RhoGDI is required for Cdc42-mediated cellular transformation

Background: Cdc42, a Rho-related small GTP binding protein, plays pivotal roles in actin cytoskeletal organization, Golgi vesicular trafficking, receptor endocytosis, and cell cycle progression. However, the target/effectors mediating these cellular activities and, in particular, those responsible for Cdc42-mediated cell growth regulation and transformation are still being determined. In this study, we set out to examine how the regulatory protein RhoGDI influences the cellular responses elicited by activated Cdc42. Results: X-ray crystallographic analysis of the Cdc42-RhoGDI complex suggested that arginine 66 of Cdc42 is essential for its interaction with RhoGDI. Here we show that mutation of either arginine 66 or arginine 68 within the Switch 11 domain of Cdc42 completely abolished the binding of Cdc42 to RhoGDI without affecting the binding of other known regulators or target/effectors of this GTP binding protein. Introduction of the RhoGDI binding-defective mutation R66A within a constitutively active Cdc42(F28L) background was accompanied by changes in cell shape and an accumulation of Cdc42 in the Golgi when these cells were compared to those expressing Cdc42(F28L). However, the most striking change was that unlike Cdc42(F28L), which was able to induce the transformation of NIH 3T3 fibroblasts as assayed by their growth in low serum or their ability to form colonies in soft-agar, the Cdc42(F28L,R66A) mutant was transformation-detective. Likewise, the introduction of RhoGDI siRNA into Cdc42(F28L)-transfected cells inhibited their transformation. Conclusions: Taken together, the results reported here indicate that despite being a negative regulator of Cdc42 activation and GTP hydrolysis, RhoGDI plays an essential role in Cdc42-mediated cellular transformation.