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bioRxiv · 10.1101/2022.11.24.515150

Crk proteins activate the Rap1 guanine nucleotide exchange factor C3G by segregated adaptor-dependent and -independent mechanisms

Rodriguez-Blazquez, A.·Carabias, A.·Moran-Vaquero, A.·de Cima, S.·Luque-Ortega, J. R.·Alfonso, C.·Schuck, P.·Manso, J. A.·Macedo-Ribeiro, S.·Guerrero, C.·de Pereda, J. M.
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Abstract

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.

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Rodriguez-Blazquez, A., Carabias, A., Moran-Vaquero, A., de Cima, S., Luque-Ortega, J. R., Alfonso, C., Schuck, P., Manso, J. A., Macedo-Ribeiro, S., Guerrero, C., de Pereda, J. M.. 2022-11-24. Crk proteins activate the Rap1 guanine nucleotide exchange factor C3G by segregated adaptor-dependent and -independent mechanisms. https://doi.org/10.1101/2022.11.24.515150

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