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

The Dbl family guanine-nucleotide exchange factors (GEFs) for Rho GTPases s hare the structural array of a Dbl homology (DH) domain in tandem with a Pl eckstrin homology (PH) domain. For oncogenic Dbl, the DH domain is responsi ble for the GEF activity, and the DH-PH module constitutes the minimum stru ctural unit required for cellular transformation. To understand the structu re-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 identif ied a panel of DH mutants made in the alpha 1, alpha 6, and alpha 9 regions and the PH junction site that suffer complete or partial loss of GEF activ ity toward Cdc42 and RhoA. Kinetic and binding analysis of these mutants re vealed that although most displayed decreased k(cat) values in the GEF reac tion, the substrate binding activities of T506A and R634A were significantl y reduced. E502A Q633A, and N673A/D674A, on the other hand, retained the bi nding 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 i n vivo Cdc42- and RhoA-activating potential, and the GEF catalytic efficien cy mirrored the transforming activity in MH 3T3 cells, Moreover, the N673A/ D674A mutant exhibited a potent dominant-negative effect on serum-induced c ell growth and caused retraction of actin structures, These studies identif y important sites of the DH domain involved in binding or catalysis of Rho proteins and demonstrate that maintaining a threshold of GEF catalytic acti vity, in addition to the Rho GTPase binding activity, is essential for effi cient transformation by oncogenic Dbl.