Mechanistic Link between PKR Dimerization, Autophosphorylation, and eIF2(alpha) Substrate Recognition

The antiviral protein kinase PKR inhibits protein synthesis by phosphorylating the translation initiation factor eIF2(alpha) on Ser51. Binding of double-stranded RNA to the regulatory domains of PKR promotes dimerization, autophosphorylation, and the functional activation of the kinase. Herein, we identify mutations that activate PKR in the absence of its regulatory domains and map the mutations to a recently identified dimerization surface on the kinase catalytic domain. Mutations of other residues on this surface block PKR autophosphorylation and eIF2(alpha) phosphorylation, while mutating Thr446, an autophosphorylation site within the catalyticdomain activation segment, impairs eIF2(alpha) phosphorylation and viral pseudosubstrate binding. Mutational analysis of catalyticdomain residues preferentially conserved in the eIF2(alpha) kinase family identifies helix (alpha)G as critical for the specific recognition of eIF2(alpha). We propose an ordered mechanism of PKR activation in which catalytic-domain dimerization triggers Thr446 autophosphorylation and specific eIF2(alpha) substrate recognition.