The C-Terminal Di-Arginine Motif of Cdc42 is Essential for Binding to Phosphatidylinositol 4,5 Bisphosphate-Containing Membranes and Inducing Cellular Transformation

Abstract

The Rho family of GTPases regulates a diverse range of processes that are dependent on the proper cellular localization of their constituent signaling proteins. The membrane localization of these GTPases is due in large part to the geranylgeranyl moiety located at their carboxyl-terminal end. In addition, most of the Rho family members contain a cluster of positive-charged residues (i.e. a ‘polybasic domain’), directly preceding their geranylgeranyl moiety, and it has been suggested that this domain serves to fine-tune their localization among different cellular membranes. Here, we have taken a closer look at the role of the polybasic domain of Cdc42 in its ability to bind to membranes and to induce the transformation of fibroblasts. A FRET assay for the binding of Cdc42 to liposomes of defined composition showed that Cdc42 associates more strongly with liposomes containing phosphatidylinositol 4,5 bisphosphate (PIP2), compared either to uncharged control membranes or liposomes containing a charge-equivalent amount of phosphatidylserine (PS). The carboxyl-terminal di-arginine motif (Arg186 and Arg187) was shown to play an essential role in the binding of Cdc42 to PIP2-containing membranes. We further showed that substitutions for the di-arginine motif, when introduced within a constitutively active (‘fast-cycling’) Cdc42(F28L) background, had little effect on the ability of the activated Cdc42 mutant to induce microspikes/filopodia in NIH 3T3 cells, whereas they eliminated the ability of Cdc42 to transform fibroblasts. Taken together, these findings suggest that the di-arginine motif within the carboxyl-terminus of Cdc42 is necessary for this GTPase to bind at membrane sites containing PIP2, where it can initiate signaling activities that are essential for the oncogenic transformation of cells.