Rho small GTPase regulates cell morphology, adhesion and cytokinesis through the actin cytoskeleton. We have identified a protein, p140mDia, as a downstream effector of Rho. It is a mammalian homolog of Drosophila diaphanous, a protein required for cytokinesis, and belongs to a family of formin‐related proteins containing repetitive polyproline stretches. p140mDia binds selectively to the GTP‐bound form of Rho and also binds to profilin. p140mDia, profilin and RhoA are co‐localized in the spreading lamellae of cultured fibroblasts. They are also co‐localized in membrane ruffles of phorbol ester‐stimulated sMDCK2 cells, which extend these structures in a Rho‐dependent manner. The three proteins are recruited around phagocytic cups induced by fibronectin‐coated beads. Their recruitment is not induced after Rho is inactivated by microinjection of botulinum C3 exoenzyme. Overexpression of p140mDia in COS‐7 cells induced homogeneous actin filament formation. These results suggest that Rho regulates actin polymerization by targeting profilin via p140mDia beneath the specific plasma membranes.

During mitosis, a ring containing actin and myosin appears beneath the equatorial surface of animal cells. This ring then contracts, forms a cleavage furrow and divides the cell, a step known as cytokinesis. The two daughter cells often remain connected by an intercellular bridge which contains a refringent structure known as the midbody. How the appearance of this ring is regulated is unclear, although the small GTPase Rho, which controls the formation of actin structures, is known to be essential. Protein kinases are also thought to participate in cytokinesis. We now show that a splice variant of a Rho target protein, named citron, contains a protein kinase domain that is related to the Rho-associated kinases ROCK14 and ROK, which regulate myosin-based contractility. Citron kinase localizes to the cleavage furrow and midbody of HeLa cells; Rho is also localized in the midbody. We find that overexpression of citron mutants results in the production of multinucleate cells and that a kinase-active mutant causes abnormal contraction during cytokinesis. We propose that citron kinase regulates cytokinesis at a step after Rho in the contractile process.

Clostridium botulinum C3 is a recently discovered exoenzyme that ADP‐ribosylates a eukaryotic GTP‐binding protein of the ras superfamily. We show now that the bacterially‐expressed product of the human rhoC gene is ADP‐ribosylated by C3 and corresponds in size, charge and behavior to the dominant C3 substrate of eukaryotic cells. C3 treatment of Vero cells results in the disappearance of microfilaments and in actinomorphic shape changes without any apparent direct effect upon actin. Thus the ADP‐ribosylation of a rho protein seems to be responsible for microfilament disassembly and we infer that the unmodified form of a rho protein may be involved in cytoskeletal control.

The Rho guanosine 5'-triphosphatase (GTPase) cycles between the active guanosine triphosphate (GTP)-bound form and the inactive guanosine diphosphate-bound form and regulates cell adhesion and cytokinesis, but how it exerts these actions is unknown. The yeast two-hybrid system was used to clone a complementary DNA for a protein (designated Rhophilin) that specifically bound to GTP-Rho. The Rho-binding domain of this protein has 40 percent identity with a putative regulatory domain of a protein kinase, PKN. PKN itself bound to GTP-Rho and was activated by this binding both in vitro and in vivo. This study indicates that a serine-threonine protein kinase is a Rho effector and presents an amino acid sequence motif for binding to GTP-Rho that may be shared by a family of Rho target proteins.

Using a mouse embryo cDNA library, we conducted a two-hybrid screening to identify new partners for the small GTPase Rho. One clone obtained by this procedure contained a novel cDNA of 291 base pairs and interacted strongly with RhoA and RhoC, weakly with RhoB, and not at all with Rac1 and Cdc42Hs. Full-length cDNAs were then isolated from a mouse brain library. While multiple splicing variants were common, we identified three cDNAs with an identical open reading frame encoding a 61-kDa protein that we named rhotekin (from the Japanese "teki," meaning target). The N-terminal part of rhotekin, encoded by the initial cDNA and produced in bacteria as a glutathione S-transferase fusion protein, exhibited in vitro binding to 35S-labeled guanosine 5'-3-O-(thio)triphosphate-bound Rho, but not to Rac1 or Cdc42Hs in ligand overlay assays. In addition, this peptide inhibited both endogenous and GTPase-activating protein-stimulated Rho GTPase activity. The amino acid sequence of this region shares approximately 30% identity with the Rho-binding domains of rhophilin and a serine/threonine kinase, PKN, two other Rho target proteins that we recently identified (Watanabe, G., Saito, Y., Madaule, P., Ishizaki, T., Fujisawa, K., Morii, N., Mukai, H., Ono, Y., Kakizuka, A., and Narumiya, S. (1996) Science 271, 645-648). Thus, not only is rhotekin a novel partner for Rho, but it also belongs to a wide family of proteins that bear a consensus Rho-binding sequence at the N terminus. To our knowledge, this is the first conserved sequence for Rho effectors, and we have termed this region Rho effector motif class 1.