¹School of Pharmaceutical, Molecular and Biomedical Sciences, University of South Australia, Adelaide, Australia

Correspondence: Dr BJ Roberts, School of Pharmaceutical, Molecular and Biomedical Sciences, University of South Australia, 4 Froome Road, Adelaide 5000, Australia. E-mail: Ben.Roberts@unisa.edu.au

Received 29 November 2005; Revised 4 May 2006; Accepted 4 May 2006; Published online 21 August 2006.

Abstract

Hypoxia-inducible factor 1 (HIF-1) degradation under normoxia is critical to modulating vascular growth. This degradation is mediated during normoxia by the von Hippel–Lindau tumour suppressor protein (VHL)-E3 ubiquitin ligase in partnership with the E2 enzyme UbcH5. In current models of the functionally similar Skp1, cullin, F-box (SCF)-E3 ligase, the E3 binds the target protein and the E2 catalyses ubiquitin transfer to lysines in an appropriately positioned domain. In the present study, we report that for efficient ubiquitination of HIF-1 to occur, three conserved lysines are required in both the HIF-1 and endothelial Per-ARNT-Sim domain protein (EPAS) sequences. The site of ubiquitin attachment via UbcH5 was mapped, and is shown to involve three HIF-1 lysines, K532, K538 and K547, and the same aligned lysines in EPAS. Only one of these lysines need to be intact for full ubiquitination to occur, analogous to the mechanism of Sic1 ubiquitination by the SCF/Cdc34 complex and further strengthening the functional link between the VHL and SCF-E3 ubiquitin ligases. We also report that lysines can be moved around the HIF-1 sequence with only minor losses in ubiquitination efficiency, thus suggesting HIF-1 and EPAS regulation by hypoxia depends primarily on an interaction with VHL per se, rather than the highly specific positioning of flanking lysine acceptors.