PP2A inactivation is a crucial step in triggering apoptin-induced tumor-selective cell killing

Abstract

Apoptin (apoptosis-inducing protein) harbors tumor-selective characteristics making it a potential safe and effective anticancer agent. Apoptin becomes phosphorylated and induces apoptosis in a large panel of human tumor but not normal cells. Here, we used an in vitro oncogenic transformation assay to explore minimal cellular factors required for the activation of apoptin. Flag-apoptin was introduced into normal fibroblasts together with the transforming SV40 large T antigen (SV40 LT) and SV40 small t antigen (SV40 ST) antigens. We found that nuclear expression of SV40 ST in normal cells was sufficient to induce phosphorylation of apoptin. Mutational analysis showed that mutations disrupting the binding of ST to protein phosphatase 2A (PP2A) counteracted this effect. Knockdown of the ST-interacting PP2A-B56γ subunit in normal fibroblasts mimicked the effect of nuclear ST expression, resulting in induction of apoptin phosphorylation. The same effect was observed upon downregulation of the PP2A-B56δ subunit, which is targeted by protein kinase A (PKA). Apoptin interacts with the PKA-associating protein BCA3/AKIP1, and inhibition of PKA in tumor cells by treatment with H89 increased the phosphorylation of apoptin, whereas the PKA activator cAMP partially reduced it. We infer that inactivation of PP2A, in particular, of the B56γ and B56δ subunits is a crucial step in triggering apoptin-induced tumor-selective cell death.

Figure 1

Transient expression of N-terminal determinants of SV40 T antigens activates apoptin. (a) Schematic representation of the domains in SV40 LT136/ST. The J domain (amino acids 1–82) is identical in both LT136 and ST. The Rb-binding domain and NLS in LT136 are also shown. ST is expressed by differential splicing and has a unique C-terminus, which contains the PP2A-binding domain. (b) Human fibroblasts (F9) were co-transfected with plasmids pcDNA-Flag-apoptin and pcDNA-LT136/ST (LT136/ST) or pcDNA-neo (neo) by AMAXA nucleofactor transfection. Twenty-four hours post transfection, cells were lysed for western blot analysis with the indicated antibodies. Mock-transfected F9 cells were used as control. Antibody α-108-P specifically recognizes phospho-apoptin at its Thr108. Flag-apoptin, LT and ST show the respective total protein amounts in the transfected cells. Actin was used as loading control. (c–e) Cells were fixed for immunofluorescence analysis at each given time-point after transfection and then stained with the indicated antibodies by indirect immunofluorescence assay. Scale bar=20 μm

Figure 2

Nuclear targeting of SV40 ST activates apoptin. In normal human fibroblasts, apoptin was co-transfected with vector only (Neo), LT136, ST or both LT136 and ST. Twenty-four hours post transfection, cells were lysed for western blot analysis with the antibodies indicated. The relative amount of phosphorylated apoptin (as compared with the total amount of apoptin) was quantified and is indicated below the a-108-P panel. The LT136+ST sample was set at 100

Figure 3

Apoptin activation via ST-mediated inhibition of PP2A. (a) pcDNA-Flag-apoptin was co-transfected with plasmids encoding the indicated proteins or vector DNA into F9 primary fibroblasts. Western blot assays were performed with the indicated antibodies at 24 h post transfection. (b) F9 primary cells were co-transfected with pcDNA-Flag-apoptin and plasmids encoding NLS-ST, NLS-ST(P101A), NLS-ST(C103S) or vector DNA as indicated. Western blot assays were performed with the indicated antibodies at 24 h post transfection. In panels a and b the relative percentage of phosphorylated apoptin in the various samples was quantified and is indicated below the a-108-P panel c. PP2A binding to ST is abolished by C103S mutation. LT136 and ST with or without the C103S mutation (LT136/ST or LT136/ST(C103S) were fused with a Strep-tag at their N terminus. Cell lysates were prepared at 24 h post transfection for protein–protein interaction assays as indicated in Materials and Methods. The final elutions were analyzed by western blot with antibodies against PP2A α subunit, LT and ST, respectively. Actin was taken as equal loading control. The first lane (input control) indicated total amount of endogenous proteins in cell lysates

Figure 4

Knockdown of PP2A–B56γ and B56δ subunits triggers apoptin phosphorylation in normal cells. (a) Downregulation of PP2A–B56γ subunit by shRNA. HeLa cells were co-transfected with pCEP-4HA-B56γ expressing 4HA-tagged B56γ, and either shB56γ or control pSuper vector and lysed 48 h post transfection, followed by western blotting analysis with the indicated antibodies. (b) Downregulation of PP2A–B56δ subunit by shRNA. F9 cells were transfected with pSuper vector encoding shRNA directed against PP2A–B56δ or pSuper vector control; 24 h post transfection, cell lysates were prepared and subsequently analyzed by western blot. (c) F9 cells were co-transfected with Flag-apoptin and either pSuper vector encoding shRNA directed against PP2A–B56γ, δ or control; 24–48 h post transfection, cell lysates were prepared and subsequently analyzed by western blot

Figure 5

BCA3 interacts with apoptin and stimulates its activity. (a) Apoptin interacts with BCA3 in a cellular background. Normal human foreskin fibroblasts were transfected with plasmids encoding myc-tagged BCA3 and Flag-tagged apoptin, or control plasmid in the indicated combinations. Total lysates (L) or protein complexes immunoprecipitated (IP) with antibody against the myc- (left panel) or Flag-tags (right panel) were separated by SDS-PAGE and analyzed by western blot. (b) Expression of myc-BCA3 together with Flag-apoptin results in increased induction of apoptosis. Human Saos-2 tumor cells were transfected with plasmids encoding Flag-tagged apoptin and myc-tagged BCA3, or vector control in the indicated combinations and grown on glass coverslips. Forty-eight hours post transfection, slides were fixed and stained with appropriate antibodies for immunofluorescence analysis. pMaxGFP (Amaxa) was used as a negative control. Apoptosis was assessed according to characteristic morphological changes following DAPI staining as shown in figure 1. Data are representative of three independent experiments, in which at least 100 cells were scored. (c) Co-expression of myc-BCA3 enhances apoptin phosphorylation. Saos-2 cells transfected with BCA3 and apoptin, or apoptin alone, were lysed 6 h post transfection and analyzed for apoptin phosphorylation by western blot analysis. (d) Inhibition of PKA results in increased apoptin phosphorylation. Saos-2 cells were transfected with Flag-tagged apoptin, and treated with the PKA inhibitor H89 (1 h, 10 μM) or activator cAMP (30 min, 10 μM H89, followed by 30 min 1 mM cAMP) 24 h post transfection. Cells were then lysed and their contents analyzed by western blot using indicated antibodies. The relative percentage of phosphorylated apoptin in the various samples was quantified and is indicated below the a-108-P panel. (e) Effect of PKA inhibitors/inducers on PP2A–B56δ phosphorylation. In a first instance, phosphorylated proteins were immunoprecipitated from Saos-2 whole-cell lysate (WCL) with six different antibodies directed against phosphoserines, as described (Materials and Methods). Western blot was performed with antibodies against B56δ. The light chain signal shows equal assay conditions. Note that only antibody 5 detects phospho- B56δ. (f) Saos-2 cells were treated with PKA inhibitor H89 or mock-treated and immunoprecipitated with the phosphoserine antibody 5 (see panel e). The amount of pulled-down B56δ was determined by western blot using an antibody directed against B56δ and quantified. The amount of phospho B56δ in the mock-treated cells was set at 100. (g) Saos-2 cells were treated with PKA-stimulator cAMP or mock-treated and immunoprecipitated with antibodies against B56δ. The amount of phosphorylated B56δ was determined by western blot using the phosphoserine antibody 5 (panel e) and quantified. The amount of phosphoB56δ in the mock-treated cells was set at 100