Multiple splice variants of the human HIF-3 alpha locus are targets of the von Hippel-Lindau E3 ubiquitin ligase complex.
Functional inactivation of the von Hippel-Lindau (VHL) tumor suppressor protein is the cause of familial VHL disease and sporadic kidney cancer. The VHL gene product (pVHL) is a component of an E3 ubiquitin ligase complex that targets the hypoxia-inducible factor (HIF) 1 and 2 alpha subunits for polyubiquitylation. This process is dependent on the hydroxylation of conserved proline residues on the alpha subunits of HIF-1/2 in the presence of oxygen. In our effort to identify orphan HIF-like proteins in the data base that are potential targets of the pVHL complex, we report multiple splice variants of the human HIF-3 alpha locus as follows: hHIF-3 alpha 1, hHIF-3 alpha 2 (also referred to as hIPAS; human inhibitory PAS domain protein), hHIF-3 alpha 3, hHIF-3 alpha 4, hHIF-3 alpha 5, and hHIF-3 alpha 6. We demonstrate that the common oxygen-dependent degradation domain of hHIF-3 alpha 1-3 splice variants is targeted for ubiquitylation by the pVHL complex in vitro and in vivo. This activity is enhanced in the presence of prolyl hydroxylase and is dependent on a proline residue at position 490. Furthermore, the ubiquitin conjugation occurs on lysine residues at position 465 and 568 within the oxygen-dependent degradation domain. These results demonstrate additional targets of the pVHL complex and suggest a growing complexity in the regulation of hypoxia-inducible genes by the HIF family of transcription factors.
Maynard
Mindy A
MA
Department of Laboratory Medicine and Pathobiology, Faculty of Medicine, University of Toronto, Toronto, Ontario M5S 1A8, Canada.
Qi
Heng
H
Chung
Jacky
J
Lee
Eric H L
EH
Kondo
Yukihiro
Y
Hara
Shuntaro
S
Conaway
Ronald C
RC
Conaway
Joan W
JW
Ohh
Michael
M
D017398:Alternative Splicing; D051017:Apoptosis Regulatory Proteins; D001483:Base Sequence; D051792:Basic Helix-Loop-Helix Proteins; D017931:DNA Primers; D006801:Humans; D008025:Ligases / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism; D011485:Protein Binding; D012097:Repressor Proteins; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D014407:Tumor Cells, Cultured; D025521:Tumor Suppressor Proteins; D044767:Ubiquitin-Protein Ligases; D051794:Von Hippel-Lindau Tumor Suppressor Protein
D051017:Apoptosis Regulatory Proteins; D051792:Basic Helix-Loop-Helix Proteins; D017931:DNA Primers; D008025:Ligases; D010100:Oxygen; D012097:Repressor Proteins; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins; D044767:Ubiquitin-Protein Ligases; D051794:Von Hippel-Lindau Tumor Suppressor Protein; C116973:HIF3A protein, human; C497446:VHL protein, human