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)

The beta 2-adrenergic receptor (beta 2AR) belongs to the large family of G protein-coupled receptors. Mutation of tyrosine residue 326 to an alanine resulted in a beta 2AR mutant (beta 2AR-Y326A) that was defective in its ability to sequester and was less well coupled to adenylyl cyclase than the wild-type beta 2AR. However, this mutant receptor not only desensitized in response to agonist stimulation but down-regulated normally. In an attempt to understand the basis for the properties of this mutant, we have examined the ability of this regulation-defective mutant to undergo agonist-mediated phosphorylation. When expressed in 293 cells, the maximal response for phosphorylation of the beta 2AR-Y326A mutant was impaired by 75%. Further characterization of this phosphorylation, using either forskolin stimulation or phosphorylation site-deficient beta 2AR-Y326A mutants, demonstrated that the beta 2AR-Y326A mutant can be phosphorylated by cAMP-dependent protein kinase (PKA) but does not serve as a substrate for the beta-adrenergic receptor kinase 1 (beta ARK1). However, overexpression of beta ARK1 led to the agonist-dependent phosphorylation of the beta 2AR-Y326A mutant and rescue of its sequestration. beta ARK1-mediated rescue of beta 2AR-Y326A sequestration could be prevented by mutating putative beta ARK phosphorylation sites, but not PKA phosphorylation sites. In addition, both sequestration and phosphorylation of the wild-type beta 2AR could be attenuated by overexpressing a dominant-negative mutant of beta ARK1 (C20 beta ARK1-K220M). These findings implicate a role for beta ARK1-mediated phosphorylation in facilitating wild-type beta 2AR sequestration.
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PMID:Role of phosphorylation in agonist-promoted beta 2-adrenergic receptor sequestration. Rescue of a sequestration-defective mutant receptor by beta ARK1. 755 96

Attenuation of receptor-mediated signal amplification in response to external stimuli, an essential step in the balance of cellular activation, may be mediated by receptor phosphorylation. We have recently shown that the carboxyl-terminal cytoplasmic domain of the N-formyl peptide receptor (FPR) interacts with G proteins and demonstrate here that this same region of the FPR is specifically phosphorylated by a neutrophil cytosolic kinase with properties similar to the G protein-coupled receptor kinase, GRK2. Both kinase activities show a lack of sensitivity toward protein kinase A, protein kinase C, and tyrosine kinase inhibitors but demonstrate almost identical sensitivity toward the kinase inhibitor heparin. Kinetic studies demonstrated that GRK2 has a Km for the carboxyl-terminal domain of the FPR of approximately 1.5 microM and that denaturation of the substrate results in an almost complete loss of phosphorylation. Comparative studies reveal that GRK3 has approximately 50% of the activity of GRK2 toward the FPR carboxyl terminus, whereas GRK5 and GRK6 have no detectable activity. Site-directed mutagenesis of numerous regions of the FPR carboxyl terminus demonstrated that, whereas Glu326/Asp327 and Asp333 are critical for phosphorylation, the carboxyl-terminal 10 amino acids are not required. Simultaneous substitution of Thr334, Thr336, Ser338, and Thr339 resulted in an approximately 50% reduction in phosphorylation, whereas simultaneous substitution of the upstream Ser328, Thr329, Thr331, and Ser332 or merely the Ser328 and Thr329 residues resulted in an approximately 80% reduction in phosphorylation. The introduction of negatively charged glutamate residues for Ser328 and Thr329 or Thr331 and Ser332 resulted in marked stimulation of phosphorylation. These results suggest a hierarchical mechanism in which phosphorylation of amino-terminal serine and threonine residues is required for the subsequent phosphorylation of carboxyl-terminal residues. These results provide the first direct evidence that an intracellular domain of a chemoattractant receptor is a high affinity substrate for GRK2 and further suggest a role for GRK2 or a closely related kinase in the attenuation of receptor-mediated activation of inflammatory cells.
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PMID:Phosphorylation of the N-formyl peptide receptor carboxyl terminus by the G protein-coupled receptor kinase, GRK2. 783 71

Olfaction is mediated by G protein-coupled receptors. In isolated rat olfactory cilia, odorants such as citralva stimulate a burst of cAMP, which peaks in 50 ms and returns almost to base-line level within 150 ms in the continuing presence of odorant. This desensitization is mediated by the cAMP dependent protein kinase and a specialized G protein-coupled receptor kinase originally termed beta ARK2 (GRK3). In vitro experiments suggest that the prenylated beta gamma-subunits of heterotrimeric G proteins target the cytosolic beta ARK1 (GRK2) enzyme to its membrane bound receptor substrate by binding to sites in its carboxyl terminus. Here we demonstrate that odorants stimulate translocation of GRK3 from cytosol to membranes in isolated rat olfactory cilia. We introduced a glutathione S-transferase-GRK3ct fusion protein, containing the carboxyl-terminal 222 amino acid residues of GRK3, which includes the beta gamma binding site, or a 28-amino acid peptide derived therefrom, into permeabilized cilia preparations. These reagents block odorant-mediated enzyme translocation and desensitization while markedly attenuating odorant-stimulated phosphorylation of olfactory proteins. These findings suggest that beta gamma-subunits may physiologically regulate a G protein-coupled receptor kinase and that enzyme translocation may be a general and required feature of the activity of some members of this enzyme family.
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PMID:Olfactory desensitization requires membrane targeting of receptor kinase mediated by beta gamma-subunits of heterotrimeric G proteins. 827 21

We have shown previously that GTP-binding regulatory protein (G protein) beta gamma subunits stimulate the agonist- or light-dependent phosphorylation of muscarinic acetylcholine receptors (mAChRs) and rhodopsin by a protein kinase partially purified from porcine brain (mAChR kinase) but not the phosphorylation of rhodopsin by rhodopsin kinase (Haga, K., and Haga, T. (1992) J. Biol. Chem. 267, 2222-2227). We report here that the mAChR kinase phosphorylates beta-adrenergic receptors (beta-ARs) purified from bovine lung in an agonist-dependent manner, and the phosphorylation is also stimulated by G protein beta gamma subunits. We also report that recombinant beta-adrenergic receptor kinase 1 (beta-ARK1) expressed in COS-7 cells phosphorylates mAChRs (human m2 subtype) and rhodopsin in an agonist- or light-dependent manner, respectively, and that this phosphorylation is stimulated by G protein beta gamma subunits. By contrast, the beta gamma subunits do not stimulate the phosphorylation of mAChRs or rhodopsin by a beta-ARK1 mutant lacking a part of the carboxyl-terminal region which is present in beta-ARKs but not in rhodopsin kinase. These results indicate that the beta-ARK1 is the same as or very similar to the mAChR kinase but is distinguished from the rhodopsin kinase with respect to activation by the beta gamma subunits and that the extra carboxyl-terminal sequence in beta-ARKs is required for the stimulation by the beta gamma subunits.
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PMID:Activation by G protein beta gamma subunits of beta-adrenergic and muscarinic receptor kinase. 846 5

A novel human G protein-coupled receptor kinase was recently identified by positional cloning in the search for the Huntington's disease locus (Ambrose, C., James, M., Barnes, G., Lin, C., Bates, G., Altherr, M., Duyao, M., Groot, N., Church, D., Wasmuth, J. J., Lehrach, H., Housman, D., Buckler, A., Gusella, J. F., and MacDonald, M. E. (1993) Hum. Mol. Genet. 1, 697-703). Comparison of the deduced amino acid sequence of GRK4 with those of the closely related GRK5 and GRK6 suggested the apparent loss of 32 codons in the amino-terminal domain and 46 codons in the carboxyl-terminal domain of GRK4. These two regions undergo alternative splicing in the GRK4 mRNA, resulting from the presence or absence of exons filling one or both of these apparent gaps. Each inserted sequence maintains the open reading frame, and the deduced amino acid sequences are similar to corresponding regions of GRK5 and GRK6. Thus, the GRK4 mRNA and the GRK4 protein can exist as four distinct variant forms. The human GRK4 gene is composed of 16 exons extending over 75 kilobase pairs of DNA. The two alternatively spliced exons correspond to exons II and XV. The genomic organization of the GRK4 gene is completely distinct from that of the human GRK2 gene, highlighting the evolutionary distance since the divergence of these two genes. Human GRK4 mRNA is expressed highly only in testis, and both alternative exons are abundant in testis mRNA. The four GRK4 proteins have been expressed, and all incorporate [3H]palmitate. GRK4 is capable of augmenting the desensitization of the rat luteinizing hormone/chorionic gonadotropin receptor upon coexpression in HEK293 cells and of phosphorylating the agonist-occupied, purified beta2-adrenergic receptor, indicating that GRK4 is a functional protein kinase.
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PMID:Characterization of the G protein-coupled receptor kinase GRK4. Identification of four splice variants. 862 39

G protein-coupled receptor kinases (GRKs) mediate agonist-dependent phosphorylation of G protein-coupled receptors (GPRs) and initiate homologous receptor desensitization. Previously, we reported that charged phospholipids directly interacted with the two GRK isoforms, GRK2 and GKR3, via a pleckstrin homology (PH) domain to regulate GRK activity (DebBurman, S. K., Ptasienski, J., Boetticher, E., Lomasney, J. W., Benovic, J. L., and Hosey, M. M. (1995) J. Biol. Chem. 270: 5742-5747). Here, evidence is provided to support the hypothesis that charged phospholipids are required for agonist-dependent phosphorylation of receptors by GRK2. In the absence of charged phospholipids, the purified human m2 muscarinic acetylcholine receptor (hm2mAChR) reconstituted in pure phosphatidylcholine vesicles or in a noninhibitory detergent was not a substrate for GRK2. However, these receptor preparations were stoichiometrically phosphorylated in an agonist-dependent manner upon addition of charged phospholipids. The known ability of G protein betagamma subunits to stimulate mAChR phosphorylation also was found to be absolutely dependent on the presence of charged phospholipids, including phosphatidylinositol 4,5-bisphosphate (PIP2). Phospholipids also regulated GRK-mediated phosphorylation of casein, a nonreceptor-soluble substrate. Among lipids tested, lipid inositol phosphates, PIP2 and phosphatidylinositol 4-monophosphate, were found to be the most potent activators of GRK2 and were the only lipids that regulated GRK2 in a complex biphasic manner. At low micro concentrations, PIP2 activated GRK2 via an interaction with the GRK pleckstrin homology domain; however, at high micro concentrations, PIP2 inhibited GRK2, apparently via another mechanism. PIP2-mediated inhibition could be partly relieved by increasing ATP. The results support the hypothesis that GRK2 is a lipid-dependent protein kinase that requires charged phospholipids for enzyme activation, for regulation by Gbetagamma subunits, and potentially for membrane association.
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PMID:G protein-coupled receptor kinase GRK2 is a phospholipid-dependent enzyme that can be conditionally activated by G protein betagamma subunits. 879 23

Although its detailed substrate specificity is not precisely known, beta-adrenergic receptor kinase 1 phosphorylates beta 2-adrenergic receptors and other G protein-coupled receptors. To elucidate the ligand recognition mechanism of the enzyme and the consensus sequence required for substrates, a three-dimensional structure of the catalytic domain of the enzyme was modeled based on the X-ray crystal structure of the catalytic subunit of cyclic AMP-dependent protein kinase A. When the phosphorylation residue of the substrate was defined as the p position in the model of beta-adrenergic receptor kinase 1, the present study suggested that the consensus sequence recognized by this enzyme would consist of a basic residue at p-3 and an acidic residue at p-2.
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PMID:Substrate recognition mechanism of human beta-adrenergic receptor kinase 1 based on a three-dimensional model structure. 901 Jun 20

Assembly of terminal complement complexes (TCC) C5b-7, C5b-8, and C5b-9 on target cells during acute and chronic inflammation induces hydrolysis of plasma membrane phospholipids and heterotrimeric G protein activation. TCC also stimulate a variety of cellular activities, which include cytokine synthesis, proto-oncogene activation, and mitotic signaling. Now we report that sublytic TCC induced Ras, Raf-1, and extracellular signal-regulated kinase (ERK) 1 activation in JY25 B cell line. When cells were exposed to C5b-9, GTP-bound Ras in anti-C5b-9 immunoprecipitates was increased 3.2-fold at 2 min, while GTP-bound Ras in anti-Ras immunoprecipitates was increased 2-fold at 10 min. Both C5b-9 and C5b-7, but not C5b6, increased Raf-1 kinase activity maximum 3.3-fold at 2 min and 2.8-fold at 5 min, respectively. ERK1 activity was 2-fold increased by C5b-9 at 2 min and by C5b-7 at 10 min, over the C5b6 level. The role of mitogen-activated protein kinase (MAPK) pathway on TCC-inducible mitotic signaling was evaluated by assessing DNA synthesis and activator protein 1 (AP-1) DNA-binding activity. The MAPK/ERK-specific inhibitor PD 098,059 abolished the C5b-9-induced DNA synthesis. Involvement of G protein in the activation of MAPK pathway by TCC was indicated by inhibition of Raf-1 and ERK1 kinase activity, as well as the DNA synthesis by pretreatment of cells with pertussis toxin. Overexpression of beta-adrenergic receptor kinase 1 carboxyl-terminal peptide in JY25 cells also inhibited Raf-1 and ERK1 activity, indicating a direct involvement of G betagamma subunits in the signal transduction generated through activation of MAPK pathway by TCC assembly in the plasma membrane.
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PMID:Activation of Ras and mitogen-activated protein kinase pathway by terminal complement complexes is G protein dependent. 912 5

Activation of the G protein-coupled receptor for parathyroid hormone (PTH)/PTH-related protein (PTHrP) produces homologous desensitization of receptor signaling. We have shown recently that the opossum PTH/PTHrP receptor stably expressed in human embryonic kidney (HEK) 293 cells is phosphorylated upon agonist binding and upon activation of serine/threonine protein kinases (PKA and PKC), an event which for some G protein-coupled receptors has been linked to desensitization. To locate the sites of phosphorylation, mutated forms of the opossum PTH/PTHrP receptor were stably expressed in HEK 293 cells, and ligand-stimulated receptor phosphorylation was evaluated. The five serine and threonine residues of the third cytoplasmic loop of the receptor were not required for receptor phosphorylation. Basal and ligand-induced phosphorylation were, however, completely abolished upon deletion of all but the 16 juxtamembrane residues of the cytoplasmic C-terminal tail of the receptor, even though this truncated receptor resembled the wild-type receptor in its level of expression based on Western blotting and radioligand binding. To identify further the phosphorylation sites, the 129 amino acid C-terminal tail of the rat PTH/PTHrP receptor was expressed in E. coli as a recombinant glutathione S-transferase fusion protein. Elimination of a single PKA consensus site in the tail (serine 491) resulted in > or = 90% loss of PKA-mediated phosphorylation, identifying this as the preferential site for PKA, with two other sites (serine 473 and/or 475) being minor sites. Phosphorylation by PKC occurred largely in the proximal portion of the tail, whereas beta-adrenergic receptor kinase 1 (beta ARK1) phosphorylated more distally in the tail. The ability of these kinases to phosphorylate the PTH/PTHrP receptor at distinct sites on the cytoplasmic tail may allow differential regulation of receptor signaling and trafficking.
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PMID:Phosphorylation of the cytoplasmic tail of the PTH/PTHrP receptor. 915 72

Asp278 of beta-adrenergic receptor kinase 1 (betaARK1) was suggested to play a key role in substrate recognition of beta2-adrenergic receptors in our previous study, in which a three-dimensional model of betaARK1 was studied in comparison with a crystal structure of PKA-PKI5-24 complex. In the present study, to confirm the molecular recognition mechanism at Asp278 of betaARK1, two mutants of betaARK1, D278R and D278A, were designed based on molecular modeling studies and produced by Sf-9 cells. As predicted by the molecular modeling study, the mutants showed no kinase activities while wild type betaARK1 phosphorylated beta2-adrenergic receptors in a concentration-dependent manner. These results strongly suggest the involvement of Asp278 in substrate recognition by betaARK1. The results also suggest a high reliability of the three-dimensional model of betaARK1.
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PMID:Asp278 of human beta-adrenergic receptor kinase 1 is essential for phosphorylation activity. 934 67


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