A model for the double-stranded RNA (dsRNA)-dependent dimerization and activation of the dsRNA-activated protein kinase PKR.
Binding of double-stranded RNA (dsRNA) to PKR induces autophosphorylation and activation. However, the requirement for dsRNA in promoting dimerization and the requirement for dimerization in PKR activation are controversial. We have studied the dsRNA binding and dimerization requirements for the activation of PKR in vivo. Co-expression and immunoprecipitation experiments detected an interaction between the K296P mutant and a bacteriophage T7-epitope-tagged K64E mutant of dsRNA binding domain. In contrast, the K64E/K296P double mutant did not form a detectable dimer with the wild-type dsRNA binding domain. These results support that dimerization of intact PKR with the isolated dsRNA binding domain requires dsRNA binding activity. Expression of the isolated PKR kinase domain (residues 228-551) reduced translation of the reporter mRNA even in the presence of PKR inhibitors. Furthermore, the isolated kinase domain (residues 228-551) undergoes autophosphorylation and sequentially transphosphorylates both mutant K296P PKR and wild-type eIF-2alpha in vitro. In contrast, the isolated kinase domain (residues 264-551) lacking the third basic region was not active. These observations lead us to propose that the dsRNA binding domains on intact PKR inhibit kinase activity and that dsRNA binding to intact PKR induces a conformational change to expose dimerization sites within the dsRNA binding domain thereby promoting dimerization and facilitating trans-phosphorylation and activation.
Wu
S
S
Howard Hughes Medical Institute, University of Michigan Medical Center, Ann Arbor 48109, USA.
Kaufman
R J
RJ
D000818:Animals; D019556:COS Cells; D019281:Dimerization; D004789:Enzyme Activation; D008956:Models, Chemical; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D012330:RNA, Double-Stranded / Q000378:metabolism*; D013539:Simian virus 40; D014162:Transfection; D014616:Vaccinia virus; D019892:eIF-2 Kinase
D012330:RNA, Double-Stranded; D017346:Protein Serine-Threonine Kinases; D019892:eIF-2 Kinase