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.
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