Functional expression and RNA binding analysis of the interferon-induced, double-stranded RNA-activated, 68,000-Mr protein kinase in a cell-free system.
Eukaryotic viruses have devised numerous strategies to downregulate activity of the interferon-induced, double-stranded (dsRNA)-activated protein kinase (referred to as p68 on the basis of its Mr of 68,000 in human cells). Viruses must exert this control to avoid extensive phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF-2) by p68 and the resultant negative effects on protein synthesis initiation. To begin to define the molecular mechanisms underlying this regulation, we optimized expression of p68 in an in vitro transcription-translation system utilizing the full-length cDNA clone. The in vitro-expressed kinase was autophosphorylated in response to dsRNAs and heparin in a manner similar to that for the native p68 provided that the kinase inhibitor, 2-aminopurine, was present during the in vitro translation reaction. Further, the activated kinase efficiently phosphorylated its natural substrate, the alpha subunit of eIF-2. Binding experiments revealed that the expressed kinase complexed with the dsRNA activator, reovirus dsRNA, as well as the adenovirus-encoded inhibitor, VAI RNA. Interestingly, both the reovirus RNAs and VAI RNA also complexed with protein kinase molecules that lacked the carboxyl terminus and all catalytic domains. Deletion analysis confirmed that the p68 amino terminus contained critical determinants for reovirus dsRNA and VAI RNA binding. Further, reovirus dsRNA efficiently bound to, but failed to activate, p68 kinase molecules containing a single amino acid substitution in the invariant lysine 295 present in catalytic domain II. Taken together, these data demonstrate that this expression system permits a detailed mutagenic analysis of the regions of p68 required for interaction with virus-encoded activators and repressors.
Katze
M G
MG
Department of Microbiology, School of Medicine, University of Washington, Seattle 98195.
Wambach
M
M
Wong
M L
ML
Garfinkel
M
M
Meurs
E
E
Chong
K
K
Williams
B R
BR
Hovanessian
A G
AG
Barber
G N
GN
D000256:Adenoviridae / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002474:Cell-Free System; D002872:Chromosome Deletion; D004279:DNA, Viral / Q000378:metabolism*; D004789:Enzyme Activation; D004790:Enzyme Induction; D015852:Eukaryotic Initiation Factor-2 / Q000378:metabolism; D006801:Humans; D007372:Interferons / Q000494:pharmacology*; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D009841:Oligonucleotides; D010766:Phosphorylation; D014176:Protein Biosynthesis; D011494:Protein Kinases / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D012330:RNA, Double-Stranded / Q000378:metabolism*; D011817:Rabbits; D012087:Reoviridae / Q000235:genetics; D012156:Reticulocytes / Q000378:metabolism; D014158:Transcription, Genetic
D004279:DNA, Viral; D015852:Eukaryotic Initiation Factor-2; D046911:Macromolecular Substances; D009841:Oligonucleotides; D012330:RNA, Double-Stranded; D007372:Interferons; D011494:Protein Kinases