Independent function of two destruction domains in hypoxia-inducible factor-alpha chains activated by prolyl hydroxylation.
Oxygen-dependent proteolytic destruction of hypoxia-inducible factor-alpha (HIF-alpha) subunits plays a central role in regulating transcriptional responses to hypoxia. Recent studies have defined a key function for the von Hippel-Lindau tumour suppressor E3 ubiquitin ligase (VHLE3) in this process, and have defined an interaction with HIF-1 alpha that is regulated by prolyl hydroxylation. Here we show that two independent regions within the HIF-alpha oxygen-dependent degradation domain (ODDD) are targeted for ubiquitylation by VHLE3 in a manner dependent upon prolyl hydroxylation. In a series of in vitro and in vivo assays, we demonstrate the independent and non-redundant operation of each site in regulation of the HIF system. Both sites contain a common core motif, but differ both in overall sequence and in the conditions under which they bind to the VHLE3 ligase complex. The definition of two independent destruction domains implicates a more complex system of pVHL-HIF-alpha interactions, but reinforces the role of prolyl hydroxylation as an oxygen-dependent destruction signal.
Masson
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N
The Henry Wellcome Building of Genomic Medicine, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, UK.
Willam
C
C
Maxwell
P H
PH
Pugh
C W
CW
Ratcliffe
P J
PJ
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002457:Cell Extracts / Q000494:pharmacology; D017124:Conserved Sequence; D003593:Cytoplasm / Q000502:physiology; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D006900:Hydroxylation; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D008025:Ligases; D051379:Mice; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000737:chemistry* / Q000378:metabolism*; D011392:Proline / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000502:physiology*; D017386:Sequence Homology, Amino Acid; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins; D044767:Ubiquitin-Protein Ligases; D014452:Ubiquitins / Q000378:metabolism*; D051794:Von Hippel-Lindau Tumor Suppressor Protein
D002457:Cell Extracts; D004268:DNA-Binding Proteins; C497443:Hif1a protein, mouse; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D009687:Nuclear Proteins; D011506:Proteins; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins; D014452:Ubiquitins; D011392:Proline; D044767:Ubiquitin-Protein Ligases; D051794:Von Hippel-Lindau Tumor Suppressor Protein; D008025:Ligases