Phosphorylation of Puma modulates its apoptotic function by regulating protein stability

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.

Figure 1

Exogenous Puma is phosphorylated on serine residues in HeLa cells in a BH3 domain-independent manner (a and c) HeLa cells were transfected with a plasmid encoding HA-Puma (a) or Flag-Puma (c) and incubated in the presence of caspase inhibitor BAF for 16–20 h before lysis. Overexpressed Puma was immunoprecipitated using anti-HA antibody and protein A beads or anti-Flag beads. Immunoprecipitated Puma was detected using an anti-Puma N-terminus antibody. For analysis of Puma phosphorylation, cells were incubated for 4 h with ³²P-orthophosphate before lysis. After SDS-PAGE, gels were stained with Coomassie to visualise protein and dried. ³²P-labelled HA-Puma (b, upper panel) and Flag-Puma (c, upper panel) were detected using a phosphorimager system. HA-Puma-WT was run in duplicate in panel b. (d) ³²P-labelled HA-Puma was transferred to PVDF membrane. The radioactive Puma band was excised from the membrane and amino acids were obtained by acid hydrolysis. ³²P-labelled amino acids were resuspended in 10 μl milliQ water including phosphoamino acid standards and this sample was run on a 1D-TLC plate. Phosphoamino acid standards were visualised by ninhydrin staining, and ³²P-labelled amino acids were detected using a phoshorimager system

Figure 2

Serine 10 is the principle site of Puma phosphorylation in HeLa cells and MEFs. (a) Alignment of PUMA protein from human (black text), mouse (grey text) and rat (italics)–conserved serines are indicated by black arrowheads and non-conserved serines by grey arrowheads. HeLa cells (b and d) or Bax/Bak double knockout (DKO) MEFs (e) were transfected with a plasmid encoding Flag-Puma containing serine-to-alanine mutations at the sites indicated above each lane at 12 h before labelling with ³²P-labelled orthophosphate. Flag-Puma was captured using anti-Flag beads, separated by SDS-PAGE and visualised by coomassie staining. ³²P-labelled proteins were detected using a phosphorimager system. (c) Western blot detection of immunoprecipitated Puma mutants using an anti-Puma antibody

Figure 3

Non-phosphorylatable Puma mutant S10A induces apoptosis at a greater rate than WT-Puma in HeLa cells. (a and b) HeLa cells were co-transfected with the indicated Puma construct and a construct encoding farnesylated GFP. At 16 h after transfection, cells were fixed with 4% PFA for 20 min, DNA was stained with Hoechst 33342 and then coverslips mounted for analysis by fluorescence microscopy (a). Cell death was quantified by examining the nuclear morphology of GFP-positive cells and calculating the percentage displaying condensed or fragmented apoptotic nuclei (white arrowheads) (b). ^**P<0.01

Figure 4

Mutation of Puma serine 10 does not affect association of Puma with Mcl-1, Bcl-2 or Bcl-xL. Cells expressing Myc-tagged Puma-WT or Puma-S10A were immunoprecipitated with anti-Myc antibody and precipitated proteins were analysed by western blotting with antibodies against: Mcl-1, Bcl-2, Bcl-Xl and Puma

Figure 5

The non-phosphorylatable Puma serine 10 mutant accumulates in a manner independent of caspases, apoptosis and autophagy. (a) DKO MEFs were lysed 24 h after transfection with the indicated constructs. Proteins were resolved by SDS-PAGE and Puma protein was detected using an antibody directed against human Puma. (b) qRT-PCR analysis of DKO MEFs transfected for 20 h with the indicated constructs. The amount of Puma mRNA was normalised in each sample against 18S RNA. (c) WT or ATG-5 KO MEFs were transfected with either WT or S10A Puma constructs in the presence or absence of pan-caspase inhibitor zVAD-fmk. At 24 h after transfection, cells were lysed and Puma expression analysed by western blot using a Puma N-terminal antibody. (d) WT and DKO MEFs were transfected with non-apoptotic Puma lacking a functional BH3 motif (ΔLRR) containing either the wild-type serine at position 10 or an alanine substitution. At 24 h after transfection, cells were lysed and Puma expression was analysed by western blot analysis using anti-Puma antibodies

Figure 6

WT-Puma is degraded faster than non-phosphorylatable Puma S10A. (a) Puma is degraded by the proteasome. DKO MEFs infected with Myc-tagged wild-type Puma were treated with cycloheximide either in the absence or presence of the proteasome inhibitor MG132. At the indicated time periods, cell lysates were prepared and analysed by western blotting with antibodies against Puma and actin. (b) DKO MEFs were transfected with Myc-tagged Puma constructs as indicated. Cells were pulsed for 1 h with 50 μCi ³⁵S-labelled methionine/cysteine before a 4 h chase step in medium containing excess cold methionine/cysteine. Cells were lysed at the indicated time points after the pulse and Myc-tagged Puma immunoprecipitated. Puma expression was tested by western blot (b, lower panel) and the amount of ³⁵S label incorporated into Puma determined using a phosphorimager system. (c) Intensities of ³⁵S-labelled-Puma from four separate experiments were quantified by measuring band intensity using ImageJ (NIH, Bethesda, MD, USA) and are expressed as a percentage of the band intensity of WT or S10A at t=0. ^**P<0.01