RIBOSOME TARGETING OF PKR IS MEDIATED BY 2 DOUBLE-STRANDED RNA-BINDING DOMAINS AND FACILITATES IN-VIVO PHOSPHORYLATION OF EUKARYOTIC INITIATION FACTOR-II

Protein kinase PKR is activated in mammalian cells during viral infect ion, leading to phosphorylation of the alpha subunit of eukaryotic ini tiation factor-2 (eIF-2 alpha) and inhibition of protein synthesis. Th is antiviral response is thought to be mediated by association of doub le-stranded RNA (ds-RNA), a by-product of viral replication, with two ds-RNA-binding domains (DRBDs) located in the amino terminus of PKR, R ecent studies have observed that expression of mammalian PKR in yeast leads to a slow growth phenotype due to hyperphosphorylation of eIF-2 alpha. In this report, we observed that while DRBD sequences are requi red for PKR to function in the yeast model system, these sequences are not required for in vitro phosphorylation of eIF-2 alpha. To explain this apparent contradiction, we proposed that these sequences are requ ired to target the kinase to the translation machinery, Using sucrose gradient sedimentation, we found that wild-type PKR was associated wit h ribosomes, specifically with 40 S particles, Deletions or residue su bstitutions in the DRBD sequences blocked kinase interaction with ribo somes. These results indicate that in addition to mediating ds-RNA con trol of PKR, the DRBD sequences facilitate PKR association with riboso mes, Targeting to ribosomes may enhance in vivo phosphorylation of eIF -2 alpha, by providing PKR access to its substrate.