Serine 48 in initiation factor 2α (eIF2α) is required for high-affinity interaction between eIF2α(p) and eIF2b

Abstract

Phosphorylation of the serine 51 residue in the α-subunit of translational initiation factor 2 in eukaryotes (eIF2α) impairs protein synthesis presumably by sequestering eIF2B, a rate-limiting pentameric guanine nucleotide exchange protein which catalyzes the exchange of GTP for GDP in the eIF2-GDP binary complex. To further understand the importance of eIF2α phosphorylation in the interaction between eIF2α(P) and eIF2B proteins and thereby the regulation of eIF2B activity, we expressed the wild type (wt) and a mutant eIF2α in which the serine 48 residue was replaced with alanine (48A mutant) in the baculovirus system. The findings reveal that the expression of both of these recombinant subunits was very efficient (15-20% of the total protein) and both proteins were recognized by an eIF2α monoclonal antibody and were phosphorylated to the same extent by reticulocyte eIF2α kinases. However, partially purified recombinant subunits (wt or 48A mutant) were not phosphorylated as efficiently as the eIF2 subunit present in the purified reticulocyte trimeric eIF2 complex and were also found to inhibit the phosphorylation of eIF2a of the trimeric complex. Furthermore, the extents of inhibition of eIF2B activity and formation of the eIF2α(P)-eIF2B complex that occurs due to eIF2α phosphorylation in poly(IC)-treated rabbit reticulocyte lysates were decreased significantly in the presence of insect cell extracts expressing the 48A mutant eIF2α compared to those for wt. These findings support the hypothesis that the serine 48 residue is required for high-affinity interaction between eIF2α(P) and eIF2B.