Question: 

The human prolyl-4-hydroxylase domain (PHD) proteins 1-3 are known as cellular oxygen sensors involved in degradation of hypoxia-inducible factor (HIF) a-subunits. In normoxia they hydroxylate specific proline residues of HIF-a followed by polyubiquitination and proteasomal degradation. All three PHDs have distinct cellular functions and target proteins, with PHD2 being the key regulator of HIF-1a stability in vivo. Previously, the nuclear localization of HIF-1a and the oxygen sensors PHD1, PHD2 and PHD3 has been localized. Here we report on the role of the intracellular localization of the oxygen sensor PHD2 regarding its role as oxygen sensor in the HIF-1 pathway.

Methodology: 

NLS (nuclear localization signal) and NES (nuclear export signal) deletion mutants of PHD2 were generated and primary structure elements essential for PHD2 localization were mapped. U2OS cells were transiently transfected with PHD2 wild-type or PHD2 NLS/NES mutants and the impact of PHD2 intracellular localization on HIF signalling was analyzed by reporter gene assays, quantitative PCR, immunoblot analysis and fluorescence resonance energy transfer (FRET).

Results: 

PHD2 amino acids 191 and 192 play a crucial role in nuclear import, while nuclear export requires amino acids 6–20. We further elucidate the role of PHD2 intracellular localization on HIF-1a transcriptional activity, HIF-1a target gene expression and HIF-1a protein stability under hypoxic conditions. FRET revealed interaction between PHD2 and HIF-1a most likely localizing oxygen sensing by PHD2 to both compartments.

Conclusion: 

We present a model by which PHD2 mediated hydroxylation of HIF-1a occurs predominantly in cell nuclei however is dependent upon dynamic subcellular trafficking of PHD2.