Complex formation and reciprocal regulation between GSK3 beta and C3G

摘要

GSK3 beta, 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 GSK3 beta. In vivo and in vitro interaction assays demonstrated that GSK3 beta and Akt are present in complex with C3G. Molecular modelling and mutational analysis identified a domain in C3G that aids interaction with GSK3 beta, and overlaps with its nuclear export sequence. GSK3 beta 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 GSK3 beta. Huntingtin aggregate formation, dependent on GSK3 beta 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-GSK3 beta phosphorylation compared to wild type cells. Co-expression of catalytically active GSK3 beta inhibited C3G induced myocyte differentiation. C3G mutant defective for GSK3 beta phosphorylation, does not alter S9-GSK3 beta phosphorylation and, is compromised for inducing myocyte differentiation. Our results show complex formation and reciprocal regulation between GSK3 beta and C3G. We have identified a novel function of C3G as a negative regulator of GSK3 beta, a property important for its ability to induce myogenic differentiation.