RIBOSOME TARGETING OF PKR IS MEDIATED BY 2 DOUBLE-STRANDED RNA-BINDING DOMAINS AND FACILITATES IN-VIVO PHOSPHORYLATION OF EUKARYOTIC INITIATION FACTOR-II
Sh. Zhu et al., RIBOSOME TARGETING OF PKR IS MEDIATED BY 2 DOUBLE-STRANDED RNA-BINDING DOMAINS AND FACILITATES IN-VIVO PHOSPHORYLATION OF EUKARYOTIC INITIATION FACTOR-II, The Journal of biological chemistry, 272(22), 1997, pp. 14434-14441
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.