Factors that Influence PKR Dimerization and Activation

Abstract

Protein kinase R (PKR) is a key component of the interferon-induced innate immunity pathway. It is expressed in a latent, inactive form and is activated upon binding dsRNA and subsequent autophosphorylation. PKR contains a C-terminal kinase domain and two tandem dsRNA binding motifs (dsRBMs) at the N-terminus. These regions are separated by a 90-residue, unstructured linker. Dimerization is believed to play a critical role in the mechanism of PKR activation by dsRNA. Activation requires dsRNAs long enough to accommodate two PKR monomers. However, there is no direct evidence for dimerization of PKR on dsRNA lattices. Under certain conditions, shorter RNAs bind multiple PKRs but fail to activate, suggesting that additional factors are involved. It is believed that binding of the second dsRBM is required for PKR activation. We have characterized the role of the second dsRBM by introducing mutations that block its interaction with dsRNA. The distance dependence of PKR dimerization and activation were probed using synthetic RNAs that function as molecular rulers. These molecules consist of two 15 bp duplex regions separated by variable length regions of 2′-O-methyl modified dsRNA that act as rigid and inert barriers. The effect of linker length was investigated using a PKR homolog containing a short, 25-residue linker. We developed a fluorescence assay to quantitatively probe PKR dimerization upon binding to RNA. The fluorescence anisotropy of a probe placed near the kinase dimer interface decreases upon PKR binding to an activating 40 bp dsRNA due to depolarization induced by homo-FRET. Thus, the kinase domains form a dimer when two PKR monomers bind to this dsRNA. Surprisingly, several non-activating dsRNAs also induce dimerization, suggesting that PKR dimerization is necessary but not sufficient for activation.