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

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