Identification of Rho GTPase-dependent sites in the Dbl homology domain of oncogenic Dbl that are required for transformation

Abstract

The Dbl family guanine-nucleotide exchange factors (GEFs) for Rho GTPases share the structural array of a Dbl homology (DH) domain in tandem with a Pleckstrin homology (PH) domain. For oncogenic Dbl, the DH domain is responsible for the GEF activity, and the DH-PH module constitutes the minimum structural unit required for cellular transformation. To understand the structure-function relationship of the DH domain, we have investigated the role of specific residues of the DH domain of Dbl in interaction with Rho GTPases and in Dbl-induced transformation. Alanine substitution mutagenesis identified a panel of DH mutants made in the alpha1, alpha6, and alpha9 regions and the PH junction site that suffer complete or partial loss of GEF activity toward Cdc42 and RhoA. Kinetic and binding analysis of these mutants revealed that although most displayed decreased k(cat) values in the GEF reaction, the substrate binding activities of T506A and R634A were significantly reduced. E502A, Q633A, and N673A/D674A, on the other hand, retained the binding capability to the Rho GTPases but lost the GEF catalytic activity. In general, the in vitro GEF activity of the DH mutants correlated with the in vivo Cdc42- and RhoA-activating potential, and the GEF catalytic efficiency mirrored the transforming activity in NIH 3T3 cells. Moreover, the N673A/D674A mutant exhibited a potent dominant-negative effect on serum-induced cell growth and caused retraction of actin structures. These studies identify important sites of the DH domain involved in binding or catalysis of Rho proteins and demonstrate that maintaining a threshold of GEF catalytic activity, in addition to the Rho GTPase binding activity, is essential for efficient transformation by oncogenic Dbl.