RT Journal Article
SR Electronic
T1 Crk proteins activate the Rap1 guanine nucleotide exchange factor C3G by segregated adaptor-dependent and -independent mechanisms
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2022.11.24.515150
DO 10.1101/2022.11.24.515150
A1 Antonio Rodríguez-Blázquez
A1 Arturo Carabias
A1 Alba Morán-Vaquero
A1 Sergio de Cima
A1 Juan R. Luque-Ortega
A1 Carlos Alfonso
A1 Peter Schuck
A1 José Antonio Manso
A1 Sandra Macedo-Ribeiro
A1 Carmen Guerrero
A1 José M de Pereda
YR 2022
UL http://biorxiv.org/content/early/2022/11/24/2022.11.24.515150.abstract
AB C3G is a guanine nucleotide exchange factor (GEF) that activates Rap1 to promote cell adhesion. Resting C3G is autoinhibited and the GEF activity is released by stimuli that signal through tyrosine kinases. Tyrosine phosphorylation of C3G and interaction with Crk adaptor proteins, whose expression is increased in multiple human cancers, participate in C3G activation. However, the molecular details of C3G activation and the interplay between C3G phosphorylation and Crk interaction are poorly understood. Here, we combine biochemical, biophysical, and cell biology approaches to elucidate the mechanisms of C3G activation. CrkL interacts through its SH3N domain with the proline-rich motifs P1 and P2 of inactive C3G in vitro and in Jurkat and HEK293T cells, and these sites are necessary to recruit C3G to the plasma membrane. However, direct stimulation of the GEF activity requires binding of Crk proteins to the P3 and P4 sites. P3 is occluded in resting C3G and is essential for activation, while P4 contributes secondarily towards complete stimulation. Tyrosine phosphorylation of C3G alone causes marginal activation. Instead, phosphorylation primes C3G lowering the concentration of Crk proteins required for activation and increasing the maximum activity. Unexpectedly, optimal activation also requires the interaction of CrkL-SH2 domain with phosphorylated C3G. Phosphorylation and Crk-binding form a two-factor mechanism that ensures tight control of C3G activation. The simultaneous SH2 and SH3N interaction of CrkL with C3G, required for the activation, reveals a novel adaptor-independent function of Crk proteins relevant to understanding their role in physiological signaling and their deregulation in diseases.Competing Interest StatementThe authors have declared no competing interest.