THE P160 RHOA-BINDING KINASE ROK-ALPHA IS A MEMBER OF A KINASE FAMILYAND IS INVOLVED IN THE REORGANIZATION OF THE CYTOSKELETON

The GTPase RhoA has been implicated in various cellular activities, in cluding the formation of stress fibers, motility, and cytokinesis, We recently reported on a p150 serine/threonine kinase (termed ROK alpha) binding RhoA only in its active GTP-bound state and on its cDNA; intr oduction of RhoA into HeLa cells resulted in translocation of the cyto plasmic kinase to plasma membranes, consistent with ROK alpha being a target for RhoA (T. Leung, E. Manser, L. Tan, and L. Lim, J. Biol. Che m. 256:29051-29054, 1995). Reanalysis of the cDNA revealed that ROK al pha contains an additional N-terminal region, We also isolated another cDNA which encoded a protein (ROK alpha) with 90% identity to ROK alp ha in the kinase domain, Both ROK alpha and ROK beta, which had a mole cular mass of 160 kDa, contained a highly conserved cysteine/histidine -rich domain located within a putative pleckstrin homology domain, The kinases bound RhoA, RhoB, and RhoC but not Rad and Cdc42. The Rho-bin ding domain comprises about 30 amino acids, Mutations within this doma in caused partial or complete loss of Rho binding, The morphological e ffects of ROK alpha were investigated by microinjecting HeLa cells wit h DNA constructs encoding various forms of ROK alpha. Full-length ROK alpha promoted formation of stress fibers and focal adhesion complexes , consistent with its being an effector of RhoA, ROK alpha truncated a t the C terminus promoted this formation and also extensive condensati on of actin microfilaments and nuclear disruption, The proteins exhibi ted protein kinase activity which was required for stress fiber format ion; the kinase-dead ROK alpha K112A and N-terminally truncated mutant s showed no such promotion, The latter mutant instead induced disassem bly of stress fibers and focal adhesion complexes, accompanied by cell spreading, These effects were mediated by the C-terminal region conta ining Rho-binding, cysteine/histidine-rich, and pleckstrin homology do mains, Thus, the multidomained ROK alpha appears to be involved in reo rganization of the cytoskeleton, with the N and C termini acting as po sitive and negative regulators, respectively, of the kinase domain who se activity is crucial for formation of stress fibers and focal adhesi on complexes.