Abstract
Puma is a potent BH3-only protein that antagonises anti-apoptotic Bcl-2 proteins, promotes Bax/Bak activation and has an essential role in multiple apoptotic models. Puma expression is normally kept very low, but can be induced by several transcription factors including p53, p73, E2F1 and FOXO3a, whereby it can induce an apoptotic response. As Puma can to bind and inactivate all anti-apoptotic members of the Bcl-2 family, its activity must be tightly controlled. We report here, for the first time, evidence that Puma is subject to post-translational control through phosphorylation. We show that Puma is phosphorylated at multiple sites, with the major site of phosphorylation being serine 10. Replacing serine 10 with alanine causes reduced Puma turnover and enhanced cell death. Interestingly, Puma turnover occurs through the proteasome, and substitution of serine 10 causes elevated Puma levels independently of macroautophagy, Bcl-2 family member binding, caspase activity and apoptotic death. We conclude, therefore, that phosphorylation of Puma at serine 10 promotes Puma turnover, represses Puma's cell death potential and promotes cell survival. Owing to the highly pro-apoptotic nature of Puma, these studies highlight an important additional regulatory step in the determination of cellular life or death.
Highlights
The anti-apoptotic Bcl-2 family proteins, including Bcl-2, Bcl-xL and Mcl-1, typically contain 3–4 Bcl homology (BH) domains, which form a groove on the protein surface through which pro-apoptotic Bcl-2 family proteins are bound
More radiolabel appeared to be incorporated into this mutant, indicating that the BH3 domain is dispensable for phosphorylation of Puma and that regions within the BH3 domain may potentially repress phosphorylation elsewhere in the protein
This specific possibility is yet to be determined, these results clearly show that exogenous Puma is phosphorylated in untreated HeLa cells in a BH3-domain-independent manner
Summary
The anti-apoptotic Bcl-2 family proteins, including Bcl-2, Bcl-xL and Mcl-1, typically contain 3–4 BH domains, which form a groove on the protein surface through which pro-apoptotic Bcl-2 family proteins are bound. Bcl-2 family, comprising at least 12 members: Bad, Bim, Bik/Nbk/Blk, Bid, Bmf, Puma, Noxa, Spike, BNIP3, Nix, Hrk/DP5 and Beclin-1.3 Given their integral role in determining cell fate, it is not surprising that the activity of BH3-only proteins is stringently controlled, both through transcriptional regulation and post-translational mechanisms such as phosphorylation.[11] Puma was originally identified during large-scale analysis for novel p53-inducible genes and Bcl-2-interacting proteins.[12,13,14] Puma is an extremely potent BH3-only protein, having the capacity to bind with high affinity to all known anti-apoptotic Bcl-2 proteins.[15] In addition, Puma may directly interact with and activate Bax/Bak, for example, through an interaction with the first a-helix of Bax.[7,8] In keeping with its highly pro-apoptotic nature, Puma has been shown to have an essential role in the induction of apoptosis in numerous in vitro and in vivo systems and cell types including neurons, lymphocytes, thymocytes, fibroblasts and cardiomyocytes.[15,16] Puma expression is regulated by transcription, and factors regulating Puma transcription include p53, p73, E2F and FOXO3a.12,13,17–19. Given the critical role of Puma downstream of p53 and in multiple additional cell death scenarios, these studies, highlight another potentially critical control point in the cellular decision between life and death
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