title: Factor inhibiting HIF-1 (FIH-1) modulates protein interactions of apoptosis-stimulating p53 binding protein 2 (ASPP2).
abstract: The asparaginyl hydroxylase factor inhibiting HIF-1 (FIH-1) is an important suppressor of hypoxia-inducible factor (HIF) activity. In addition to HIF-α, FIH-1 was previously shown to hydroxylate other substrates within a highly conserved protein interaction domain, termed the ankyrin repeat domain (ARD). However, to date, the biological role of FIH-1-dependent ARD hydroxylation could not be clarified for any ARD-containing substrate. The apoptosis-stimulating p53-binding protein (ASPP) family members were initially identified as highly conserved regulators of the tumour suppressor p53. In addition, ASPP2 was shown to be important for the regulation of cell polarity through interaction with partitioning defective 3 homolog (Par-3). Using mass spectrometry we identified ASPP2 as a new substrate of FIH-1 but inhibitory ASPP (iASPP) was not hydroxylated. We demonstrated that ASPP2 asparagine 986 (N986) is a single hydroxylation site located within the ARD. ASPP2 protein levels and stability were not affected by depletion or inhibition of FIH-1. However, FIH-1 depletion did lead to impaired binding of Par-3 to ASPP2 while the interaction between ASPP2 and p53, apoptosis and proliferation of the cancer cells were not affected. Depletion of FIH-1 and incubation with the hydroxylase inhibitor dimethyloxalylglycine (DMOG) resulted in relocation of ASPP2 from cell-cell contacts to the cytosol. Our data thus demonstrate that protein interactions of ARD-containing substrates can be modified by FIH-1-dependent hydroxylation. The large cellular pool of ARD-containing proteins suggests that FIH-1 can affect a broad range of cellular functions and signalling pathways under certain conditions, for example, in response to severe hypoxia.
authors.affiliation: Institute of Physiology, Faculty of Medicine, University Duisburg-Essen, 45147 Essen, Germany.
authors.forename: Kirsten
authors.initials: K
authors.lastname: Janke
authors.forename: Ulf
authors.initials: U
authors.lastname: Brockmeier
authors.forename: Katja
authors.initials: K
authors.lastname: Kuhlmann
authors.forename: Martin
authors.initials: M
authors.lastname: Eisenacher
authors.forename: Jan
authors.initials: J
authors.lastname: Nolde
authors.forename: Helmut E
authors.initials: HE
authors.lastname: Meyer
authors.forename: Heimo
authors.initials: H
authors.lastname: Mairbäurl
authors.forename: Eric
authors.initials: E
authors.lastname: Metzen
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D017209:Apoptosis / Q000502:physiology*; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism*; D002448:Cell Adhesion / Q000502:physiology; D018797:Cell Cycle Proteins / Q000378:metabolism; D048708:Cell Growth Processes / Q000502:physiology; D015687:Cell Hypoxia / Q000502:physiology; D002460:Cell Line; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000502:physiology; D016764:Cell Polarity / Q000502:physiology; D003600:Cytosol / Q000378:metabolism / Q000502:physiology; D045325:HCT116 Cells; D057809:HEK293 Cells; D006801:Humans; D006900:Hydroxylation; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D008969:Molecular Sequence Data; D011485:Protein Binding; D060066:Protein Interaction Maps; D017434:Protein Structure, Tertiary / Q000502:physiology; D012097:Repressor Proteins / Q000378:metabolism*; D016415:Sequence Alignment; D015398:Signal Transduction; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D051017:Apoptosis Regulatory Proteins; D018797:Cell Cycle Proteins; C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; C509689:PARD3 protein, human; C119612:PPP1R13L protein, human; D012097:Repressor Proteins; C495901:TP53 protein, human; C090468:TP53BP2 protein, human; D016159:Tumor Suppressor Protein p53; D006899:Mixed Function Oxygenases; C439790:HIF1AN protein, human
keywords: Cell polarity; Hydroxylation; Hypoxia; Protein interaction