REGULATION OF EIF-2 ALPHA-SUBUNIT PHOSPHORYLATION IN RETICULOCYTE LYSATE

An eIF-2 associated 67-kDa protein (p(67)) protects eIF-2 alpha-subunit from eIF-2 kinase(s) catalyzed phosphorylation and promotes protein synthesis in the presence of active eIF-2 kinase(s). p(67) is a glycoprotein and contains multiple O-linked GlcNAc moieties. We have now studied the roles of hemin, p(67), and the glycosyl residues on p(67) in the regulation of eIF-2 alpha-subunit phosphorylation in reticulocyte lysates. The results are as follows: (i) Both hemin and p(67) inhibited HRI (heme-regulated protein synthesis inhibitor) and dsI (double-stranded RNA activated protein synthesis inhibitor) catalyzed phosphorylation of eIF-2 alpha-subunit in vitro. However, only hemin, and not p(67), inhibited casein kinase catalyzed phosphorylation of eIF-2 beta-subunit. (ii) Only p(67), and not hemin, inhibited eIF-2 alpha-subunit phosphorylation by eIF-2 kinase(s) in reticulocyte lysate. Significant eIF-2 alpha-subunit phosphorylation was observed even in the presence of hemin when p(67) in the reticulocyte lysate was removed by treatment with p(67) antibodies. (iii) Reticulocyte lysate contains a p(67)-deglycosylase in latent form, and hemin prevents activation of this deglycosylase. In the absence of hemin, this p(67)-deglycosylase is activated. Once activated in the absence of hemin, the activated deglycosylase deglycosylates p(67), even in the presence of hemin. This inactivates p(67) and allows eIF-2 kinase to phosphorylate eIF-2 alpha-subunit and inhibit protein synthesis. Protein synthesis in reticulocyte lysate is thus regulated by two novel cascades of covalent modifications: protein deglycosylation leading to protein phosphorylation.