Substrate Binding Relieves Autoinhibition of the Mammalian GCN2 Kinase

Cells respond to amino acid starvation and translational stress through the integrated stress response (ISR) kinase GCN2. Current models maintain that GCN2 senses these stresses either by binding deacylated tRNAs that accumulate during amino acid starvation or by recognizing ribosome collisions that arise from perturbation of translation elongation. As tRNA charging is inherently coupled to translation elongation, assessing the relative contributions of these two pathways has proven difficult. To fully separate the roles of these potential agonists, we reconstituted the regulation of GCN2 in vitro using recombinant proteins. GCN2 has low basal activity, which is stimulated by the addition of ribosomes or the ribosomal P-stalk. By contrast, deacylated tRNAs fail to activate GCN2 both in vitro and in vivo and instead counteract activation by the ribosome and inhibit GCN2 auto- and substrate phosphorylation. This inhibition of autophosphorylation requires the presence of GCN2's substrate, eIF2α, suggesting that the substrate plays a role in kinase regulation. Indeed, eIF2α binding to a newly defined autoinhibitory loop in the GCN2 kinase domain relieves its repression and stimulates autophosphorylation of the kinase's activation loop. Together, these data redefine the role of tRNAs in the regulation of GCN2 and reveal a mechanism novel to kinases in general by which substrate binding regulates activation loop phosphorylation.