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

Shuhao Zhu, Alexander Y. Sobolev, Ronald C. Wek

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Abstract

In yeast, starvation for amino acids stimulates GCN2 phosphorylation of the α subunit of eukaryotic initiation factor-2 (eIF-2). Phosphorylation of eIF-2α 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-2α 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-2α. Phosphorylation of the recombinant elF-2α substrate was dependent on both GCN2 kinase activity and the eIF-2α phosphorylation site, serine 51. Mutations in the HisRS-related domain of GCN2, which have been shown to block phosphorylation of eIF-2α in vivo and the subsequent stimulation of the general control pathway, also greatly reduced eIF-2α phosphorylation in the in vitro assay. These results indicate that the HisRS-related sequences are required for activation of GCN2 kinase function.

Original languageEnglish (US)
Pages (from-to)24989-24994
Number of pages6
JournalJournal of Biological Chemistry
Volume271
Issue number40
DOIs
StatePublished - Oct 15 1996

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ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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