Interaction of recombinant human eIF2 subunits with eIF2B and eIF2α kinases

Abstract

The heterotrimeric eukaryotic initiation factor 2 (eIF2) plays a critical role in the mechanics and regulation of protein synthesis. Unlike yeast and archaeal eIF2, the purified baculovirus-expressed recombinant human eIF2 subunits used in these studies reveal that the α- and β-subunits interact with each other. Consistent with this observation, the β-subunit specifically interacts with the purified eIF2B in ELISA studies and this interaction is enhanced when wt eIF2α in the recombinant trimeric complex is phosphorylated or replaced by a mutant phosphomimetic eIF2α (S51D). These findings together with other observations raise the possibility that the β-subunit plays a key role in the regulation and function of mammalian eIF2 complex. PERK, an eIF2α kinase, is found to interact with wt and mutants of eIF2α in which the serine 51 or 48 residue is replaced by alanine or aspartic acid thereby suggesting that the phosphorylation site in the substrate is not important for interaction. Fluorescence spectroscopic and fluorescence resonance energy transfer analyses reveal that the energy transfer occurs from PERK to eIF2α. The dissociation constant of α-subunit-PERK complex (Kd α-subunit) is 0.74 μM and the interaction is stoichiometric. © 2005 Elsevier Inc. All rights reserved.