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)

We have reported that platelets exposed to thrombin or thrombin receptor-directed ligand activate phospholipase C and rapidly accumulate phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P3) and phosphatidylinositol (3,4)-bisphosphate (PtdIns(3,4)P2) as a function of the activation of phosphoinositide (PI) 3-kinases in a GTP-binding protein-dependent manner. In such platelets, serine- and threonine-directed phosphorylation of pleckstrin also occurs and has been attributed to protein kinase C activation. We now report that the phosphorylation of pleckstrin is partially dependent upon PI 3-kinase. Pleckstrin phosphorylation in response to thrombin receptor stimulation is progressively susceptible to inhibition by wortmannin, a potent and specific inhibitor of platelet PI 3-kinases. PI 3-kinase thus seems to play a gradually increasing role in promoting pleckstrin phosphorylation. The IC50 for wortmannin in inhibiting SFLLRN-stimulated 3-phosphorylated phosphoinositide accumulation is 10 nM, and that (i.e. 50% of maximum inhibition) for inhibiting pleckstrin phosphorylation is 15 nM. Synthetic PtdIns(3,4,5)P3, when added to saponin-permeabilized (but not intact) platelets, causes wortmannin-insensitive phosphorylation of pleckstrin. PtdIns(3,4,5)P3 also overcomes the inhibition by wortmannin of thrombin- or guanosine 5'-3-O-(thio)trisphosphate-stimulated pleckstrin phosphorylation. In contrast, PtdIns(4,5)P2 or inositol (1,3,4,5)-tetrakisphosphate are ineffective in these respects. The pattern of phosphorylation of pleckstrin activated by PtdIns(3,4,5)P3 is not distinguishable from that of pleckstrin phosphorylated in intact platelets exposed to protein kinase C-activating beta-phorbol myristate acetate, mimicking diacylglycerol. Activation of protein kinase(s) by PtdIns(3,4,5)P3 thus offers a route for pleckstrin phosphorylation in vivo that is an alternative to activation of phospholipase C-->diacylglycerol-->protein kinase C.
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PMID:Phosphatidylinositol (3,4,5)-trisphosphate stimulates phosphorylation of pleckstrin in human platelets. 755 10

The brain-enriched p21cdc42/rac1-activated serine/threonine kinase, p65PAK, was identified and purified on the basis of overlays with [gamma-32P]GTP-Cdc42 onto SDS-fractionated proteins (Manser, E., Leung, T., Salihuddin, H., Zhao, Z.-S., and Lim, L. (1994) Nature 367, 40-46). In this study, the ubiquitously expressed p21cdc42/rac1 binding protein with relative molecular weight of 62,000 was purified from rat testes and shown to contain peptides related to PAK. It has thus been designated as the gamma-PAK isoform (alpha- and beta-isoforms being brain enriched). Isolation of gamma-PAK cDNAs show that the kinase is highly conserved with alpha-PAK in both the p2 binding and kinase domains. The purified protein exhibited kinase activity that was activated by GTP-Cdc42 or GTP-Rac1 in vitro. In platelets, a p62 in situ renaturable kinase was recognized by antibodies raised against gamma-PAK. This thrombin-activated protein kinase appears to coprecipitate with another kinase of M(r) 86,000, suggesting that PAK may be part of a thrombin-responsive signaling complex.
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PMID:Identification and molecular cloning of a p21cdc42/rac1-activated serine/threonine kinase that is rapidly activated by thrombin in platelets. 759 96

The link between increased usage of beta-adrenoceptor agonists and worsening of asthma symptoms has raised interest in the effects of agents such as salbutamol on airway wall remodelling, and particularly airway smooth muscle proliferation. In the present study we have investigated the role of increases in intracellular cAMP in the inhibitory effect of salbutamol on airway smooth muscle proliferation. The inhibitory effects of a combination of submaximally effective concentrations of salbutamol (10 nM) and the non-selective phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX, 100 microM) on thrombin (0.3 U/mL)-induced mitogenesis in human cultured airway smooth muscle cells was greater than that for either agent alone. In addition, agents known to increase cAMP-dependent protein kinase activity including forskolin (10 microM), 8-bromoadenosine-3',5'-cyclic monophosphate (100 microM), and prostaglandin E2 (1 microM) have an inhibitory effect on thrombin (0.3 U/mL)-induced induced proliferation. Furthermore, the cAMP antagonist, 8-bromoadenosine-3',5'-cyclic monophosphorothioate, Rp-isomer (300 microM) significantly reduced the inhibitory effect of salbutamol (10 nM) on thrombin (0.3 U/mL)-induced DNA synthesis. In IBMX (100 microM)-pretreated cells, salbutamol (100 nM) increased intracellular cAMP levels via stimulation of a beta 2-adrenoceptor. Salbutamol (10 microM), at concentrations supramaximally effective for inhibition of mitogenesis, had no effect on thrombin (0.3 U/mL)-induced increases in intracellular calcium levels. Therefore, our results suggest that the previously reported inhibition of mitogen-induced proliferation in human cultured airway smooth muscle cells by the beta 2-adrenoceptor agonist, salbutamol (100 nM), is at least partly due to elevation of intracellular cAMP, while there is no effect of salbutamol on initial mitogen-induced increases in intracellular calcium.
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PMID:Salbutamol inhibits the proliferation of human airway smooth muscle cells grown in culture: relationship to elevated cAMP levels. 759 43

A potent, proteinaceous inducer of platelet aggregation designated as IVa, has been purified to homogeneity from Cerastes cerastes venom by molecular sieve and ion exchange chromatography. It is composed of 2 subunits with total M(r) of 62,000 as shown by native gel chromatography and chemical cross-linking with disuccinimidyl suberate. It is not clear at the present time whether both subunits are identical gene products, however, both have identical N-terminal sequences for the first 15 amino acids. The protein has a pI above 9.6. IVa (0.1 micrograms/ml) could aggregate platelets up to 80% and was inhibited by p-APMSF, leupeptin, iodoacetamide, protein kinase C inhibitor, phosphatase inhibitor, ATP and PGE1, while it was insensitive to acetylsalicylic acid, ADP scavenger system, protein kinase A inhibitor and hirudin. Protein IVa is a serine proteinase with thrombin-like activity as it hydrolysed thrombin chromogenic substrate CBS 34.47, its aggregatory activity was partially inhibited by monoclonal antibodies against GPIb and the thrombin receptor, as was the thrombin, and its ability to induce intracellular Ca2+ release was blocked by pretreating platelets with thrombin. Unlike thrombin, the IVa protein showed very weak coagulant activity as indicated by plasma recalcification time and fibrinogen clotting time although it could hydrolyse fibrinogen alpha-chains.
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PMID:Characterization of a potent platelet aggregation inducer from Cerastes cerastes (Egyptian sand viper) venom. 761 60

We studied the participation of the protein kinase C pathway in thrombin-induced cytoskeletal alterations in confluent cultured bovine corneal endothelial (BCE) cells. Cultured BCE cells were exposed to alpha-thrombin (0.1-10 U/ml for 15-60 min) and the distribution of F-actin and vinculin plaques was examined using immunofluorescent staining and electron microscopy. Phorbol 12-myristate 13-acetate (PMA, 10 nM for 15 min), the broad spectrum protein kinase inhibitors staurosporine (10 nM) and H-7 (10 nM), and highly specific PKC inhibitor calphostin C (10 nM) were used to evaluate the role of PKC/phosphorylation in this phenomenon. HA-1004 (10 nM) was used as a negative control for these inhibitors. In a parallel experiment, PKC activity of cytosol and membrane of BCE cells was also evaluated. In control samples, F-actin was distributed mainly at the periphery of cells, where it formed dense peripheral bundles; vinculin plaques were also present at the cell boundary. Exposure of BCE cells to thrombin changed the distribution of F-actin and vinculin into a diffuse pattern; a similar alteration was also induced by incubation with PMA. These phenomena were blocked by incubation with H-7, staurosporine and calphostin C. Both cytosolic and membrane PKC activity was increased after 5 to 30 min exposure of alpha-thrombin and returned to the control level after 1 h. alpha-Thrombin induces alteration in the cytoskeleton of BCE cells, and this message is transduced at least in part by PKC dependent pathways. PKC/phosphorylation may thus play an important role in physiological processes that involve alterations of the cytoskeleton.
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PMID:Thrombin induced cytoskeletal change in cultured bovine corneal endothelial cells mediated via protein kinase C pathway. 772 Apr 4

cGMP-dependent protein kinase (cGMP kinase) has been implicated in the regulation of the cytosolic calcium level ([Ca2+]i). In Chinese hamster ovary (CHO) cells stably transfected with the cGMP kinase I alpha (CHO-cGK cells), cGMP kinase suppressed the thrombin-induced increase in inositol 1,4,5-trisphosphate and [Ca2+]i (Ruth, P., Wang, G.-X., Boekhoff, I., May, B., Pfeifer, A., Penner, R., Korth, M., Breer, H., and Hofmann, F. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 2623-2627). Cholecystokinin activated intracellular calcium release via a pertussis toxin (PTX)-insensitive pathway in CHO-cGK cells. cGMP kinase did not attenuate the CCK-stimulated [Ca2+]i. In contrast, cGMP kinase suppressed calcium influx stimulated by insulin-like growth factors 1 and 2 (IGF-1 and IGF-2) via PTX-sensitive pathways. The effects of PTX and cGMP kinase on [Ca2+]i were not additive. 8-Bromo-cGMP had no effect on [Ca2+]i stimulated by IGF-1 or IGF-2 in wild type CHO cells. These results suggested that cGMP kinase inhibited the different signaling pathways by the phosphorylation of a PTX-sensitive G protein. cGMP kinase phosphorylated the alpha subunits of Gi1, Gi2, and Gi3 in vitro. Phosphorylation stoichiometry was 0.4 mol of phosphate/mol of G alpha i1 after reconstitution of heterotrimeric Gi1 in phospholipid vesicles. The alpha subunit of Gi was also phosphorylated in vivo. These results show that cGMP kinase blocks transduction of distinct hormone pathways that signal via PTX-sensitive Gi proteins.
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PMID:Cyclic GMP-dependent protein kinase blocks pertussis toxin-sensitive hormone receptor signaling pathways in Chinese hamster ovary cells. 772 18

Thrombin stimulates synthesis and secretion of endothelin-1 (ET-1), a vasoactive peptide that triggers responses in the vascular endothelium and smooth muscle. We investigated the signal transduction pathways by which thrombin stimulates preproET-1 gene expression and ET-1 peptide secretion in macrovascular cells (human umbilical vein endothelial cells [HUVECs] and bovine pulmonary artery endothelial cells [BPAECs]) and microvascular cells (human microvascular endothelial cell line [HMEC-1]). Thrombin (4 U/mL) stimulated maximal induction of ET-1 peptide secretion and preproET-1 mRNA after 2 hours in HUVECs and BPAECs and after 1 hour in HMEC-1. A synthetic thrombin receptor activator peptide confirmed ligand-specific receptor actions to induce preproET-1 mRNA. Protein kinase C (PKC) activation by phorbol ester transiently induced preproET-1 mRNA but had no effect on ET-1 peptide synthesis. PKC inhibitors sangivamycin and calphostin C and PKC depletion failed to suppress thrombin-stimulated preproET-1 mRNA. Adenylate cyclase and cAMP-dependent protein kinase did not participate in thrombin-induced preproET-1 gene activation. Thrombin stimulated a rapid increase in phosphotyrosine-containing proteins, suggesting a role for tyrosine phosphorylation in thrombin signaling. These data demonstrate that thrombin induces the preproET-1 gene and ET-1 peptide synthesis by a PKC-independent PTK-dependent pathway in macrovascular and microvascular endothelial cells. Protein tyrosine kinase inhibitors herbimycin A and genistein blocked thrombin-stimulated preproET-1 mRNA and peptide secretion, whereas daidzein, which lacks inhibitory activity, did not suppress thrombin-induced ET-1.
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PMID:Thrombin induces the preproendothelin-1 gene in endothelial cells by a protein tyrosine kinase-linked mechanism. 775 70

Endothelial cell (EC) contraction results in intercellular gap formation and loss of the selective vascular barrier to circulating macromolecules. We tested the hypothesis that phosphorylation of regulatory myosin light chains (MLC) by Ca2+/calmodulin-dependent myosin light chain kinase (MLCK) is critical to EC barrier dysfunction elicited by thrombin. Thrombin stimulated a rapid (< 15 sec) increase in [Ca2+]i which preceded maximal MLC phosphorylation (60 sec) with a 6 to 8-fold increase above constitutive levels of phosphorylated MLC. Dramatic cellular shape changes indicative of contraction and gap formation were observed at 5 min with maximal increases in albumin permeability occurring by 10 min. Neither the Ca2+ ionophore, A23187, nor phorbol myristate acetate (PMA), a direct activator of protein kinase C (PKC), alone or in combination, produced MLC phosphorylation. The combination was synergistic, however, in stimulating EC contraction/gap formation and barrier dysfunction (3 to 4-fold increase). Down-regulation or inhibition of PKC activity attenuated thrombin-induced MLC phosphorylation (approximately 40% inhibition) and both thrombin- and PMA-induced albumin clearance (approximately 50% inhibition). Agents which augmented [cAMP]i partially blocked thrombin-induced MLC phosphorylation (approximately 50%) and completely inhibited both thrombin- and PMA-induced EC permeability (100% inhibition). Furthermore, cAMP produced significant reduction in the basal levels of constitutive MLC phosphorylation. Finally, MLCK inhibition (with either ML-7 or KT 5926) or Ca2+/calmodulin antagonism (with either trifluoperazine or W-7) attenuated thrombin-induced MLC phosphorylation and barrier dysfunction. These results suggest a model wherein EC contractile events, gap formation and barrier dysfunction occur via MLCK-dependent and independent mechanisms and are significantly modulated by both PKC and cAMP-dependent protein kinase A activities.
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PMID:Regulation of endothelial cell gap formation and barrier dysfunction: role of myosin light chain phosphorylation. 777 94

ATP and thrombin both induced Ca2+ mobilization from intracellular Ca2+ store site of megakaryocyte, the progenitor cell of platelet (Uneyama C., Uneyama H. and Akaike N. (1993) J. Physiol. (Lond.), 470, 73-749). Since in platelet, thrombin is known as a strong agonist and ADP is known as a weak agonist, we further investigated the effect of these agonists on megakaryocyte. Thrombin induced Ca2+ mobilization, 5-hydroxy tryptamine (5-HT) release and aggregatory morphological changes in megakaryocyte, but ATP induced only Ca2+ mobilization. Thrombin-induced 5-HT release was inhibited by adenylate cyclase-activating drugs, and the morphological changes could be induced by H-8, an inhibitor of cAMP-dependent protein kinase. These results suggest that the Ca2+ mobilization is not sufficient to induce morphological changes, and the signal to cause morphological changes in megakaryocyte may be cAMP.
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PMID:Not Ca2+ but CAMP is the second messenger for morphological changes in rat megakaryocyte. 777 97

Subcellular fractions were prepared from human platelet membranes by sucrose density gradient centrifugation and the localization of a low M(r) GTP-binding protein, rap1 protein (Rap1) was analysed by immunoblotting using a specific antibody. Rap1, which has been purified from human platelets, was found to be located in plasma membrane and alpha-granule fractions in resting platelets. Treatment of isolated alpha-granules with pronase led to proteolysis of Rap1, indicating that this protein is exposed to the cytoplasmic face of the granules. Degranulation of alpha-granules consists of translocation and subsequent fusion of the granules with the open canalicular system. Activation of this process by thrombin induced the redistribution of Rap1 on the alpha-granules to plasma membranes. On the other hand, Rap1 is known to be phosphorylated by cyclic AMP-dependent protein kinase (A-kinase) in vitro and in vivo. In intact human platelets, phosphorylation of Rap1 by A-kinase in response to prostaglandin E1 (PGE1) was observed only in Rap1 localized in plasma membranes and not on alpha-granules, although Rap1 was phosphorylated in a cell-free system when plasma membranes and alpha-granule membranes were exposed to A-kinase as substrates. These results strongly suggest that Rap1 in plasma membranes and the protein on alpha-granules are regulated by different mechanisms, and have different functions.
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PMID:A low M(r) GTP-binding protein, Rap1, in human platelets: localization, translocation and phosphorylation by cyclic AMP-dependent protein kinase. 778 83


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