Characterization of regulatory mechanisms of CdGAP, a negative regulator of the small GTPases Rac1 and Cdc42

The Rho GTPases form a diverse family of proteins which regulates numerous cell processes from cytoskeletal reorganization to control of gene expression. They are negatively regulated by GTPase-activating proteins (GAPs). Cdc42-GTPase-Activating Protein (CdGAP) is a negative regulator of the Rho GTPases Rad and Cdc42, whose aberrant signaling is involved in pathological processes such as tumorigenesis and metastasis. In this study, we set out to identify regulatory mechanisms of CdGAP. We show that CdGAP exists as a long isoform of 1425 amino acids in addition to the shorter 820 amino acid isoform that was previously described. We also identify RSK-1, ERK1 and GSK-3 as interacting partners of CdGAP, with ERK1 and GSK-3 interacting with the proline-rich domain (PRD) of CdGAP, a region known to be necessary for CdGAP regulation. We characterize the phosphorylation of CdGAP and show that in vivo CdGAP is mostly phosphorylated on serine residues. RSK-1, ERK1 and GSK-3 are all able to phosphorylate CdGAP in the PRD, and, in the case of ERK1 and GSK-3, we have identified Thr-776 as a site phosphorylated by both kinases. The site can be phosphorylated in vivo, and its phosphorylation leads to decreased CdGAP activity. We have also identified a novel potential mechanism of RhoGAP protein regulation, namely the serum-induced upregulation of CdGAP mRNA levels. We characterize the mechanism by which this occurs and demonstrate that Rho GTPase activity is necessary for this upregulation to occur. In summary, we have identified and characterized novel regulatory mechanisms for CdGAP, a negative regulator of Rac1 and Cdc42.