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Integrin-dependent translocation of p160ROCK to cytoskeletal complex in thrombin-stimulated human platelets.

ABSTRACT: p160(ROCK) is a protein serine/threonine kinase that binds to GTP-Rho and is activated by this binding. We have recently found that the expression of p160(ROCK) induces focal adhesions and stress fibres in HeLa cells, whereas a dominant-negative form of this kinase suppresses Rho-induced formation of these structures, suggesting that this kinase is a downstream target of Rho in this process [Ishizaki, Naito, Fujisawa, Maekawa, Watanabe, Saito and Narumiya (1997) FEBS Lett. 404, 118-124]. To find out the mode of action of p160(ROCK), we developed immunoblotting with an anti-p160(ROCK) antibody and investigated the subcellular localization of p160(ROCK) during platelet aggregation. In resting human platelets, more than 90% of p160(ROCK) was present in the Triton X-100-soluble fraction. When platelets were stimulated with thrombin, approx. 10% of p160(ROCK) was translocated to the Triton X-100-insoluble fraction. This translocation was detected as early as 20 s after stimulation and reached a maximum at 5 min; it was suppressed by the addition of EDTA or an Arg-Gly-Asp-Ser peptide (RGDS), both of which inhibit integrin alphaIIbbeta3-mediated platelet aggregation. Using [32P]Pi-loaded platelets, we found that p160(ROCK) was phosphorylated in response to stimulation by thrombin. This phosphorylation, however, was not affected by the addition of EDTA and RGDS. These results suggest that p160(ROCK) translocates to cytoskeleton in a manner dependent on integrin ligation and works in an early stage of cytoskeletal reorganization in thrombin-stimulated platelets.

SUBMITTER: Fujita A

PROVIDER: S-EPMC1218985 | biostudies-other | 1997 Dec

REPOSITORIES: biostudies-other

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Project description:Platelets derive most of their energy from anaerobic glycolysis; during activation this requirement rises approx. 3-fold. To accommodate the high glucose flux, platelets express extremely high concentrations (155+/-18 pmol/mg of membrane protein) of the most active glucose transporter isoform, GLUT3. Thrombin, a potent platelet activator, was found to stimulate 2-deoxyglucose transport activity 3-5-fold within 10 min at 25 degrees C, with a half-time of 1-2 min. To determine the mechanism underlying the increase in glucose transport activity, an impermeant photolabel, [2-3H]2N-4-(1-azi-2,2,2-trifluoethyl)benzoyl-1,3, -bis-(d-mannose-4-ylozy)-2-propylamine, was used to covalently bind glucose transporters accessible to the extracellular milieu. In response to thrombin, the level of transporter labelling increased 2.7-fold with a half-time of 1-2 min. This suggests a translocation of GLUT3 transporters from an intracellular site to the plasma membrane in a manner analogous to that seen for the translocation of GLUT4 in insulin-stimulated rat adipose cells. To investigate whether a similar signalling pathway was involved in both systems, platelets and adipose cells were exposed to staurosporin and wortmannin, two inhibitors of GLUT4 translocation in adipose cells. Thrombin stimulation of glucose transport activity in platelets was more sensitive to staurosporin inhibition than was insulin-stimulated transport activity in adipose cells, but it was totally insensitive to wortmannin. This indicates that the GLUT3 translocation in platelets is mediated by a protein kinase C not by a phosphatidylinositol 3-kinase mechanism. In support of this contention, the phorbol ester PMA, which specifically activates protein kinase C, fully stimulated glucose transport activity in platelets and was equally sensitive to inhibition by staurosporin. This study provides a cellular mechanism by which platelets enhance their capacity to import glucose to fulfil the increased energy demands associated with activation.

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Integrin-dependent translocation of p160ROCK to cytoskeletal complex in thrombin-stimulated human platelets.

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