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

Shuhao Zhu, Alexander Y. Sobolev, Ronald Wek

Research output: Contribution to journalArticle

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

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Histidine-tRNA Ligase
Eukaryotic Initiation Factor-2
Phosphorylation
Protein Kinases
Phosphotransferases
Amino Acids
Yeast
Assays
Yeasts
Sequence Homology
Starvation
Transfer RNA
In Vitro Techniques
Serine
Chemical activation
Mutation

ASJC Scopus subject areas

  • Biochemistry

Cite this

Histidyl-tRNA synthetase-related sequences in GCN2 protein kinase regulate in vitro phosphorylation of eIF-2. / Zhu, Shuhao; Sobolev, Alexander Y.; Wek, Ronald.

In: Journal of Biological Chemistry, Vol. 271, No. 40, 1996, p. 24989-24994.

Research output: Contribution to journalArticle

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