Functional and structural characterization of the c-Cbl-CrkL-C3G interaction
Ana Dávila Hidalgo
Centro de Investigación del Cáncer, Campus Miguel de Unamuno s/n
c-Cbl is a RING-type E3 ubiquitin ligase that regulates multiple signaling pathways by targeting proteins for degradation and acting as a multifunctional adaptor protein. Mutations that impair c- Cbl E3 ubiquitin ligase activity are associated with hematologic malignancies and solid tumors. C3G is a guanine nucleotide exchange factor that activates the small GTPase Rap1 and regulates cell adhesion, migration, proliferation, and apoptosis. c-Cbl and C3G are regulated by overlapping sets of tyrosine kinases, and reciprocal regulation between c-Cbl and C3G has been reported. They interact indirectly through adaptor proteins of the Crk family, including CrkL. In these complexes, the SH2 domain of CrkL binds to phosphotyrosine sites in c-Cbl, whereas its SH3 domain binds to proline-rich motifs in C3G. Despite the potential importance of the c-Cbl–CrkL–C3G interaction in cell signaling, the molecular organization and functional consequences of these complexes remain poorly understood.
Here, we combined cellular models with in vitro biophysical and structural approaches to investigate the functional relationship between c-Cbl and C3G and the molecular basis of their association. We reconstituted c-Cbl–CrkL–C3G complexes in vitro using recombinant proteins, including phosphorylated c-Cbl. This approach allowed us to characterize the stoichiometry and binding affinities governing complex assembly, focusing on the contributions of multivalency in phosphorylated c-Cbl and C3G, as well as c-Cbl dimerization. We also investigated the structural organization of the assembled complexes. This work provides a framework to understand how c-Cbl and C3G regulate each other and engage with other signaling proteins.