HIF-1α and EPAS ubiquitination mediated by the VHL tumour suppressor involves flexibility in the ubiquitination mechanism, similar to other RING E3 ligases

Abstract

Hypoxia-inducible factor 1a (HIF-1a) 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-1a to occur, three conserved lysines are required in both the HIF-1a 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-1a 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-1a sequence with only minor losses in ubiquitination efficiency, thus suggesting HIF-1a and EPAS regulation by hypoxia depends primarily on an interaction with VHL per se, rather than the highly specific positioning of flanking lysine acceptors.

Imaging of HIF-1-Active Tumor Hypoxia Using a Protein Effectively Delivered to and Specifically Stabilized in HIF-1-Active Tumor Cells

“…A positive charge is important for the cellular membrane permeability of PTD. Moreover, lysine residues are required for efficient ubiquitination and after degradation of HIF-1a (29). Therefore, we combined a poly-lysine sequence with a hydrophobic polypeptide to construct a unique PTD, PTD3.…”

Cyclin A Degradation Employs Preferentially Used Lysines and a Cyclin Box Function other than Cdk1 Binding

“…36 However, there are examples of other proteins with selectively ubiquitinated lysines, such as the Hypoxia Inducible Factor (HIF-1a), Interferon a receptor (IFNAR1), Fanconi anemia protein (FANCD2) and the yeast Cdk inhibitor, Sic1. [45][46][47][48] Stable CycA constructs described so far all have N-terminal deletions removing several lysine residues. Moreover, our previous work had shown the construct CycA DKEN1 D40-70 to be not as stable as CycA DKEN1 D40-86, 27 although from this study it is clear both constructs lacked D70.…”

Hypoxia-Inducible Factor Prolyl Hydroxylase Inhibition: Robust New Target or Another Big Bust for Stroke Therapeutics?

“…Hydroxylation of HIF-1a allows it to bind to the VHL (Von Hippel Lindau) tumor suppressor protein that acts as a recognition component of E3 ubiquitin ligase complex. Hydroxylated HIF-1a is polyubiquitinated at three lysines in the central ODD domain and directed to the 26S proteosome for degradation (Paltoglou and Roberts, 2007). The half-life of HIF-1a is < 5 minutes in normoxia (Huang et al, 1996).…”