Complex formation and reciprocal regulation between GSK3β and C3G

GSK3β, a ubiquitously expressed Ser/Thr kinase, regulates cell metabolism, proliferation and differentiation. Its activity is spatially and temporally regulated dependent on external stimuli and interacting partners, and its deregulation is associated with various human disorders. In this study, we identify C3G (RapGEF1), a protein essential for mammalian embryonic development as an interacting partner and substrate of GSK3β. In vivo and in vitro interaction assays demonstrated that GSK3β and Akt are present in complex with C3G. Molecular modelling and mutational analysis identified a domain in C3G that aids interaction with GSK3β, and overlaps with its nuclear export sequence. GSK3β phosphorylates C3G on primed as well as unprimed sites, and regulates its subcellular localization. Over-expression of C3G resulted in activation of Akt and inactivation of GSK3β. Huntingtin aggregate formation, dependent on GSK3β inhibition, was enhanced upon C3G overexpression. Stable clones of C2C12 cells generated by CRISPR/Cas9 mediated knockdown of C3G, that cannot differentiate, show reduced Akt activity and S9-GSK3β phosphorylation compared to wild type cells. Co-expression of catalytically active GSK3β inhibited C3G induced myocyte differentiation. C3G mutant defective for GSK3β phosphorylation, does not alter S9-GSK3β phosphorylation and, is compromised for inducing myocyte differentiation. Our results show complex formation and reciprocal regulation between GSK3β and C3G. We have identified a novel function of C3G as a negative regulator of GSK3β, a property important for its ability to induce myogenic differentiation.