C3G is a guanine nucleotide exchange factor (GEF) that regulates cell adhesion and migration by activating the GTPase Rap1. The GEF activity of C3G is stimulated by the adaptor proteins Crk and CrkL and by tyrosine phosphorylation. Here, we uncovered mechanisms of C3G autoinhibition and activation. Specifically, we found that two intramolecular interactions regulate the activity of C3G. First, an autoinhibitory region (AIR) within the central domain of C3G binds to and blocks the catalytic Cdc25H domain. Second, the binding of the protein's N-terminal domain to its Ras exchanger motif (REM) is required for its GEF activity. CrkL activated C3G by displacing the AIR/Cdc25HD interaction. Two missense mutations in the AIR found in non-Hodgkin's lymphomas, Y554H and M555K, disrupted the autoinhibitory mechanism. Expression of C3G-Y554H or C3G-M555K in Ba/F3 pro-B cells caused constitutive activation of Rap1 and, consequently, the integrin LFA-1. Our findings suggest that sustained Rap1 activation by deregulated C3G might promote progression of lymphomas and that designing therapeutics to target C3G might treat these malignancies.
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