c-Abl binds directly to Rad9. (A) U-937 cell lysates were incubated with GST, GST-c-Abl SH2, or GST-c-Abl SH3 bound to glutathione beads. The adsorbates were analyzed by immunoblotting (IB) with anti-Rad9 (top) or anti-GST (bottom). (B) Column-purified His-Rad9 and His-Rad9 (1-265) were incubated with glutathione beads containing GST or GST-c-Abl. The adsorbates were analyzed by immunoblotting with anti-Rad9. (C) His-Rad9 (266-391) was incubated with GST or GST-c-Abl SH3 bound to glutathione beads. The adsorbates were subjected to immunoblot analysis with anti-His.

c-Abl-dependent phosphorylation of Rad9 in response to DNA damage. (A) U-937 cells were treated with 10 μM ara-C or 15 Gy of IR and collected at 2 h. Anti-Rad9 immunoprecipitates were analyzed by immunoblotting (IB) with anti-P-Tyr (top) or anti-Rad9 (lower panel). Levels of Rad9 phosphorylation were quantitated by densitometric scanning of the signals. (B) c-abl^+/+, c-abl^−/−, and c-abl⁺ cells were analyzed by immunoblotting with anti-c-Abl (top) or antitubulin (bottom). (C) c-abl^+/+, c-abl^−/−, and c-abl⁺ cells were treated with 10 μM ara-C for the indicated times. Anti-Rad9 immunoprecipitates (IP) were analyzed by immunoblotting with anti-P-Tyr (top) or anti-Rad9 (bottom). (D) U-937 (left) and c-abl⁺ (right) cells were left untreated or treated with 1 μM STI-571 for 24 h followed by treatment with 10 μM ara-C for 2 h. Anti-Rad9 immunoprecipitates were analyzed by immunoblotting with anti-P-Tyr (top) or anti-Rad9 (bottom). (E) 293T cells were transfected with 3 μg of GFP-Rad9 or GFP-Rad9(Y28-F) and treated with 10 μM ara-C for 2 h. Anti-GFP immunoprecipitates were analyzed by immunoblotting with anti-P-Tyr (top) or anti-GFP (bottom).

DNA damage-induced binding of Rad9 to Bcl-x_L is a c-Abl-dependent mechanism. (A) Nuclear extracts from U-937/Bcl-x_L cells were layered onto a 10 to 35% glycerol gradient. After fractionation, the indicated fractions were subjected to SDS-PAGE and immunoblotting (IB) with anti-Rad9 (top) or anti-Bcl-x_L (bottom). (B) U-937/Bcl-x_L cells were treated with 10 μM ara-C or 15 Gy of IR for 2 h. Anti-Rad9 immunoprecipitates (IP) were analyzed by immunoblotting with anti-Bcl-x_L (top) or anti-Rad9 (middle). Cell lysates were also analyzed by immunoblotting with anti-Bcl-x_L (bottom). (C) c-abl^+/+, c-abl^−/−, and c-abl⁺ cells were treated with 10 μM ara-C for 2 h. Cell lysates were immunoprecipitated with anti-Bcl-x_L (top) or anti-Rad9 (second from top). The immunoprecipitates were subjected to immunoblotting with the indicated antibodies. Cell lysates were also subjected to immunoblotting with anti-Rad9 and anti-Bcl-x_L (bottom two blots). (D) c-abl⁺ cells were left untreated or were treated with 1 μM STI-571 for 24 h and then exposed to 10 μM ara-C for 2 h. Anti-Rad9 and preimmune rabbit serum (PIRS) immunoprecipitates were analyzed by immunoblotting with anti-Bcl-x_L (top) or anti-Rad9 (bottom).

Association of c-Abl with Rad9. (A) 293T cells were transfected with 3 μg of pSRαMSV/c-Abl and/or pHM6/Rad9. Transfection efficiency as determined with pcDNA3-LacZ and β-galactosidase staining was 81.7% ± 9.2% (mean ± standard deviation of three independent experiments). Cell lysates were immunoprecipitated with anti-HA (left) or anti-c-Abl (right). Immune complexes were subjected to immunoblotting (IB) with anti-c-Abl and anti-HA. (B) Lysates from U-937 cells were immunoprecipitated with preimmune rabbit serum (PIRS), anti-c-Abl, or anti-Rad9. The precipitates were subjected to immunoblotting with anti-c-Abl (top) or anti-Rad9 (bottom). (C) U-937 cells were treated with 10 μM ara-C or 15 Gy of IR and collected at 2 h. Anti-c-Abl immunoprecipitates were analyzed by immunoblotting with anti-Rad9 (top) or anti-c-Abl (bottom). (D) Nuclear extracts from U-937 cells were layered onto a 10 to 35% glycerol gradient. After fractionation, the indicated fractions were subjected to SDS-PAGE and immunoblotting with anti-c-Abl (top) or anti-Rad9 (bottom).

c-Abl phosphorylates Rad9 on tyrosine 28. (A) Recombinant c-Abl, c-Abl(K-R), and c-Abl with SH3-deleted (c-AblΔSH3) were incubated with [γ-³²P]ATP and GST-Rad9 (top) or GST-Crk (120-225) (bottom). The reaction products were analyzed by SDS-PAGE and autoradiography. (B) GST-Rad9, GST-Rad9(Y28-F) (left), and GST-Rad9 (1-265) (right) were incubated with recombinant c-Abl and [γ-³²P]ATP. GST-Crk (120-225) was used as a positive control. The reaction products were analyzed by SDS-PAGE and autoradiography (top) or Coomassie brilliant blue (CBB) staining (bottom). (C) GST-Rad9 and GST-Crk (120-225) were incubated with recombinant c-Abl in the presence or absence of 50 nM STI-571. The reaction products were analyzed by SDS-PAGE and autoradiography. (D) 293T cells were cotransfected with 3 μg of c-Abl or c-Abl(K-R) and GFP-Rad9 or GFP-Rad9(Y28-F). Anti-GFP immunoprecipitates (IP) were analyzed by immunoblotting (IB) with anti-P-Tyr (top) or anti-GFP (middle). Cell lysates were also subjected to immunoblotting with anti-c-Abl (bottom). Levels of c-Abl expression were quantitated by densitometric scanning of the signals.

Tyrosine phosphorylation of Rad9 by c-Abl induces binding of Rad9 to Bcl-x_L in vitro. (A) Glutathione beads containing GST, GST-Rad9, or GST-Rad9(Y28-F) were incubated with or without recombinant c-Abl and ATP for 30 min. The reaction mixtures were incubated with purified His-Bcl-x_L protein for an additional 1 h. The beads (top) and supernatants (bottom) were analyzed by SDS-PAGE and immunoblotting (IB) with anti-Bcl-x_L. Levels of Bcl-x_L binding to GST-Rad9 were quantitated by densitometric scanning of signals. (B) Recombinant c-Abl and c-Abl(K-R) were incubated with GST-Rad9 or GST-Rad9 (1-265) for 30 min followed by incubation with the purified His-Bcl-x_L protein for 1 h. Reaction products were separated by SDS-PAGE and analyzed by immunoblotting with anti-Bcl-x_L. (C) 293T cells were cotransfected with 1 μg of pcDNA3-Bcl-x_L and 3 μg of c-Abl, c-Abl(K-R), GFP vector, or GFP-Rad9. Anti-GFP immunoprecipitates (IP) were subjected to immunoblotting with anti-Bcl-x_L (top). Cell lysates were also subjected to immunoblotting with anti-GFP (second from top), anti-c-Abl (third from top), or anti-Bcl-x_L (bottom). (D) 293T cells were cotransfected with 1 μg of pcDNA3-Bcl-x_L and 3 μg of c-Abl, GFP-Rad9, or GFP-Rad9(Y28-F). Anti-GFP immunoprecipitates were subjected to immunoblotting with anti-Bcl-x_L (top) or anti-GFP (middle). Cell lysates were also subjected to immunoblotting with anti-Bcl-x_L (lower). Levels of Bcl-x_L binding to GFP-Rad9 were quantitated by densitometric scanning of the signals.

c-Abl regulates Rad9-induced apoptosis in response to DNA damage. (A and B) 293T cells were transfected with 3 μg of c-Abl, c-Abl(K-R), or GFP-Rad9. (A) At 36 h posttransfection, cell lysates were subjected to immunoblot (IB) analysis with anti-c-Abl (top) or anti-GFP (bottom). (B) Cells were also fixed in ethanol and stained with propidium iodide. DNA content was analyzed by flow cytometry. The results (means ± standard deviations [SD] of two experiments, each performed in duplicate) are presented as the percentages of cells with sub-G₁ DNA. (C) c-abl^+/+, c-abl^−/−, and c-abl⁺ cells were infected with a vector control or retrovirus vectors expressing Rad9 or Rad9(Y28-F). At 24 h postinfection, cell lysates were subjected to immunoblot analysis with anti-Rad9 (top). Infection efficiency as determined with pLXSN-GFP was 74.8% ± 3.6% (mean ± SD of three independent experiments). At 24 h postinfection, cells were left untreated or treated with 10 μM ara-C for 12 h and 24 h. The results (means ± SD of three independent experiments) are presented as the percentage of apoptotic cells with sub-G₁ DNA (bottom). wt, wild type. (D) c-abl⁺ cells were left untreated (solid bar) or treated with 1 μM STI-571 (open bar) for 24 h and then exposed 10 μM ara-C for 24 h. The results (means ± SD) of three independent experiments are presented as the percentages of apoptotic cells with sub-G₁ DNA.