A Glycosylation Site, 60SGTS63, of p67 Is Required for Its Ability To Regulate the Phosphorylation and Activity of Eukaryotic Initiation Factor 2α

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Abstract
Eukaryotic initiation factor 2- (eIF2-) associated glycoprotein p67 blocks eIF2α phosphorylation by kinases, and its N-terminal 1−97 amino acid segment can induce efficient translation. To investigate whether glycosylation at the serine/threonine clusters at this region is important in protein synthesis, we selected 27TSST30 and 60SGTS63 clusters for further analysis. By site-directed mutagenesis, 27TSST30 and 60SGTS63 clusters were substituted with 27AAGA30 and 60AGAA63 amino acid residues in full-length p67, and their EGFP fusions were constitutively expressed in rat tumor hepatoma cells (KRC-7). The 60AGAA63 mutant blocked eIF2α phosphorylation less than either wild-type p67 or the 27AAGA30 mutant. The 60AGAA63 mutant also showed a low level of protein synthesis rate, a lower level of glycosylation, increased turnover rate, and weaker binding to eIF2α. These results suggest that glycosylation within the 60SGTS63 sequence of p67 plays an important role in its stability and thus its regulation of protein synthesis by modulating the phosphorylation of the α-subunit of eIF2.