Fig 2: PP4 promotes recovery from the G2/M checkpoint arrest. (A) Schematic diagram of the checkpoint recovery experiment. S-phase cells were first arrested by incubation with aphidicolin, then mock-treated (No IR) or irradiated with an X-ray dose of 3 Gy. At 1 h post-treatment, nocodazole was added to the media and cells were fixed at various time points for processing by flow cytometry. (B) U2OS cells transfected with siRNAs against PP4C or a non-targeting control sequence were collected 1 and 8 h after mock (No IR) or 3 Gy irradiation and processed for flow cytometry with an antibody against phospho-histone H3 and propidium iodide (PI). The boxes represent the mitotic cells. (C) U2OS cells transfected with siRNAs against PP4C or a non-targeting control sequence were mock-treated (No IR) or irradiated (3Gy), and processed as described in (A). The percentage of mitotic cells (as determined by phospho-histone H3 staining) was calculated for each indicated time point. The experiment was repeated three times and the error bars represent the standard deviation. (D) Percentage of mitotic cells (as determined by phospho-histone H3 staining) calculated for each time point for the mock-irradiated (No IR, left) or irradiated (3 Gy, right) samples. The onset of mitosis represented by the intersecting lines was arbitrarily fixed at 0.4%. (E) Model of action of PP4 and dephosphorylation of ?H2AX. IR, irradiation; siRNA, small interfering RNA.

Fig 3: PP4 is active toward pSer666 and pThr806 and negatively regulated by Cdk9.a PP4R2 was immunoprecipitated from HCT116 cell extracts, incubated in vitro with purified, recombinant Cdk9/cyclin T1 and ATP, and immunoblotted with antibodies to PP4R2-pThr173, total PP4R2, or PP4C, as indicated at right. b HCT116 cells were mock treated (DMSO) or treated with inhibitors of Cdk9 (NVP-2) or Cdk1 (RO-3306), as indicated, for 1 h before lysis and extract preparation. Anti-PP4R2, anti-PP4C, or control IgG immunoprecipitates, as indicated, were incubated with Spt5-derived phosphopeptides containing pSer666 or pThr806, as indicated, and phosphate release was measured colorimetrically. Error bars indicate ±s.d. of six biological replicates (n = 6). c Anti-PP4R2 immunoprecipitates were incubated with 5 ng purified, recombinant Cdk9/cyclin T1 and ATP or mock treated (as indicated), washed and tested for phosphatase activity toward an Spt5-derived phosphopeptide containing pSer666 or pThr806, as in b. Activity of Cdk9-treated samples is plotted as a fraction of activity of the corresponding, mock-treated immunoprecipitate (defined as 1.0). Error bars indicate ±s.d. from three biological replicates (n = 3). Individual data points from biological replicates are shown in the plots (b and c). d HCT116 cells were infected with lentivirus expressing shRNA targeting PP4C (three different hairpins, sh1-3) or a nontargeted control vector (V), and chromatin fractions were immunoblotted for Spt5-pSer666, Spt5-pThr806, total Spt5 (to ensure equal loading),and total PP4C (to assess efficiency of depletion). Source data are provided as a Source data file.

Fig 4: Protein phosphatase 2A (PP2A) specifically dephosphorylates cytoplasmic ATR at S428 and its action involves direct binding to ATR. (A) A549 cells were treated with siRNAs against three protein phosphatases (PP2Ac, PP4c, and PP5c) followed by UV treatment at 40 J/m2 with a 2 h recovery. Analysis of cytoplasmic extracts reveals that PP2A is required to dephosphorylate cytoplasmic pATR (S428). (B) siRNA knockdown of PP2Ac in A549 cells was followed by UV treatment at 40 J/m2 with a 2 h recovery. pATR (S428) levels are increased in cytoplasm with PP2Ac knockdown while the phosphorylation status of ATR in the nucleus remains unchanged. WB of whole cell extracts shows PP2A siRNA knockdown efficiency in A549 cells (C) HCT 116 ATRflox/– cells were transfected with N-terminal Flag-tagged wild-type (WT) or S428A mutant ATR expression constructs and UV treated at 40 J/m2 with a 2 h recovery. Analysis of the cytoplasmic extracts reveals that PP2A specifically targets the pATR (S428) residue. The pATR (S428) observed in the S428A cells reflects some phosphorylation of the residual endogenous ATR remaining in the ATRflox/– cells (right panel). (D) PLA revealing that in A549 cells there is a direct interaction between ATR and PP2A, especially after UV irradiation at 40 J/m2. A DAPI-staining overlay is used to show location of the nuclei. The bar graph represents a statistical analysis of the PLA images. (E) A549 cells treated with UV at 40 J/m2 were lysed, followed by co-immunoprecipitation of PP2A using anti-ATR antibody. *Stands for p value < 0.01.

Fig 5: PP4C and PP2ACa dephosphorylate H2AX in vitro. (A–D) Purified histone substrates isolated from cycling (N) or irradiation-treated U2OS cells (I) were incubated with varying amounts of immunopurified wild-type (WT) Flag-PP4C (A,C,D), Flag-PP2ACa (B), Flag-PP1C (A,B), phosphatase-dead Flag-PP4C-H56Q and PP4C-R86A mutants (C) and Flag-PP4R2 (D). Phosphatase reactions were followed by immunoblotting and probed with the indicated antibodies. H3, histone H3.