Binding of double-stranded RNA to protein kinase PKR is required for dimerization and promotes critical autophosphorylation events in the activation loop.
Protein kinase PKR is activated by double-stranded RNA (dsRNA) and phosphorylates translation initiation factor 2alpha to inhibit protein synthesis in virus-infected mammalian cells. PKR contains two dsRNA binding motifs (DRBMs I and II) required for activation by dsRNA. There is strong evidence that PKR activation requires dimerization, but the role of dsRNA in dimer formation is controversial. By making alanine substitutions predicted to remove increasing numbers of side chain contacts between the DRBMs and dsRNA, we found that dimerization of full-length PKR in yeast was impaired by the minimal combinations of mutations required to impair dsRNA binding in vitro. Mutation of Ala-67 to Glu in DRBM-I, reported to abolish dimerization without affecting dsRNA binding, destroyed both activities in our assays. By contrast, deletion of a second dimerization region that overlaps the kinase domain had no effect on PKR dimerization in yeast. Human PKR contains at least 15 autophosphorylation sites, but only Thr-446 and Thr-451 in the activation loop were found here to be critical for kinase activity in yeast. Using an antibody specific for phosphorylated Thr-451, we showed that Thr-451 phosphorylation is stimulated by dsRNA binding. Our results provide strong evidence that dsRNA binding is required for dimerization of full-length PKR molecules in vivo, leading to autophosphorylation in the activation loop and stimulation of the eIF2alpha kinase function of PKR.
Zhang
F
F
Laboratory of Gene Regulation and Development, NICHHD, National Institutes of Health, Bethesda, MD 20892, USA.
Romano
P R
PR
Nagamura-Inoue
T
T
Tian
B
B
Dever
T E
TE
Mathews
M B
MB
Ozato
K
K
Hinnebusch
A G
AG
D000595:Amino Acid Sequence; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010766:Phosphorylation; D010957:Plasmids; D011070:Poly I-C / Q000378:metabolism; D012330:RNA, Double-Stranded / Q000378:metabolism*; D013912:Threonine / Q000378:metabolism; D015003:Yeasts / Q000201:enzymology; D019892:eIF-2 Kinase / Q000235:genetics / Q000378:metabolism*
D012330:RNA, Double-Stranded; D013912:Threonine; D019892:eIF-2 Kinase; D011070:Poly I-C