Intra- and inter-molecular interactions regulate the activity of the Rac-GAPs alpha2- and beta2-chimaerins

Chimaerins are a family of GTPase activating proteins (GAPs) for the small G-protein Rac that have gain recent attention due to their important roles in development, cancer, neuritogenesis, and T-cell function. Chimaerins possess a C1 domain capable of binding phorbol esters and the lipid second messenger diacylglycerol (DAG) in vitro. Here we identified the N- and C-termini of alpha2-chimaerin as autoinhibitory domains that restrict ligand binding to the C1 domain and activation of its Rac-GAP activity. Using biochemical and modeling analysis we identified residues that are involved in intramolecular interactions and are key to stabilize the protein in its inactive state. Mutation of these sites renders alpha2-chimaerin hypersensitive to C1 ligands, as reflected by its enhanced ability to translocate in response to PMA and to inhibit Rac activity and cell migration. Furthermore, we demonstrate for the first time that alpha2-chimaerin is an effector of the EGFR. EGF treatment induces alpha2-chimaerin translocation to the plasma membrane in a DAG-dependent manner. A C1 domain mutant with reduced affinity for DAG is unable to translocate in response to EGF. alpha2-chimaerin expression in HeLa cells blocks EGF-induced Rac activation, indicating that EGF-mediated activation of alpha2-chimaerin serves to self-limit Rac activation. In addition, we present evidence showing that alpha2- and beta2-chimaerins bind to the adaptor protein Nck1. Deletional and mutational analysis allowed us to identify a novel non-typical proline rich region conserved in alpha2- and beta2-chimaerin that interacts with the third SH3 domain of Nck1. Treatment with PMA at concentrations that translocate chimaerins induces the dissociation of the Nck-chimaerin complex. Moreover, preliminary data indicates that overexpression of Nck1 inhibits PMA-induced translocation of alpha2-chimaerin. Modeling analysis of beta2-chimaerin in complex with the third SH3 domain of Nck suggests that Nck binding is specific to the inactive conformation. This was supported by the observation that hyperactivated mutants of alpha2- and beta2-chimaerin show decreased binding to Nck. Based on our results we propose, that the activation of alpha2- and beta2-chimaerins is regulated both by intra- and inter-molecular interactions and by DAG binding to the C1 domain.