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)

Stimulation of platelets by thrombin leads to an increased association of activated phosphoinositide 3-kinase (PI 3-K) with a membrane cytoskeletal fraction (CSK). Activation of PI 3-K is dependent upon GTP-binding protein(s), since PI 3-K in permeabilized platelets is stimulated by GTP gamma S (guanosine 5'-3-O-(thio)triphosphate), and stimulation of platelet cytosolic PI 3-K by GTP gamma S requires a functional small G-protein, Rho. Recent reports indicate that cytosolic PI 3-Ks can also be activated by the beta gamma subunits of heterotrimeric G-proteins (G beta gamma). We now report that the activated PI 3-K that is associated with CSK can be inhibited by a recombinant protein containing the G beta gamma-binding pleckstrin homology domain of beta-adrenergic receptor kinase 1 (beta ARK-PH). Inhibition is blocked by G beta gamma. PI 3-K in nonactivated platelet CSK is activated by GTP gamma S but unaffected by beta ARK-PH or G beta gamma. Western blots indicate that activated platelet CSK contains a novel 110-kDa PI 3-K(gamma) that has been shown to be stimulated by G beta gamma and to lack binding sites for the 85-kDa subunit of conventional PI 3-K. PI 3-K in immunoprecipitates obtained via p85 subunit-directed antibodies can be activated by GTP gamma S but not by G beta gamma. PI 3-K that is stimulatable by G beta gamma remains soluble, as does PI 3-K(gamma), and is unaffected by Rho. In contrast, ADP-ribosylation of Rho present in p85 immunoprecipitates is inhibitory. Further, activation of PI 3-K in permeabilized platelets exposed to thrombin or GTP gamma S is inhibited by beta ARK-PH and/or Rho-specific ADP-ribosylating enzymes. We conclude that Rho and G beta gamma each, respectively, contributes to the activation of different PI 3-Ks (p85-containing heterodimer and PI 3-K (gamma)) in thrombin-stimulated platelets.
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PMID:Sequestration of a G-protein beta gamma subunit or ADP-ribosylation of Rho can inhibit thrombin-induced activation of platelet phosphoinositide 3-kinases. 789 97

Integrin-mediated adhesion is known to stimulate production of phosphatidylinositol 4,5-bisphosphate (4,5-PIP2) and increase 4,5-PIP2 hydrolysis in response to platelet-derived growth factor (PDGF). We now show that treatment of cells with lovastatin, which inhibits modification of small GTP-binding proteins, reduced PIP2 levels and decreased calcium mobilization in response to PDGF and thrombin. In cell lysates, GTP gamma S stimulated PIP 5-kinase activity, and this effect was blocked by botulinum C3 exoenzyme, suggesting that Rho was responsible. GTP-bound recombinant Rho stimulated PIP 5-kinase activity, whereas GDP-Rho was much less potent and GTP-bound Rac was ineffective. Microinjected botulinum C3 exoenzyme caused diminished calcium mobilization in response to PDGF or thrombin. Conversely, microinjection of activated Rho reversed the decrease in calcium mobilization normally seen in nonadherent cells. These data demonstrate that Rho regulates 4,5-PIP2 synthesis and, indirectly, 4,5-PIP2 hydrolysis. They also raise the possibility that PIP2 synthesis could mediate the effects of Rho on the actin cytoskeleton.
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PMID:The small GTP-binding protein Rho regulates a phosphatidylinositol 4-phosphate 5-kinase in mammalian cells. 795 16

The small GTP-binding protein Rho regulates the assembly of actin stress fibers and focal adhesions in cells responding to growth factors. ADP-ribosylation of Rho by C3 transferase blocks this function; however, an enzymatic target for Rho has not yet been defined. We now report that Rho activates phosphatidylinositide 3-kinase in soluble preparations of platelets. Activation of phosphatidylinositide 3-kinase by GTP gamma S is blocked by ADP-ribosylation of endogenous Rho, and Rho shifts to the cytoskeleton in platelets exposed to thrombin. The inhibitory effects of ADP-ribosylation are overcome by exogenous recombinant Rho but not by recombinant Rac, another member of the Ras superfamily. Exposure of platelets to thrombin has been reported to lead to activation of phosphatidylinositide 3-kinase, a shift of this enzyme to the platelet membrane skeleton, and rapid cytoskeletal reorganization. In other studies, ADP-ribosylation of Rho has been found to inhibit thrombin-induced platelet aggregation, a cytoskeletally linked event. We suggest that Rho may exert its effects on cytoskeletal reorganization via phosphatidylinositide 3-kinase.
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PMID:Activation of platelet phosphatidylinositide 3-kinase requires the small GTP-binding protein Rho. 822 30

The Escherichia coli transcription factor NusA and the bacteriophage lambda antiterminator Q proteins were expressed as inducible glutathione S-transferase (GST) fusion proteins. The fusion proteins were purified under nondenaturing conditions by affinity chromatography on glutathione agarose. Thrombin cleavage of the glutathione agarose-bound fusion proteins yielded homogeneously pure NusAN+15 (5 mg/g cells) and almost homogeneously pure QN+13 protein (0.7 mg/g cells), where N+x indicates the presence of x additional amino acids at the N-terminus of the protein. The purified NusAN+15 exhibited the same activities as wildtype NusA in enhancement of transcriptional pausing, enhancement of termination at Rho-independent terminators, and enhancement of Q-mediated antitermination in vitro. The QN+13 protein exhibited both anti-pausing and antitermination activities in Q-mediated transcription antitermination. However, the antitermination activity of QN+13 was lost gradually during storage if the thrombin used for cleavage of the GST fusion protein was not removed. This was due to cleavage by thrombin after Arg22 within the Q protein itself, at a noncanonical thrombin cleavage site, so the truncated protein (QN+22) lacked the first 22 amino acids at the N-terminus of Q. The expression vectors described here can be used to rapidly produce large quantities of these proteins, and the truncated Q protein can be used to evaluate the requirement for the N-terminus of Q in antitermination, anti-pausing, interactions with the DNA template (qut site), and interaction with RNA polymerase itself.
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PMID:Expression and functional characterization of Escherichia coli NusA and lambda Q as glutathione S-transferase fusion proteins. 853 55

Serum stimulation of quiescent fibroblasts leads to a dramatic depolarization of the plasma membrane; however, the identity of the active serum factor(s) and the underlying mechanism are unknown. We find that this serum activity is attributable to albumin-bound lysophosphatidic acid (LPA) acting on its own G protein-coupled receptor, and that membrane depolarization is due to activation of an anion conductance mediating Cl- efflux. This depolarizing Cl- current can also be activated by thrombin and neuropeptide receptors; it is distinct from volume-regulated Cl- currents. Activation of the Cl- current consistently follows stimulation of phospholipase C and coincides with remodelling of the actin cytoskeleton, which is regulated by the Ras-related GTPase Rho. However, the response is not due to Ca2+/protein kinase C signalling and requires neither Rho nor Ras activation. The results indicate that in quiescent fibroblasts, LPA and other G protein-coupled receptor agonists evoke membrane depolarization by activating a new type of Cl- channel through a signalling pathway that is closely associated with phosphoinositide hydrolysis, yet independent of known second messengers.
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PMID:Serum-induced membrane depolarization in quiescent fibroblasts: activation of a chloride conductance through the G protein-coupled LPA receptor. 859 7

We have investigated thrombin-stimulated morphological changes and the activation of phosphoinositide 3-kinase (PI 3-K), as manifested by the accumulation of PtdIns(3,4)P2 and PtdIns(3,4,5)P3 (labelled with 32P or myo-[3H]inositol), in CHRF-288 cells, a leukaemic cell line derived from a platelet progenitor cell. We report that these cells, when exposed to thrombin or SFLLRN (the peptide Ser-Phe-Leu-Leu-Arg-Asn, a thrombin-receptor ligand) rapidly change shape, forming membrane 'blebs', detectable by differential interference contrast or confocal microscopy, as well as labelled 3-phosphorylated phosphoinositides. The 'blebs' are distinguishable from 'ruffles' or lamellae, since they do not contain phalloidin-detectable actin. Studies with permeabilized cells indicate that PI 3-K is activated synergistically by thrombin+guanosine 5'[gamma-thio]triphosphate. Two forms of PI 3-K, i.e. PI 3-K(gamma) and p85/PI 3-K, regulated by G beta gamma subunits of heterotrimeric G-protein and the small G-protein Rho, respectively, are present in these cells, as is true for platelets. Wortmannin, a known potent and specific inhibitor of PI 3-K activities, inhibits thrombin-stiumlated accumulation of 3-phosphorylated phosphoinositides in a dose-dependent manner (IC50 approximately 10nM), without affecting phospholipase C activation. Pretreatment of CHRF-288 cells with either wortmannin (100 nM) or an unrelated synthetic PI 3-K inhibitor, LY294002 (50 microM), abolishes thrombin-receptor-stimulated blebbing. These results suggest that thrombin-stimulated accumulation of 3-phosphorylated phosphoinositide(s) is required for the shape-change response in CHRF-288 cells.
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PMID:Thrombin stimulates wortmannin-inhibitable phosphoinositide 3-kinase and membrane blebbing in CHRF-288 cells. 861 73

The vav proto-oncogene product, p95vav or Vav, is primarily expressed in hematopoietic cells and has been shown to be a substrate for tyrosine kinases. Although its function is unknown, Vav shares a region of homology with DBL, an exchange factor for the Rho family of GTP-binding proteins. The presence of this domain and the observation that cells transformed with Vav display prominent stress fibers and focal adhesions similar to those that are observed in RhoA transformed cells suggests that Vav may play a role in regulating the actin cytoskeleton. We have, therefore, examined Vav phosphorylation in platelets, which undergo dramatic cytoskeletal reorganization in response to agonists. Two potent platelet agonists, thrombin (via its G protein-coupled receptor) and collagen (via its interaction with the alpha2beta1 integrin), caused Vav to become phosphorylated on tyrosine. Weaker platelet agonists, including ADP, epinephrine and the thromboxane A2 analog, U46619, did not. The phosphorylation of Vav in response to thrombin was maximal within 15 s and was unaffected by aspirin, inhibitors of aggregation, or the presence of the ADP scavenger, apyrase. Vav phosphorylation was also observed when platelets became adherent to immobilized collagen (via integrin alpha2beta1), fibronectin (via integrin alpha5beta1), and fibrinogen (via integrin alphaIIbbeta3). These results show that Vav phosphorylation by tyrosine kinases 1) occurs during platelet activation by potent agonists, 2) also occurs when platelets adhere to biologically relevant matrix proteins, 3) requires neither platelet aggregation nor the release of secondary agonists such as ADP and TxA2, and 4) can be initiated by at least some members of two additional classes of receptors, G protein-coupled receptors and integrins, providing further evidence that both of these can couple to tyrosine kinases.
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PMID:Thrombin receptor activation and integrin engagement stimulate tyrosine phosphorylation of the proto-oncogene product, p95vav, in platelets. 863 86

The endothelial cytoskeleton is important for the regulation of endothelial barrier function. Small GTP-binding Rho proteins play a central role in the organization of the microfilament system. Clostridium difficile toxin B (TcdB) inactivates Rho proteins by glucosylation at Thr-37. We used TcdB as a probe to study the role of Rho proteins in the regulation of endothelial barrier function. TcdB time (50-170 min) and dose (10-100 ng/ml) dependently increased the hydraulic conductivity of cultured porcine pulmonary artery endothelial cell monolayers approximately 10-fold. Simultaneously, the albumin reflection coefficient decreased substantially from 0.8 to 0.15. Before endothelial hyperpermeability, TcdB reduced F-actin content in a dose-dependent manner, whereas G-actin content remained unchanged. Finally, we proved that TcdB caused dose (5-100 ng/ml)- and time-dependent glucosylation of Rho proteins in endothelial cells. Phalloidin, which stabilizes filamentous actin, prevented the effect of TcdB on endothelial permeability. In contrast to thrombin-, hydrogen peroxide-, or Escherichia coli hemolysin-induced hyperpermeability, the elevation of cyclic nucleotides did not block TcdB-related permeability. The data demonstrate a central role of small GTP-binding Rho proteins for the control of endothelial barrier function.
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PMID:Glucosylation of small GTP-binding Rho proteins disrupts endothelial barrier function. 903

In some cell systems muscarinic receptor stimulation can induce proliferation or transformation. This phenomenon is subtype-specific (only m1 and m3 receptors are effective) and cell type dependent. In 1321N1 astrocytoma cells activation of m3 receptors stimulates phospholipase C, but does not induce DNA synthesis. In contrast the thrombin receptor, which also couples to phospholipase C, is strongly mitogenic and induces AP-1-dependent gene expression. Various experimental findings indicate that this discrepancy is not due to muscarinic receptor desensitization or blockade of growth stimulatory pathways. Muscarinic receptor number may be limiting, in particular for receptor coupling to the pertussis toxin-insensitive G-protein G12. This G-protein is required for thrombin-induced mitogenesis in 1321N1 cells and may couple selectively to the thrombin versus muscarinic receptor. In cardiomyocytes hypertrophic cell growth is induced by heterologously expressed m1 or m3 receptors but not by the endogenous m2 receptors. Studies using chimeric receptors confirm that induction of hypertrophy requires signalling through phospholipase C, but indicate that additional signals are needed to induce the morphological features of this response. We suggest that small G-proteins of the Rho subfamily, in addition to G12, mediate growth responses to G-protein-coupled receptors.
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PMID:Pathways and roadblocks in muscarinic receptor-mediated growth regulation. 912 50

The signal transduction systems that mediate growth factor receptor-induced cellular shape change have not been fully elucidated, but are known to involve alterations in the state of actin filaments, termed stress fibres. It now appears from several studies that the GTP-binding protein, Rho, is involved. However, the mechanisms by which Rho is activated, and what effectors Rho in turn stimulates are largely matters of conjecture. The present work shows that thrombin is an effective stimulant of stress fibre formation in Swiss 3T3 cells. In addition, we show the 70 kDa form of S6 kinase (p70s6k) to colocalise with stress fibres in both unstimulated and thrombin-activated cells. Coincident with the thrombin-induced formation of stress fibres is the elevated association p70s6k with the fibres. Pretreatment of cells with rapamycin, to inhibit p70s6k activation, inhibits thrombin-induced stress fibre formation and the associated presence of p70s6k on the fibres, supporting a role for p70s6k in thrombin-stimulated stress fibre formation. Thrombin is also shown to stimulate p70s6k activity and that this is inhibited by rapamycin. Thus, the data presented show that thrombin activates stress fibre formation through stimulation of p70s6k via a non-Gi pathway.
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PMID:Regulation of thrombin-induced stress fibre formation in Swiss 3T3 cells by the 70-kDa S6 kinase. 914 21


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