Abstract
In the present study, the role of the C-terminal α-helical domain (amino acid (aa) 195–208) of the von Hippel–Lindau (VHL) tumour suppressor was investigated. Deletions of the VHL C-terminus up to the naturally occurring 195-Gln-Term resulted in hypoxia-inducible factor (HIF)-1α downregulation in renal cell carcinoma (RCC)4 cells during normoxia, suggesting that this domain is not an absolute requirement for the ubiquitination of HIF-1α. However, detailed investigation of the ubiquitin protein isopeptide ligase ubiquitin ligase properties of VHL revealed C-terminal deletions to cause a significant impairment of HIF-1α ubiquitination, which is shown to be due to a loss in high-affinity binding to the target substrate. When VHL regulation of both HIF-1α N- and C-terminal oxygen-dependent degradation domains (HIF-ODDD) was investigated, it was found that only ubiquitination of the C-terminal HIF-ODDD was affected by the deletion of the VHL C-terminus. When RCC4 cells expressing C-terminal truncations of VHL were exposed to graded hypoxia, differences in the induction of HIF-1α were observed in comparison with full-length VHL, with a shift in the maximal induction of HIF-1α to a higher oxygen tension. These changes were accompanied by increased glucose transporter 1 expression, p300 CH1 domain binding and HIF-mediated reporter activity. We have thus defined a role for the C-terminal α-helical domain of VHL in the regulation of HIF-1α.
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Abbreviations
- VHL:
-
von Hippel–Lindau
- HIF:
-
hypoxia-inducible factor
- HLF:
-
HIF-like factors
- Cul-2:
-
Cullin-2
- VEGF:
-
vascular endothelial growth factor
- GLUT1:
-
glucose transporter 1
- E3:
-
ubiquitin protein isopeptide ligase
- aa:
-
amino acids
- TIMP:
-
tissue inhibitors of metalloproteinases
- MCS:
-
multiple cloning site
- DMSO:
-
dimethyl sulphoxide
- RCC:
-
renal cell carcinoma
- HRE:
-
HIF response element
- EF-IRES:
-
elongation factor promoter-internal ribosome entry site
- NES:
-
nuclear export signal
- NLS:
-
nuclear localization signal
- DRB:
-
5,6-dichlorobenzimidazole ribososide
- DMEM:
-
Dulbecco's modified Eagle's medium
- FCS:
-
foetal calf serum
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Acknowledgements
This work was supported in part by the National Health and Medical Research Council of Australia, Project Grant: 10365, awarded to BJ Roberts.
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Lewis, M., Roberts, B. Role of the C-terminal α-helical domain of the von Hippel–Lindau protein in its E3 ubiquitin ligase activity. Oncogene 23, 2315–2323 (2004). https://doi.org/10.1038/sj.onc.1207384
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DOI: https://doi.org/10.1038/sj.onc.1207384
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