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:3.4.21.5 (thrombin)
33,306 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Somatostatin regulates multiple biological functions by acting through a family of five G protein-coupled receptors, somatostatin receptors (SSTRs) 1-5. Although all five receptor subtypes inhibit adenylate cyclase activity and decrease intracellular cAMP levels, specific receptor subtypes also couple to additional signaling pathways. In CCL39 fibroblasts expressing either human SSTR1 or SSTR2, we demonstrate that activation of SSTR1 (but not SSTR2) attenuated both thrombin- and integrin-stimulated Rho-GTP complex formation. The reduction in Rho-GTP formation in the presence of somatostatin was associated with decreased translocation of Rho and LIM kinase to the plasma membrane and fewer focal contacts. Activation of Rho resulted in the formation of intracellular actin stress fibers and cell migration. In CCL39-R1 cells, somatostatin treatment prevented actin stress fiber assembly and attenuated thrombin-stimulated cell migration through Transwell membranes to basal levels. To show that native SSTR1 shares the ability to inhibit Rho activation, we demonstrated that somatostatin treatment of human umbilical vein endothelial cells attenuated thrombin-stimulated Rho-GTP accumulation. These data show for the first time that a G protein-coupled receptor, SSTR1, inhibits the activation of Rho, the assembly of focal adhesions and actin stress fibers, and cell migration.
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PMID:Somatostatin, acting at receptor subtype 1, inhibits Rho activity, the assembly of actin stress fibers, and cell migration. 1204 95

Soluble mediators such as thrombin and sphingosine-1-phosphate regulate morphological changes in endothelial cells that affect vascular permeability and new blood vessel formation. Although these ligands activate a similar set of heterotrimeric G proteins, thrombin causes cell contraction and rounding whereas sphingosine-1-phosphate induces cell spreading and migration. A functional requirement for Rho family GTPases in the cytoskeletal responses to both ligands has been established, yet the dynamics of their regulation and additional signaling mechanisms that lead to such opposite effects remain poorly understood. Using a pull-down assay to monitor the activity of Rho GTPases in human umbilical vein endothelial cells, we find significant temporal and quantitative differences in RhoA and Rac1 activation. High levels of active RhoA rapidly accumulate in cells in response to thrombin whereas Rac1 is inhibited. In contrast, sphingosine-1-phosphate addition leads to comparatively weak and delayed activation of RhoA and it activates Rac1. In addition, we show here that sphingosine-1-phosphate treatment activates a Src family kinase and triggers recruitment of the F-actin-binding protein cortactin to sites of actin polymerization at the rim of membrane ruffles. Both Src and Rac pathways are essential for lamellipodia targeting of cortactin. Further, Src plays a determinant role in sphingosine-1-phosphate-induced cell spreading and migration. Taken together these data demonstrate that the thrombin-induced contractile and immobile phenotype in endothelial cells reflects both robust RhoA activation and Rac inhibition, whereas Src- and Rac-dependent events couple sphingosine-1-phosphate receptors to the actin polymerizing machinery that drives the extension of lamellipodia and cell migration.
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PMID:Distinct signals via Rho GTPases and Src drive shape changes by thrombin and sphingosine-1-phosphate in endothelial cells. 1204 18

Adenosine triphosphate and diphosphate that activate platelet, leukocyte, and endothelium functions are hydrolyzed by endothelial CD39/ATPDase. Because CD39/ATPDase is downregulated in endothelial cells by inflammation and this may be affected by HMG-CoA reductase inhibitors, we examined the role of cerivastatin and simvastatin in regulation of endothelial CD39/ATPDase expression, metabolism of ATP/ADP, and function in platelets. Thrombin-stimulated endothelial cells in vitro were treated with the statins, and hydrolysis of exogenous ADP and ATP was assessed by high-performance liquid chromatography and malachite green assay. Platelet aggregation studies were performed with endothelial cell supernatants as triggers. CD39/ATPDase surface expression by endothelial cells was determined immunologically by fluorescence-activated cell sorter, mRNA expression by RT-PCR, and thrombin-induced dissociation of Rho-GTPases by Western blotting. Treatment by simvastatin or cerivastatin restored impaired metabolism of exogenous ATP and ADP in thrombin-activated endothelial cells by preventing thrombin-induced downregulation of CD39/ATPDase. In platelet aggregation studies, ATP and ADP supernatants of thrombin-activated endothelial cells were less stimulatory in the presence of statins than in their absence. Data show that statins preserve CD39/ATPDase activity in thrombin-treated endothelial cells involving alterations by statins of Rho-GTPase function and CD39/ATPDase expression. Preservation of adenine nucleotide metabolism may directly contribute to the observed anti-thrombotic and anti-inflammatory actions of statins.
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PMID:Reversal of thrombin-induced deactivation of CD39/ATPDase in endothelial cells by HMG-CoA reductase inhibition: effects on Rho-GTPase and adenosine nucleotide metabolism. 1206 95

Platelet activation and aggregation is considered a crucial step in the initiation and aggravation of arterial thrombosis. ADP from activated platelets is recognized as major factor in thrombus formation and is a potent stimulator of oxygen-free radical release from neutrophils. The aim of the present investigation was to determine in vitro the direct effects of statins on ATP and ADP secretion by platelets and its impact on subsequent oxidative burst activity in neutrophils. Human neutrophils and platelets were isolated from peripheral blood. Levels of platelet-derived ATP and ADP were measured by high-performance liquid chromatography, oxygen-free radical release of neutrophils was measured fluorometrically, and chemotaxis experiments were performed. Rho-GTPases were studied by Western blot analysis. Thrombin-activated platelets primed neutrophils for enhanced oxygen-free radical release on triggering with formyl-Met-Leu-Phe, reduced by cerivastatin and simvastatin treatment of platelets. The two statins decreased the amount of adenosine-derivative release in these cells. Rho-GTPases, required for the thrombin signaling in platelets and neutrophils, were decreased after coincubation with statins. Data demonstrate that inhibition of Rho-GTPases by statins inhibit platelet ADP and ATP release and the consecutive augmentation of neutrophil oxygen-free radical release. Statins affect platelet-neutrophil interactions by altering Rho-GTPase-dependent adenosine nucleotide function.
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PMID:Rho-GTPase-dependent platelet-neutrophil interaction affected by HMG-CoA reductase inhibition with altered adenosine nucleotide release and function. 1206 16

Recent studies from our laboratory have shown that insulin induces relaxation of vascular smooth muscle cells (VSMCs) via stimulation of myosin phosphatase and inhibition of Rho kinase activity. In this study, we examined the mechanism whereby insulin inhibits Rho signaling and its impact on actin cytoskeleton organization. Incubation of confluent serum-starved VSMCs with thrombin or phenylephrine (PE) caused a rapid increase in glutathione S-transferase-Rhotekin-Rho binding domain-associated RhoA, Rho kinase activation, and actin cytoskeleton organization, which was blocked by preincubation with insulin. Preexposure to N(G)-monomethyl L-arginine acetate (L-NMMA), a nitric oxide synthase inhibitor, and Rp-8 CPT-cyclic guanosine monophosphate (RpcGMP), a cyclic guanosine monophosphate (cGMP) antagonist, attenuated the inhibitory effect of insulin on RhoA activation and restored thrombin-induced Rho kinase activation, and site-specific phosphorylation of the myosin-bound regulatory subunit (MBS(Thr695)) of myosin-bound phosphatase (MBP), and caused actin fiber reorganization. In contrast, 8-bromo-cGMP, a cGMP agonist, mimicked the inhibitory effects of insulin and abolished thrombin-mediated Rho activation. Insulin inactivation of RhoA was accompanied by inhibition of isoprenylation via reductions in geranylgeranyl transferase-1 activity as well as increased RhoA phosphorylation, which was reversed by pretreatment with RpcGMP and L-NMMA. We conclude that insulin may inhibit Rho signaling by affecting posttranslational modification of RhoA via nitric oxide/cGMP signaling pathway to cause MBP activation, actin cytoskeletal disorganization, and vasodilation.
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PMID:Negative regulation of rho signaling by insulin and its impact on actin cytoskeleton organization in vascular smooth muscle cells: role of nitric oxide and cyclic guanosine monophosphate signaling pathways. 1208 58

To reveal the possible role of the amino-terminal domain of G protein-coupled receptor kinases(GRKs)in receptor phosphorylation and/or modulation of its kinase activity, a truncated mutant of GRK-2 lacking the amino-terminal domain(deltaN-GRK2)was made. deltaN-GRK2 was expressed effectively in E.coli as a GST fusion protein and was purified by affinity chromatography on a GSH-Sepharose column. deltaN-GRK2 was then separated from GST tag by thrombin cleavage and recovered. Although deltaN-GRK2 had nearly identical activity with wild-type GRK-2 in phosphorylation of peptide substrate, it completely lost the ability to phosphorylate the light-activated receptor rhodopsin. Furthermore, deletion of the amino-terminal domain rendered GRK-2 unresponsive to the regulation of kinase activity by a truncated form of rhodopsin, (329)G-Rho(*) and beta gamma subunits of G protein. These results demonstrated that the amino-terminal domain was necessary to GRK2 for both the phosphorylation of receptor and the regulation of its kinase activity by the receptor. It was reasonable to postulate that this domain has little, if any effect on the catalytic domain of natural form of GRK2.
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PMID:Effects of Deleting the Amino-terminal Domain of GRK-2 on Its Function. 1211 Sep 29

The signaling pathways that lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) use to activate Akt in ovarian cancer cells are investigated here. We show for the first time, with the use of both pharmacological and genetic inhibitors, that the kinase activity and S473 phosphorylation of Akt induced by LPA and S1P requires both mitogen-activated protein (MAP) kinase kinase (MEK) and p38 MAP kinase, and MEK is likely to be upstream of p38, in HEY ovarian cancer cells. The requirement for both MEK and p38 is cell type- and stimulus-specific. Among 12 cell lines that we tested, 11 respond to LPA and S1P and all of the responsive cell lines require p38 but only nine of them require MEK. Among different stimuli tested, platelet-derived growth factor stimulates S473 phosphorylation of Akt in a MEK- and p38-dependent manner. However, epidermal growth factor, thrombin, and endothelin-1-stimulated Akt S473 phosphorylation require p38 but not MEK. Insulin, on the other hand, stimulates Akt S473 phosphorylation independent of both MEK and p38 in HEY cells. T308 phosphorylation stimulated by LPA/S1P requires MEK but not p38 activation. MEK and p38 activation were sufficient for Akt S473 but not T308 phosphorylation in HEY cells. In contrast to S1P and PDGF, LPA requires Rho for Akt S473 phosphorylation, and Rho is upstream of phosphatidylinositol 3-kinase (PI3-K). LPA/S1P-induced Akt activation may be involved in cell survival, because LPA and S1P treatment in HEY ovarian cancer cells results in a decrease in paclitaxel-induced caspase-3 activity in a PI3-K/MEK/p38-dependent manner.
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PMID:Akt activation induced by lysophosphatidic acid and sphingosine-1-phosphate requires both mitogen-activated protein kinase kinase and p38 mitogen-activated protein kinase and is cell-line specific. 1218 43

We have investigated the role of the Rho and Rac family small guanine triphosphate (GTP) exchange factors (RhoGEFs), Vav1 and Vav2, in the activation of platelets by the immunoreceptor tyrosine-based activation motif (ITAM)-coupled collagen receptor GPVI and by the G protein-coupled receptor agonist thrombin. The glycoprotein VI (GPVI)-specific agonist collagen-related peptide (CRP) and thrombin stimulated tyrosine phosphorylation of Vav1 but not Vav2 in human platelets. Surprisingly, however, CRP did not activate the low-molecular-weight G protein Rac and stimulated only a small increase in activity of p21-associated kinase 2 (PAK2), despite the fact that both proteins are regulated downstream of Vav1 in other cells. Further, activation of Rac and PAK2 by thrombin was maintained in platelets from mice deficient in Vav1. Activation of phospholipase C (PLC) by GPVI and thrombin was unaltered in Vav1-, Vav2-, and Vav1/Vav2-deficient platelets. A weak inhibition of late-stage aggregation to CRP and thrombin was observed in platelets deficient in Vav1 but not Vav2, whereas spreading on fibrinogen was not changed. The present results demonstrate that neither Vav1 nor Vav2 lie upstream of PLC or Rac in platelets, highlighting an important difference in their role in signaling by ITAM-coupled receptors in other cell types. The present study has provided evidence for a possible role of Vav1 but not Vav2 in the later stages of platelet aggregation.
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PMID:Vav1, but not Vav2, contributes to platelet aggregation by CRP and thrombin, but neither is required for regulation of phospholipase C. 1241 20

Endothelial nitric oxide synthase (eNOS) is an important regulator of cardiovascular homeostasis by production of nitric oxide (NO) from vascular endothelial cells. It can be activated by protein kinase B (PKB)/Akt via phosphorylation at Ser-1177. We are interested in the role of Rho GTPase/Rho kinase (ROCK) pathway in regulation of eNOS expression and activation. Using adenovirus-mediated gene transfer in human umbilical vein endothelial cells (HUVECs), we show here that both active RhoA and ROCK not only downregulate eNOS gene expression as reported previously but also inhibit eNOS phosphorylation at Ser-1177 and cellular NO production with concomitant suppression of PKB activation. Moreover, coexpression of a constitutive active form of PKB restores the phosphorylation but not gene expression of eNOS in the presence of active RhoA. Furthermore, we show that thrombin inhibits eNOS phosphorylation, as well as expression via Rho/ROCK pathway. Expression of the active PKB reverses eNOS phosphorylation but has no effect on downregulation of eNOS expression induced by thrombin. Taken together, these data demonstrate that Rho/ROCK pathway negatively regulates eNOS phosphorylation through inhibition of PKB, whereas it downregulates eNOS expression independent of PKB.
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PMID:Rho GTPase/Rho kinase negatively regulates endothelial nitric oxide synthase phosphorylation through the inhibition of protein kinase B/Akt in human endothelial cells. 1244 67

Platelet activation by thrombin or thrombin receptor-activating peptide (TRAP) results in extensive actin reorganization that leads to filopodia emission and lamellae spreading concomitantly with activation of the Rho family small G proteins, Cdc42 and Rac1. Evidence has been provided that direct binding of Cdc42-guanosine triphosphate (GTP) and Rac1-GTP to the N-terminal regulatory domain of the p21-activated kinase (PAK) stimulates PAK activation and actin reorganization. In the present study, we have investigated the relationship between shape change and PAK activation. We show that thrombin, TRAP, or monoclonal antibody (MoAb) anti-Fc(gamma)RIIA IV.3 induces an activation of Cdc42 and Rac1. The GpVI ligand, convulxin (CVX), that forces platelets to lamellae spreading efficiently activates Rac1. Thrombin, TRAP, MoAb IV.3, and CVX stimulate autophosphorylation and kinase activity of PAK. Inhibition of Cdc42 and Rac1 with clostridial toxin B inhibits PAK activation and lamellae spreading. The cortical-actin binding protein, p80/85 cortactin, is constitutively associated with PAK in resting platelets and dissociates from PAK after thrombin stimulation. Inhibition of PAK autophosphorylation by toxin B prevents the dissociation of cortactin. These results suggest that Cdc42/Rac1-dependent activation of PAK may trigger early platelet shape change, at least in part through the regulation of cortactin binding to PAK.
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PMID:Cdc42/Rac1-dependent activation of the p21-activated kinase (PAK) regulates human platelet lamellipodia spreading: implication of the cortical-actin binding protein cortactin. 1245 77


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