title: Leu-574 of human HIF-1alpha is a molecular determinant of prolyl hydroxylation.
abstract: Hypoxia-inducible factor (HIF)-1alpha, a master regulator of oxygen homeostasis, regulates genes crucial for cell growth and survival. In normoxia, HIF-1alpha is constantly degraded via the ubiquitin-proteasome pathway. The von Hippel-Lindau (VHL) E3 ubiquitin ligase binds HIF-1alpha through specific recognition of hydroxylated Pro-402 or Pro-564, both of which are modified by the oxygen-dependent HIF prolyl hydroxylases (PHDs/HPHs). Despite the identification of a conserved Leu-X-X-Leu-Ala-Pro motif, the molecular requirement of HIF-1alpha for PHDs/HPHs binding remains elusive. Recently, we demonstrated that Leu-574 of human HIF-1alpha--10 residues downstream of Pro-564--is essential for VHL recognition. We show here that the role of Leu-574 is to recruit PHD2/HPH2 for Pro-564 hydroxylation. An antibody specific for hydroxylated Pro-564 has been used to determine the hydroxylation status; mutation or deletion of Leu-574 results in a significant decrease in the ratio of the hydroxylated HIF-1alpha to the total amount. The nine-residue spacing between Pro-564 and Leu-574 is not obligatory for prolyl hydroxylation. Furthermore, mutation of Leu-574 disrupts the binding of PHD2/HPH2, a key prolyl hydroxylase for oxygen-dependent proteolysis of HIF-1alpha. Hence, our findings indicate that Leu-574 is essential for recruiting PHD2/HPH2, thereby providing a molecular basis for modulating HIF-1alpha activity.
authors.affiliation: Laboratory of Human Carcinogenesis, NCI, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: Yukio
authors.initials: Y
authors.lastname: Kageyama
authors.forename: Minori
authors.initials: M
authors.lastname: Koshiji
authors.forename: Kenneth K W
authors.initials: KK
authors.lastname: To
authors.forename: Ya-Min
authors.initials: YM
authors.lastname: Tian
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Ratcliffe
authors.forename: L Eric
authors.initials: LE
authors.lastname: Huang
mesh_terms: D000595:Amino Acid Sequence; D000906:Antibodies / Q000276:immunology; D002460:Cell Line; D045744:Cell Line, Tumor; D006801:Humans; D006900:Hydroxylation; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D007930:Leucine / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D011393:Procollagen-Proline Dioxygenase / Q000378:metabolism; D011392:Proline / Q000378:metabolism*; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D013816:Thermodynamics; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic / Q000235:genetics
chemical_list: D000906:Antibodies; C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D014157:Transcription Factors; D011392:Proline; D011393:Procollagen-Proline Dioxygenase; D007930:Leucine