Mechanism of PKR activation: Dimerization and kinase activation in the absence of double-stranded RNA

The kinase PKR is a central component of the interferon antiviral pathway. PKR is activated upon binding double-stranded (ds) RNA to undergo autophosphorylation. Although PKR is known to dimerize, the relationship between dimerization and activation remains unclear. Here, we directly characterize dimerization of PKR in free solution using analytical ultracentrifugation and correlate self-association with autophosphorylation. activity. Latent, unphosphorylated PKR exists predominantly as a monomer at protein concentrations below 2 mg/ml. A monomer sedimentation coefficient of s(20,w)(0) = 3.58 S and a frictional ratio of f/f(0) = 1.62 indicate an asymmetric shape. Sedimentation equilibrium measurements indicate that PKR undergoes a weak, reversible monomer-dimer equilibrium with K-d = 450 muM. This dimerization reaction serves to initiate a previously unrecognized dsRNA-independent autophosphorylation reaction. The resulting activated enzyme is phosphorylated on the two critical threonine residues present in the activation loop and is competent to phosphorylate the physiological substrate, eIF2alpha. Dimer stability is enhanced by similar to500-fold upon autophosphorylation. We propose a chain reaction model for PKR dsRNA-independent activation where dimerization of latent enzyme followed by intermolecular phosphorylation serves as the initiation step. Subsequent propagation steps likely involve phosphorylation of latent PKR monomers by activated enzyme within high-affinity heterodimers. Our results support a model whereby dsRNA functions by bringing PKR monomers into close proximity in a manner that is analogous to the dimerization of free PKR. (C) 2004 Elsevier Ltd. All rights reserved.