Chimaerins are a family of
GTPase activating proteins (GAPs) for the
small G-protein Rac that have gained recent attention due to their important roles in development,
cancer, neuritogenesis, and T-cell function. Like
protein kinase C isozymes, chimaerins possess a C1 domain capable of binding
phorbol esters and the
lipid second messenger
diacylglycerol (DAG) in vitro. Here we identified an autoinhibitory mechanism in alpha2-chimaerin that restricts access of
phorbol esters and DAG, thereby limiting its activation. Although
phorbol 12-myristate 13-acetate (PMA) caused limited translocation of wild-type alpha2-chimaerin to the plasma membrane, deletion of either N- or C-terminal regions greatly sensitize alpha2-chimaerin for intracellular redistribution and activation. Based on modeling analysis that revealed an occlusion of the
ligand binding site in the alpha2-chimaerin C1 domain, we identified key
amino acids that stabilize the inactive conformation. 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. Notably, in contrast to PMA,
epidermal growth factor promotes full translocation of alpha2-chimaerin in a
phospholipase C-dependent manner, but not of a C1 domain mutant with reduced affinity for DAG (P216A-alpha2-chimaerin). Therefore, DAG generation and binding to the C1 domain are required but not sufficient for
epidermal growth factor-induced alpha2-chimaerin membrane association. Our studies suggest a role for DAG in anchoring rather than activation of alpha2-chimaerin. Like other DAG/
phorbol ester receptors, including
protein kinase C isozymes, alpha2-chimaerin is subject to autoinhibition by intramolecular contacts, suggesting a highly regulated mechanism for the activation of this Rac-GAP.