Construction and expression of an enzymatically active human-mouse chimeric double-stranded RNA-dependent protein kinase.
The interferon (IFN)-inducible double-stranded (ds) RNA-activated protein kinase (p68 kinase) is a physiologically important enzyme that regulates the rate of cellular and viral protein synthesis by phosphorylating and thereby inactivating the peptide chain initiation factor 2. We have generated a partial cDNA clone, which probably represents the murine p68 kinase, by reverse transcription-polymerase chain reaction (RT-PCR) using sequence information of the human p68 kinase. The 725-bp cDNA clone encoded the carboxyl-terminal 238 amino acid residues of the mouse kinase. It has 67% overall identity with the corresponding region of the human kinase. All the protein kinase catalytic domains are conserved in the mouse protein. Moreover, there are additional stretches of residues that are totally conserved between the two proteins. The functional equivalence of the two proteins was tested by constructing a chimeric cDNA that encoded a protein whose amino-terminal 364 residues were of human origin and carboxyl-terminal 187 residues were of mouse origin. The chimeric protein was as efficient as the human p68 kinase in binding to the dsRNA, autophosphorylating and phosphorylating exogenous substrate.
Department of Molecular Biology, Cleveland Clinic Foundation, OH 44195-5285.
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002678:Chimera / Q000235:genetics*; D003001:Cloning, Molecular; D006801:Humans; D066298:In Vitro Techniques; D051379:Mice; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis / Q000235:genetics; D011494:Protein Kinases / Q000096:biosynthesis* / Q000378:metabolism; D012330:RNA, Double-Stranded / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014158:Transcription, Genetic / Q000235:genetics; D019892:eIF-2 Kinase
D012330:RNA, Double-Stranded; D011494:Protein Kinases; D019892:eIF-2 Kinase