HIF-1a Stabilization by Bcl-2 Figure 3. bcl-2 interacts with HIF-1a. (A) Analysis of HIF-1a/bcl-2 protein interaction in M14 control (puro) and stably bcl-2 overexpressing (Bcl2/ 5, Bcl2/37) clones exposed to hypoxia for 24 h. Whole cell lysates were immunoprecipitated (IP) with anti-bcl-2 or control (IgG) antibodies and then the Western blot analysis was performed using anti-HIF-1a and anti-bcl-2 antibodies. Analysis of HIF-1a/bcl-2 protein interaction in (B) M14 control (puro) and stably bcl-2 overexpressing (Bcl2/5, Bcl2/37) clones or (C,D) in PLF2 and JR8 control cells (PLF2/puro, JR8/puro) and stably bcl-2 overexpressing (PLF2/Bcl-2, JR8/Bcl-2) cells, exposed to MG132 (10 mM, 6 h). Whole cell lysates were immunoprecipitated with anti-HIF-1a or control (IgG) antibodies and then the Western blot analysis was performed using anti-HIF-1a and anti-bcl-2 antibodies. (A–D) b-actin protein amounts are used to check equal loading and transfer of proteins. Western blot analyses representative of two independent experiments with similar results are shown. doi:10.1371/journal.pone.0011772.g003 immunoprecipitated HIF-1a, bcl-2 protein was well detectable within the immunoprecipitates in bcl-2 transfectants but only weakly in control cells, suggesting that HIF-1a interaction with bcl-2 protein was stronger in bcl-2 overexpressing clones. Similar results were obtained when immunoprecipitations were performed using different antibodies recognizing different epitopes on the bcl- 2 and HIF-1a proteins (data not shown). Immunoprecipitation experiments of HIF-1a protein were also perfomed in two other melanoma cell lines, JR8 and PLF2, and their bcl-2 derivative stably clones treated with MG132 obtaining similar results (Figure 3C,D) and thus generalizing the ability of bcl-2 protein to interact with HIF-1a protein. bcl-2 protein interacts with HIF-1a protein in the nucleoplasm bcl-2 is primarily localized in the outer mitochondrial membrane with minor expression in the nucleus and in the endoplasmatic reticulum [20]. Recent reports indicate that bcl-2 also resides in the nuclear membrane and may even function within the nucleus [21–24]. On the other hand, HIF-1a protein induced by hypoxic conditions mainly localizes and elicits its transcriptional activity in the nucleus [1]. Given that bcl-2 is able to interact with HIF-1a, we examined the effect of hypoxia on the intracellular localization of HIF-1a and bcl-2 by using biochemical fractionation and confocal microscopy. As reported in Figure 4A, hypoxic conditions induced HIF-1a protein translocation in the nuclear fraction of both control cells and bcl-2 transfectants, even though HIF-1a protein expression was higher in bcl-2 transfec- tants. By contrast, overexpressed bcl-2 protein was expressed in nuclear and mainly in cytoplasmic compartments, and hypoxia did not modulate both bcl-2 expression or its cellular localization. Confocal microscopy (Figure 4B) confirmed that bcl-2 protein is mainly cytoplasmic but it is also localized in the nuclear envelope, and hypoxia does not modify bcl-2 localization. As expected, HIF- 1a is mainly localized into the nucleus, it was found to be organized in spots which co-localized with chromatin, correlated to an enhanced transcriptional activity of HIF-1a under hypoxia. Given that hypoxia-induced HIF-1a is mainly localized in the nuclear compartment, we formulated the hypothesis that bcl-2 may regulate HIF-1a protein stability through the formation of a protein complex localized in the nucleus. Immunoprecipitation experiments on isolated nuclear protein extracts showed that bcl-2 was associated with HIF-1a, while undetectable levels of HIF-1a/ bcl-2 complexes were observed in the cytosolic fraction, indicating that under hypoxia HIF-1a/bcl-2 interaction may only occur in the nucleus (Figure 4C). Thus, the finding of an interaction between HIF-1a/bcl-2 proteins in the nucleus suggests that bcl-2 may act on the stabilization of HIF-1a in this cellular compartment. bcl-2 regulates HIF-1a protein stability in a prolyl hydroxylation-independent manner Under normoxia, the proline to alanine mutation of residues 402 and 564 of human HIF-1a makes the protein resistant to PHD-dependent hydroxylation and subsequent VHL-dependent ubiquitination and degradation [25]. Besides, PHD2 can be active in the degradation of HIF-1a even under hypoxic conditions [26,27]. In order to study the impact of bcl-2 on PHD-mediated PLoS ONE | www.plosone.org 5 July 2010 | Volume 5 | Issue 7 | e11772