Enhanced Interaction between Pseudokinase and Kinase Domains in Gcn2 stimulates eIF2α Phosphorylation in Starved Cells

Sebastien Lageix, Stefan Rothenburg, Thomas E. Dever, Alan G. Hinnebusch

Research output: Contribution to journalArticle

15 Citations (Scopus)

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.

Original languageEnglish (US)
Article numbere1004326
JournalPLoS Genetics
Volume10
Issue number5
DOIs
StatePublished - 2014
Externally publishedYes

Fingerprint

phosphorylation
phosphotransferases (kinases)
substitution
Phosphotransferases
Phosphorylation
cells
yeast
Transfer RNA
amino acid
Histidine-tRNA Ligase
Yeasts
protein
Allosteric Site
yeasts
Amino Acids
Peptide Initiation Factors
amino acids
ligand
Heat-Shock Proteins
mitogen-activated protein kinase

ASJC Scopus subject areas

  • Genetics
  • Molecular Biology
  • Ecology, Evolution, Behavior and Systematics
  • Cancer Research
  • Genetics(clinical)
  • Medicine(all)

Cite this

Enhanced Interaction between Pseudokinase and Kinase Domains in Gcn2 stimulates eIF2α Phosphorylation in Starved Cells. / Lageix, Sebastien; Rothenburg, Stefan; Dever, Thomas E.; Hinnebusch, Alan G.

In: PLoS Genetics, Vol. 10, No. 5, e1004326, 2014.

Research output: Contribution to journalArticle

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