Serine 48 in initiation factor 2 alpha (eIF2 alpha) is required for high-affinity interaction between eIF2 alpha(P) and eIF2B

Phosphorylation of the serine 51 residue in the alpha-subunit of translatio nal initiation factor 2 in eukaryotes (eIF2 alpha) impairs protein synthesi s presumably by sequestering eTF2B, a rate-limiting pentameric guanine nucl eotide exchange protein which catalyzes the exchange of GTP for CDP in the eIF2-GDP binary complex, To further understand the importance of eIF2 alpha phosphorylation in the interaction between eIF2 alpha(P) and eIF2B protein s and thereby the regulation of eTF2B activity, we expressed the wild type (wt) and a mutant eIF2 alpha in which the serine 48 residue was replaced wi th alanine (48A mutant) in the baculovirus system. The findings reveal that the expression of both of these recombinant subunits was very efficient (1 5-20% of the total protein) and both proteins were recognized by an eIF2 al pha monoclonal antibody and were phosphorylated to the same extent by retic ulocyte eIF2 alpha kinases. However, partially purified recombinant subunit s (wt or 48A mutant) were not phosphorylated as efficiently as the eIF2 alp ha subunit present in the purified reticulocyte trimeric eIF2 complex and w ere also found to inhibit the phosphorylation of eIF2 alpha of the trimeric complex. Furthermore, the extents of inhibition of eIF2B activity and form ation of the eIF2 alpha(P)-eIF2B complex that occurs due to eIF2 alpha phos phorylation in poly(IC)treated rabbit reticulocyte lysates were decreased s ignificantly in the presence of insect cell extracts expressing the 48A mut ant eIF2 alpha compared to those for wt. These findings support the hypothe sis that the serine 48 residue is required for high-affinity interaction be tween eIF2 alpha(P) and eIF2B.