HISTIDYL-TRANSFER-RNA SYNTHETASE-RELATED SEQUENCES IN GCN2 PROTEIN-KINASE REGULATE IN-VITRO PHOSPHORYLATION OF EIF-2

In yeast, starvation for amino acids stimulates GCN2 phosphorylation o f the cu subunit of eukaryotic initiation factor-2 (eIF-2). Phosphoryl ation of eIF-2 alpha induces the translational expression of GCN4, a t ranscriptional 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 a mino acid limitation and stimulate the activity of GCN2 kinase. In thi s report we address whether the HisRS-related sequences are required f or GCN2 phosphorylation of eIF-2 alpha in an in vitro assay. To measur e the activity of GCN2 kinase in cellular extracts, we expressed and p urified a truncated form of yeast eIF-2 alpha. Phosphorylation of the recombinant eIF-2 alpha substrate was dependent on both GCN2 kinase ac tivity and the eIF-2 alpha phosphorylation site, serine 51. Mutations in the HisRS-related domain of GCN2, which have been shown to block ph osphorylation of eIF-2 alpha in vivo and the subsequent stimulation of the general control pathway, also greatly reduced eIF-2 alpha phospho rylation in the in. vitro assay. These results indicate that the HisRS -related sequences are required for activation of GCN2 kinase function .