Oligomerization of DH domain is essential for Dbl-induced transformation

The dbl oncogene product (onco-Dbl) is the prototype member of a family of guanine nucleotide exchange factors (GEFs) for Rho GTPases. The Dbl homolog y (DH) domain of onco-Dbl is responsible for the GEF catalytic activity, an d the DH domain, together with the immediately adjacent pleckstrin homology (PH) domain, constitutes the minimum module bearing transforming function. In the present study, we demonstrate that the onco-Dbl protein exists in o ligomeric form in vitro and in cells. The oligomerization is mostly hemophi lic in nature and is mediated by the DH domain. Mutagenesis studies mapped the region involved in oligomerization to the conserved region 2 of the DH domain, which is located at the opposite side of the Rho GTPase interacting surface. Residue His556 of this region, in particular, is important for th is activity, since the H556A mutant retained the GEF catalytic capability a nd the binding activity toward Cdc42 and RhoA in vitro but was deficient in oligomer formation. Consequently, the Rho GTPase activating potential of t he H556A mutant was significantly reduced in cells. The focus-forming and a nchorage-independent growth activities of onco-Dbl were completely abolishe d by the His556-to-Ala mutation, whereas the abilities to stimulate fell gr owth, activate Jun N-terminal kinase, and cause actin cytoskeletal changes were retained by the mutant. The ability of onco-Dbl to oligomerize allowed multiple Rho GTPases to be recruited to the same signaling complex, and su ch an ability is defective in the H556A mutant. Taken together, these resul ts suggest that oligomerization of onco-Dbl through the DH domain is essent ial for cellular transformation by providing the means to generate a signal ing complex that further augments and/or coordinates its Rho GTPase activat ing potential.