A7
The role of hypoxia-induced p53 in apoptosis
Rachel Poole, Zuzana Bencokova, Isabel Pires, Ester Hammond
CRUK-MRC Gray Institute for Radiation Oncology and Biology, University of Oxford, UK
Background
In low oxygen environments p53 is stabilised but fails to activate transcription of known target genes and instead causes apoptosis via a transrepression mechanism. Here, we investigate hypoxia-specific post-translational modifications of p53 and compare them to those seen following DNA damage. In addition, we aim to identify hypoxia-specific p53 binding partners and to characterise the role of post-translational modifications in p53-dependent apoptosis.
Method
Immunoprecipitation of p53 was carried out followed by western blotting and mass spectrometry to identify post-translational modifications and binding partners. Cells were treated with SB203580, a p38 inhibitor, and extracts were probed for phosphorylated serine 46. To evaluate the role of serine 46 in hypoxia-induced apoptosis we have used site directed mutagenesis to generate a hypoxia-inducible p53 construct which is mutated at this residue.
Results
The phosphorylation status of hypoxia-induced p53 has been determined for serine residues 6, 9, 15, 20, 37, 46 and 392. p53 was found to be robustly phosphorylated at serine 46 in response to hypoxia. Phosphorylation at serine 46 has previously been implicated in p53-dependent apoptosis, but it is not known whether hypoxia-induced apoptosis occurs in the same way. We have investigated this modification further to determine the kinase responsible and the role it plays in hypoxia-induced apoptosis.
Conclusion
It is clear that hypoxia-induced p53 behaves differently to that induced by DNA damage. In this work we have set up and optimised a variety of systems that will enable the further study of p53 in hypoxia and the elucidation of the mechanism of apoptosis.
