Leu-574 of HIF-1alpha is essential for the von Hippel-Lindau (VHL)-mediated degradation pathway.

Oxygen homeostasis is crucial for a myriad of developmental, physiological, and pathophysiological processes. Hypoxia-inducible factor 1alpha (HIF-1alpha) plays a pivotal role in response to hypoxia by transcriptionally activating target genes involving oxygen uptake, transport, delivery, and consumption. HIF-1alpha activity is regulated primarily through the ubiquitin-proteasome degradation pathway, which targets the oxygen-dependent degradation domain (ODD) of HIF-1alpha. In particular, the von Hippel-Lindau (VHL) protein complex, an E3 ubiquitin ligase, binds to the ODD upon hydroxylation of HIF-1alpha Pro-564. Here, we show that in vivo VHL interacts with the N-terminal as well as the C-terminal ODD independently, supporting the notion of functional redundancy within the ODD. Moreover, we demonstrate that Leu-574 of HIF-1alpha is essential for VHL binding to the C-terminal ODD. Despite the presence of Pro-564, deletion or mutation of Leu-574 resulted in a loss of VHL binding and a gain of protein stability. Furthermore, the identification of Leu-574 redefines the N-terminal activation domain of HIF-1alpha to be constitutively active. Taken together, this study provides new insight into the mechanisms underlying VHL-mediated HIF-1alpha degradation and transcriptional activation, and a molecular basis for drug targeting.