Wip1 Phosphatase Modulates ATM-Dependent Signaling Pathways Sathyavageeswaran Shreeram, Oleg N. Demidov, Weng Kee Hee, Hiroshi Yamaguchi, Nobuyuki Onishi, Calvina Kek, Oleg N. Timofeev, Crissy Dudgeon, Albert J. Fornace, Carl W. Anderson, Yasuhiro Minami, Ettore Appella, Dmitry V. Bulavin  Molecular Cell  Volume 23, Issue 5, Pages 757-764 (September 2006) DOI: 10.1016/j.molcel.2006.07.010 Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 1 Modulation of ATM Signaling by the Wip1 Phosphatase in Mouse Cells (A) E1A+Ras-expressing wt and Wip1 null MEFs were either mock treated or irradiated with 5, 10, or 20 Gy of IR; cells were harvested 1 hr later. The levels of phosphorylation of ATM, p53, H2AX, Rad17, Nbs1, p38, and HSP27 were analyzed by Western immunoblotting using appropriate phosphospecific antibodies. The level of total p38, used as a loading control, was determined with the C20 antibody (Santa Cruz). (B) E1A+Ras-expressing wt and Wip1 null MEFs were irradiated with 10 Gy IR, and cells were harvested at different time points. The levels of Ser1987 phosphorylation and total ATM were determined by using appropriate antibodies. (C) Cells were irradiated as in (B) and harvested 1 hr later. ATM kinase activity was analyzed as described in Experimental Procedures. The quantitative analysis of ATM activity, based on three independent experiments, is shown in the right panel. PS stands for preimmune serum. (D) Ras-expressing p53−/− and p53−/− and Wip1−/− MEFs were treated as in (A) and analyzed for phosphorylation of ATM. (E) E1A+Ras-expressing wt MEFs were infected with either PINCO-neo or PINCO-neo-Wip1 retroviruses. After selection in the presence of G418, the cells were treated with IR and harvested 1 hr later. Phosphorylation at ATM Ser1987 and p53 Ser18 was determined with appropriate antibodies; p38 MAPK served as a loading control. Molecular Cell 2006 23, 757-764DOI: (10.1016/j.molcel.2006.07.010) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 2 Modulation of ATM Signaling by the Wip1 Phosphatase in Human Cells (A) Saos-2 cells expressing a tet-inducible Wip1 phosphatase were treated overnight with 5 μg/ml doxycycline (Dox) to induce expression of Wip1 and then were treated with IR (as in Figure 1A). The levels of phosphorylation of ATM and Chk2 were analyzed using phosphospecific antibodies. (B) Saos-2 cells were either treated with Dox or left untreated, and then ATM was immunoprecipitated (H-248 antibody, Santa Cruz). The levels of Wip1 and ATM in whole cell extracts (WCE) and in control (-) and ATM immunoprecipitates were analyzed using appropriate antibodies (see Experimental Procedures). NSS stands for nonspecific serum. (C) Association of endogenous ATM with Wip1. Extracts from MCF7 cells were immunoprecipitated with anti-Wip1 antibody and immunoblotted with anti-ATM or anti-Wip1 antibodies. Preimmune serum was used as a negative control. (D) In vitro association of ATM with Wip1. Extracts from HEK293T cells transfected with Flag-ATM were incubated with purified GST, GST-Wip1 (wt), or GST-Wip1 (D314A). Flag-ATM bound to GST or GST-Wip1 was detected after pull-down with glutathione-Sepharose using an anti-Flag antibody. GST and GST-Wip1 were detected by amido black staining. Note that, in addition to the full-length GST-Wip1, multiple degradation products also are present. (E) Flag-tagged, wt ATM was expressed in 293T cells, which were then irradiated (10 Gy), and the ATM was immunoprecipitated using M2-Flag agarose. Dephosphorylation of ATM Ser1981 was analyzed at different time points in the presence of phosphatase buffer only (lanes 1–4), with recombinant wt human Wip1 (lanes 5–8) or with a phosphatase-impaired human Wip1 (lanes 9–12). No signal for phosphorylated Ser1981 was observed in a similar experiment with Ser1981A ATM (data not shown). Molecular Cell 2006 23, 757-764DOI: (10.1016/j.molcel.2006.07.010) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 3 Control of ATM Phosphorylation at Ser1981 by Wip1, and Their Binding after IR (A) MCF7 cells were exposed to IR (10 Gy, 1 hr), and Wip1 was immunoprecipitated 1 hr later with polyclonal antibody (Fujimoto et al., 2006). The level of ATM in precipitates was analyzed using a monoclonal antibody (2C1). MCF7 cells treated with Wip1 siRNA were used as a control (lane 4). NSS stands for nonspecific serum. (B) HeLa cells were transfected with either a Flag-tagged wt Wip1 or D314A-Wip1. After 28 hr, cells were irradiated (10 Gy), and 1 hr later Wip1 was precipitated with M2 Flag-agarose. The level of ATM and phospho-ATM was analyzed. (C) Control or Wip1 siRNA-transfected MCF7 cells were exposed to IR (10 Gy) and incubated for the indicated times. Extracts were then analyzed by immunoblotting with the indicated antibodies. Molecular Cell 2006 23, 757-764DOI: (10.1016/j.molcel.2006.07.010) Copyright © 2006 Elsevier Inc. Terms and Conditions