title: Oxygen sensing by the prolyl-4-hydroxylase PHD2 within the nuclear compartment and the influence of compartmentalisation on HIF-1 signalling.
abstract: Hypoxia-inducible factors (HIFs) regulate more than 200 genes involved in cellular adaptation to reduced oxygen availability. HIFs are heterodimeric transcription factors that consist of one of three HIF-α subunits and a HIF-β subunit. Under normoxic conditions the HIF-α subunit is hydroxylated by members of a family of prolyl-4-hydroxylase domain (PHD) proteins, PHD1, PHD2 and PHD3, resulting in recognition by von-Hippel-Lindau protein, ubiquitylation and proteasomal degradation. It has been suggested that PHD2 is the key regulator of HIF-1α stability in vivo. Previous studies on the intracellular distribution of PHD2 have provided evidence for a predominant cytoplasmic localisation but also nuclear activity of PHD2. Here, we investigated functional nuclear transport signals in PHD2 and identified amino acids 196-205 as having a crucial role in nuclear import, whereas amino acids 6-20 are important for nuclear export. Fluorescence resonance energy transfer (FRET) showed that an interaction between PHD2 and HIF-1α occurs in both the nuclear and cytoplasmic compartments. However, a PHD2 mutant that is restricted to the cytoplasm does not interact with HIF-1α and shows less prolyl hydroxylase activity for its target HIF-1α than wild-type PHD2 located in the nucleus. Here, we present a new model by which PHD2-mediated hydroxylation of HIF-1α predominantly occurs in the cell nucleus and is dependent on very dynamic subcellular trafficking of PHD2.
authors.affiliation: Institute of Physiology, Center for Structural and Cell Biology in Medicine, University of Lübeck,  Lübeck, Germany.
authors.forename: Friederike Katharina
authors.initials: FK
authors.lastname: Pientka
authors.forename: Jun
authors.initials: J
authors.lastname: Hu
authors.forename: Susann Gaby
authors.initials: SG
authors.lastname: Schindler
authors.forename: Britta
authors.initials: B
authors.lastname: Brix
authors.forename: Anika
authors.initials: A
authors.lastname: Thiel
authors.forename: Olaf
authors.initials: O
authors.lastname: Jöhren
authors.forename: Joachim
authors.initials: J
authors.lastname: Fandrey
authors.forename: Utta
authors.initials: U
authors.lastname: Berchner-Pfannschmidt
authors.forename: Reinhard
authors.initials: R
authors.lastname: Depping
mesh_terms: D021581:Active Transport, Cell Nucleus; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000201:enzymology*; D015870:Gene Expression; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D006900:Hydroxylation; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics / Q000378:metabolism*; D064799:Hypoxia-Inducible Factor-Proline Dioxygenases; D049409:Luciferases, Firefly / Q000096:biosynthesis / Q000235:genetics; D008856:Microscopy, Fluorescence; D019913:Nuclear Localization Signals; D010100:Oxygen / Q000378:metabolism*; D011393:Procollagen-Proline Dioxygenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017384:Sequence Deletion; D015398:Signal Transduction; D014158:Transcription, Genetic
chemical_list: C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D019913:Nuclear Localization Signals; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D049409:Luciferases, Firefly; C490190:EGLN1 protein, human; D011393:Procollagen-Proline Dioxygenase; D064799:Hypoxia-Inducible Factor-Proline Dioxygenases; D010100:Oxygen