Characterization of functional domains of mDia1, a link between the small GTPase Rho and the actin cytoskeleton

The widely expressed diaphanous proteins, a subclass of formins, comprise l inks between the Rho GTPases and the actin-based cytoskeleton. They contain several functional domains that are thought to be responsible for interact ion with different ligands: the FH1 domain for binding the actin-associated protein profilin; the RBD for targeting activated Rho; and the C-terminal CIID module for autoregulation of the overall diaphanous activity. Using de letion constructs of the murine mDia1, we have analyzed the functional prop erties of these three domains separately in in vitro assays and in transien tly and stably transfected cell lines. We show that the proline-rich FH1 do main effectively binds to profilins in vitro as well as in cells, that the RBD complexes with the CIID in a species-restricted manner and that overexp ression of RBD causes spontaneous ruffling and loss of stress fibers, toget her with loss of directional motility. Supertransfection of cells stably ex pressing the RBD with dominant negative Rac effectively suppresses ruffling . Our data contribute to the understanding of the function of these domains in linking the actin cytoskeleton with the Rho-signaling cascade. Furtherm ore, they suggest that inactivation of Rho by exogenous RBD causes upregula tion of Rac activity in the transfected cells.