Characterization of the interaction between the interferon-induced protein P56 and the Int6 protein encoded by a locus of insertion of the mouse mammary tumor virus.
For determining cellular functions of the interferon-inducible human cytoplasmic protein P56, we undertook a Saccharomyces cerevisiae two-hybrid screen that identified Int6 as a P56-interacting protein. That the interaction also occurs in human cells was confirmed by coimmunoprecipitation and the observed cytoplasmic displacement of nuclear Int6 upon coexpression of P56. Because Int6 has been claimed to be both a cytoplasmic and a nuclear protein, we investigated the structural basis of this discrepancy. By mutational analyses, we showed that the Int6 protein contains a bipartite nuclear localization signal and a nuclear export signal at the far end of the amino terminus. The 20 amino-terminal residues of Int6, when they were attached to a different nuclear protein, were sufficient to translocate that protein to the cytoplasm. Within this region, replacement of any of the three leucine residues with alanine destroyed the function of the export signal. The specific domain of P56 that is required for its interaction with Int6 was mapped using the yeast two-hybrid assay and a mammalian coimmunoprecipitation assay. Both assays demonstrated that the C-terminal region of P56 containing three specific tetratricopeptide motifs is required for this interaction. In contrast, removal of an internal domain of P56 enhanced the interaction, as quantified by the two-hybrid assay.
Guo
J
J
Department of Molecular Biology, The Lerner Research Institute, The Cleveland Clinic Foundation, Cleveland, Ohio 44195, USA.
Sen
G C
GC
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D001665:Binding Sites; D002874:Chromosome Mapping; D039621:Eukaryotic Initiation Factor-3; D005814:Genes, Viral; D006801:Humans; D016899:Interferon-beta / Q000494:pharmacology; D008324:Mammary Tumor Virus, Mouse / Q000235:genetics; D016254:Mutagenesis, Insertional; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins; D011817:Rabbits; D013347:Subcellular Fractions; D014407:Tumor Cells, Cultured
D048868:Adaptor Proteins, Signal Transducing; D039621:Eukaryotic Initiation Factor-3; C484290:IFIT1 protein, human; D009687:Nuclear Proteins; D011506:Proteins; D011518:Proto-Oncogene Proteins; D016601:RNA-Binding Proteins; D016899:Interferon-beta