The eukaryotic initiation factor 2-associated 67-kDa polypeptide (p⁶⁷) plays a critical role in regulation of protein synthesis initiation in animal cells

The eukaryotic initiation factor 2 (eIF-2)-associated 67-kDa polypeptide (p⁶⁷) isolated from reticulocyte lysate protects the eIF-2 α subunit from eIF-2 kinase-catalyzed phosphorylation and promotes protein synthesis in the presence of active eIF-2 kinases. We have now studied the roles of p⁶⁷ and eIF-2 kinases in regulation of protein synthesis using several animal cell lysates and an animal cell line (KRC-7) in culture under various growth conditions. The results are as follows. (i) Both p⁶⁷ and eIF-2 kinase(s) are present in active forms in all animal cells under normal growth conditions and p⁶⁷ protects the eIF-2 α subunit from eIF-2 kinase-catalyzed phosphorylation, thus promoting protein synthesis in the presence of active eIF-2 kinases. (ii) In heme-deficient reticulocyte lysates and in serum-starved KRC-7 cells in culture, p⁶⁷ is deglycosylated and subsequently degraded. This leads to eIF-2 kinase-catalyzed eIF-2 α-subunit phosphorylation and thus to protein synthesis inhibition. (iii) Addition of a mitogen (namely, phorbol 12-myristate 13-acetate) to serum-starved KRC-7 cells in culture induces an increase of p⁶⁷ and thus increases protein synthesis. These results suggest the following conclusions. (i) Protein synthesis inhibition in a heme-deficient reticulocyte lysate is not due to the activation of an eIF-2 kinase (heme-regulated inhibitor), as is generally believed, but is due to degradation of p⁶⁷. The heme-regulated inhibitor is present in an active form and possibly in equal amounts in both heme-deficient and heme-supplemented reticulocyte lysates but cannot phosphorylate eIF-2 α subunit because of the presence of p⁶⁷. (ii) p⁶⁷ is essential for protein synthesis as it protects the eIF-2 α subunit from eIF-2 kinase-catalyzed phosphorylation and promotes protein synthesis in the presence of one or more active eIF-2 kinases present in all animal cells. (iii) p⁶⁷ is both degradable and inducible. Only the p⁶⁷ level correlates directly with the protein synthesis activity of the cell, indicating that p⁶⁷ is a critical factor in protein synthesis regulation in animal cells. (.