RHOTEKIN, A NEW PUTATIVE TARGET FOR RHO-BEARING HOMOLOGY TO A SERINE THREONINE KINASE, PKN, AND RHOPHILIN IN THE RHO-BINDING DOMAIN/

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 intera cted strongly with RhoA and RhoC, weakly with RhoB, are not at all wit h Rac1 and Cdc42Hs. Full-length cDNAs were then isolated from a mouse brain library. While multiple splicing variants were common, we identi fied three cDNAs with an identical open reading frame encoding a 61-kD a 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 S-35-labeled guanosine 5'-3'O-(thio)tr iphosphate-bound Rho, but not to Rac1 of Cdc42Hs in ligand overlay ass ays. In addition, this peptide inhibited both endogenous and GTPase-ac tivating proteins-stimulated Rho GTPase activity. The amino acid seque nce of this region shares similar to 30% identity with the Rho-binding domains of rhophilin and a serine/threonine kinase, PKN, two other Rh o target proteins that we recently identified (Watanabe, G., Saito, Y. , Madaula, P., Ishizuki, 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 a t the N terminus. To our knowledge, this is the first conserved sequen ce for Rho effectors, and we have termed this region Rho effector moti f class 1.