Inhibition of ATM abolishes DNA damage-induced Thr55 dephosphorylation. (A, B, and C) U2OS cells were pretreated with ATM inhibitor caffeine (A), wortmannin (B), or KU55933 (C) and then subjected to IR. Cells were harvested at the indicated time points (', min) and subjected to immunoblotting to detect Thr55, Ser15, and Ser20 phosphorylation (P) levels with the corresponding antibody. p53 protein levels were normalized by treating the cells with MG132. (D) A control empty vector (EV), Flag-tagged wild-type ATM (ATM), or KD mutant was transfected into U2OS cells. The presence of overexpressed ATM was verified by immunoblotting using anti-Flag antibody. The p53 protein and the Thr55, Ser15, Ser20, and Ser37 phosphorylation levels were detected by immunoblotting with the corresponding antibody. (E) U2OS cells were transfected with a control empty vector (control), Flag-tagged wild-type ATM (ATM), or KD mutant and then treated with IR and harvested at the indicated time points after treatment. ATM and p53, as well as the Ser15, Thr55, Ser20, and Ser37 phosphorylation levels, were detected by immunoblotting with the corresponding antibody. (F) Normal and ATM-deficient (A=T) human lymphoblasts were subjected to IR and harvested 1 h after treatment. The B56γ-p53 association was analyzed by immunoprecipitation with anti-B56γ antibody and immunoblotting (IB) with anti-p53 antibody. The p53 and B56γ protein levels, as well as the Thr55, Ser15, and Ser20 phosphorylation levels, were assayed with the corresponding antibody. IgG, immunoglobulin G; vinc, vinculin.

The S15A mutant is unable to cooperate with PP2A in inhibition of cell proliferation. (A) The left four panels show results representative of cell proliferation of the H1299 human lung cancer cell line transfected with either B56γ3 (HA-B56γ3) or a control empty vector (Control) along with an empty vector (EV), wild-type p53, the S15A mutant, or the S20A mutant. Error bars show the averages ± standard deviations of the results from triplicate plates in one representative experiment. The right panels show immunoblots of transfected HA-B56γ3, endogenous B56γ3/B56γ2, transfected wild-type (WT) or S15A mutant p53, and endogenous p21 from the cell proliferation assay. M, mock; vinc, vinculin. (B) H1299 cells were transfected with B56γ3 or empty vector (EV) control along with wild-type p53, the S15A mutant, or the S20A mutant. The cell cycle profile was analyzed 60 h after transfection. (C) Cell-doubling times. The values are the averages ± standard deviations of the results from three independent experiments. (D) Increases (positive values) and decreases (negative values) of percentages in each cell cycle distribution compared to the percentages in the corresponding cell cycle stage of the EV control. +, present; −, absent.

Proposed model of the sequential modification of p53. Upon DNA damage, activated ATM phosphorylates p53 at Ser15, thereby promoting interaction with B56γ-PP2A and dephosphorylation of Thr55. A circled P indicates phosphorylation.

Modifications of p53 may be important for DNA damage-induced p53-B56γ interaction. (A) U2OS cells were subjected to IR and the association of endogenous B56γ3/B56γ2 with p53 and with the PP2A core after DNA damage was detected by immunoprecipitation with anti-B56γ antibody and immunoblotting with anti-PP2A A, anti-PP2A C, and anti-p53 antibodies. Five percent of the input was loaded on the gel. (B) U2OS cells were pretreated with MG132 and subjected to IR, and the association of p53 and the PP2A core with GST-B56γ3 was assayed in GST pull-down assays. Five percent of the input was loaded on the gel. (C and D) Cell lysates were prepared from U2OS cells treated either with IR and (C) or UV(D) for the indicated times and then subjected to immunoblotting to detect Thr55, Ser15, and Ser20 phosphorylation, as well as Lys373 acetylation, with the corresponding antibody. p53 protein levels were normalized by treating cells with MG132. IgG, immunoglobulin G; vinc, vinculin; P, phosphorylation; DMSO, dimethyl sulfoxide.

Ser15 phosphorylation is required for the B56γ-p53 interaction and Thr55 dephosphorylation. (A and B) Whole-cell extracts of H1299 cells transfected with either wild-type (WT) p53 or p53 mutants S15A, S15D, and S20A were immunoprecipitated with either p53 (A) or B56γ (B) antibody. The precipitated proteins were then analyzed for the presence of p53, endogenous B56γ3/B56γ2, PP2A A, PP2A C, or vinculin (vinc) by immunoblotting (IB) as indicated. (C) H1299 cells were transfected with wild-type p53, the S15A mutant, or the S20A mutant and then treated with IR. The B56γ-p53 association was analyzed by immunoprecipitation with anti-B56γ antibody and immunoblotting with anti-p53 antibody. The p53 and B56γ protein levels, as well as the Thr55, Ser15, Ser20, and Ser37 phosphorylation (P), were assayed with the corresponding antibody. (D) Double modifications on p53 were assayed by immunoprecipitation with either Lys373 acetyl-specific (KAc) or Thr55 phosphospecific antibody at 0 (Mock), 20, or 80 min after IR, followed by immunoblotting with anti-p53 antibody, as well as Ser15 or Ser20 phosphospecific antibodies, of cell lysates from U2OS cells. α, anti; IgG, immunoglobulin G.

The S15A mutant is unable to cooperate with PP2A in inhibition of cell transformation. (A) Anchorage-independent growth of H1299 or H1437 cells transfected with empty vector (EV), wild-type (WT) p53, the S15A mutant, or the S20A mutant in the absence (Control) or presence of overexpressed B56γ3 (HAB56γ3). (B) Colony numbers. The values are the averages ± standard deviations of the results of three representative experiments. (C) Immunoblots of the transfected HA-B56γ3 and p53 and endogenous B56γ3/B56γ2 and p21 proteins, along with Thr55 phosphorylation (P) levels, in H1299 (left) and H1437 (right) cells. Ser37 phosphorylation in H1299 cells was also assayed with the corresponding antibody. +, present; −, absent; vinc, vinculin.