Volume 22, Issue 3, Pages 317-327 (May 2006) Interaction between c-Abl and Arg Tyrosine Kinases and Proteasome Subunit PSMA7 Regulates Proteasome Degradation  Xuan Liu, Wei Huang, Chufang Li, Ping Li, Jing Yuan, Xiaorong Li, Xiao-Bo Qiu, Qingjun Ma, Cheng Cao  Molecular Cell  Volume 22, Issue 3, Pages 317-327 (May 2006) DOI: 10.1016/j.molcel.2006.04.007 Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 1 c-Abl and Arg Associate with PSMA7 (A) Left, lysates from 293T cells were subjected to immunoprecipitation with anti-c-Abl or IgG, fractionated by SDS-PAGE, and subsequently analyzed by immunoblotting with indicated antibodies. Right, lysates were fractionated by gel filtration chromatography, and selected fractions were blotted with antibodies as noted. Fractions 14–16 contain proteasomes, whereas fractions 24–26 contain free subunits. (B) Left, 293T cells were cotransfected with Myc-c-Abl and Flag-PSMA7 expression plasmids or Flag-vector, and anti-Flag or IgG immunoprecipitates were analyzed by immunoblotting with anti-Myc or anti-Flag antibody. Right, proteasome complexes were prepared from lysates of 293T cells cotransfected with Myc-c-Abl and Flag-PSMA7 expressing plasmids by gel filtration chromatography; the fraction 15 containing proteasome complexes was subjected to anti-Flag or IgG immunoprecipitation and analyzed by immunoblotting with indicated antibodies. (C) Left, 293T cells were cotransfected with Myc-PSMA7 and Flag-c-Abl or Flag-Arg expressing plasmids. The immunoprecipitates were analyzed by immunoblotting with anti-Myc or anti-Flag antibody as above. Right, the immunoprecipitates of Figure 1C were tested for proteasomal activity as follows. Flag peptide was used to elute Flag-c-Abl from the anti-Flag immunoprecipitates. The proteasomal peptidase substrate Suc-Leu-Leu-Val-Tyr-AMC was then incubated with the eluted protein. A 10% V/V lysate was employed as an activity control. The results are expressed as the mean ± SD of three independent experiments. (D) Left, 293T cells were cotransfected with Flag-PSMA7 and Myc-c-Abl or Myc-c-Abl(K290R) expressing plasmids. The immunoprecipitates with anti-Flag or IgG were analyzed by immunoblotting with anti-Myc or anti-Flag antibody. Right, lysates of 293T cells transfected with Flag-c-Abl or Flag-c-Abl(K290R) expressing plasmids were fractionated by gel filtration chromatography. Anti-Flag immunoprecipitates prepared from the fraction 15 were analyzed by immunoblotting with indicated antibodies. Molecular Cell 2006 22, 317-327DOI: (10.1016/j.molcel.2006.04.007) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 2 c-Abl and Arg Bind Directly to PSMA7 (A) Lysates from 293T cells transfected with Flag-c-Abl expressing plasmid were incubated with a GST or GST-PSMA7 fusion protein for 2 hr. The absorbates were analyzed by immunoblotting with anti-c-Abl (top). Loading of the GST proteins was assessed by Coomassie blue staining (bottom). (B) Anti-Flag or IgG immunoprecipitates prepared from cells transfected with Flag-c-Abl or Flag-vector expressing plasmids were subjected to SDS-PAGE and blotted onto nitrocellulose membrane. The nitrocellulose membrane was incubated with soluble GST-PSMA7 or IgG for 2 hr and then analyzed with anti-GST, anti-IgG, or anti-Flag antibody. (C) 293T cells were transfected with Flag-PSMA7 expressing plasmid. The GST fusion protein absorbates from cell lysates were analyzed by immunoblotting with anti-Flag antibody (top). (D) Purified 20S proteasomes were incubated with GST-c-Abl SH3 fusion proteins or GST proteins (as shown in Figure 2C, bottom). The absorbates were analyzed by immunoblotting with indicated antibodies. Molecular Cell 2006 22, 317-327DOI: (10.1016/j.molcel.2006.04.007) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 3 c-Abl and Arg Phosphorylate PSMA7 (A) Left, MCF-7 cells were treated with or without 10 μM STI571 for 12 hr. Anti-PSMA7 immunoprecipitates were analyzed by immunoblotting with anti-P-Tyr or anti-PSMA7 antibody. Right, lysates prepared from 293T cells cotransfected with Flag-PSMA7 and Myc-c-Abl expressing plasmids were subjected to gel filtration chromatography. Anti-Flag or IgG immunoprecipitates of the proteasome complexes fraction in the 15th elution volume were analyzed by immunoblotting with anti-P-Tyr or anti-Flag antibody. (B) c-abl−/−arg−/− MEFs (left and middle) or 293T cells (right) were cotransfected with indicated vectors and treated with or without 10 μM STI571 for 12 hr. Anti-Flag immunoprecipitates were analyzed by immunoblotting with anti-P-Tyr or anti-Flag antibody. (C) MEFs, c-abl−/− MEFs, or c-abl−/−arg−/− MEFs were cotransfected with Flag-PSMA7 and Myc-c-Abl or pCMV-Myc expressing plasmids. Anti-Flag immunoprecipitates were analyzed by immunoblotting with anti-P-Tyr or anti-Flag antibody. (D) Recombinant GST-PSMA7 or GST-PSMA7 (Y153F) was incubated with purified Flag-c-Abl in the presence of [γ-32P]-ATP. The reaction products were analyzed by SDS-PAGE and autoradiography. (E) Purified c-Abl (Upstate Biotechnology) was incubated with 26S proteasome for 30 or 90 min in the presence of ATP. The reaction products were analyzed by immunoblotting with anti-P-Tyr or anti-20S proteasome antibody. (F) The 90 min reaction products in (E) were separated by 2D electrophoresis, and the 2D gel was subjected to immunoblotting with anti-P-Tyr or anti-PSMA7 antibody, or analyzed by Coomassie Brilliant Blue staining. (G) 293T cells were cotransfected with indicated plasmids, and anti-Flag immunoprecipitates were analyzed by immunoblotting with anti-P-Tyr or anti-Flag antibody. Molecular Cell 2006 22, 317-327DOI: (10.1016/j.molcel.2006.04.007) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 4 Degradation Activity of the Proteasome Is Inhibited by c-Abl and Arg (A) Suc-Leu-Leu-Val-Tyr-AMC substrate was incubated with 20S (left) or 26S (right) proteasome in the presence of c-Abl, Arg, or imidazole eluates prepared from an uninfected Sf9 cell lysate for 1 hr, and the fluorescence signal of released AMC was measured. The effect of treatment with the proteasome inhibitor MG132 is also shown. The results are expressed as the mean ± SD of three independent experiments. (B) Equivalent proteasome fractions (fraction 15 of gel filtration chromatography) from cells expressing Flag-c-Abl, Flag-c-Abl(K290R) (left), or MCF-7, MCF-7/c-Abl(K290R) (right) were monitored for proteasomal peptidase activity assayed as above. MG132 treatment is also shown. The results are expressed as the mean ± SD of three independent experiments. (C) 26S proteasomes were incubated with c-Abl for 30 or 90 min in the presence of 2 mM ATP. Then [35S] labeled proteins prepared from MG132-treated c-abl−/−arg−/− MEFs were incubated with the pretreatment proteasome mix for 4 hr, and the acid soluble fraction was quantitated by liquid scintillation counting. The results are expressed as the mean ± SD of three independent experiments. (D) Left, 293T cells expressing PSMA7 or PSMA7(Y153F) mutant were treated with (gray hatched boxes) or without 10 μM STI571 (open boxes) for 12 hr. After being pulsed with [35S]-methionine for 45 min, cells were washed and incubated with DMEM medium for 2 hr. Cell lysates together with medium were precipitated by 13% TCA, and the acid soluble fraction was quantitated by liquid scintillation counting (top). Also, proteasome fraction 15 was blotted with anti-Flag antibody (bottom). Right, normalized cell lysates of c-abl−/−arg−/− MEFs transfected with Flag-PSMA7 or Flag-PSMA7(Y153F) expressing plasmids were subjected to proteasomal activity assay as described in (A). The results are expressed as the mean ± SD of three independent experiments. (E) MCF-7/ZsGreen-ODC cells were treated with or without 2.5 μM STI571 for 12 hr, and cellular fluorescence intensity was evaluated by flow cytometry. (F) 293T cells cotransfected with indicated plasmids were treated with or without 10 μM MG132 for 2 hr, and cells lysates were analyzed by immunoblotting with anti-Flag or anti-β-actin antibody. Molecular Cell 2006 22, 317-327DOI: (10.1016/j.molcel.2006.04.007) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 5 Interaction of PSMA7 and c-Abl Is Regulated by Oxidative Stress and Ionizing Irradiation (A) MCF-7 cells were treated with the indicated concentrations of H2O2 for 1 hr. The anti-c-Abl immunoprecipitates were analyzed by immunoblotting with anti-PSMA7, anti-c-Abl, or anti-β-actin antibody. (B) MCF-7 cells were treated with 0.5 mM H2O2 for the indicated times. Anti-PSMA7 immunoprecipitates were analyzed by immunoblotting with the indicated antibodies. (C) 293T cells cotransfected with Myc-c-Abl and Flag-PSMA7 expressing plasmids were treated with indicated concentrations of H2O2 for 1 hr or with 0.5 mM H2O2 for indicated times. Anti-Flag immunoprecipitates were analyzed by immunoblotting with anti-P-Tyr or anti-Flag antibody. (D) MCF-7/ZsGreen-ODC cells were treated with or without 2.5 μM STI571 for 12 hr followed by treatment with the indicated concentration of H2O2 for 3 hr. The fluorescence intensity of cells was assayed by a fluorescence spectrophotometer. The results are expressed as the mean ± SD of three independent experiments. As in flow cytometry, a reduction in the relative fluorescence units (RFU) is indicative of increased degradation of ZsGreen-ODC. (E) MCF-7 cells were treated with 0, 0.5, and 2 mM H2O2 for 3 hr in the presence of 0 or 10 μM MG132. Lysates were analyzed by immunoblotting with anti-c-Abl or anti-β-actin antibody. (F) Left, MCF-7 cells transfected with Flag-PSMA7 were treated with 10 Gy γ-irradiation. Two hours after the treatment, anti-c-Abl immunoprecipitates prepared from lysates were analyzed by immunoblotting with indicated antibodies. Right, MCF-7/ZsGreen-ODC cells were treated with or without 2.5 μM STI571 for 12 hr followed by treatment with 10 Gy γ-irradiation. The fluorescence intensity of cells was assayed by fluorescence spectrophotometer. The results are expressed as the mean ± SD of three independent experiments. Molecular Cell 2006 22, 317-327DOI: (10.1016/j.molcel.2006.04.007) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 6 Expression of PSMA7 Y153F Mutant Induces a G1/S Cell Cycle Arrest (A) Lysates of G1/S-synchronized 293T cells transfected with PSMA7 or PSMA7 Y153F expressing plasmids were analyzed by immunoblotting with antibodies against the cell cycle transition mediator p27, cyclin A, and cyclin E. (B) 293T cells expressing PSMA7 or PSMA7 Y153F mutant were cultured to 80% confluence. Cell cycle was monitored by flow cytometry analysis after DNA staining with propidium iodide. The relative percentages of cells in G1, S, and G2 are as listed at the top of each panel. (C) 293T cells expressing PSMA7 or PSMA7 Y153F mutant were treated with 2 mM thymidine for G1/S synchronization. Cells were collected at the indicated time point after withdrawal of thymidine, and cell cycle was analyzed by flow cytometry. (D) The same experiment as in Figure 6C was carried out for c-abl−/−arg−/− MEFs. Molecular Cell 2006 22, 317-327DOI: (10.1016/j.molcel.2006.04.007) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 7 A Model Depicting the Functional Consequences of c-Abl-Mediated Phosphorylation on Proteasomal Activity Molecular Cell 2006 22, 317-327DOI: (10.1016/j.molcel.2006.04.007) Copyright © 2006 Elsevier Inc. Terms and Conditions