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 ROK␣) 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 ROK␣ being a target for RhoA (T. Leung, E. Manser, L. Tan, and L. Lim, J. Biol. Chem. 256:29051-29054, 1995). Reanalysis of the cDNA revealed that ROK␣ contains an additional N-terminal region. We also isolated another cDNA which encoded a protein (ROK␤) with 90% identity to ROK␣ in the kinase domain. Both ROK␣ and ROK␤, 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 ROK␣ were investigated by microinjecting HeLa cells with DNA constructs encoding various forms of ROK␣. Full-length ROK␣ promoted formation of stress fibers and focal adhesion complexes, consistent with its being an effector of RhoA. ROK␣ 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 ROK␣K112A 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 ROK␣ 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.In mammals, the Ras-related Rho subfamily includes RhoA, -B, and -C, Rac1 and -2, and Cdc42, which play pivotal roles in cytoskeletal control and cell morphology. RhoA has been implicated in stress fiber formation (33, 34), whereas Rac1 (36) and Cdc42 (15,29) are involved in lamellipodial and filopodial formation, respectively. Fibroblasts injected with these GTPases display a set of distinctive morphological changes, suggestive of probable hierarchy in the order Cdc42, Rac, and Rho. These changes demand a high level of flexibility in the dynamic reorganization of actin microfilaments in cells. RhoA, -B, and -C have about 85% identity and appear to have different cellular localizations (1). Although their exact roles in cells have not been clearly defined, these GTPases have been implicated in a variety of cellular activities either directly by overexpression of wil...

G12V, which themselves colocalize to these sites. By deletion analysis, the N terminus of PAK is shown to contain targeting sequences for focal adhesions which indicate that these complexes are the site of kinase function in vivo. Cdc42 and Rac1 cause ␣-PAK autophosphorylation and kinase activation. Mapping ␣-PAK autophosphorylation sites has allowed generation of a constitutively active kinase mutant. By fusing regions of Cdc42 to the C terminus of PAK, activated chimeras were also obtained. Plasmids encoding these different constitutively active ␣-PAKs caused loss of stress fibers when introduced into both HeLa cells and fibroblasts, which was similar to the effect of introducing Cdc42 G12V or Rac1 G12V. Significantly dramatic losses of focal adhesions were also observed. These combined effects resulted in retraction of the cell periphery after plasmid microinjection. These data support our previous suggestions of a role for PAK downstream of both Cdc42 and Rac1 and indicate that PAK functions include the dissolution of stress fibers and reorganization of focal complexes.

Two isolates (WR1 and WR2) of Blastocystis from laboratory-bred Wistar rats were axenized by a method of colony growth in soft agar combined with antibiotic treatment. The colonies were cultured in Iscove's modified Dulbecco's medium (IMDM) and Bacto agar mixture supplemented with 10% horse serum in the presence of thioglycollate. The cells from the colonies had an ameboid outline with a central body. Large inclusions were seen in the central body of some cells. Some granular forms were also found. In the axenic culture of isolate WR2, about one-third of the organisms were granular forms. Cysts were found in the axenic culture of both isolates. This is the first report of such cyst formation in in vitro culture. The karyotypic patterns of both isolates of the rat Blastocystis were analyzed by pulsed-field gel electrophoresis (PFGE). A total of 13 chromosomal bands were separated, ranging from 1.86 Mb to 295 kb. The karyotypic patterns of the rat Blastocystis were different from those of B. hominis and reptilian Blastocystis. On the basis of the above-mentioned differences, the rat Blastocystis is assigned as B. ratti sp. nov.