Negative regulation of the protection of eIF2alpha phosphorylation activity by a unique acidic domain present at the N-terminus of p67.

Eukaryotic initiation factor 2 (eIF2)-associated glycoprotein, p67, has protection of eIF2alpha phosphorylation (POEP) activity, and this activity requires lysine-rich domains I and II of p67. Another unique acidic residue-rich domain is also present at the N-terminus of p67. In this study we analyzed the role of this acidic residue-rich domain in POEP activity. Our data revealed that constitutive expression of a mutant form of p67 (D6/2) in mammalian cells resulted in increased POEP activity, and this activity was partially inhibited when second-site alanine substitutions at the conserved amino acids D251, D262, E364, and E459 were introduced in the D6/2 mutant. In contrast, a similar mutation at the conserved H331 position did not show any effect on POEP activity. Individual alanine substitutions at the above conserved amino acids in wild-type p67 did not show any significant effect on POEP activity except the E459 position where alanine substitution caused approximately 50% increase in POEP activity as compared to the wild type. Although, the levels of endogenous p67 and p67-deglycosylase did not correlate with the POEP activity, we found that the D6/2 mutant of p67 was glycosylated at a higher level in mammalian cells as compared to wild-type p67. The increased POEP activity of the D6/2 mutant also correlated with the higher rate of overall protein synthesis in mammalian cells constitutively expressing this mutant form of p67. Taken together, these data suggest that the acidic residue-rich domain present at the N-terminus of p67 may have a negative role in POEP activity.

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