DRBP76, a double-stranded RNA-binding nuclear protein, is phosphorylated by the interferon-induced protein kinase, PKR.
The interferon-induced double-stranded RNA-activated protein kinase PKR is the prototype of a class of double-stranded (dsRNA)-binding proteins (DRBPs) which share a dsRNA-binding motif conserved from Drosophila to humans. Here we report the purification of DRBP76, a new human member of this class of proteins. Sequence from the amino terminus of DRBP76 matched that of the M phase-specific protein, MPP4. DRBP76 was also cloned by the yeast two-hybrid screening of a cDNA library using a mutant PKR as bait. Analysis of the cDNA sequence revealed that it is the full-length version of MPP4, has a bipartite nuclear localization signal, two motifs that can mediate interactions with both dsRNA and PKR, five epitopes for potential M phase-specific phosphorylation, two potential sites for phosphorylation by cyclin-dependent kinases, a RG2 motif present in many RNA-binding proteins and predicts a protein of 76 kDa. DsRNA and PKR interactions of DRBP76 were confirmed by analysis of in vitro translated and purified native proteins. Cellular expression of an epitope-tagged DRBP76 demonstrated its nuclear localization, and its co-immunoprecipitation with PKR demonstrated that the two proteins interact in vivo. Finally, purified DRBP76 was shown to be a substrate of PKR in vitro, indicating that this protein's cellular activities may be regulated by PKR-mediated phosphorylation.
Department of Molecular Biology, Lerner Research Institute, The Cleveland Clinic Foundation, Cleveland, Ohio 44195, USA.
R C
RC
Patel
D J
DJ
Vestal
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Xu
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Bandyopadhyay
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W
Guo
S M
SM
Erme
B R
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Williams
G C
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Sen
D000595:Amino Acid Sequence; D001483:Base Sequence; D002453:Cell Cycle; D018076:DNA, Complementary; D004790:Enzyme Induction; D006367:HeLa Cells; D006801:Humans; D007372:Interferons / Q000494:pharmacology; D008969:Molecular Sequence Data; D050779:Nuclear Factor 90 Proteins; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010750:Phosphoproteins; D010766:Phosphorylation; D011485:Protein Binding; D012330:RNA, Double-Stranded / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019892:eIF-2 Kinase / Q000096:biosynthesis / Q000378:metabolism*
D018076:DNA, Complementary; C429643:ILF3 protein, human; D050779:Nuclear Factor 90 Proteins; D009687:Nuclear Proteins; D010750:Phosphoproteins; D012330:RNA, Double-Stranded; D016601:RNA-Binding Proteins; D007372:Interferons; D019892:eIF-2 Kinase