Oligomerization of DH Domain Is Essential for Dbl-Induced Transformation

Abstract

The dbl oncogene product (onco-Dbl) is the prototype member of a family of guanine nucleotide exchange factors (GEFs) for Rho GTPases. The Dbl homology (DH) domain of onco-Dbl is responsible for the GEF catalytic activity, and 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 oligomeric form in vitro and in cells. The oligomerization is mostly homophilic 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 this activity, since the H556A mutant retained the GEF catalytic capability and the binding activity toward Cdc42 and RhoA in vitro but was deficient in oligomer formation. Consequently, the Rho GTPase activating potential of the H556A mutant was significantly reduced in cells. The focus-forming and anchorage-independent growth activities of onco-Dbl were completely abolished by the His556-to-Ala mutation, whereas the abilities to stimulate cell growth, 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 such an ability is defective in the H556A mutant. Taken together, these results suggest that oligomerization of onco-Dbl through the DH domain is essential for cellular transformation by providing the means to generate a signaling complex that further augments and/or coordinates its Rho GTPase activating potential.The dbl oncogene product (onco-Dbl) was originally isolated from a diffuse B-cell lymphoma (16). Over the past decade, a large group of proteins has joined the Dbl family by virtue of their structural similarity with onco-Dbl in an approximately 300-amino-acid region consisting of a Dbl homology (DH) domain and a pleckstrin homology (PH) domain. Many members of this family, including Vav, Ect2, Tim, Ost, Dbs, Lbc, Lfc, Lsc, and Net, possess a transformation or invasion capability like onco-Dbl has. Other members include proteins identified as gene products of sequences that are rearranged in human diseases (Bcr or FGD1) or as proteins with other catalytic functions, such as the Sos or RasGRF Ras guanine nucleotide exchange factors (GEFs) (for reviews, see references 8 and 58).Onco-Dbl and the related yeast protein Cdc24 were among the first to be realized to function as Rho GTPase GEFs, i.e., to stimulate the replacement of bound GDP by GTP on specific members of the Rho family small GTPases (25, 62). Subsequent studies of individual Dbl-like molecules have found that Lbc, Lfc, and Lsc oncoproteins act as specific GEFs for Rho and cause cellular transformation through the Rho signaling pathway (19,57,64), the ost oncogene product sho...

Homo- and hetero-oligomerization of PDZ-RhoGEF, LARG and p115RhoGEF by their C-terminal region regulates their in vivo Rho GEF activity and transforming potential

“…Deletion of the leucine zipper domain and the resulting loss of homo-oligomerization prevented, rather than stimulating, Pix function in vivo (Kim et al, 2001). In other cases, oligomerization has also been reported to occur through DH-DH domain interactions, such as for RasGRF1, RasGRF2, and Dbl (Anborgh et al, 1999;Zhu et al, 2001). In these cases, inhibition of oligomerization diminishes the in vivo GEF activity of Dbl for Cdc42 and Rho, and that of RasGEF1 and RasGEF2 for Ras.…”

The Novel Cdc42 Guanine Nucleotide Exchange Factor, Zizimin1, Dimerizes via the Cdc42-binding CZH2 Domain

“…Interestingly, the DH domains of Tiam1, RasGRF1, RasGRF2, Dbs, and Dbl also form oligomers (23)(24)(25)(26). OncoDbl monomers and oligomers have similar in vitro activity, but activation of Rho GTPases in vivo by monomers was significantly reduced, and transformation ability was completely abolished (24).…”

Homo-oligomerization of ALS2 through Its Unique Carboxyl-terminal Regions Is Essential for the ALS2-associated Rab5 Guanine Nucleotide Exchange Activity and Its Regulatory Function on Endosome Trafficking

“…These findings fuel speculation that the Rab5GEFs might function as a multiple protein complex in vivo, regardless of whether homo-or heterophilic complexes are formed. By contrast, several RhoGEFs, including ␤1PIX (36, 37), Dbl (38), RasGRF1 (39), Ras-GRF2 (39), p115RhoGEF (40,41), LARG (41), and PDZ-Rho-GEF (41), have already been shown to dimerize or oligomerize. Inhibition of oligomerization diminishes the in vivo GEF activity of Dbl (38) and abolishes ␤1PIX function in vivo (37).…”

“…By contrast, several RhoGEFs, including ␤1PIX (36, 37), Dbl (38), RasGRF1 (39), Ras-GRF2 (39), p115RhoGEF (40,41), LARG (41), and PDZ-Rho-GEF (41), have already been shown to dimerize or oligomerize. Inhibition of oligomerization diminishes the in vivo GEF activity of Dbl (38) and abolishes ␤1PIX function in vivo (37). In the cases of p115RhoGEF, LARG, and PDZ-RhoGEF, deletion of the C-terminal parts that were required for oligomerization had no significant effect on the in vitro GEF activities but resulted in the drastic stimulation of in vivo functions (40,41).…”