title: Interaction between the tRNA-binding and C-terminal domains of Yeast Gcn2 regulates kinase activity in vivo.
abstract: The stress-activated protein kinase Gcn2 regulates protein synthesis by phosphorylation of translation initiation factor eIF2α. Gcn2 is activated in amino acid-deprived cells by binding of uncharged tRNA to the regulatory domain related to histidyl-tRNA synthetase, but the molecular mechanism of activation is unclear. We used a genetic approach to identify a key regulatory surface in Gcn2 that is proximal to the predicted active site of the HisRS domain and likely remodeled by tRNA binding. Mutations leading to amino acid substitutions on this surface were identified that activate Gcn2 at low levels of tRNA binding (Gcd- phenotype), while other substitutions block kinase activation (Gcn- phenotype), in some cases without altering tRNA binding by Gcn2 in vitro. Remarkably, the Gcn- substitutions increase affinity of the HisRS domain for the C-terminal domain (CTD), previously implicated as a kinase autoinhibitory segment, in a manner dampened by HisRS domain Gcd- substitutions and by amino acid starvation in vivo. Moreover, tRNA specifically antagonizes HisRS/CTD association in vitro. These findings support a model wherein HisRS-CTD interaction facilitates the autoinhibitory function of the CTD in nonstarvation conditions, with tRNA binding eliciting kinase activation by weakening HisRS-CTD association with attendant disruption of the autoinhibitory KD-CTD interaction.
authors.affiliation: Laboratory of Gene Regulation and Development, Eunice K. Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, United States of America.
authors.forename: Sebastien
authors.initials: S
authors.lastname: Lageix
authors.affiliation: National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland, United States of America.
authors.forename: Jinwei
authors.initials: J
authors.lastname: Zhang
authors.affiliation: Division of Biology, Kansas State University, Manhattan, Kansas, United States of America.
authors.forename: Stefan
authors.initials: S
authors.lastname: Rothenburg
authors.affiliation: Laboratory of Gene Regulation and Development, Eunice K. Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, United States of America.
authors.forename: Alan G
authors.initials: AG
authors.lastname: Hinnebusch
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics; D018360:Crystallography, X-Ray; D015852:Eukaryotic Initiation Factor-2 / Q000235:genetics; D006643:Histidine-tRNA Ligase / Q000737:chemistry / Q000235:genetics*; D009154:Mutation; D010766:Phosphorylation; D011487:Protein Conformation; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D017434:Protein Structure, Tertiary; D012343:RNA, Transfer / Q000235:genetics* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014349:Trypanosoma cruzi
chemical_list: D015852:Eukaryotic Initiation Factor-2; D029701:Saccharomyces cerevisiae Proteins; D012343:RNA, Transfer; C512912:GCN2 protein, S cerevisiae; D017346:Protein Serine-Threonine Kinases; D006643:Histidine-tRNA Ligase