Mechanisms of Rho GTPase activation by Dbl family guanine nucleotide exchange factors

The Dbl family guanine nucleotide exchange factors (GEFs) mediate guanine nucleotide exchange of Rho GTPases. This large family of proteins contains a catalytic Dbl homology (DH) domain in tandem with a pleckstrin homology (PH) domain. We investigated how the PH domain modulated the activity of Dbl. Our data showed that the Dbl PH domain binded to phosphatidylinositol phosphates, resulting in the inhibition of GEF activity of Dbls. PH domain binding to PIPs is also important for targeting Dbl proteins to the plasma membrane. The Dbl mutants unable to interact with phosphatidylinositol phosphates displayed lower cellular transforming activity. These results suggest that through interaction with PH domain phosphatidylinositol phosphates regulate the biologic activity of Dbl.; We also studied the mechanism underlying the GEF substrate specificity. Trio and Tiam1 are a subset of GEFs that specifically activate Rac1 but not the closely related Cdc42. We have analyzed a panel of Rac1/Cdc42 chimeras and point mutants of Rac1 for their ability to interact with the GEFs. The results reveal that Rac1 Trp56 in β₃ appears to be the critical determinant for specific recognition by the GEFs. Introduction of Trp56 to Cdc42 renders it fully responsive to the Rac-specific GEF. Further, a polypeptide derived from Rac1 Trp56 serves as a specific inhibitor for Rac1 interaction with the GEFs. Taken together, these results indicate that Trp56 is the necessary and sufficient determinant of Rac1 for discrimination by the subset of Rac1-specific GEFs.; Directed by computer-based rational drug design, we identified and characterized a first generation inhibitor targeting Rac1 activation by specific interference of the GEF-Rac interaction. In vitro the compound effectively inhibited Rac1 binding and activation by TrioN (IC₅₀ at ∼40 μM) but did not interfere with Cdc42 activation by Intersectin. In NIH 3T3 cells, it potently blocked serum or PDGF induced Rac1 activation but not Cdc42. When applied to human prostate cancer cells, it was able to significantly inhibit the tumor cell proliferation and invasion. We believe that this compound may act as a Rac activation-specific inhibitor and can be employed to study the role of Rac in various cellular systems.