Characteristics of the Eukaryotic Initiation Factor 2 Associated 67-kDa Polypeptide

A eukaryotic initiation factor 2 (eIF-2) associated 67-kDa polypeptide (p⁶⁷) protects the eIF-2 α-subunit from eIF-2 kinase(s) catalyzed phosphorylation, and this promotes protein synthesis in the presence of active eIF-2 kinase(s), [Datta, B., et al. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 3324–3328]. This report presents the results of studies related to characteristics of p⁶⁷ action and the mechanism of p⁶⁷:eIF-2 interaction: (1) p⁶⁷ antibodies inhibited protein synthesis in hemin-supplemented rabbit reticulocyte lysates, and such inhibition was reversed by preincubation of the antibodies, specifically with p⁶⁷. (2) p⁶⁷ inhibited HRI- and dsI-catalyzed phosphorylations of the eIF-2 α-subunit and histones, but it did not inhibit casein kinase catalyzed phosphorylation of the eIF-2 β-subunit. (3) p⁶⁷ bound specifically to the eIF-2 γ-subunit. p⁶⁷ co-immunoprecipitated with the eIF-2 subunits when a p⁶⁷/eIF-2 mixture was treated with p⁶⁷ or eIF-2 subunit antibodies and protein A agarose. However, when eIF-2 was preincubated specifically with the eIF-2 γ-subunit antibodies, subsequent co-immunoprecipitation of p⁶⁷ with the eIF-2 subunits was completely inhibited. Similarly, preincubation of p⁶⁷ and p⁶⁷ antibodies prevented subsequent p⁶⁷ binding to eIF-2. Preincubation of eIF-2, with either eIF-2 α- or β-subunit antibodies, had no effect on p⁶⁷ co-immunoprecipitation with the eIF-2 subunits. (4) p⁶⁷:eIF-2 interaction is necessary for p⁶⁷ activity to protect the eIF-2 α-subunit from eIF-2 kinase(s) catalyzed phosphorylation, p⁶⁷ bound to eIF-2 only at low Mg²⁺ concentrations (0.5–2 mM) and protected the eIF-2 α-subunit at these Mg²⁺ concentrations. High Mg²⁺ (3–5 mM) completely inhibited p⁶⁷ binding to eIF-2 and also inhibited p⁶⁷ activity to protect the eIF-2 α-subunit. These results suggest that p⁶⁷ binding to eIF-2 is necessary for p⁶⁷ activity to protect the eIF-2 α-subunit from inhibitory phosphorylation. Several factors, such as the p⁶⁷ level in the cell, may affect the equilibrium between p⁶⁷-bound eIF-2 and free eIF-2 and thus regulate protein synthesis.