Activation of Rac1 by a Crk SH3-binding protein, DOCK180

The Rho family of low-molecular-weight G proteins, which consists of the Rho, Rac, and Cdc42 subfamilies, regulates the actin cytoskeleton (Hall 1998). Microinjection of constitutive active forms of these proteins into serum-starved fibroblasts demonstrated that Rho stimulates the organization of actin stress fibers, Rac stimulates the formation of lamellipodia or membrane ruffling, and Cdc42 induces the formation of filopodia (Nobes and Hall 1995). Like other G proteins, the Rho-family proteins are activated by guanine nucleotide exchange proteins (GEPs) and inactivated by GTPase-activating proteins (GAPs) (Lamarche and Hall 1994; Nobes and Hall 1994). The catalytic domains of GEPs for Rho share amino-acid sequences with the Dbl-oncogene product; thus, this domain is designated as the Dbl-homology domain (Cerione and Zheng 1996). It has been also reported that PIP2 and lipid kinases bind to and activate Rho family members (Tolias et al. 1995, 1998; Zheng et al. 1996). A third group of Rho regulator is RhoGDI, a GDP-dissociation inhibitor of Rho (Sasaki and Takai 1998). RhoGDI binds to GDP-bound Rho proteins and retains them in the cytoplasm in an inactive form (Sasaki et al. 1993). CrkII is a cellular homolog of the v-Crk oncogene product and consists almost entirely of SH2 and SH3 domains (Matsuda et al. 1992). Two major CrkII SH2-binding proteins are paxillin and p130Cas, both of which are components of focal adhesions (for review, see Kiyokawa et al. 1997). More recently, it has been shown that CrkII–p130Cas complexes regulate integrin-mediated cell movement and spreading (Vuori et al. 1996; Klemke et al. 1998). The amino-terminal SH3 domain of CrkII binds to several proteins; among the most prominent are C3G and DOCK180 (Matsuda and Kurata 1996). Homologs of DOCK180 have recently been isolated from Drosophila melanogaster and Caenorhabditis elegans and designated as mbc and ced-5, respectively (Erickson et al. 1997; Wu and Horvitz 1998). ced-5 mutants are defective in the engulfment of cell corpses and the migration of the gonadal distal tip cells. The latter phenotype was rescued by the expression of human DOCK180. mbc was originally isolated as a gene that was essential for muscle development, suggesting that mbc is also involved in cellular motility. Human DOCK180 also appears to be involved in cytoskeletal reorganization. Expression of membrane-targeted DOCK180 induced spreading of NIH-3T3 cells (Hasegawa et al. 1996). DOCK180 forms a complex with CrkII and p130Cas only after integrin stimulation of NIH-3T3 cells. Expression of DOCK180 with CrkII and p130Cas induces cell spreading and accumulation of DOCK180 at focal adhesions (Kiyokawa et al. 1998). However, the biochemical function of DOCK180 is unknown. Here, we demonstrate that DOCK180 activates and associates with the Rac1 GTPase and that DOCK180 requires Rac1 for the induction of cell spreading in NIH-3T3 cells.