Biochemical and biophysical characterization of the activation of human protein kinase R

Date of Completion

January 2006


Biology, Molecular|Chemistry, Biochemistry|Biophysics, General




The human protein kinase R (PKR) is a central component of the interferon antiviral pathway. PKR undergoes autophosphorylation at multiple serine and threonine residues resulting in activation. The activated enzyme phosphorylates the eukaryotic initiation factor (eIF2α) resulting in inhibition of protein synthesis in the cell. However, the critical protein-protein and protein-ligand interactions that govern PKR activation are poorly understood. We have developed a high-yield prokaryotic expression and purification system for PKR that has enabled us to carry out biophysical experiments and correlate the data with functional activation assays. Sedimentation equilibrium and activation measurements indicate that PKR undergoes a weak, reversible monomer-dimer equilibrium (K d = 500 μM) that serves to initiate a previously unrecognized dsRNA-independent autophosphorylation reaction. Contrary to previous observations that were widely believed, PKR exists in an open conformation accessible to ATP binding (Kd = 34 μM) in the absence of dsRNA. Dimer stability is enhanced by ∼500-fold upon autophosphorylation. We propose a chain reaction model for PKR dsRNA-independent activation where dimerization of the latent enzyme followed by intermolecular phosphorylation serves as the initiation step. Subsequent propagation steps likely involve phosphorylation of latent PKR monomers by the activated enzyme. PKR is activated only by dsRNA that is long enough to promote functional dimerization of PKR. 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.^