title: The function of hypoxia-inducible factor (HIF) is independent of the endoplasmic reticulum protein OS-9.
abstract: The protein "amplified in osteosarcoma-9" (OS-9) has been shown previously to interact with the prolyl hydroxylases PHD2 and PHD3. These enzymes initiate oxygen-dependent degradation of the α-subunit of hypoxia-inducible factor (HIF), a transcription factor that adapts cells to insufficient oxygen supply (hypoxia). A new model has been proposed where OS-9 triggers PHD dependent degradation of HIF-α. It was the aim of our study to define the molecular mode of action of OS-9 in the regulation of PHD and HIF activity. Although initial co-immunoprecipitation experiments confirmed physical interaction between OS-9 and PHD2, neither overexpression nor lentiviral inhibition of OS-9 expression affected HIF regulation. Subcellular localization experiments revealed a distinct reticular staining pattern for OS-9 while PHD2 was mainly localized in the cytoplasm. Further cell fractionation experiments and glycosylation tests indicated that OS-9 is a luminal ER protein. In vivo protein interaction analysis by fluorescence resonance energy transfer (FRET) showed no significant physical interaction of overexpressed PHD2-CFP and OS-9-YFP. We conclude that OS-9 plays no direct functional role in HIF degradation since physical interaction of OS-9 with oxygen sensing HIF prolyl hydroxylases cannot occur in vivo due to their different subcellular localization.
authors.affiliation: Institute of Physiology, University of Duisburg-Essen, Essen, Germany.
authors.forename: Ulf
authors.initials: U
authors.lastname: Brockmeier
authors.forename: Corinna
authors.initials: C
authors.lastname: Platzek
authors.forename: Kirsten
authors.initials: K
authors.lastname: Schneider
authors.forename: Pauline
authors.initials: P
authors.lastname: Patak
authors.forename: André
authors.initials: A
authors.lastname: Bernardini
authors.forename: Joachim
authors.initials: J
authors.lastname: Fandrey
authors.forename: Eric
authors.initials: E
authors.lastname: Metzen
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry; D045744:Cell Line, Tumor; D003593:Cytoplasm / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005347:Fibroblasts / Q000378:metabolism; D005786:Gene Expression Regulation; D015972:Gene Expression Regulation, Neoplastic; D006031:Glycosylation; D006367:HeLa Cells; D006801:Humans; D000860:Hypoxia; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000378:metabolism*; D037102:Lectins / Q000378:metabolism*; D008954:Models, Biological; D009363:Neoplasm Proteins / Q000378:metabolism*; D010100:Oxygen / Q000737:chemistry; D017434:Protein Structure, Tertiary
chemical_list: D000911:Antibodies, Monoclonal; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D037102:Lectins; D009363:Neoplasm Proteins; C098898:OS9 protein, human; D010100:Oxygen