Molecular basis for the functions of dominantly active Y35N and inactive D60K Rheb mutants in mTORC1 signaling

摘要

Mammalian target of rapamycin complex 1 (mTORC1) serves as a central regulator of cell growth and proliferation by integrating signals from growth factors, nutrients, energy status, and cellular stress (Saxton and Sabatini, 2017). A small GTPase, called Ras homolog enriched in brain (Rheb), is a positive regulator of mTORC1. Like other small GTPases, the function of Rheb is dictated by its guanine nucleotide binding states: it is active in the GTP-bound form and inactive in the GDP-bound form (Aspuria and Tamanoi, 2004). Crystal structures of Rheb in complexes with GDP, GTP, and GppNHp (a nonhydrolysable GTP analog) revealed that major conformational change takes place in the switch I region during the GTP–GDP transition (Yu et al., 2005). Cryo-EM structure of mTORC1 in complex with GTP-bound Rheb suggested an allosteric mechanism for mTORC1 activation by Rheb (Yang et al., 2017). Intriguingly, the interaction of Rheb with mTOR is relatively weak and in a nucleotide-independent manner, which is different from the interactions of classical Ras proteins with their effectors (Long et al., 2005). Nevertheless, the functional data showed that only the GTP-bound Rheb can activate mTOR (Yang et al., 2017). So far, multiple Rheb mutants have been identified (Heard et al., 2014). Among them, the Y35A mutant exhibited increased intrinsic GTPase activity and its overexpression reduced the activation of mTORC1 by growth factor availability, and thus is deemed as a loss-of-function mutant (Mazhab-Jafari et al., 2012; Heard et al., 2014). Surprisingly, the Y35N mutant, which was initially identified in several human cancers (Lawrence et al., 2014), could significantly increase the phosphorylation level of mTORC1 substrate S6K1 compared to the wild-type (WT) Rheb, and thus is regarded as a constitutively active mutant (Grabiner et al., 2014; Heard et al., 2014). On the other hand, the D60K mutant was shown to be unable to bind to Mg2t or the nucleotide (either GTP or GDP) and hence to assume a nucleotide-free form; thus, it is regarded as a dominantly inactive mutant and widely used as a negative control in the functional study of mTORC1 activation (Tabancay et al., 2003). However, how the Y35N and D60K mutations alter the proper function of Rheb in the activation of mTORC1 signaling remains unclear.