The EMBO Journal (2006) 25, 3774–3783, doi:10.1038/sj.emboj.7601263
Published online 3 August 2006
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| Figures and tables |
| MUC1 oncoprotein blocks nuclear targeting of c-Abl in the apoptotic response to DNA damage |
| Deepak Raina, Rehan Ahmad, Shailendra Kumar, Jian Ren, Kiyotsugu Yoshida, Surender Kharbanda and Donald Kufe |
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| Figures |
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Figure 1 Exogenous MUC1 attenuates nuclear targeting of c-Abl in HeLa cells. (A) Lysates from HeLa cells were subjected to immunoblotting with anti-MUC1-N, anti-MUC1-C, anti-c-Abl or anti- -actin. (B, C). HeLa/vector and HeLa/MUC1 cells were treated either with adriamycin (ADR) (B) or cisplatin (CDDP) (C) for 2 h. Nuclear fractions were subjected to immunoblot analysis with anti-c-Abl, anti-lamin B or anti-I B . (D) HeLa/vector and HeLa/MUC1 cells were treated with CDDP for 2 h. Cytosolic extracts were subjected to immunoblot analysis with anti-c-Abl or anti--actin.
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 | Figure 2 c-Abl is targeted to the nucleus by silencing MUC1 in ZR-75-1 cells. (A) Lysates from ZR-75-1/vector and ZR-75-1/MUC1siRNA cells were subjected to immunoblotting with anti-MUC1-N, anti-MUC1-C, anti-c-Abl or anti--actin. (B and C) ZR-75-1/vector and ZR-75-1/MUC1siRNA cells were treated with CDDP for 2 and 4 h. Nuclear (B) and cytosolic (C) fractions were subjected to immunoblot analysis with the indicated antibodies.
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Figure 3 MUC1 forms complexes with c-Abl at the cell membrane. (A, B). Lysates from HeLa/MUC1 (A) and ZR-75-1/vector (B) cells were subjected to immunoprecipitation (IP) with mouse IgG or anti-MUC1-N. The precipitates were immunoblotted with anti-c-Abl or anti-MUC1-N. (C) Cell membrane fractions from HeLa/vector and HeLa/MUC1 cells were subjected to immunoblot analysis with the indicated antibodies. (D) Cell membrane fractions from ZR-75-1/vector or ZR-75-1/MUC1siRNA cells were subjected to immunoblot analysis with the indicated antibodies. Immunoblots from three separate experiments were subjected to densitometric scanning to determine the effects of MUC1 silencing on localization of c-Abl to the cell membrane. The results are expressed as the percentage (mean s.d.) of c-Abl localization for ZR-75-1/vector cells (assigned a value of 100%) and ZR-75-1/MUC1siRNA cells. (E) Confocal microscopy of ZR-75/vector cells stained with anti-MUC1-C or anti-c-Abl. Nuclei were stained with TO-PRO-3.
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 | Figure 4 c-Abl phosphorylates the MUC1 cytoplasmic domain. (A) Lysates from HCT116/vector and HCT116/Flag-MUC1-CD cells were immunoblotted with anti-MUC1-C, anti-c-Abl or anti--actin. (B) Lysates from HCT116/Flag-MUC1-CD cells were immunoprecipitated with mouse IgG or anti-Flag. The precipitates were immunoblotted with anti-c-Abl or anti-Flag. (C) Purified GST-MUC1-CD was cleaved with thrombin to remove the GST tag and then incubated with GST-c-Abl or GST-c-Abl(K-R) and [ -32P]ATP for 15 min at 30°C. The reaction products were analyzed by SDS–PAGE and autoradiography (top panel) or by immunoblotting with anti-MUC1-C (lower panel). (D) Amino-acid sequence of MUC1-CD (upper panel). Purified MUC1-CD or MUC1-CD(Y60F) was incubated with GST-c-Abl or GST-c-Abl(K-R) and [-32P]ATP for 15 min at 30°C. Reaction products were analyzed by SDS–PAGE and autoradiography or by immunoblotting with anti-MUC1-C (lower panels).
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Figure 5 c-Abl-SH2 domain binds to the MUC1 pY60TNP site. (A) Lysates from HCT116/MUC1 (clones A and B) and HCT116/MUC1(Y60F) (clones A and B) were subjected to immunoblotting with anti-MUC1-N, anti-MUC1-C, anti-c-Abl or anti--actin. (B) Lysates from HCT116/MUC-1 and HCT116/MUC1(Y60F) cells were immunoprecipitated with mouse IgG and anti-MUC1-N. The precipitates were immunoblotted with anti-c-Abl, anti-MUC1-N and anti-MUC1-C. (C) Cell membrane fractions from HCT116/vector, HCT116/MUC1 and HCT116/MUC1(Y60F) cells were immunoblotted with the indicated antibodies. (D) 293T cells were transfected with GFP and MUC1, GFP-c-Abl and MUC1, and GFP-c-Abl and MUC1(Y60F). Lysates were subjected to immunoprecipitation with anti-MUC1-N or control IgG and immunoblotting of the precipitates with anti-GFP and anti-MUC1-N. (E) Purified MUC1-CD and MUC1-CD (Y60F) were incubated in the presence or absence of recombinant truncated Abl (no SH2 domain) and 200  M ATP for 30 min at 30°C. GST or GST-c-Abl-SH2 bound to GST-beads was then added and incubated for 1 h at 4°C. Adsorbed (upper panel) and input (lower panel) proteins were subjected to immunoblot analysis with anti-MUC1-C.
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 | Figure 6 MUC1 blocks c-Abl Thr-735 phosphorylation and binding of c-Abl to 14-3-3. (A–C) Lysates from HeLa/vector, HeLa/MUC1 (A), ZR-75-1/vector, ZR-75-1/MUC1siRNA (B), HCT116/vector, HCT116/MUC1 and HCT116/MUC1(Y60F) (C) cells were immunoprecipitated with anti-c-Abl. The precipitates were immunoblotted with anti-phospho-c-Abl-Thr-735 and anti-c-Abl. (D–F) Cytoplasmic fractions of HeLa/vector, HeLa/MUC1 (D), ZR-75-1/vector, ZR-75-1/MUC1siRNA (E), HCT116/vector, HCT116/MUC1 and HCT116/MUC1(Y60F) (F) cells were immunoprecipitated with anti-14-3-3. The precipitates were immunoblotted with anti-c-Abl and anti-14-3-3.
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Figure 7 Binding of MUC1 and c-Abl attenuates DNA damage-induced apoptosis. (A) HCT116/vector, HCT116/MUC1 and HCT116/MUC1(Y60F) cells were treated with CDDP for 2 and 4 h. Nuclear lysates were immunoblotted with anti-c-Abl, anti-lamin B and anti-IB. Intensity of the signals was determined by densitometric scanning and compared to that for untreated cells. (B) Anti-MUC1-N precipitates from HCT116/MUC1 cells treated with CDDP for the indicated times were immunoblotted with anti-c-Abl and anti-MUC1-N. (C) HCT116/vector, HCT116/MUC1 and HCT116/MUC1(Y60F) cells were treated with CDDP for indicated times. Lysates were immunoblotted with anti-PKC and anti--actin. FL: full-length. CF: cleaved fragments. (D) The results from sub-G1 DNA analysis of HCT116/vector, HCT116/MUC1 and HCT116/MUC1(Y60F) cells treated with CDDP for 0 h (open bars), 12 h (solid bars) or 24 h (hatched bars) are expressed as the percentage apoptosis (means.d. of three separate experiments). (E) The results from sub-G1 DNA analysis of HCT116/vector, HCT116/MUC1 and HCT116/MUC1(Y60F) cells left untreated (open bars) or treated with CDDP alone for 12 h (solid bars), STI571 alone for 12 h (shaded bars) or both CDDP and STI571 for 12 h (hatched bars) are expressed as the percentage apoptosis (means.d. of three separate experiments). (F) The results from sub-G1 DNA analysis of HCT116/vector, HCT116/MUC1 and HCT116/MUC1(Y60F) cells transfected with the control CsiRNA or c-AblsiRNA and left untreated (open bars) or treated with CDDP for 12 h (solid bars) are expressed as the percentage of apoptosis (mean+s.d. of three separate experiments). NS, not significant.
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