Inhibition of PP2A abolishes DNA damage-induced Thr55 dephosphorylation. (A) U2OS cells were treated with OA for 2 h at different concentrations. p53 protein as well as Thr55, Ser15, Ser37 and Ser20 phosphorylation levels were detected with the indicated antibodies. (B) Thr55 phosphorylation in cells treated with calyculin A (CA). (C) U2OS whole-cell extracts were immunoprecipitated with anti-p53 antibody and immunoblotted with anti-PP2A C, anti-PP2A A, anti-cyclin G and anti-vinculin antibodies. (D) U2OS lysates were incubated with microcystin beads in the absence (−) or presence (+) of 50 nM OA. Proteins associated with beads were analyzed by Western blot analysis with the indicated antibodies. (E, F) U2OS cells were exposed to IR (E) or UV (F) radiation in the absence (DMSO control) or presence of 200 nM okadaic acid (OA). The cells in (E) were treated with OA for 1 h and the cells in (F) were treated for 3 h. To normalize the p53 protein level, cells were treated with MG132 before irradiation. Cells were harvested after DNA damage at the times indicated and Thr55 and Ser15 phosphorylation was assayed.

Specific B regulatory subunits B56γ1 and B56γ3 interact with p53 and are responsible for Thr55 dephosphorylation. (A) HA-tagged B55α, B56α, B56γ1 and B56γ3 were overexpressed in U2OS cells. Cell lysates were immunoprecipitated with anti-p53 antibody and immunoblotted with anti-HA antibody. (B) Thr55 phosphorylation levels were analyzed by phospho-specific antibody for Thr55 under B55α, B56α, B56γ1 or B56γ3 overexpression conditions in the absence (DMSO) or presence of OA. (C) HA-tagged B55α-, B56α-, B56γ1- or B56γ3-overexpressed cell lysates were immunoprecipitated with anti-HA antibody and immunoblotted with anti-PP2A A and C antibodies. (D) p53 and p21 protein levels were tested in the absence of MG132. (E) Affinity-purified baculovirus-expressed p53 that is enriched for phosphorylation at Thr55 via TAF1 coinfection. (F) The Thr55 phosphorylated p53 protein was dephosphorylated by immunoprecipitated B56γ3-containing complexes of PP2A in the absence (−) or presence (+) of OA.

B56γ mediates Thr55 dephosphorylation in response to DNA damage. (A) U2OS cells were transfected with siRNA oligos targeted to B56γ or a control nonspecific oligo (NS). The protein levels of B56γ, B56α, B56β, p53, Mdm2, vinculin and Thr55 phosphorylation of p53 were analyzed. (B) U2OS cells were subjected to UV or IR radiation with (B56γ KD) or without (control) B56γ protein ablation. Phosphorylation levels of p53 at Thr55 and Ser37 were tested in the presence of MG132. The p53 and Bax protein levels were also assayed in the absence of MG132 (C). (D) Reduction of the DNA damage-induced cell apoptotic response in U2OS cells by RNAi-mediated B56γ ablation. (E) SV40 t ag was overexpressed in U2OS cells. (F) U2OS cells were subjected to UV radiation with (small t ag) or without (control) SV40 small t antigen overexpression. Thr55 phosphorylation of p53 was assayed in the absence (mock) or presence of UV radiation.

Increases in B56γ protein levels and in the association with p53 correlate with Thr55 dephosphorylation. (A) Whole-cell extracts of γ-irradiated U2OS cells were subjected to immunoblotting with anti-B56γ, anti-B55α, anti-PP2A C, anti-PP2A A, anti-p53, anti-phospho-Ser37 and anti-vinculin antibodies. Thr55 phosphorylation was assayed with anti-phospho-p53 antibody as described. (B) U2OS cells were exposed to IR and association of p53 and B56γ3 with PP2A C was detected by microcystin bead pull-down assay. (C) Association of p53 with B56γ3-containing PP2A complex after DNA damage was detected by p53 immunoprecipitation (IP) and PP2A A, PP2A C and B56γ immunoblotting (IB).

B56γ3 inhibits cell proliferation in a p53-dependent manner. Representatives of cell proliferation of the HCT116 human colon cancer cell lines (A) or the H1299 human lung cancer cell line (B) transfected with either B56γ3 or a control CMV empty vector. H1299+p53: H1299 transfected with wild-type p53; H1299+T55D: H1299 transfected with T55D. Error bars show average±s.d. from triplicate plates in one representative experiment. (C) Immunoblotting of the transfected HA-B56γ3, endogenous B56γ3/B56γ2, p53 and p21 proteins in HCT116 and H1299 cells. M: empty vector-transfected cell lysate at seeding. (D) Cell doubling times show average±s.d. from three independent experiments. EV: empty vector.

B56γ3 functions in cell G1 arrest and transformation via Thr55 dephosphorylation. (A) H1299 cells were transfected with B56γ3 together with wild-type p53 or T55D. The cell-cycle profile was analyzed 60 h after transfection. (B) Anchorage-independent growth of HCT116 cells in the presence or absence of B56γ3 as indicated. EV: empty vector. (C) Anchorage-independent growth of H1299 cells in the presence or absence of p53, T55D and B56γ3 as indicated.

A proposed model for the dynamics of Thr55 phosphorylation by TAF1 and PP2A.