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 (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

The GTPase RhoA has been implicated in various cellular activities, including the formation of stress fibers, motility, and cytokinesis. We recently reported on a p150 serine/threonine kinase (termed ROK alpha) binding RhoA only in its active GTP-bound state and on its cDNA; introduction of RhoA into HeLa cells resulted in translocation of the cytoplasmic kinase to plasma membranes, consistent with ROK alpha being a target for RhoA (T. Leung, E. Manser, L. Tan, and L. Lim, J. Biol. Chem. 256:29051-29054, 1995). Reanalysis of the cDNA revealed that ROK alpha contains an additional N-terminal region. We also isolated another cDNA which encoded a protein (ROK beta) with 90% identity to ROK alpha in the kinase domain. Both ROK alpha and ROK beta, which had a molecular mass of 160 kDa, contained a highly conserved cysteine/histidine-rich domain located within a putative pleckstrin homology domain. The kinases bound RhoA, RhoB, and RhoC but not Rac1 and Cdc42. The Rho-binding domain comprises about 30 amino acids. Mutations within this domain caused partial or complete loss of Rho binding. The morphological effects of ROK alpha were investigated by microinjecting HeLa cells with DNA constructs encoding various forms of ROK alpha. Full-length ROK alpha promoted formation of stress fibers and focal adhesion complexes, consistent with its being an effector of RhoA. ROK alpha truncated at the C terminus promoted this formation and also extensive condensation of actin microfilaments and nuclear disruption. The proteins exhibited protein kinase activity which was required for stress fiber formation; the kinase-dead ROK alpha K112A and N-terminally truncated mutants showed no such promotion. The latter mutant instead induced disassembly of stress fibers and focal adhesion complexes, accompanied by cell spreading. These effects were mediated by the C-terminal region containing Rho-binding, cysteine/histidine-rich, and pleckstrin homology domains. Thus, the multidomained ROK alpha appears to be involved in reorganization of the cytoskeleton, with the N and C termini acting as positive and negative regulators, respectively, of the kinase domain whose activity is crucial for formation of stress fibers and focal adhesion complexes.
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PMID:The p160 RhoA-binding kinase ROK alpha is a member of a kinase family and is involved in the reorganization of the cytoskeleton. 881 43

Protein kinase C mu (PKC mu) displays unusual structural features like a pleckstrin homology domain and an amino-terminal hydrophobic region with a putative leader peptide and transmembrane sequence. As a discrete location often is a direct clue to the potential biological function of a kinase, antibodies directed against unique amino- and carboxy-terminal domains of PKC mu were used to localize the protein within intracellular compartments in immunofluorescence and subcellular fractionation studies. Confocal laser scanning microscopy showed colocalization of PKC mu with the resident Golgi marker protein beta 1,4 galactosyltransferase in PKC mu transfectants and in the human hepatocellular carcinoma cell line HepG2, expressing endogenous PKC mu. Long-term treatment of cells with brefeldin A, which disintegrates the Golgi apparatus, disrupted PKC mu-specific staining. Cosegregation of PKC mu with beta 1,4 galactosyltransferase, but not with the endosomal marker rab5, upon density gradient fractionation and Western blot analysis of HepG2 cell extracts, provides independent evidence for a Golgi localization of PKC mu. Moreover, cellular sulfate uptake and Golgi-specific glycosaminoglycan sulfation was enhanced in PKC mu transfectants. Together, these data suggest that PKC mu is a resident protein kinase of the core Golgi compartment and is involved in basal transport processes.
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PMID:Protein kinase C mu is located at the Golgi compartment. 883 Jul 70

Insulin receptor substrate-1 (IRS-1), a substrate of various receptor tyrosine kinases transmits mitogenic signals initiated by extracellular ligands. This protein is involved in normal hepatocyte growth and has been found to be overexpressed in human hepatocellular carcinoma. Expression of a carboxy-terminal truncated IRS-1 molecule containing the pleckstrin homology and phosphotyrosine-binding domains associates with the insulin receptor and prevents tyrosyl phosphorylation of endogenous IRS-1 and Shc proteins. Thus, subsequent activation of downstream signaling molecules induced by insulin and IGF-1 such as phosphatidylinositol-3 kinase and mitogen activated protein kinase is inhibited. The morphologic features of transformed human hepatocellular carcinoma cells change to a differentiated hepatocyte appearance and characteristics of the malignant phenotype as manifested by anchorage independent cell growth and tumor formation in nude mice are lost. These studies demonstrate that signal transduction pathways mediated through or by IRS-1 are important in hepatocyte and human hepatocellular carcinoma cell growth.
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PMID:A carboxy-terminal truncated insulin receptor substrate-1 dominant negative protein reverses the human hepatocellular carcinoma malignant phenotype. 890 30

Phosphatidylinositol (PI) 3-kinase is a cytoplasmic signaling molecule that is recruited to activated growth factor receptors after growth factor stimulation of cells. Activation of PI 3-kinase results in increased intracellular levels of 3' phosphorylated inositol phospholipids and the induction of signaling responses, including the activation of the protein kinase Akt, which is also known as RAC-PK or PKB. We tested the possibility that the phospholipid products of PI 3-kinase directly mediate the activation of Akt. We have previously described a constitutively active PI 3-kinase, p110, which can stimulate Akt activity. We used purified p110 protein to generate a series of 3' phosphorylated inositol phospholipids and tested whether any of these lipids could activate Akt in vitro. Phospholipid vesicles containing PI3,4 bisphosphate (P2) specifically activated Akt in vitro. By contrast, the presence of phospholipid vesicles containing PI3P or PI3,4,5P3 failed to increase the kinase activity of Akt. Akt could also be activated by synthetic dipalmitoylated PI3,4P2 or after enzymatic conversion of PI3,4,5P3 into PI3,4P2 with the signaling inositol polyphosphate 5' phosphatase SIP. We show that PI3,4P2-mediated activation is dependent on a functional pleckstrin homology domain in Akt, since a point mutation in the pleckstrin homology domain abrogated the response to PI3,4P2. Our findings show that a phospholipid product of PI 3-kinase can directly stimulate an enzyme known to be an important mediator of PI 3-kinase signaling.
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PMID:A specific product of phosphatidylinositol 3-kinase directly activates the protein kinase Akt through its pleckstrin homology domain. 897 14

The small GTPases Cdc42 and Rac regulate a variety of biological processes, including actin polymerization, cell proliferation, and JNK/mitogen-activated protein kinase activation, conceivably via distinct effectors. Whereas the effector for mitogen-activated protein kinase activation appears to be p65PAK, the identity of effector(s) for actin polymerization remains unclear. We have found a putative effector for Drosophila Cdc42, Genghis Khan (Gek), which binds to Dcdc42 in a GTP-dependent and effector domain-dependent manner. Gek contains a predicted serine/threonine kinase catalytic domain that is 63% identical to human myotonic dystrophy protein kinase and has protein kinase activities. It also possesses a large coiled-coil domain, a putative phorbol ester binding domain, a pleckstrin homology domain, and a Cdc42 binding consensus sequence that is required for its binding to Dcdc42. To study the in vivo function of gek, we generated mutations in the Drosophila gek locus. Egg chambers homozygous for gek mutations exhibit abnormal accumulation of F-actin and are defective in producing fertilized eggs. These phenotypes can be rescued by a wild-type gek transgene. Our results suggest that this multidomain protein kinase is an effector for the regulation of actin polymerization by Cdc42.
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PMID:Genghis Khan (Gek) as a putative effector for Drosophila Cdc42 and regulator of actin polymerization. 937 83

Protein kinase D (PKD) is a serine/threonine protein kinase that contains a cysteine-rich repeat sequence homologous to that seen in the regulatory domain of protein kinase C (PKC) and a catalytic domain with only a low degree of sequence similarity to PKCs. PKD also contains a pleckstrin homology (PH) domain inserted between the cysteine-rich motifs and the catalytic domain that is not present in any of the PKCs. To investigate the function of the PH domain in the regulation of PKD activity, we determined the kinase activity of several PKD PH domain mutants immunoprecipitated from lysates of transiently transfected COS-7 cells. Deletion of the entire PH domain (amino acids 429-557) markedly increased the basal activity of the enzyme as assessed by autophosphorylation ( approximately 16-fold) and exogenous syntide-2 peptide substrate phosphorylation assays (approximately 12-fold). Mutant PKD proteins with partial deletions or single amino acid substitutions within the PH domain (e. g. R447C and W538A) also exhibited increased basal kinase activity. These constitutive active mutants of PKD were only slightly further stimulated by phorbol-12,13-dibutyrate treatment of intact cells. Our results demonstrate, for the first time, that the PKD PH domain plays a negative role in the regulation of enzyme activity.
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PMID:Protein kinase D activation by mutations within its pleckstrin homology domain. 941 97

The pleckstrin homology domains (PH domains) derived from four different proteins, the N-terminal part of pleckstrin, RAC-protein kinase, diacylglycerol kinase and the 130 kDa protein originally cloned as an inositol 1,4,5-trisphosphate binding protein, were analysed for binding of inositol phosphates and derivatives of inositol lipids. The PH domain from pleckstrin bound inositol phosphates according to a number of phosphates on the inositol ring, i.e. more phosphate groups, stronger the binding, but a very limited specificity due to the 2-phosphate was also observed. On the other hand, the PH domains from RAC-protein kinase and diacylglycerol kinase specifically bound inositol 1,3,4,5,6-pentakisphosphate and inositol 1,4,5,6-tetrakisphosphate most strongly. The PH domain from the 130 kDa protein, however, had a preference for inositol 1,4,5-trisphosphate and 1,4,5,6-tetrakisphosphate. Comparison was also made between binding of inositol 1,4,5-trisphosphate, inositol 1,3,4,5-tetrakisphosphate and soluble derivatives of their corresponding phospholipids. The PH domains examined, except that from pleckstrin, showed a 8- to 42-times higher affinity for inositol 1,4,5-trisphosphate than that for corresponding phosphoinositide derivative. However, all PH domains had similar affinity for inositol 1,3,4,5-tetrakisphosphate compared to the corresponding lipid derivative. The present study supports our previous proposal that inositol phosphates and/or inositol lipids could be important ligands for the PH domain, and therefore inositol phosphates/inositol lipids may have the considerable versatility in the control of diverse cellular function. Which of these potential ligands are physiologically relevant would depend on the binding affinities and their cellular abundance.
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PMID:Distinct specificity in the binding of inositol phosphates by pleckstrin homology domains of pleckstrin, RAC-protein kinase, diacylglycerol kinase and a new 130 kDa protein. 943 33

Phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] has been known to bind to the pleckstrin homology domain and the phosphotyrosine-binding domain as well as actin-binding proteins, and to regulate their functions. We have tried to find new PtdIns(4,5)P2-binding proteins and to clarify the physiological effects of PtdIns(4,5)P2 on their function. We report here that histones H1 and H3 are PtdIns(4,5)P2-binding proteins which were identified using antibodies specific to PtdIns(4,5)P2, H1, and H3. This binding was further confirmed by extracting PtdIns(4,5)P2 from purified histone H1 and H3. Furthermore, the binding site of PtdIns(4,5)P2 in histone H1 was found in the carboxyl-terminal 103 amino acids. It was also shown that the amounts of PtdIns(4,5)P2 bound to H1 decrease when histone H1 is phosphorylated by protein kinase C but not by protein kinase A or cdc2 kinase, in vitro. The protein kinase C phosphorylation site is localized close to the PtdIns(4,5)P2-binding site, suggesting that phosphorylation of histone H1 by protein kinase C interferes stereostructurally with PtdIns(4,5)P2 binding. We further noticed that PtdIns(4,5)P2 binding to H1 counteracts the histone H1-mediated repression of basal transcription by RNA polymerase II in a Drosophila transcription system in vitro. Phosphatidylinositol 4-phosphate and phosphatidylinositol 3,4,5-trisphosphate affect this transcription activity more weakly than PtdIns(4,5)P2, but PtdIns and other acidic lipids have no effect on this activity. These data indicate that PtdIns(4,5)P2 bound to nuclear protein histone H1 may contribute to the regulation of transcription in eukaryotic cells.
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PMID:Phosphatidylinositol 4,5-bisphosphate reverses the inhibition of RNA transcription caused by histone H1. 949 95

A wide variety of biological activities including the major metabolic actions of insulin is regulated by phosphatidylinositol (PI) 3-kinase. However, the downstream effectors of the various signaling pathways that emanate from PI 3-kinase remain unclear. Akt (protein kinase B), a serine-threonine kinase with a pleckstrin homology domain, is thought to be one such downstream effector. A mutant Akt (Akt-AA) in which the phosphorylation sites (Thr308 and Ser473) targeted by growth factors are replaced by alanine has now been shown to lack protein kinase activity and, when overexpressed in CHO cells or 3T3-L1 adipocytes with the use of an adenovirus vector, to inhibit insulin-induced activation of endogenous Akt. Akt-AA thus acts in a dominant negative manner in intact cells. Insulin-stimulated protein synthesis, which is sensitive to wortmannin, a pharmacological inhibitor of PI 3-kinase, was abolished by overexpression of Akt-AA without an effect on amino acid transport into the cells, suggesting that Akt is required for insulin-stimulated protein synthesis. Insulin activation of p70 S6 kinase was inhibited by approximately 75% in CHO cells and approximately 30% in 3T3-L1 adipocytes, whereas insulin-induced activation of endogenous Akt was inhibited by 80 to 95%, by expression of Akt-AA. Thus, Akt activity appears to be required, at least in part, for insulin stimulation of p70 S6 kinase. However, insulin-stimulated glucose uptake in both CHO cells and 3T3-L1 adipocytes was not affected by overexpression of Akt-AA, suggesting that Akt is not required for this effect of insulin. These data indicate that Akt acts as a downstream effector in some, but not all, of the signaling pathways downstream of PI 3-kinase.
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PMID:Requirement for activation of the serine-threonine kinase Akt (protein kinase B) in insulin stimulation of protein synthesis but not of glucose transport. 963 53


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