Abstract
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.
Highlights
A family of serine/threonine protein kinases regulate protein synthesis by phosphorylation of the ␣ subunit of eukaryotic initiation factor-21 [1,2,3,4]
The histidyltRNA synthetases (HisRS)-related sequences have been proposed to enhance GCN2 phosphorylation of eukaryotic initiation factor-2 (eIF-2)␣ in response to interaction with different uncharged tRNAs that accumulate in cells starving for amino acids (14 –16)
This increase in eIF-2␣ phosphorylation appeared to be directly controlled by uncharged tRNA levels because mutant cells containing a defective aminoacyl-tRNA synthetase showed elevated levels of phosphorylation of eIF-2␣ in the absence of amino acid limitation [16]
Summary
A family of serine/threonine protein kinases regulate protein synthesis by phosphorylation of the ␣ subunit of eukaryotic initiation factor-2 (eIF-2)1 [1,2,3,4]. GCN2 phosphorylation of eIF-2␣ is thought to lead to lowered eIF-2-GTP levels, reducing the inhibitory effects of the upstream open reading frames and allowing for increased GCN4 translation [11, 13, 14]. Class II aminoacyltRNA synthetases, including HisRS [17, 18], share three motifs, and mutations in the motif 2 sequence of GCN2 abolished phosphorylation of eIF-2␣ during these limiting conditions This suggests that the HisRS-related sequences measure starvation for many different amino acids by interaction with their cognate uncharged tRNAs. In vitro binding studies showed that recombinant protein containing the HisRS-related domain of GCN2 can bind uncharged tRNA by a process requiring motif 2 sequences [16]. We observed that amino-terminal sequences in GCN2 that contain homology to a portion of a second kinase domain are required for in vitro phosphorylation of the eIF-2␣
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