulation by forskolin of intracellular cyclic AMP accumulation. These compounds caused much smaller or no increases in receptor-mediated stimulation of cyclic AMP accumulation induced by isoproterenol and by prostaglandin E1. Carbachol and histamine, agonists acting at receptors coupled to polyphosphoinositide turnover in these cells, induced less sensitization of subsequent stimulation by forskolin but greater sensitization of stimulation by isoproterenol and by prostaglandin E1. The specificities of various analogs of phorbol 12-myristate-13-acetate, for induction of sensitization of forskolin stimulation were consistent with involvement of protein kinase C. The effects of protein kinase inhibitors and of down-regulation of protein kinase C activity also indicated involvement of protein kinase C in sensitization of forskolin stimulation, although additional mechanisms are likely to be involved in sensitization of isoproterenol stimulation. Neither pertussis toxin pretreatment nor inclusion of isobutylmethylxanthine during assays of cyclic AMP accumulation were able to prevent or mimic these sensitization phenomena, suggesting that the primary site of modification responsible for sensitization is neither the inhibitory guanine nucleotide-binding protein nor cyclic AMP phosphodiesterase. Sensitization was only observed in assays with intact cells. These results, together with those from our previous study describing protein kinase C-mediated desensitization of broken cell adenylate cyclase activity, indicate that activation of protein kinase C leads to multiple changes in the receptor-stimulated adenylate cyclase signal transduction pathway of these cells.
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PMID:Protein kinase C activators sensitize cyclic AMP accumulation by intact 1321N1 human astrocytoma cells. 168 54

Cyclic GMP mediates vascular smooth muscle relaxation to a variety of drugs and naturally-occurring substances. The reduction of intracellular Ca2+ levels is believed to underlie this action, but the mechanism of this effect is unknown. In order to test the hypothesis that inhibition of guanine nucleotide-binding protein function is involved in the actions of cGMP, the effects of cGMP-dependent protein kinase on the phosphorylation of both pertussis toxin-sensitive (Gi/Go) and insensitive (Gz) G-proteins were examined in vitro. None of these proteins were effective substrates for either cGMP- or cAMP-dependent protein kinases, despite the fact that assay conditions were designed to detect poorly phosphorylated substrate proteins. In line with these observations, atriopeptin II did not inhibit angiotensin II-treated inositol phosphate formation in cultured vascular smooth muscle cells. These results suggest that phosphorylation by cGMP-dependent protein kinase of these G-proteins is not the major mechanism by which cGMP reduces intracellular Ca2+ levels in vascular smooth muscle.
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PMID:Pertussis toxin-sensitive and insensitive guanine nucleotide binding proteins (G-proteins) are not phosphorylated by cyclic GMP-dependent protein kinase. 183 99

The integrity of coupling of the beta 2-adrenergic receptor (beta 2AR) to its guanine nucleotide-binding protein, Gs, and phosphorylation events on the receptor molecule have been proposed to be important determinants in the processes of receptor sequestration and down-regulation. However, little is known about the molecular mechanisms underlying these processes, and the regions of the receptor molecule that specifically subserve sequestration and down-regulation have yet to be delineated. To address these questions, we stably transfected eight mutant beta 2AR genes into Chinese hamster fibroblasts and evaluated the coupling, sequestration, and down-regulation properties of the mutated receptors. These mutant receptors have been previously demonstrated either to exhibit abnormal coupling to Gs or to lack functionally important phosphorylation sites for either the cAMP-dependent protein kinase or the agonist-dependent beta-adrenergic receptor kinase. All eight mutants exhibited receptor sequestration equivalent in extent to that of the beta 2AR, regardless of their coupling or phosphorylation status. However, four mutants that exhibited various degrees of impairment in coupling to Gs showed blunted receptor down-regulation patterns. Simultaneous treatment with isoproterenol and dibutyryl-cAMP did not improve the abilities of the mutant receptors to undergo down-regulation. These findings demonstrate that a variety of mutant beta 2AR with impaired coupling to Gs are, nevertheless, able to be sequestered normally. In contrast, agonist-induced down-regulation appears to require coupling of the beta 2AR to Gs but is largely independent of the generation of cAMP. Our results also suggest that molecular determinants of the beta 2AR involved in receptor sequestration are distinct from those participating in the down-regulation process.
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PMID:Mutations of the human beta 2-adrenergic receptor that impair coupling to Gs interfere with receptor down-regulation but not sequestration. 184 93

To determine the role of the stimulatory guanine nucleotide-binding protein, Gs, and adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase in the basal metabolism of beta-adrenergic receptors in S49 lymphoma cells, we measured the return of receptor number and function after irreversible blockade of receptors. After inactivation of receptors with the irreversible ligand N8-(bromoacetyl)-N'-[3-(4-indolyoxy)-2-hydroxypropyl]-(2)-1,8-diam ino-p- methane (BIM), beta-adrenergic receptors (defined as [125I]iodocyanopindolol binding sites) reappeared in a biphasic manner, the faster phase having a half-time (t 1/2) of 3-8 h (approximately 50% of the sites) and the slower phase greater than 40 h. Although the slow phase is not readily explained, recovery of binding sites during the first 10 h matched recovery of receptor function after BIM treatment (as measured by stimulation of cAMP accumulation) and recovery of receptor sites after downregulation induced by the agonist isoproterenol. Thus quantifying receptor recovery during the first 10 h after BIM treatment appears to be a reasonable method for examining basal receptor metabolism in S49 cells. Measured in this way, metabolism of beta-adrenergic receptors is very similar in wild-type S49 and the following variant clones: cyc- (absent Gs alpha), UNC and H21a (defective Gs alpha), and kin- (lacking cAMP-dependent protein kinase activity). Although previous data have demonstrated that agonist-promoted downregulation of beta-adrenergic receptors requires functional receptor-Gs coupling, the current data suggest that neither Gs nor cAMP-dependent protein kinase activity plays an important role in the regulation of basal metabolism of beta-adrenergic receptors.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Beta-adrenoceptor metabolism in wild-type, Gs, and protein kinase A-variant S49 cells. 197

Various pharmacological effectors were used to investigate the mechanism of arachidonic acid release by N-formyl-methionyl-leucyl-phenylalanine (fMLP) and platelet-activating factor (PAF) in guinea pig alveolar macrophages. The fMLP- and PAF-stimulated arachidonic acid release (i) was mimicked by sodium fluoride and inhibited by Bordetella pertussis toxin, suggesting the participation of a guanine nucleotide-binding protein; ii) was mimicked by A23187 but was insensitive to the calmodulin inhibitor R24571, making the involvement of a calmodulin-dependent pathway unlikely; and (iii) was mimicked by 12-O-tetra-decanoyl phorbol 13 acetate (TPA) and was, like the TPA-stimulated release, markedly decreased when protein kinase C (PKC) had been down-regulated by TPA (65% decrease) or inhibited by sphingosine, a diacylglycerol-competitive PKC inhibitor shown to completely abolish the enzyme activity from alveolar macrophages at 40 microM. Moreover, PAF and fMLP, under conditions where they stimulated arachidonic acid release, promoted an appreciable, albeit transient, translocation of PKC, suggesting a possible involvement of the enzyme in the agonist-stimulated process. However, staurosporine, another PKC inhibitor decreasing PKC activity from alveolar macrophages by 60% at 20 nM, failed to alter fMLP- and PAF-stimulated release. These data lead us to suggest that fMLP- and PAF-stimulated arachidonic acid release is mediated by mechanisms involving either a staurosporine-insensitive PKC isoform or a sphingosine-sensitive coupling between a pertussis toxin-sensitive guanine nucleotide-binding protein and phospholipase A2. Finally, the fMLP- and PAF-stimulated arachidonic acid release was inhibited by cholera toxin and was, like A23187-stimulated release, potentiated by N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H8), an exclusive protein kinase A inhibitor in alveolar macrophages, suggesting a negative regulation by protein kinase A.
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PMID:Mechanism of N-formyl-methionyl-leucyl-phenylalanine- and platelet-activating factor-induced arachidonic acid release in guinea pig alveolar macrophages: involvement of a GTP-binding protein and role of protein kinase A and protein kinase C. 211 77

In the neuroblastoma X glioma hybrid cell line NG108-15, bradykinin (BK) receptor stimulation induced a rapid and concentration-dependent rise in cytosolic free Ca2+ levels, as measured with the Ca2(+)-sensitive fluorescent dye fura-2. The Ca2+ transient was present in the absence of extracellular Ca2+ and was associated with a concentration-dependent production of inositol phosphates, particularly inositol trisphosphate (InsP3). Pretreatment of intact NG108-15 cells with forskolin or dibutyryl-cAMP plus isobutylmethylxanthine reduced BK-stimulated InsP3 production and the increase in cytosolic free Ca2+. Membranes prepared from forskolin- and [3H]inositol-pretreated NG108-15 cells also showed a diminished production of InsP3 elicited by guanosine 5'-[gamma-thio]triphosphate, NaF, or BK plus GTP. On the other hand, the Ca2+ sensitivity of membrane-associated phosphoinositide-specific phospholipase C (PI-PLC) was unaffected by forskolin pretreatment of intact NG108-15 cells. Collectively, these results suggest that A-kinase may inhibit receptor-mediated and postreceptor stimulation of PI-PLC in neuron-like cells, perhaps by impairing the coupling between a guanine nucleotide-binding protein and PI-PLC.
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PMID:Cyclic AMP inhibits inositol polyphosphate production and calcium mobilization in neuroblastoma X glioma NG108-15 cells. 216 7


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