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Fig. 3 PPM1A Interacts with Smad2 (A) Coimmunoprecipitation of Flag-PPM1A and HA-Smad2 in 293T cells. IP, immunoprecipitation; IB, Western blotting; WCL, whole-cell lysates. (B) Endogenous PPM1A-Smad2 interaction is TGFβ inducible. HaCaT cells were treated with TGFβ1 (1 hr), and cell lysates were subjected to IP using anti-PPM1A (lane 5 and 6) or a control mouse antibody (lane 4). (C) Smad2(V398R) mutant interacts with PPM1A in 293T cells. (D) Subcellular colocalization of PPM1A and Smad2 in HaCaT cells. Smad2 and PPM1A proteins were detected by using anti-Smad2 (FITC) and anti-PPM1A (Texas red) antibodies. DNA was stained using DAPI dye. (E) PPM1A is primarily localized in the nucleus. Left: HaCaT cells treated with TGFβ1 (2 ng/ml for 1 hr) were crosslinked with formaldehyde before fractionation and Western blotting analysis (Supplemental Data). C, cytoplasmic fraction; N, nuclear fraction. Right: NIH 3T3 cells. (F) Direct interaction of PPM1A with Smad2/3. Purified recombinant His-PPM1A or His-D239N protein bound to recombinant GST-Smad2 (lanes 4 and 7) or Smad3 (lanes 5 and 8) but not GST alone (lanes 3 and 6) was detected by anti-PPM1A Western blotting. (G) PPM1A exclusively binds to P-Smad2. Recombinant Smad2 MH2 (aa 241–467), phosphorylated (lane 1) or unphosphorylated (lane 2), bound to recombinant His-PPM1A protein was detected by Western blotting.
Reprinted from Cell, 125(5), Lin, X., Duan, X., Liang, Y.Y., Su, Y., Wrighton, K.H., Long, J., Hu, M., Davis, C.M., Wang, J., Brunicardi, F.C., Shi, Y., Chen, Y.G., Meng, A., and Feng, X.H., PPM1A functions as a Smad phosphatase to terminate TGFbeta signaling, 915-928, Copyright (2006) with permission from Elsevier. Full text @ Cell