title: Heterologous dimerization domains functionally substitute for the double-stranded RNA binding domains of the kinase PKR.
abstract: The protein kinase PKR (dsRNA-dependent protein kinase) phosphorylates the eukaryotic translation initiation factor eIF2alpha 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 double-stranded RNA, mediate dimerization of the protein and target PKR to the ribosome. To investigate further the importance of dimerization for PKR activity, fusion proteins were generated linking the PKR kinase domain to heterologous dimerization domains. Whereas the isolated PKR kinase domain (KD) was non-functional in vivo, expression of a glutathione S-transferase-KD fusion, or co-expression of KD fusions containing the heterodimerization domains of the Xlim-1 and Ldb1 proteins, restored PKR activity in yeast cells. Finally, coumermycin-mediated dimerization of a GyrB-KD fusion protein increased eIF2alpha phosphorylation and inhibited reporter gene translation in mammalian cells. These results demonstrate the critical importance of dimerization for PKR activity in vivo, and suggest that a primary function of double-stranded RNA binding to the dsRBDs of native PKR is to promote dimerization and activation of the kinase domain.
authors.affiliation: Laboratory of Gene Regulation and Development, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: T L
authors.initials: TL
authors.lastname: Ung
authors.forename: C
authors.initials: C
authors.lastname: Cao
authors.forename: J
authors.initials: J
authors.lastname: Lu
authors.forename: K
authors.initials: K
authors.lastname: Ozato
authors.forename: T E
authors.initials: TE
authors.lastname: Dever
mesh_terms: D016475:3T3 Cells; D000818:Animals; D001665:Binding Sites; D019281:Dimerization; D004789:Enzyme Activation; D051379:Mice; D012330:RNA, Double-Stranded / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D019892:eIF-2 Kinase / Q000737:chemistry / Q000378:metabolism*
chemical_list: D012330:RNA, Double-Stranded; D019892:eIF-2 Kinase