Histidyl-tRNA synthetase-related sequences in GCN2 protein kinase regulate in vitro phosphorylation of eIF-2

J Biol Chem. 1996 Oct 4;271(40):24989-94. doi: 10.1074/jbc.271.40.24989.

Abstract

In yeast, starvation for amino acids stimulates GCN2 phosphorylation of the alpha subunit of eukaryotic initiation factor-2 (eIF-2). Phosphorylation of eIF-2alpha induces the translational expression of GCN4, a transcriptional activator of the general amino acid control pathway. It has been proposed that GCN2 sequences containing homology to histidyl-tRNA synthetases (HisRS) bind uncharged tRNA that accumulate during amino acid limitation and stimulate the activity of GCN2 kinase. In this report we address whether the HisRS-related sequences are required for GCN2 phosphorylation of eIF-2alpha in an in vitro assay. To measure the activity of GCN2 kinase in cellular extracts, we expressed and purified a truncated form of yeast eIF-2alpha. Phosphorylation of the recombinant eIF-2alpha substrate was dependent on both GCN2 kinase activity and the eIF-2alpha phosphorylation site, serine 51. Mutations in the HisRS-related domain of GCN2, which have been shown to block phosphorylation of eIF-2alpha in vivo and the subsequent stimulation of the general control pathway, also greatly reduced eIF-2alpha phosphorylation in the in vitro assay. These results indicate that the HisRS-related sequences are required for activation of GCN2 kinase function.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Eukaryotic Initiation Factor-2 / genetics
  • Eukaryotic Initiation Factor-2 / metabolism*
  • Histidine-tRNA Ligase / genetics*
  • Mutation
  • Phosphorylation
  • Precipitin Tests
  • Protein Kinases / genetics
  • Protein Kinases / metabolism*
  • Protein Serine-Threonine Kinases
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / metabolism
  • Saccharomyces cerevisiae Proteins

Substances

  • Eukaryotic Initiation Factor-2
  • Recombinant Proteins
  • Saccharomyces cerevisiae Proteins
  • Protein Kinases
  • GCN2 protein, S cerevisiae
  • Protein Serine-Threonine Kinases
  • Histidine-tRNA Ligase