Abstract
Although Puma (p53 upregulated modulator of apoptosis) was known as a principal mediator of cell death in response to diverse apoptotic signals, the molecular mechanism underlying its proapoptotic regulation remains largely uncharacterized. Here we reported that myeloid cell leukemia-1 (Mcl-1), an antiapoptotic member of the Bcl-2 family with a rapid turnover rate, interacts with Puma. The Puma/Mcl-1 interaction was verified by both yeast two-hybrid assay and co-immunoprecipation studies. Their binding sites were mapped to BH3 (Bcl-2 homology) domain of Puma and BH1 domain of Mcl-1, respectively. Mcl-1 and Puma was shown to colocalize at the mitochondria by immunostaining. The level of Mcl-1 was increased when coexpressed with Puma, indicating Puma is able to stabilize Mcl-1. Puma binding to Mcl-1 via its BH3 domain is the prerequisite for this effect, which is further supported by the finding that Puma mutant lacking BH3 domain no longer promotes Mcl-1 protein stability. This Puma-enhanced Mcl-1 stabilization was validated in vivo under nonoverexpression conditions. We also showed that BH1 domain is essential for Mcl-1 to inhibit Puma-induced apoptosis, since Mcl-1 mutant lacking BH1 domain completely abrogates its protective function. In addition, we concluded that binding of Puma to BH1 domain of Mcl-1 is necessary, but not sufficient to prevent rapid degradation of Mcl-1. In addition to PEST (proline, glutamic acid, serine, and threonine) and BH1 domain, some additional degradation signal is expected to reside in the C-terminal region of Mcl-1. In conclusion, our results provide the first evidence that the interaction between Mcl-1 and Puma may represent a novel mechanism by which Mcl-1 prevents apoptosis by increasing its stability through binding to Puma.
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Acknowledgements
We are grateful to Professor Bert Vogelstein for Puma cDNA, Puma wild type and Puma knockout HCT116 cell lines. We also thank Dr Lin Zhang for his constructive suggestion. This research was supported by a 973 grant (2002CB713702) from Ministry of Science and Technology of China; grants from the National Natural Science Foundation of China (90208027, 30370308 and 30121001) and a grant SBS/SUG/22/04 (to Wu Mian) from School of Biological Sciences, Nanyang Technological University, Singapore.
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Mei, Y., Du, W., Yang, Y. et al. Puma*Mcl-1 interaction is not sufficient to prevent rapid degradation of Mcl-1. Oncogene 24, 7224–7237 (2005). https://doi.org/10.1038/sj.onc.1208873
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DOI: https://doi.org/10.1038/sj.onc.1208873
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