Molecular imaging of hypoxia-inducible factor 1 alpha and von Hippel-Lindau interaction in mice.
Tumor hypoxia plays a crucial role in tumorigenesis. Under hypoxia, hypoxia-inducible factor 1 alpha (HIF-1 alpha) regulates activation of genes promoting malignant progression. Under normoxia, HIF-1 alpha is hydroxylated on prolines 402 and 564 and is targeted for ubiquitin-mediated degradation by interacting with the von Hippel-Lindau protein complex (pVHL). We have developed a novel method of studying the interaction between HIF-1 alpha and pVHL using the split firefly luciferase complementation-based bioluminescence system in which HIF-1 alpha and pVHL are fused to amino-terminal and carboxy-terminal fragments of the luciferase, respectively. We demonstrate that hydroxylation-dependent interaction between the HIF-1 alpha and pVHL leads to complementation of the two luciferase fragments, resulting in bioluminescence in vitro and in vivo. Complementation-based bioluminescence is diminished when mutant pVHLs with decreased affinity for binding HIF-1 alpha are used. This method represents a new approach for studying interaction of proteins involved in the regulation of protein degradation.
Choi
Clara Y H
CY
Department of Radiation Oncology, Stanford University School of Medicine, Stanford, CA 94305-5152, USA.
Chan
Denise A
DA
Paulmurugan
Ramasamy
R
Sutphin
Patrick D
PD
Le
Quynh-Thu
QT
Koong
Albert C
AC
Zundel
Wayne
W
Gambhir
Sanjiv S
SS
Giaccia
Amato J
AJ
D000818:Animals; D045744:Cell Line, Tumor; D003952:Diagnostic Imaging / Q000379:methods*; D045325:HCT116 Cells; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics / Q000378:metabolism*; D049409:Luciferases, Firefly / Q000235:genetics / Q000378:metabolism*; D008163:Luminescent Measurements / Q000379:methods*; D051379:Mice; D008819:Mice, Nude; D008958:Models, Molecular; D009154:Mutation; D011392:Proline / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D051794:Von Hippel-Lindau Tumor Suppressor Protein / Q000235:genetics / Q000378:metabolism*; D051598:Whole Body Imaging / Q000379:methods
C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D011993:Recombinant Fusion Proteins; D011392:Proline; D049409:Luciferases, Firefly; D051794:Von Hippel-Lindau Tumor Suppressor Protein; C497446:VHL protein, human