Volume 271, Number 40, Issue of October 4, 1996 pp. 24989-24994
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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Histidyl-tRNA Synthetase-related Sequences in GCN2 Protein Kinase Regulate in Vitro Phosphorylation of eIF-2

(Received for publication, March 28, 1996, and in revised form, June 12, 1996)

From the Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana 46202-5122

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

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