Functional interaction of RacF2 and the WASp family protein, scar, in the Rab8 signaling pathway of the social amoeba, Dictyostelium discoideum

The small GTPase, Rab8, has been shown to play a role in cell-cell adhesion and restructuring of the actin cytoskeleton in both mammalian cells and the lower eukaryote, Dictyostelium discoideum. In D. discoideum, cells expressing constitutively activated Rab8 (Rab8CA) display reduced cell-cell adhesion and increased actin-rich protrusions as well as delayed aggregation. Rab8 has been implicated in the restructuring of the actin cytoskeleton, but no specific pathway for this action has been identified. In other systems, actin-rich membrane extension formation is regulated by WASp family proteins, including SCAR. Here we provide evidence of a functional relationship between the WASp family protein, SCAR, and Rab8. This provides the first genetic evidence in any cell system of a functional interaction between Rab8 and a WASp family protein.;SCAR is known to be directly activated by Rac. Our results indicate that Rab8 interacts directly with RacF2 to rescue aggregation in cells expressing Rab8CA. While we were unable to demonstrate that RacF2 interacts directly with SCAR, we have demonstrated that RacF2 likely interacts directly with Rab8 to control cell-cell adhesion.;Additionally, we have begun to conduct similar experiments in mammalian cells. To this end, we have developed and expressed EGFP (enhanced green fluorescent protein) chimeras of wildtype as well as constitutively active and dominant negative mutant forms of Rab8 in mammalian cells. In addition, we have designed a Rab8 activation assay based on its interaction with GCK, a germinal center kinase, which interacts directly with the active, GTP-bound form of Rab8. We have also investigated the effect of expression of mutant versions of Rab8 on GCK intracellular levels.