title: Enhanced interaction between pseudokinase and kinase domains in Gcn2 stimulates eIF2α phosphorylation in starved cells.
abstract: The stress-activated protein kinase Gcn2 regulates protein synthesis by phosphorylation of translation initiation factor eIF2α, from yeast to mammals. The Gcn2 kinase domain (KD) is inherently inactive and requires allosteric stimulation by adjoining regulatory domains. Gcn2 contains a pseudokinase domain (YKD) required for high-level eIF2α phosphorylation in amino acid starved yeast cells; however, the role of the YKD in KD activation was unknown. We isolated substitutions of evolutionarily conserved YKD amino acids that impair Gcn2 activation without reducing binding of the activating ligand, uncharged tRNA, to the histidyl-tRNA synthetase-related domain of Gcn2. Several such Gcn- substitutions cluster in predicted helices E and I (αE and αI) of the YKD. We also identified Gcd- substitutions, evoking constitutive activation of Gcn2, mapping in αI of the YKD. Interestingly, αI Gcd- substitutions enhance YKD-KD interactions in vitro, whereas Gcn- substitutions in αE and αI suppress both this effect and the constitutive activation of Gcn2 conferred by YKD Gcd- substitutions. These findings indicate that the YKD interacts directly with the KD for activation of kinase function and identify likely sites of direct YKD-KD contact. We propose that tRNA binding to the HisRS domain evokes a conformational change that increases access of the YKD to sites of allosteric activation in the adjoining KD.
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: Kansas State University, Division of Biology, 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: Thomas E
authors.initials: TE
authors.lastname: Dever
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: D000595:Amino Acid Sequence; D015852:Eukaryotic Initiation Factor-2 / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010766:Phosphorylation; D010770:Phosphotransferases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
chemical_list: D015852:Eukaryotic Initiation Factor-2; D029701:Saccharomyces cerevisiae Proteins; D010770:Phosphotransferases; C512912:GCN2 protein, S cerevisiae; D017346:Protein Serine-Threonine Kinases