Oxygen sensing by the prolyl-4-hydroxylase PHD2 within the nuclear compartment and the influence of compartmentalisation on HIF-1 signalling

Hypoxia-inducible factors (HIFs) regulate more than 200 genes involved in cellular adaptation to reduced oxygen availability. HIFs areheterodimeric transcription factors that consist of one of three HIF-a subunits and a HIF-b subunit. Under normoxic conditions the HIFasubunit is hydroxylated by members of a family of prolyl-4-hydroxylase domain (PHD) proteins, PHD1, PHD2 and PHD3, resulting inrecognition by von-Hippel-Lindau protein, ubiquitylation and proteasomal degradation. It has been suggested that PHD2 is the keyregulator of HIF-1a stability in vivo. Previous studies on the intracellular distribution of PHD2 have provided evidence for apredominant cytoplasmic localisation but also nuclear activity of PHD2. Here, we investigated functional nuclear transport signals inPHD2 and identified amino acids 196-205 as having a crucial role in nuclear import, whereas amino acids 6-20 are important fornuclear export. Fluorescence resonance energy transfer (FRET) showed that an interaction between PHD2 and HIF-1a occurs in both thenuclear and cytoplasmic compartments. However, a PHD2 mutant that is restricted to the cytoplasm does not interact with HIF-1a andshows less prolyl hydroxylase activity for its target HIF-1a than wild-type PHD2 located in the nucleus. Here, we present a new modelby which PHD2-mediated hydroxylation of HIF-1a predominantly occurs in the cell nucleus and is dependent on very dynamicsubcellular trafficking of PHD2.