In vitro phosphorylation of initiation factor 2α (aIF2α) from hyperthermophilic archaeon Pyrococcus horikoshii OT3

Eukaryotic initiation factor 2 (eIF2) is a heterotrimeric protein composed of α, β, and γ subunits, of which the α subunit (eIF2α) plays a crucial role in regulation of protein synthesis through phosphorylation at Ser51. All three subunit genes are conserved in Archaea. To examine the properties of archaeal initiation factor 2α (aIF2α), three genes encoding α, β, and γ subunits of aIF2 from the hyperthermophilic archaeon Pyrococcus horikoshii OT3 were expressed in Escherichia coli cells, and the resulting proteins, aIF2α, aIF2β, and aIF2γ, were characterized with reference to the properties of eIF2. aIF2α preferentially interacts with aIF2γ, but does not interact with aIF2β, which is consistent with data obtained with eIF2, of which eIF2γ serves as a core subunit, interacting with eIF2α and eIF2β. It was found that aIF2α was, albeit to a lower degree, phosphorylated by double-stranded RNA-dependent protein kinase (hPKR) from human, and a primary target site was suggested to be Ser48 within aIF2α. This finding led us to the search for a putative aIF2 specific kinase gene (PH0512) in the P. horikoshii genome. The gene product Ph0512p unambiguously phosphorylated aIF2α, and Ser48, as in the phosphorylation by hPKR, was suggested to be a target amino acid residue for the PKR homologue Ph0152p in P. horikoshii. These findings suggest that aIF2α, like eIF2α in eukaryotes, plays a role in regulation of the protein synthesis in Archaea through phosphorylation and dephosphorylation.