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)

G protein-coupled receptor kinases (GRK), such as the beta-adrenergic receptor kinase (beta ARK) and rhodopsin kinase, specifically phosphorylate the activated form of G protein-coupled receptors. To identify additional members of the GRK family, we screened a human heart cDNA library by low stringency hybridization using the catalytic domains of two beta ARK isoforms. Here we report the isolation of a cDNA that encodes a 576-amino-acid protein kinase, termed GRK6, that has significant homology with GRK5 (70.1% amino acid identity), IT11 (68.5%), rhodopsin kinase (47.1%), and beta ARK (37.4%). RNA blot analysis of GRK6 with selected human tissues reveals two distinct mRNAs of 3 and 2.4 kilobases with a distribution very similar to that of beta ARK (i.e. brain, skeletal muscle > pancreas > heart, lung, kidney, placenta > liver). GRK6, overexpressed in Sf9 insect cells using the baculovirus system, was able to phosphorylate both the beta 2-adrenergic receptor and rhodopsin in a stimulus-dependent fashion, although it was significantly less active then beta ARK on these substrates. These data extend the family of GRKs and suggest that GRK6 may have a substrate specificity quite distinct from beta ARK and rhodopsin kinase.
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PMID:Molecular cloning and expression of GRK6. A new member of the G protein-coupled receptor kinase family. 836 96

Pleckstrin homology (PH) domains are difficult to find in protein sequence databases with widely used computer programs. A simple program developed to overcome this difficulty identified three proteins containing previously unrecognized PH domains; the beta-adrenergic receptor kinase (beta-ARK), the tecA protein kinase and the insulin receptor substrate protein IRS-1. The region of beta-ARK containing the novel PH domain coincides with that previously shown to bind the beta gamma subunits of trimeric G-proteins, suggesting a general hypothesis for PH domain function. PH domains were then found at the N-termini of the tecA homologues Btk and itk. In line with the hypothesis a point mutation in the PH domain of Btk is associated with defects in signal transduction.
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PMID:Identification of novel pleckstrin homology (PH) domains provides a hypothesis for PH domain function. 837 91

Exposure of beta 2-adrenergic receptors to agonists causes a rapid desensitization of the receptor-stimulated adenylyl cyclase, associated with an increased phosphorylation of the receptor. Agonist-promoted phosphorylation of the beta 2-adrenergic receptor (beta 2AR) by protein kinase A and the beta-adrenergic receptor kinase (beta ARK) is believed to promote a functional uncoupling of the receptor from the guanyl nucleotide regulatory protein Gs. More recently, palmitoylation of Cys341 of the receptor has also been proposed to play an important role in the coupling of the beta 2-adrenergic receptor to Gs. Here we report that substitution of the palmitoylated cysteine by a glycine (Gly341 beta 2 AR) using site directed mutagenesis leads to a receptor being highly phosphorylated and largely uncoupled from Gs. In Chinese hamster fibroblasts (CHW), stably transfected with the human receptor cDNAs, the basal phosphorylation level of Gly341 beta 2AR was found to be approximately 4 times that of the wild type receptor. This elevated phosphorylation level was accompanied by a depressed ability of the receptor to stimulate the adenylyl cyclase and to form a guanyl nucleotide-sensitive high affinity state for agonists. Moreover, exposure of this unpalmitoylated receptor to an agonist did not promote any further phosphorylation or uncoupling. A modest desensitization of the receptor-stimulated adenylyl cyclase response was observed but resulted from the agonist-induced sequestration of the unpalmitoylated receptor and could be blocked by concanavalin A. This contrasts with the agonist-promoted phosphorylation and uncoupling of the wild type receptor.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Altered phosphorylation and desensitization patterns of a human beta 2-adrenergic receptor lacking the palmitoylated Cys341. 838 52

Receptor-specific or homologous desensitization of beta 2-adrenergic receptors is thought to be effected via phosphorylation of the receptor by the beta-adrenergic receptor kinase (beta ARK), followed by binding of beta-arrestin. We have generated stably transfected Chinese hamster ovary cell lines overexpressing either of the two regulatory proteins and also expressing low or high levels of beta 2-adrenergic receptors (approximately 80 and approximately 600 fmol/mg of membrane protein). In these cells, we studied the process of desensitization induced by the beta-adrenergic receptor agonist isoproterenol. In cells expressing high levels of beta 2-adrenergic receptors, desensitization to high concentrations of isoproterenol (previously shown to be mediated by both beta ARK and protein kinase A) amounted to approximately 50% in control cells, approximately 80% in beta ARK-overexpressing cells, and approximately 90% in beta-arrestin-overexpressing cells. In cells expressing low levels of beta 2-adrenergic receptors, these values were approximately 50, approximately 60, and approximately 60%, respectively. Desensitization to low concentrations of isoproterenol (previously shown to be essentially protein kinase A-mediated and not receptor-specific, i.e. heterologous) was not affected by overexpression of either beta ARK or beta-arrestin. These data suggest that in cells expressing high levels of beta 2-adrenergic receptors, beta-arrestin and beta ARK become limiting for homologous receptor desensitization. They provide further support for the involvement of these two proteins in the regulation of beta 2-adrenergic receptor function.
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PMID:Overexpression of beta-arrestin and beta-adrenergic receptor kinase augment desensitization of beta 2-adrenergic receptors. 838 21

We have previously shown that second-messenger-dependent kinases (cAMP-dependent kinase, protein kinase C) in the olfactory system are essential in terminating second-messenger signaling in response to odorants. We now document that subtype 2 of the beta-adrenergic receptor kinase (beta ARK) is also involved in this process. By using subtype-specific antibodies to beta ARK-1 and beta ARK-2, we show that beta ARK-2 is preferentially expressed in the olfactory epithelium in contrast to findings in most other tissues. Heparin, an inhibitor of beta ARK, as well as anti-beta ARK-2 antibodies, (i) completely prevents the rapid decline of second-messenger signals (desensitization) that follows odorant stimulation and (ii) strongly inhibits odorant-induced phosphorylation of olfactory ciliary proteins. In contrast, beta ARK-1 antibodies are without effect. Inhibitors of protein kinase A and protein kinase C also block odorant-induced desensitization and phosphorylation. These data suggest that a sequential interplay of second-messenger-dependent and receptor-specific kinases is functionally involved in olfactory desensitization.
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PMID:A beta-adrenergic receptor kinase-like enzyme is involved in olfactory signal termination. 838 66

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

Phosphorylation of G-protein-linked receptors is thought to play a central role in receptor regulation and desensitization. Unlike the case of the extensively studied beta-adrenergic receptor/adenylate cyclase pathway, in which receptor-specific phosphorylation is known to be mediated by beta-adrenergic receptor kinase ( beta-ARK), the kinases responsible for phosphorylation of phospholipase C-linked receptors have yet to be identified, although a role for beta-ARK has been implicated. This study describes the purification of a novel 40-kDa receptor kinase from porcine cerebellum that is able to phosphorylate the phospholipase C-linked m3-muscarinic receptor in an agonist-dependent manner. The assay for kinase activity was based on the ability of the kinase to phosphorylate a bacterial fusion protein, Ex-m3, containing amino acids Ser345-Leu463 of the third intracellular loop of the m3-muscarinic receptor. Purification of the muscarinic receptor kinase from a high speed supernatant fraction of porcine cerebellum was achieved using the following steps: (i) 30-60% ammonium sulfate cut and successive chromatography on (ii) butyl-Sepharose (iii) Resource Q, (iv) Resource S, and (v) heparin-Sepharose. The purified protein kinase represented an approximately 18,600-fold purification and was a single polypeptide with a molecular weight of approximately 40 kDa. Based on the chromatographic mobility, molecular weight, and kinase inhibitor studies, the kinase, designated MRK, was shown to be distinct from previously characterized second messenger regulated protein kinases, beta-ARK, and other members of the G-protein-linked receptor kinase family. It therefore represents a new class of receptor kinase.
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PMID:Identification of a novel receptor kinase that phosphorylates a phospholipase C-linked muscarinic receptor. 863 12

Protein phosphorylation is central to agonist-induced attenuation of the function of G-protein-linked receptors. Stable expression of RNA antisense to specific protein kinase mRNAs permitted analysis of loss-of-function mutants of A431 human epidermoid carcinoma cells, lacking protein kinase A, protein kinase C, or beta-adrenergic receptor kinase. Deficiency of protein kinase C, but not the others, amplified rather than attenuated agonist-induced desensitization. In wild-type cells, the t1/2 for recovery from desensitization was approximately 25 min following removal of agonist. In the protein kinase C-deficient cells, no resensitization was observed even 60 min after agonist removal. Like protein kinase C-deficiency, inhibition of protein kinase C with bisindolylmaleimide or calphostin C blocked resensitization. Resensitization was suppressed by FK506, an inhibitor of protein phosphatase 2B, mimicking protein kinase C-deficiency, but in a non-additive manner. The data reveal protein kinase C and protein phosphatase 2B to be critical elements of resensitization.
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PMID:Protein kinase C deficiency blocks recovery from agonist-induced desensitization. 870 31

Phosducin is a member of the large group of proteins that bind to G-protein betagamma-subunits (Gbetagamma) and whose biological functions are often unknown. Human A431 cells do not contain detectable amounts of phosducin. We generated A431 cells expressing phosducin at a level of approximately 1 pmol/mg of cytosolic protein, which is approximately 10% of the phosducin level in brain. cAMP accumulation in response to beta2-adrenergic receptor agonists was enhanced at early times in phosducin-expressing cells, but reached a lower plateau than in control cells. Permeabilization of the cells with digitonin did not change this pattern, but allowed the introduction of specific inhibitors: antibodies to phosducin abolished all differences between the two cell lines. Inhibitors of the beta-adrenergic receptor kinase abolished the differences at early time points. An almost complete loss of beta2-adrenergic receptor desensitization in the phosducin-expressing cells was also observed when intact cells were desensitized and receptor function was then determined in membrane preparations. Inhibition of protein kinase A accentuated the effects of phosducin, suggesting that also in vivo phosducin is regulated by this kinase. These data indicate that phosducin affects G-protein-mediated signaling in at least two ways: it dampens the overall responsiveness, and it impairs the rapid desensitization mediated by the beta-adrenergic receptor kinase.
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PMID:Expression of phosducin in a phosducin-negative cell line reveals functions of a Gbetagamma-binding protein. 879 22

We have previously demonstrated that the phospholipase C-coupled m3-muscarinic receptor is phosphorylated in an agonist-sensitive manner by a protein kinase of approximately 40 kDa purified from porcine cerebellum (Tobin, A. B., Keys, B., and Nahorski, S. R. (1996) J. Biol Chem. 271, 3907-3916). This kinase, called muscarinic receptor kinase (MRK), is distinct from second messenger-regulated protein kinases and from beta-adrenergic receptor kinase and other members of the G-protein-coupled receptor kinase family. In the present study we propose that MRK is casein kinase 1alpha (CK1alpha) based on the following evidence: 1) the amino acid sequence from two proteolytic peptide fragments derived from purified MRK corresponded exactly to sequences within CK1alpha. 2) Casein kinase activity co-eluted with MRK activity from the final two chromatography steps in the purification of porcine brain MRK. 3) Recombinant CK1alpha expressed in Sf9 cells is able to phosphorylate both casein and the bacterial fusion protein, Ex-m3, that contains a portion of the third intracellular loop of the m3-muscarinic receptor downstream of glutathione S-transferase. 4) Partially purified CK1alpha increased the level of muscarinic receptor phosphorylation in an agonist-sensitive manner when reconstituted with membranes from Chinese hamster ovary-m3 cells expressing the human recombinant m3-muscarinic receptor. 5) Partially-purified CK1alpha phosphorylated rhodopsin, contained in urea-treated bovine rod outer segment membranes, and the extent of phosphorylation was increased in the presence of light. These data demonstrate that the kinase previously called MRK is CK1alpha, and that CK1alpha offers an alternative protein kinase pathway from that of the G-protein-coupled receptor kinase family for the stimulus-dependent phosphorylation of the m3-muscarinic receptor, rhodopsin, and possibly other G-protein-coupled receptors.
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PMID:Stimulus-dependent phosphorylation of G-protein-coupled receptors by casein kinase 1alpha. 925 10


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