Heterologous dimerization domains functionally substitute for the double-stranded RNA binding domains of the kinase PKR

The protein kinase PKR (dsRNA-dependent protein kinase) phosphorylates the eukaryotic translation initiation factor eIF2 alpha to downregulate protein synthesis in virus-infected cells. Two double-stranded RNA binding domains (dsRBDs) in the N-terminal half of PKR are thought to bind the activator d ouble-stranded RNA, mediate dimerization of the protein and target PKR to t he ribosome. To investigate further the importance of dimerization for PKR activity, fusion proteins were generated linking the PKR kinase domain to h eterologous dimerization domains. Whereas the isolated PKR kinase domain (K D) was non-functional in vivo, expression of a glutathione S-transferase-KD fusion, or co-expression of KD fusions containing the heterodimerization d omains of the Xlim-1 and Ldb1 proteins, restored PKR activity in yeast cell s. Finally, coumermycin-mediated dimerization of a GyrB-KD fusion protein i ncreased eIF2 alpha phosphorylation and inhibited reporter gene translation in mammalian cells. These results demonstrate the critical importance of d imerization for PKR activity in vivo, and suggest that a primary function o f double-stranded RNA binding to the dsRBDs of native PKR is to promote dim erization and activation of the kinase domain.