title: Human hypoxic signal transduction through a signature motif in hepatocyte nuclear factor 4.
abstract: We identified a human hypoxic signal transduction pathway acting through a signature motif in the carboxyl terminal of hepatocyte nuclear factor 4 (HNF-4), by functional comparison of the transcriptional and protein-protein interaction activities of the wild type and mutants. It was previously shown that HNF-4 functions as a tissue-specific and hypoxia-activated transcription factor for the erythropoietin (Epo) gene. Human HNF-4 (465 amino acid residues) has DNA-binding, ligand-binding, and transactivation domains. The deletion mutant without the carboxyl terminal transactivation domain (amino acids 369-465) has been shown to be a dominant-negative mutant that repressed Epo transcriptional activity in hypoxia. Further characterization of the hypoxia-responsive domain by site-directed mutagenesis indicated that a TKQE motif of the carboxyl terminal (amino acids 460-463) in HNF-4 was essential for hypoxia-inducible Epo gene expression. We also found, by means of immunoprecipitation and a mammalian two-hybrid system, direct interactions between HNF-4 and hypoxia-inducible factor 1 (HIF-1), a heterodimer composed of alpha and beta subunits. HNF-4 was observed to interact with HIF-1alpha and HIF-1beta (arylhydrocarbon receptor nuclear translocator, ARNT) during hypoxia. In addition, the TKQE motif of HNF-4 was essential for protein-protein interactions with HIF-1alpha and ARNT. These results indicate that the human hypoxic signal of HIF-1 is transduced through interactions with the signature TKQE motif of the carboxyl terminal of HNF-4, resulting in Epo gene expression as a response to hypoxia.
authors.affiliation: Department of Molecular Medicine, Research Center for Molecular Medical Science, 35-1 Shimo, Fussa, Tokyo 197-0023, Japan. rcmms@v004.vaio.ne.jp
authors.forename: Terumasa
authors.initials: T
authors.lastname: Tsuchiya
authors.forename: Yoshihiko
authors.initials: Y
authors.lastname: Kominato
authors.forename: Masatsugu
authors.initials: M
authors.lastname: Ueda
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D051784:Aryl Hydrocarbon Receptor Nuclear Translocator; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D015153:Blotting, Western; D015687:Cell Hypoxia / Q000502:physiology; D002846:Chromatography, Affinity; D004268:DNA-Binding Proteins / Q000378:metabolism*; D051557:Hepatocyte Nuclear Factor 4; D022781:Hepatocytes / Q000378:metabolism*; D006639:Histidine / Q000378:metabolism; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D018336:Receptors, Aryl Hydrocarbon; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*; D013329:Structure-Activity Relationship; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D015533:Transcriptional Activation; D014407:Tumor Cells, Cultured
chemical_list: C497396:ARNT protein, human; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D004268:DNA-Binding Proteins; C497442:HIF1A protein, human; D051557:Hepatocyte Nuclear Factor 4; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; C403128:MLX protein, human; D010750:Phosphoproteins; D018336:Receptors, Aryl Hydrocarbon; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D051784:Aryl Hydrocarbon Receptor Nuclear Translocator; D006639:Histidine