| The Rho GTPases form a family of enzymes that control numerous cellular processes including cell migration and proliferation through effects on the cytoskeleton, membrane trafficking and cell adhesion. The activity of these molecular switches is modulated by GTPase-activating proteins (GAPs), a group of negative-regulators which includes Cdc42-GTPase-Activating Protein (CdGAP). This protein specifically negatively-regulates the Rho GTPases Cdc42 and Rac1. In this study, we show that CdGAP is regulated by lipid-, protein- and intramolecular-interactions. First, we demonstrate that a polybasic region (PBR) of CdGAP preceding the GAP domain and found in numerous Rho family GAPs is required for CdGAP specific association with phosphatidilinositol-3,4,5-trisphosphate (PI(3,4,5)P3). We show that the binding of PI(3,4,5)P3 is required for CdGAP-mediated GAP activity in vitro, and that an intact PBR is required for its CdGAP-mediated GAP activity in vivo. Second, we characterize the binding site for the negative-regulator of CdGAP Intersectin-1 located in the Basic-Rich (BR) domain of CdGAP. We present evidence that this interaction mediated by the SH3D domain of Intersectin requires one to three lysine residues located in the Basic-Rich (BR) domain of CdGAP. Thirdly, we show that CdGAP is negatively-regulated by its C-terminal domain. This observation is part of a study that links two human CdGAP gene mutations to a syndrome which presents a combination of aplasia cutis congenita (ACC) and terminal transverse limb defects (TTLD). In this syndrome, the deletion-mutant gene products which lack the residual amino-acid of CdGAP at its C-terminus have an increased activity compared to wild-type proteins. We show that this C-terminus can bind to the GAP domain of CdGAP, providing a model to explain how the absence of the C-terminus induces this syndrome. In summary, this work provides novel insight into understanding the mechanisms of regulation of CdGAP, a protein involved in cell migration and adhesion with unexpected roles related to human diseases. |
