Enzymatic Activity of Prolyl-4-Hydroxylase PHD2 is predominantly effective in the nucleus

HIFs (hypoxia-inducible factors) are major transcription factors involved in cellular adaptation and response to reduced oxygen availability. HIFs are composed of one of three O2-labile α-subunits (HIF-1α, -2α or -3α) and a constitutive β-subunit (HIF-1β or ARNT). Specific proline residues (Pro564 and Pro402) of HIF-a are oxygen dependent hydroxylated by three distinct non-heme, Fe2+ and α-ketoglutarate-dependent HIF-α prolyl hydroxylases (PHD1, PHD2, and PHD3) which strictly require oxygen as a co-substrate. After hydroxylation, HIF-α is polyubiquitinated by the von Hippel-Lindau protein followed by proteasomal degradation. It has been discussed that all three PHDs have distinct cellular functions, with PHD2 being the key regulator of HIF-1α stability in vivo. Studies on the intracellular localisation of the PHDs revealed that PHD1 is exclusively located in the nucleus, PHD2 is mainly cytoplasmic and PHD3 is detected in both compartments. Here, we report on the intracellular distribution of PHD2 regarding its role as oxygen sensor in the HIF-1 pathway. By immunofluorescence analysis, primary structure elements essential for PHD2 localization were mapped indicating that PHD2 amino acids 196 and 205 play a crucial role in nuclear import, while nuclear export requires amino acids 6-20. We further show that PHD2 intracellular localisation is involved in the regulation of HIF-1α transcriptional activity and HIF-1α target gene expression under hypoxic conditions. Finally, we present a model by which PHD2-mediated hydroxylation of HIF-1α depends upon dynamic subcellular trafficking of PHD2.