Translational regulation by the interferon-induced double-stranded-RNA-activated 68-kDa protein kinase.
Activation of the interferon-inducible 68-kDa protein kinase (referred to as P68) by double-stranded RNA catalyzes phosphorylation of the alpha subunit of eukaryotic protein synthesis initiation factor 2. We have analyzed the transient expression of mutant and wild-type kinase molecules in transfected COS cells to examine the effects of the kinase on gene expression in the absence of other interferon-induced gene products. The wild-type P68 kinase was expressed inefficiently whereas a catalytically inactive P68 was expressed at 30- to 40-fold higher levels. Protein stability measurements and primer-extension analysis of human kinase-specific mRNA levels provided evidence that kinase expression was regulated at the level of mRNA translation. Further, cotransfection experiments revealed that the domain II catalytically inactive mutant could stimulate reporter gene protein synthesis in a transdominant manner. We also examined the expression of mutants with deletions in the N-terminal double-stranded RNA binding domains and found that a kinase construct lacking aa 156-243 was expressed at levels comparable to the wild type whereas a P68 construct lacking aa 91-243 was expressed at levels 70-fold higher. Both the inactive domain II P68 mutant and the deletion mutant lacking aa 91-243 were less inhibitory to growth in yeast due to the reduced ability to phosphorylate initiation factor 2 alpha in vivo. In conclusion we have demonstrated that the P68 kinase can regulate mRNA translation primarily of its own mRNA and to a lesser extent of a heterologous mRNA and that this regulation is notably affected by mutations in either the catalytic or N-terminal regulatory domains.
Barber
G N
GN
Department of Microbiology, School of Medicine, University of Washington, Seattle 98195.
Wambach
M
M
Wong
M L
ML
Dever
T E
TE
Hinnebusch
A G
AG
Katze
M G
MG
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002522:Chlorocebus aethiops; D005786:Gene Expression Regulation; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D010766:Phosphorylation; D014176:Protein Biosynthesis; D011494:Protein Kinases / Q000378:metabolism*; D012330:RNA, Double-Stranded / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D013329:Structure-Activity Relationship; D014162:Transfection; D019892:eIF-2 Kinase
D009838:Oligodeoxyribonucleotides; D012330:RNA, Double-Stranded; D012333:RNA, Messenger; D011494:Protein Kinases; D019892:eIF-2 Kinase