Mechanism of interferon action: cDNA structure, expression, and regulation of the interferon-induced, RNA-dependent P1/eIF-2 alpha protein kinase from human cells.
A molecular cDNA clone (P1 KIN) was isolated that encodes the human RNA-dependent P1/eIF-2 alpha protein kinase. The complete cDNA sequence of the P1 KIN cDNA was determined; the longest open reading frame (ORF) encoded a 551 amino acid protein with a deduced molecular weight of 62055 Da. Transcripts prepared from the P1 KIN cDNA by transcription in vitro with T7 RNA polymerase programmed the cell-free synthesis of a protein indistinguishable by immunoprecipitation and immunoblot gel analyses from the authentic 67-kDa P1 protein synthesized in human U cells treated with interferon (IFN). Furthermore, by use of a sensitive primer extension assay with T7 DNA polymerase, the major site of translation initiation within the deduced ORF of the P1 KIN cDNA was directly identified. Northern RNA gel-blot analysis revealed that the P1 KIN cDNA strongly hybridized to two IFN-induced mRNAs present in both human amnion U cells and HeLa cells; their sizes were 2.5 and 6 kb. Both transcripts were efficiently induced by IFN-alpha, but poorly by IFN-gamma. Polyclonal antibody was prepared against the product of the P1 KIN cDNA expressed in Escherichia coli. In Western blot analysis the antibody recognized a 67-kDa protein induced in human cells by IFN-alpha and, in addition, a 90-kDa protein whose level was not greatly altered by IFN treatment. The IFN-induced 67-kDa protein was found associated with the ribosomal salt-wash fraction of IFN-treated human cells, whereas the 90-kDa protein was predominantly in the S100 soluble fraction. The time course for the induction by IFN-alpha of RNA-dependent protein P1 kinase activity measured by immunoprecipitation was comparable to the time course for protein P1 induction measured by Western immunoblot analysis. The amino acid sequence of P1/eIF-2 alpha protein kinase deduced from the cDNA was 62% identical with the 518-residue murine TIK kinase and contained, within the carboxy-terminal half of the protein, the motifs commonly conserved among protein-serine/threonine kinases. The amino-terminal half of the P1 protein did not possess conserved kinase motifs, but did show extensive homology with vaccinia virus-predicted protein E3L.
Thomis
D C
DC
Department of Biological Sciences, University of California, Santa Barbara 93106.
Doohan
J P
JP
Samuel
C E
CE
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry / Q000235:genetics; D015852:Eukaryotic Initiation Factor-2 / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects*; D006367:HeLa Cells; D006801:Humans; D007372:Interferons / Q000494:pharmacology*; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D014176:Protein Biosynthesis; D011494:Protein Kinases / Q000235:genetics* / Q000378:metabolism; D012313:RNA / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D014764:Viral Proteins / Q000235:genetics; D019892:eIF-2 Kinase
D015852:Eukaryotic Initiation Factor-2; D014764:Viral Proteins; D012313:RNA; D004247:DNA; D007372:Interferons; D011494:Protein Kinases; D019892:eIF-2 Kinase