The p160 RhoA-binding kinase ROKα is a member of a kinase family and is involved in the reorganization of the cytoskeleton

Abstract

The GTPase RhoA has been implicated in various cellular activities, including the formation of stress fibers, motility, and cytokinesis. We recently reported on a p150 serine/threonine kinase (termed ROKα) binding RhoA only in its active GTP-bound state and on its cDNA; introduction of RhoA into HeLa cells resulted in translocation of the cytoplasmic kinase to plasma membranes, consistent with ROKα being a target for RhoA (T. Leung, E. Manser, L. Tan, and L. Lira, J. Biol. Chem. 256:29051-29054, 1995). Reanalysis of the cDNA revealed that ROKα contains an additional N-terminal region. We also isolated another cDNA which encoded a protein (ROKβ) with 90% identity to ROKα in the kinase domain. Both ROKα and ROKβ, which had a molecular 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 Rac1 and Cdc42. The Rho-binding domain comprises about 30 amino acids. Mutations within this domain caused partial or complete loss of Rho binding. The morphological effects of ROKα were investigated by microinjecting HeLa cells with DNA constructs encoding various forms of ROKα. Full-length ROKα promoted formation of stress fibers and focal adhesion complexes, consistent with its being an effector of RhoA. ROKα truncated at the C terminus promoted this formation and also extensive condensation of actin microfilaments and nuclear disruption. The proteins exhibited protein kinase activity which was required for stress fiber formation; the kinase-dead ROKαK112A and N-terminally truncated mutants showed no such promotion. The latter mutant instead induced disassembly of stress fibers and focal adhesion complexes, accompanied by cell spreading. These effects were mediated by the C-terminal region containing Rho-binding, cysteine/histidine-rich, and pleckstrin homology domains. Thus, the multidomained ROKα appears to be involved in reorganization of the cytoskeleton, with the N and C termini acting as positive and negative regulators, respectively, of the kinase domain whose activity is crucial for formation of stress fibers and focal adhesion complexes.