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 alpha subunit of eukaryotic initiation factor-2 (eIF-2). Phosphorylation of eIF-2alpha 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-2alpha 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-2alpha. Phosphorylation of the recombinant eIF-2alpha substrate was dependent on both GCN2 kinase activity and the eIF-2alpha phosphorylation site, serine 51. Mutations in the HisRS-related domain of GCN2, which have been shown to block phosphorylation of eIF-2alpha in vivo and the subsequent stimulation of the general control pathway, also greatly reduced eIF-2alpha phosphorylation in the in vitro assay. These results indicate that the HisRS-related sequences are required for activation of GCN2 kinase function.
Zhu
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Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana 46202-5122, USA.
Sobolev
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Wek
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D015852:Eukaryotic Initiation Factor-2 / Q000235:genetics / Q000378:metabolism*; D006643:Histidine-tRNA Ligase / Q000235:genetics*; D009154:Mutation; D010766:Phosphorylation; D011233:Precipitin Tests; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins
D015852:Eukaryotic Initiation Factor-2; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D011494:Protein Kinases; C512912:GCN2 protein, S cerevisiae; D017346:Protein Serine-Threonine Kinases; D006643:Histidine-tRNA Ligase