The Borgs, a new family of Cdc42 and TC10 GTPase-interacting proteins

The Rho family of GTPases plays key roles in the regulation of cell motilit y and morphogenesis. They also regulate protein kinase cascades, gene expre ssion, and cell cycle progression. This multiplicity of roles requires that the Rho GTPases interact with a wide variety of downstream effector protei ns. An understanding of their functions at a molecular level therefore requ ires the identification of the entire set of such effecters. Towards this e nd, we performed a two-hybrid screen using the TC10 GTPase as bait and iden tified a family of putative effector proteins related to MSE55, a murine st romal and epithelial cell protein of 55 kDa. We have named this family the Borg (binder of Rho GTPases) proteins. Complete open reading frames have be en obtained for Borg1 through Borg3. We renamed MSE55 as Borg5. Borg1, Borg 2, Borg4, and Borg5 bind both TC10 and Cdc42 in a GTP-dependent manner. Sur prisingly, Borg3 bound only to Cdc42. An intact CRIB (Cdc42, Rac interactiv e binding) domain was required for binding. No interaction of the Borgs wit h Rad or RhoA was detectable. Three-hemagglutinin epitope (HA(3))-tagged Bo rg3 protein was mostly cytosolic when expressed ectopically in NIH 3T3 cell s, with some accumulation in membrane ruffles. The phenotype induced by Bor g3 was reminiscent of that caused by an inhibition of Rho function and was reversed by overexpression of Rho. Surprisingly, it was independent of the ability to bind Cdc42. Borg3 also inhibited Jun kinase activity by a mechan ism that was independent of Cdc42 binding. HA(3)-Borg3 expression caused su bstantial delays in the spreading of cells on fibronectin surfaces after re plating, and the spread cells lacked stress fibers. We propose that the Bor g proteins function as negative regulators of Rho GTPase signaling.