Objective: 

HIF-1a (hypoxia inducible factor-1a) is a major transcription factor composed of two subunits, HIF-1a and HIF-1b. Specific proline residues (Pro564 and Pro402) of HIF-1a are oxygen dependent modified by three distinct HIF-a prolyl hydroxylases (PHD1, PHD2, and PHD3) leading to polyubiquitination by von Hippel-Lindau protein and proteasomal degradation. It has been discussed that all three PHDs have distinct cellular functions, PHD2 being the key regulator of HIF-1a stability in vivo. Studies on intracellular distribution patterns revealed that PHD1 is exclusively nuclear, PHD2 is mainly detected in the cytoplasm and PHD3 is located in both compartments. Here we report on the role of the intracellular distribution of PHD2 regarding its role as oxygen sensor in the HIF-1 pathway.

Methods: 

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. HeLa cells were transiently transfected with PHD2 wild-type or PHD2 NLS/NES mutants and the impact of PHD2 intracellular localization was analyzed by reporter gene assay, qPCR and immunoblot analysis.

Results: 

We could show that 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-a transcriptional activity, HIF-a target gene expression and HIF-1a protein stability under hypoxic conditions.

Conclusion: 

Intracellular localization of PHD2 plays a pivotal role in the regulation of HIF-a providing new insights into the molecular mechanisms underlying the oxygen sensor cascade.