INTEGRIN-DEPENDENT TRANSLOCATION OF P160(ROCK) TO CYTOSKELETAL COMPLEX IN THROMBIN-STIMULATED HUMAN PLATELETS

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 expr ession 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) FEES Lett. 404, 118-124 ]. To find out the mode of action of p160(ROCK), We developed immunobl otting with an anti-p160(ROCK) antibody and investigated the subcellul ar 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-insolu ble fraction. This translocation was detected as early as 20 s after s timulation and reached a maximum at 5 min; it was suppressed by the ad dition of EDTA or an Arg-Gly-Asp-Ser peptide (RGDS), both of which inh ibit integrin alpha IIb beta 3-mediated platelet aggregation. Using [P -32]P-i-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 sugg est that p160(ROCK) translocates to cytoskeleton in a manner dependent on integrin ligation and works in an early stage of cytoskeletal reor ganization in thrombin-stimulated platelets.