Fig 1. Interaction of PP5 with ASK1 in non-stressed cells. (A and B) 293 cells were transiently co-transfected with the indicated plasmids. Lysates were divided and immunoprecipitated with anti-Flag antibody (M2 gel). Immunoprecipitates were subjected to immunoblot analysis with anti-HA antibody (A, top) or with anti-Myc antibody (B, top). The presence of expressed proteins in the same lysates was verified by the indicated combination of immunoprecipitation (IP) and immunoblotting (WB).

Fig. 3. PP5 dephosphorylates and inactivates ASK1 in vivo and in vitro. (A) PP5 reduces ASK1 activity in vivo. HeLa cells were transfected with the indicated plasmids. Thirty-six h later, immunocomplex kinase assay for ASK1 was performed as described in Materials and methods. ASK1 activity was measured using GST–MKK6 as a substrate (top). Consistent expression of Flag-ASK1 was confirmed by immunoblotting (bottom). (B) PP5 reduces ASK1-induced activation of JNK. 293 cells were transfected with the indicated plasmids. JNK activity was measured by immunoblotting using phospho-specific antibody to SAPK/JNK (Thr183/Tyr185). Expression of JNK-HA, Flag-ASK1 and Flag-PP5 was confirmed by immunoblotting using the indicated antibodies. (C) PP5 does not inhibit MEKK1-induced activation of JNK. 293 cells were transfected with the indicated plasmids. Lysates were immunoprecipitated with anti-JNK antibody, and JNK activity was measured by immunocomplex kinase assay using GST–c-jun as a substrate. Expression of JNK-HA, ASK1-HA, HA-MEKK1 and Flag-PP5 was confirmed by immunoblotting using the indicated antibodies. (D) PP5 does not inhibit the ERK pathway. 293 cells were transfected with the indicated plasmids. Cells were then stimulated with 20% FBS for 20 min, and ERK activity was measured by immunecomplex kinase assay using MBP as a substrate (top). Expression of transfected plasmids were confirmed by immunoblotting using the indicated antibodies. (E) PP5 directly dephosphorylates a critical phospho-threonine residue of ASK1. PAE cells stably transfected with ASK1-HA were treated with 1 mM H₂O₂ for 30 min. ASK1 was immunoprecipitated with anti-HA, incubated with recombinant full-length PP5 or with truncated PP5 (TPR domain only) for 20 min in the presence (+) or absence (–) of arachidonic acid. The samples were subjected to immunoblotting analysis with anti-phospho-ASK1 antibody (P-ASK). The presence of ASK1 and GST fusion proteins was verified by immunoblotting (WB) and staining with Coomassie Brilliant Blue dye (C.B.B stain), respectively.

Fig. 5. PP5 inhibits ASK1-dependent apoptosis. (A) PP5 inhibits ASK1-dependent activation of caspase-3-like protease. Indicated plasmids were transiently transfected into 293 cells and caspase-3-like protease activity was measured as described in Materials and methods. Results are the means of duplicate determinations ± SE from one of more than three representative experiments. (B) PP5 inhibits ASK1-dependent cell death. The indicated plasmids were transiently transfected into HeLa cells with pEGFP, and apoptotic cell death was determined by a morphological analysis as described in Materials and methods. Results are the means of duplicate determinations ± SE from one of two representative experiments.

Fig. 2. Oxidative stress enhances the interaction between PP5 and ASK1. (A) H₂O₂- and TNF-induced interaction of PP5 and ASK1. HeLa cells were transiently co-transfected with HA-PP5 and Flag-ASK1. Thirty-six hours later, the cells were treated with 1 mM H₂O₂ or 200 ng/ml TNF for 20 min, and lysates were subjected to co-immunoprecipitation analysis as described in Figure 1A. (B) H₂O₂ dose-dependent interaction of PP5 and ASK1. HeLa cells and COS7 were transfected as in (A), treated with increasing concentrations of H₂O₂ for 30 min and analyzed by co-immunoprecipitation analysis. (C) Time course of the H₂O₂-induced interaction of PP5 and ASK1. HeLa cells were transfected as in (A), treated with indicated concentrations of H₂O₂ for the indicated periods and analyzed by co-immunoprecipitation analysis. (D) Specific interaction of ASK1 with PP5 but not PP2A. 293 cells were transiently co-transfected with ASK1-HA and Flag-PP5 or Flag-PP2A. Cells were treated with 5 mM H₂O₂ for 30 min, and lysates were subjected to co-immunoprecipitation analysis as described in Figure 1A. (E) Interaction of endogenous PP5 and ASK1. Approximately 5 × 10⁷ of A549 cells were treated with 5 mM H₂O₂ for 30 min. Cell lysates were divided and immunoprecipitated with normal rabbit IgG or anti-ASK1 polyclonal antibody (DAV) and were immunoblotted with anti-PP5 monoclonal antibody. The presence of ASK1 and PP5 in the same lysates was verified by immunoblotting (WB). Ig indicates non-specific reactions derived from rabbit IgG. (F) Subcellular localization of endogenous PP5 in A549 cells. A subcellular fractionation was performed as described in Materials and methods, and PP5 was detected by immunoblotting. Anti-PML antibody was used as a positive control for the nuclear protein. (G) Subcellular localization of transfected ASK1 and PP5 in HeLa cells. HeLa cells were transfected with Flag-ASK1 and HA-PP5, or with HA-PP5 alone, and the cells were subjected to an immunofluorescence staining as described in Materials and methods.

Fig. 4. PP5 inhibits H₂O₂-induced sustained activation of ASK1 in vivo. (A) PP5 can participate in the complex of ASK1 and Trx. 293 cells were transiently co-transfected with the indicated plasmids. Lysates were divided and immunoprecipitated with anti-Flag antibody (M2 gel). Immunoprecipitates were subjected to immunoblot analysis with anti-HA antibody (top panel) or with anti-Myc antibody (second panel). The presence of expressed proteins in the same lysates was verified by the indicated combination of immunoprecipitation (IP) and immunoblotting (WB). (B) Opposing effects of H₂O₂ on the interactions of ASK1 with PP5 and Trx. 293 cells were transfected with the indicated plasmids, treated with 5 mM H₂O₂ for 30 min and analyzed by co-immunoprecipitation analysis. (C) PP5 inhibits H₂O₂-induced sustained activation of ASK1. Flag-ASK1 was transiently transfected with or without HA-PP5 into HeLa cells. Thirty-six h later, the cells were treated with 5 mM H₂O₂ for the indicated periods. Lysates were divided, immunoprecipitated or immunoblotted, and the kinase activity of ASK1 (top panel), phosphorylation status of Thr845 of ASK1 (third panel) and co-immunoprecipitated HA-PP5 (second panel) were analyzed. The presence of HA-PP5 and Flag-ASK1 in the same lysates was verified by immunoblotting (WB). (D) The intensity of GST–MKK6 phosphorylation (scale in the left) and the amount of co-immunoprecipitated PP5 (scale in the right) in (C) were quantified and represented by a graph. Relative values of activation and interaction were calculated by dividing the intensities of phosphorylated GST–MKK6 or co-immunoprecipitated HA-PP5 (Co-IP PP5) at different time points by those at time zero.