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Role of the C-terminal α-helical domain of the von Hippel–Lindau protein in its E3 ubiquitin ligase activity

Oncogene volume 23, pages 2315–2323 (2004)Cite this article

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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  1. School of Pharmaceutical, Molecular and Biomedical Sciences, Reid Building, University of South Australia, Frome Rd., Adelaide, 5000, Australia

    Martin D Lewis & Ben J Roberts

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Correspondence to Ben J 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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