Rekha Datta

Protection of translation initiation factor eIF2 phosphorylation correlates with eIF2-associated glycoprotein p67 levels and requires the lysine-rich domain I of p67.

The rate of protein synthesis in mammals is largely regulated by phosphorylation of the alpha-subunit of eukaryotic initiation factor 2 (eIF2) that is modulated by the cellular glycoprotein, p67, due to its protection of eIF2alpha phosphorylation (POEP) activity. At the N-terminus of p67, there are three unique domains, and at the C-terminus there is a conserved amino acid sequence. To analyze the importance of these domains, C-terminal deletion mutants of rat p67 were expressed constitutively in KRC-7 cells. In these cells, the phosphorylation level of the alpha-subunit of eIF2 was determined, and it was found that expression of the 1-97 amino acid segment of rat p67 increases POEP activity in vivo, and induces the endogenous levels of p67. These cells also show increased growth rate, and efficient translation of chloramphenicol acetyltransferase and beta-galactosidase reporter genes. At the N-terminus of p67, there are two unique domains: a lysine-rich domain I with the sequence (36)KKKRRKKKK(44), and an acidic residue-rich domain with the sequence (77)EEKEKDDDDEDGDGD(91). Substitution of lysine-rich domain I with (36)NMKSGNKTQ(44) in rat recombinant p67 resulted in the inhibition of its POEP activity, and substitution of the acidic residue-rich domain with (77)QNIQKALEPEAGDGA(91), resulted in no inhibition of POEP activity in KRC-7 cells. Taken together, our data suggest that protection of translation initiation factor eIF2 phosphorylation correlates with eIF2-associated glycoprotein p67 levels and requires the lysine-rich domain I of p67.

Negative regulation of the protection of eIF2α 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.

Mutation at the acidic residue-rich domain of eukaryotic initiation factor 2 (eIF2α)-associated glycoprotein p67 increases the protection of eIF2α phosphorylation during heat shock

Eukaryotic initiation factor 2 (eIF2)-associated glycoprotein p67 protects eIF2alpha phosphorylation from kinases. The N-terminal lysine-rich domains increase this activity and the acidic residue-rich domain inhibits it. Conserved amino acid residues D251, D262, E364, and E459 are involved in this inhibition. During heat shock, the overall protein synthesis rate decreases due to the increased levels of eIF2alpha phosphorylation. In this study, we examined whether the above inhibition is also found during heat shock. Indeed, the acidic residue-rich domain mutant (D6/2) showed a decreased level of eIF2alpha phosphorylation, and its second-site alanine substitutions at D251, D262, and E459 reversed this effect, whereas second-site alanine substitution at H331 and E364 residues further augmented it. A high-molecular-weight phosphoprotein and at least two faster-migrating phosphoproteins were detected by the monospecific polyclonal antibody against eIF2alpha(P) form in rat tumor hepatoma cells constitutively expressing the double mutant D6/2+D251A. Although the levels of p67 mutants were unaffected during heat shock, those of p67 and p67-deactivating enzyme varied. Furthermore, the overall rate of protein synthesis correlated with the level of eIF2alpha phosphorylation. Taken together, these results suggest that the lysine-rich domains and conserved amino acid residues of p67 are involved in the regulation of eIF2alpha phosphorylation during heat shock.