A glycosylation site, 60SGTS63, of p67 is required for its ability to regulate the phosphorylation and activity of eukaryotic initiation factor 2alpha.

Eukaryotic initiation factor 2- (eIF2-) associated glycoprotein p67 blocks eIF2alpha 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 (27)TSST(30) and (60)SGTS(63) clusters for further analysis. By site-directed mutagenesis, (27)TSST(30) and (60)SGTS(63) clusters were substituted with (27)AAGA(30) and (60)AGAA(63) amino acid residues in full-length p67, and their EGFP fusions were constitutively expressed in rat tumor hepatoma cells (KRC-7). The (60)AGAA(63) mutant blocked eIF2alpha phosphorylation less than either wild-type p67 or the (27)AAGA(30) mutant. The (60)AGAA(63) mutant also showed a low level of protein synthesis rate, a lower level of glycosylation, increased turnover rate, and weaker binding to eIF2alpha. These results suggest that glycosylation within the (60)SGTS(63) sequence of p67 plays an important role in its stability and thus its regulation of protein synthesis by modulating the phosphorylation of the alpha-subunit of eIF2.