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 alpha, beta, and gamma subunits, of which the alpha subunit (eIF2alpha) 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 2alpha (aIF2alpha), three genes encoding alpha, beta, and gamma subunits of aIF2 from the hyperthermophilic archaeon Pyrococcus horikoshii OT3 were expressed in Escherichia coli cells, and the resulting proteins, aIF2alpha, aIF2beta, and aIF2gamma, were characterized with reference to the properties of eIF2. aIF2alpha preferentially interacts with aIF2gamma, but does not interact with aIF2beta, which is consistent with data obtained with eIF2, of which eIF2gamma serves as a core subunit, interacting with eIF2alpha and eIF2beta. It was found that aIF2alpha was, albeit to a lower degree, phosphorylated by double-stranded RNA-dependent protein kinase (hPKR) front human, and a primary target site was suggested to be Ser48 within aIF2alpha. 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 aIF2alpha, 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 aIF2alpha, like eIF2alpha in eukaryotes, plays a role in regulation of the protein synthesis in Archaea through phosphorylation and dephosphorylation.