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 2-adrenergic receptor (beta 2AR) is the initial event that underlies rapid agonist-promoted desensitisation. However, the role of phosphorylation in mediating long-term beta 2AR desensitisation is not known. To investigate this possibility, we performed intact cell phosphorylation studies with COS-7 cells transiently expressing an epitope tagged wild-type beta 2AR and found that receptor phosphorylation in cells treated with 1 microM isoproterenol for 24 h was approximately 4-fold over the basal state. This finding suggested that persistent phosphorylation of the receptor might contribute to functional long-term desensitisation which we further explored with mutated beta 2AR lacking the determinants of phosphorylation by the beta AR kinase (beta ARK), PKA or both. In CHW cells expressing the WT beta 2AR, pretreatment with 1 microM isoproterenol for 24 h reduced the isoproterenol-stimulated cAMP response by 82 +/- 5%. Substitution of the PKA sites with alanines had no effect on the extent of desensitisation (77 +/- 6%, P = NS compared to WT). In contrast, desensitisation was only 49 +/- 4% (P < 0.001 compared to WT) when the beta ARK sites were similarly substituted. Removal of both the beta ARK and PKA sites impaired desensitisation to the same extent as the beta ARK mutant. The extent of receptor loss (downregulation) was the same among all of the cell lines used and therefore could not account for the observed differences in desensitisation. Cellular beta ARK activity, assessed by a rhodopsin phosphorylation assay, was equivalent in all cell lines and was unaffected by agonist treatment. PKA activity, however, was dynamically regulated, increasing 4-fold over basal levels after 15 min of isoproterenol and returning to near basal levels after 24 h. The lower level of PKA activity after long-term agonist exposure may therefore have contributed to the apparent lack of effect of removing PKA sites. Nonetheless, long-term desensitisation was clearly attenuated with beta 2AR lacking beta ARK phosphorylation sites. These findings show that in addition to its role in regulating short-term desensitisation, beta ARK-mediated phosphorylation is an important mechanism underlying long-term desensitisation of the beta 2AR as well.
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PMID:Role of beta ARK in long-term agonist-promoted desensitisation of the beta 2-adrenergic receptor. 960 43

The study of signaling pathways has begun to uncover the mechanism by which cells respond and adapt to extracellular stimuli. It has become increasingly clear that the signaling pathways interact with one another to form a complex network through which regulation occurs. Here, we focus on three mechanisms by which signaling pathways interact and the physiological consequences of these interactions. Coincident signaling in long-term depression of synaptic responses in the cerebellum, protein kinase A gating of Ras to mitogen-activated protein kinase signal flow in proliferative responses, and a modified gating mechanism by phosducin resulting in feedback regulation of signal flow from rhodopsin to the cGMP phosphodiesterase in retinal light adaptation are analyzed as examples of different types of interactions between signaling pathways. These interactions allow the cell to spatially and temporally integrate complex information and respond in an appropriate and defined manner.
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PMID:Modes of interactions between signaling pathways. 1007 24

A comparative map of human chromosome 3 (HSA 3) and pig chromosome 13 (SSC 13) was constructed using physically assigned pig sequence-tagged sites (STSs). Pig STSs representing 11 HSA 3 genes, including v-Raf-1 murine leukemia viral oncogene homolog 1 (RAF1), retinoic acid beta receptor (RARB), cholecystokinin (CCK), pituitary transcription factor 1 (POU1F1), ceruloplasmin (CP), guanine nucleotide binding protein, alpha-inhibiting polypeptide 2 (GNAI2), sucrase-isomaltase (SI), rhodopsin (RHO), dopamine receptor D3 (DRD3), growth-associated protein 43 (GAP43), and somatostatin (SST), were developed. Ten pig STSs were regionally mapped using a somatic cell hybrid panel (SCHP) to SSC 13 with 80-100% concordance. Large-insert probes were obtained by screening a pig yeast artificial chromosome (YAC) library with primers for each STS. Several YACs were identified for DRD3, GAP43, POU1F1, RHO, SI, and SST for fluorescence in situ hybridization (FISH) mapping. Single gene and bi-color FISH with each pairwise combination were used to further define the gene order on SSC 13. While these data confirm chromosome painting results showing that HSA 3 probes hybridize to a major portion of SSC 13, they also demonstrate extensive gene-order differences between man and pig within this large conserved synteny group. Interestingly, several conserved chromosomal regions have been detected between pig and mouse that are not conserved between man and mouse, suggesting that the SSC 13 gene arrangement may be the closest to that of the ancestral eutherian chromosome.
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PMID:Human chromosome 3 and pig chromosome 13 show complete synteny conservation but extensive gene-order differences. 1044 17

Retinal cGMP phosphodiesterase (PDE) is regulated by Pgamma, the regulatory subunit of PDE, and GTP/Talpha, the GTP-bound alpha subunit of transducin. In the accompanying paper (Matsuura, I., Bondarenko, V. A., Maeda, T., Kachi, S., Yamazaki, M., Usukura, J., Hayashi, F., and Yamazaki, A. (2000) J. Biol. Chem. 275, 32950-32957), we have shown that all known Pgammas contain a specific phosphorylation motif for cyclin-dependent protein kinase 5 (Cdk5) and that the unknown kinase is Cdk5 complexed with its activator. Here, using frog rod photoreceptor outer segments (ROS) isolated by a new method, we show that Cdk5 is involved in light-dependent Pgamma phosphorylation in vivo. Under dark conditions only negligible amounts of Pgamma were phosphorylated. However, under illumination that bleached less than 0.3% of the rhodopsin, approximately 4% of the total Pgamma was phosphorylated in less than 10 s. Pgamma dephosphorylation occurred in less than 1 s after the light was turned off. Analysis of the phosphorylated amino acid, inhibition of Pgamma phosphorylation by Cdk inhibitors in vivo and in vitro, and two-dimensional peptide map analysis of Pgamma phosphorylated in vivo and in vitro indicate that Cdk5 phosphorylates a Pgamma threonine in the same manner in vivo and in vitro. These observations, together with immunological data showing the presence of Cdk5 in ROS, suggest that Cdk5 is involved in light-dependent Pgamma phosphorylation in ROS and that the phosphorylation is significant and reversible. In an homogenate of frog ROS, PDE activated by light/guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) was inhibited by Pgamma alone, but not by Pgamma complexed with GDP/Talpha or GTPgammaS/Talpha. Under these conditions, Pgamma phosphorylated by Cdk5 inhibited the light/GTPgammaS-activated PDE even in the presence of GTPgammaS/Talpha. These observations suggest that phosphorylated Pgamma interacts with and inhibits light/GTPgammaS-activated PDE, but does not interact with GTPgammaS/Talpha in the homogenate. Together, our results strongly suggest that after activation of PDE by light/GTP, Pgamma is phosphorylated by Cdk5 and the phosphorylated Pgamma inhibits GTP/Talpha-activated PDE, even in the presence of GTP/Talpha in ROS.
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PMID:Phosphorylation by cyclin-dependent protein kinase 5 of the regulatory subunit of retinal cGMP phosphodiesterase. II. Its role in the turnoff of phosphodiesterase in vivo. 1088 79

Inactivation of the visual G protein transducin, during recovery from photoexcitation, is regulated by RGS9-1, a GTPase-accelerating protein of the ubiquitous RGS protein family. Incubation of dark-adapted bovine rod outer segments with [gamma-(32)P]ATP led to RGS9-1 phosphorylation by an endogenous kinase in rod outer segment membranes, with an average stoichiometry of 0.2-0.45 mol of phosphates/mol of RGS9-1. Mass spectrometry revealed a single major site of phosphorylation, Ser(475). The kinase responsible catalyzed robust phosphorylation of recombinant RGS9-1 and not of an S475A mutant. A synthetic peptide corresponding to the region surrounding Ser(475) was also phosphorylated, and a similar peptide with the S475A substitution inhibited RGS9-1 phosphorylation. The RGS9-1 kinase is a peripheral membrane protein that co-purifies with rhodopsin in sucrose gradients and can be extracted in buffers of high ionic strength. It is not inhibited or activated significantly by a panel of inhibitors or activators of protein kinase A, protein kinase G, rhodopsin kinase, CaM kinase II, casein kinase II, or cyclin-dependent kinase 5, at concentrations 50 or more times higher than their reported IC(50) or K(i) values. It was inhibited by the protein kinase C inhibitor bisindolylmaleimide I and by lowering Ca(2+) to nanomolar levels with EGTA; however, it was not stimulated by the addition of phorbol ester, under conditions that significantly enhanced rhodopsin phosphorylation. A monoclonal antibody specific for the Ser(475)-phosphorylated form of RGS9-1 recognized RGS9-1 in immunoblots of dark-adapted mouse retina. Retinas from light-adapted mice had much lower levels of RGS9-1 phosphorylation. Thus, RGS9-1 is phosphorylated on Ser(475) in vivo, and the phosphorylation level is regulated by light and by [Ca(2+)], suggesting the importance of the modification in light adaptation.
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PMID:Phosphorylation of RGS9-1 by an endogenous protein kinase in rod outer segments. 1129 25

In vertebrate photoreceptors, photoexcited rhodopsin interacts with the G protein transducin, causing it to bind GTP and stimulate the enzyme cGMP phosphodiesterase. The rapid termination of the active state of this pathway is dependent upon a photoreceptor-specific regulator of G protein signaling RGS9-1 that serves as a GTPase activating protein (GAP) for transducin. Here, we show that, in preparations of photoreceptor outer segments (OS), RGS9-1 is readily phosphorylated by an endogenous Ser/Thr protein kinase. Protein kinase C and MAP kinase inhibitors reduced labeling by about 30%, while CDK5 and CaMK II inhibitors had no effect. cAMP-dependent protein kinase (PKA) inhibitor H89 reduced RGS9-1 labeling by more than 90%, while dibutyryl-cAMP stimulated it 3-fold, implicating PKA as the major kinase responsible for RGS9-1 phosphorylation in OS. RGS9-1 belongs to an RGS subfamily also including RGS6, RGS7, and RGS11, which exist as heterodimers with the G protein beta subunit Gbeta5. Phosphorylated RGS9-1 remains associated with Gbeta5L, a photoreceptor-specific splice form, which itself was not phosphorylated. RGS9-1 immunoprecipitated from OS was in vitro phosphorylated by exogenous PKA. The PKA catalytic subunit could also phosphorylate recombinant RGS9-1, and mutational analysis localized phosphorylation sites to Ser(427) and Ser(428). Substitution of these residues for Glu, to mimic phosphorylation, resulted in a reduction of the GAP activity of RGS9-1. In OS, RGS9-1 phosphorylation required the presence of free Ca(2+) ions and was inhibited by light, suggesting that RGS9-1 phosphorylation could be one of the mechanisms mediating a stronger photoresponse in dark-adapted cells.
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PMID:Phosphorylation of the regulator of G protein signaling RGS9-1 by protein kinase A is a potential mechanism of light- and Ca2+-mediated regulation of G protein function in photoreceptors. 1160 86

G-protein coupled receptor kinase 2 (GRK2) regulates the activity of many receptors. Because potent inhibitors of GRK2 are thus far limited to polyanionic compounds like heparin, we searched for new inhibitors with the aid of a molecular model of GRK2. We used the available crystal structure of cAMP dependent protein kinase (cAPK) as a template to construct a 3D homology model of GRK2. Known cAPK and GRK2 inhibitors were docked into the active sites of GRK2 and cAPK using DOCK v3.5. H8 docked into the hydrophobic pocket of the adenosine 5'-triphosphate (ATP) binding site of cAPK, consistent with its known competitive cAPK inhibition relative to ATP. Similarly, 3 of 4 known GRK2 inhibitors docked into the ATP binding pocket of GRK2 with good scores. Screening the Fine Chemicals Directory (FCD, containing the 3D structures of 13,000 compounds) for docking into the active sites of GRK2 identified H8 and the known GRK2 inhibitor trifluoperazine as candidates. Whereas H8 indeed inhibited light-dependent phosphorylation of rhodopsin by GRK2, but with low potency, 3 additional FCD compounds with promising GRK2 scores failed to inhibit GRK2. This result demonstrates limitations of the GRK2 model in predicting activity among diverse chemical structures. Docking suramin, an inhibitor of protein kinase C (not present in FCD) yielded a good fit into the ATP binding site of GRK2 over cAPK. Suramin did inhibit GRK2 with IC50 32 microM (pA26.39 for competitive inhibition of ATP). Suramin congeners with fewer sulfonic acid residues (NF062, NF503 [IC50 14 microM]) or representing half of the suramin molecule (NF520) also inhibited GRK2 as predicted by docking. In conclusion, suramin and analogues are lead compounds in the development of more potent and selective inhibitors of GRK2.
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PMID:Molecular modeling of G-protein coupled receptor kinase 2: docking and biochemical evaluation of inhibitors. 1174 Dec 18

Cyclic GMP phosphodiesterase (PDE6) is a key enzyme in vertebrate retinal phototransduction. After GTP/GDP exchange on the a subunit of transducin (Talpha) by illuminated rhodopsin, the GTP-bound form Talpha (GTP/Talpha) interacts with the regulatory subunit (Pgamma) of PDE6 to activate cGMP hydrolytic activity. The regulatory mechanism of PDE6 has been believed to be a typical G protein-mediated signal transduction process. We found that cyclin-dependent protein kinase 5 (Cdk5) phosphorylates Pgamma complexed with GTP/Talpha in vitro and in vivo. Phosphorylated Py dissociates from GTP/Talpha without GTP hydrolysis and interacts effectively with catalytic subunits of PDE6 to inhibit the enzyme activity. These observations provide new twists to the current model of retinal phototransduction. In this article, in addition to the details of Py phosphorylation by Cdk5, we review previous studies implying the Pgamma phosphorylation and the turnoff of PDE6 without GTP hydrolysis and indicate the direction for future studies of Py phosphorylation, including the possible involvement of Ca2+/Ca2+-binding proteins.
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PMID:Phosphorylation by cyclin-dependent protein kinase 5 of the regulatory subunit (Pgamma) of retinal cgmp phosphodiesterase (PDE6): its implications in phototransduction. 1259 20

Phosphorylation of G protein-coupled receptors is a critical step in the rapid termination of G protein signaling. In rod cells of the vertebrate retina, phosphorylation of rhodopsin is mediated by GRK1. In cone cells, either GRK1, GRK7, or both, depending on the species, are speculated to initiate signal termination by phosphorylating the cone opsins. To compare the biochemical properties of GRK1 and GRK7, we measured the K(m) and V(max) of these kinases for ATP and rhodopsin, a model substrate. The results demonstrated that these kinases share similar kinetic properties. We also determined that cAMP-dependent protein kinase (PKA) phosphorylates GRK1 at Ser(21) and GRK7 at Ser(23) and Ser(36) in vitro. These sites are also phosphorylated when FLAG-tagged GRK1 and GRK7 are expressed in HEK-293 cells treated with forskolin to stimulate the endogenous production of cAMP and activation of PKA. Rod outer segments isolated from bovine retina phosphorylated the FLAG-tagged GRKs in the presence of dibutyryl-cAMP, suggesting that GRK1 and GRK7 are physiologically relevant substrates. Although both GRKs also contain putative phosphorylation sites for PKC and Ca(2+)/calmodulin-dependent protein kinase II, neither kinase phosphorylated GRK1 or GRK7. Phosphorylation of GRK1 and GRK7 by PKA reduces the ability of GRK1 and GRK7 to phosphorylate rhodopsin in vitro. Since exposure to light causes a decrease in cAMP levels in rod cells, we propose that phosphorylation of GRK1 and GRK7 by PKA occurs in the dark, when cAMP levels in photoreceptor cells are elevated, and represents a novel mechanism for regulating the activities of these kinases.
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PMID:Phosphorylation of GRK1 and GRK7 by cAMP-dependent protein kinase attenuates their enzymatic activities. 1594 41

Phosphorylation of the agonist-activated form of G-protein-coupled receptors (GPCRs) by a protein kinase from the G-protein-coupled receptor kinase (GRK) family initiates, with arrestin proteins, a negative feedback process known as desensitization. Because these receptors are involved in so many vital functions, it seems likely that disorders affecting GRK- or arrestin-mediated regulation of GPCRs would contribute to, if not engender, disease. Traditionally, it is believed that the desensitization process protects the cell against an overstimulation; however, in certain situations, this process is maladjusted and participes in disease progression. For example, in Oguchi disease, excessive rhodopsin stimulation due to a functional loss of GRK1 or arrestin 1 leads to light sensitization and stationary night blindness. Also, transgenic mice with vascular smooth muscle-targeted overexpression of GRK2 showed an elevated resting blood pressure, suggesting that increase in GRK2 level in humans is involved in hypertension associated with a decreased effect of beta-adrenergic receptor-mediated vasorelaxation. The restoration of normal GPCR function in modulating the desensitization process has been successfully demonstrated in animal models of heart failure, which indicates that targeting GRKs or arrestins may open a novel therapeutic strategy in human diseases with GPCR dysregulation. However, the few effective pharmacological compounds in this domain currently preclude human clinical tests.
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PMID:[GRKs and arrestins: the therapeutic pathway?]. 1668 24


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