Figures & data
Figure 1. Apaf-1 main isoforms translocate to the nucleus and mediate cell-cycle arrest upon genotoxic stress. (A) Schematic diagram of the Apaf-1 isoforms examined in this study. The CARD domain, the CED-4 homology domain and the WD-repeats are indicated. (B) Expression of indicated Apaf-1 constructs in transduced apaf-1−/− MEFs as determined by immunoblotting with an anti-Apaf-1 antibody. (C) Cytochrome c-dependent processing of procaspase-9 by the different Apaf-1 isoforms.Citation35S-labeled procaspase-9 was incubated with S100 extracts of apaf-1−/− MEFs cells expressing the indicated constructs for 30 min at 30°C in the presence or absence of 8 μg/ml cytochrome c and 1 mM dATP. The samples were then analyzed by SDS-PAGE and autoradiography. (D) Nuclear translocation of Apaf-1 isoforms upon DNA damage. WT MEFs or apaf-1−/− MEFs expressing the indicated constructs, were treated or not with cisplatin (20 μM, 24 h), fixed in Triton X-100 and immunolabeled with anti-Apaf-1 antibody (red). Nuclei were stained with DAPI. Numbers indicate percentage of cells with nuclear Apaf-1 (means ± s.e.m.); n = 3. (E, F) All Apaf-1 isoforms complement defective genotoxic stress-induced Chk1 activating phosphorylation and S phase arrest in apaf-1−/− MEFs. (E) WT MEFs or apaf-1−/− MEFs cells expressing the indicated constructs were treated or not with cisplatin (20 μM, 24 h) and the phosphorylation of Chk1 (serine 317) was assayed by western blotting. (F) WT MEFs or apaf-1−/− MEFs cells expressing the indicated constructs were treated or not with cisplatin (20 μM, 24 h) and the S phase arrest was assessed by cell-cycle analysis.
Figure 2. Apaf-1 associates with the nucleoporin Nup107. (A) Apaf-1 nuclear translocation upon genotoxic stress is independent of p53. A549 (p53+/+) and H1299 (p53−/−) cells were treated or not with cisplatin (20 μM, 24 h) and immunolabeled with anti-Apaf-1 antibody (red). Nuclei were stained with DAPI. Numbers indicate percentage of cells with nuclear Apaf-1 (means ± s.e.m.); n = 3. (B) Top: Schematic representation of Apaf-1 XL (1-591), which was used as bait for the yeast 2-hybrid screening. Bottom: Overlapping fragments of the nucleoporin Nup107 (NM_020401) that interact with Apaf-1 in the yeast-2-hybrid assay. Black lines indicate the fragments of Nup107 that interact with Apaf-1. The number of times the fragments were identified in the screen are indicated. The minimal interacting domain is from aa 457 to aa 618. (C) Nup107 (457–618) interacts with the CED-4 domain of Apaf-1. 293T cells were cotransfected with T7-Nup107 (457–618) together with the indicated Apaf-1 Flag-tagged constructs. Cells were lysed and the lysates were subjected to anti-Flag immunoprecipitation (IP) followed by immunoblotting (IB) with anti-T7. The expression of different T7- or Flag-tagged constructs was determined by direct immunoblotting. (D) In vitro interaction of Apaf-1 with Nup107. 35S-labeled Nup107 or Nup107 (457-618) (lane 1, 10% input) were incubated with an equal amount of glutathione Sepharose beads bound to an equal amount of GST (lane 2) or GST- Apaf-1 (87-420) (lane 3). Bound proteins were eluted and analyzed by SDS-PAGE followed by autoradiography. (E) Endogenous Apaf-1 interacts with endogenous Nup107. Whole cell extracts derived from A549 cells treated or not with cisplatin were immunoprecipitated with a control antibody or anti-Apaf-1 antibody and analyzed by immunoblotting with anti-Nup107 antibody.
Figure 3 (See previous page). ATR-dependent Apaf-1 phosphorylation increases its association with Nup107. (A) Apaf-1 is phosphorylated at serine residue(s) following genotoxic stress. Whole cell extracts derived from A549 cells treated or not with cisplatin were immunoprecipitated with an anti-Apaf-1 antibody and analyzed by immunoblotting with anti-phosphoserine, anti-phosphothreonine or anti-Apaf-1 (as an immunoprecipitation control) antibody. (B) The kinase inhibitor staurosporine prevents Apaf-1 nuclear translocation upon genotoxic stress. MDA-MB436 cells were treated or not with cisplatin (20 μM, 24 h) in the presence or absence of staurosporine (4 nM). After fixation, cells were immunolabeled with anti-Apaf-1 antibody (red). Nuclei were stained with DAPI. Numbers indicate percentage of cells with nuclear Apaf-1 (means ± s.e.m.); n = 3. (C) Staurosporine prevents Apaf-1 phosphorylation upon genotoxic stress. Lysates of cells treated as in (B) were immunoprecipitated with anti-Apaf-1 antibody and analyzed by immunoblotting with anti-phosphoserine or anti-Apaf-1 (as an immunoprecipitation control) antibody. (D) Staurosporine reduces Apaf-1-Nup107 interaction. Lysates of cells treated as in (B) were immunoprecipitated with anti-Apaf-1 antibody and analyzed by immunoblotting with anti-Nup107 antibody. (E) Inhibition of ATR drastically reduces Apaf-1 nuclear translocation upon cisplatin treatment. A549 cells were left untreated or were pretreated for 1 h with DMSO, 10 μM NU-7026, 10 μM KU-55933, 10 μM VE-821, 20 μM U0126, 20 μM SP600125, 50 μM LY294002 or 10 μM BI-D1870. Cells were then treated or not with cisplatin (20 μM, 24 h), fixed and nuclear Apaf-1 assessed by immunofluorescence (means ± s.e.m., n = 3). (F) Inhibition of ATR prevents Apaf-1 phosphorylation upon genotoxic stress. A549 cells pretreated for 1 h with vehicle or 10 μM VE-821 were treated or not with cisplatin (20 μM, 24 h), lysed and the lysates immunoprecipitated and analyzed as in (C). (G) Inhibition of ATR reduces Apaf-1-Nup107 interaction. Lysates of cells treated as in (F) were immunoprecipitated with anti-Apaf-1 antibody and analyzed as in (D). (H) Deregulation of ATR prevents Apaf-1 phosphorylation upon genotoxic stress. A549 cells were transfected with mock or ATR specific siRNAs. After 48 h, cells were treated or not with cisplatin (20 μM, 24 h), lysed and the lysates immunoprecipitated and analyzed as in (C). (I) Deregulation of ATR reduces Apaf-1-Nup107 interaction. A549 cells were transfected with mock or ATR specific siRNAs. After 48 h, cells were treated or not with cisplatin (20 μM, 24 h), lysed and the lysates immunoprecipitated with anti-Apaf-1 antibody and analyzed as in (D). (J) Deregulation of ATR prevents Apaf-1 nuclear translocation upon genotoxic stress. A549 cells were transfected with mock or ATR specific siRNAs. After 48 h, cells were treated or not with cisplatin (20 μM, 24 h). After fixation, cells were immunolabeled with anti-Apaf-1 antibody (red). Nuclei were stained with DAPI. Numbers indicate percentage of cells with nuclear Apaf-1 (means ± s.e.m.); n = 3.
Figure 4. Deregulation of Nup107 prevents Apaf-1 role in DNA damage checkpoint. (A) A549 cells transfected with 2 rounds of control or Nup107 siRNAs were harvested and lysed 48 h after second round of siRNA transfection. Cell lysates were analyzed by immunoblotting with anti-Nup107 or anti-β-actin antibody. (B) Silencing of Nup107 prevents Apaf-1 nuclear translocation upon genotoxic stress. A549 cells transfected with 2 rounds of control siRNA or Nup107 siRNAs were treated or not with cisplatin (20 μM, 24 h) 24 h after second round of siRNA transfection. After fixation, cells were immunolabeled with anti-Apaf-1 antibody (red) or anti-Nup107 antibody (green). Nuclei were stained with DAPI. Numbers indicate percentage of cells with nuclear Apaf-1 (means ± s.e.m.); n = 3. (C) Expression of Nup107 (457–618) prevents Apaf-1 nuclear translocation upon genotoxic stress. A549 cells expressing Flag-Nup107 (457-618) were treated or not with cisplatin (20 μM, 24 h), fixed in Triton X-100 and immunolabeled with anti-Apaf-1 antibody (red) or anti-Flag antibody (green). Nuclei were stained with DAPI. Numbers indicate percentage of cells with nuclear Apaf-1 (means ± s.e.m.); n = 3. (D, E) Expression of Nup107 (457-618) prevents S phase arrest and Chk1 activating phosphorylation upon genotoxic stress. (D) A549 cells expressing Flag-Nup107 (457–618) were treated or not with cisplatin (20 μM, 24 h) and the cell-cycle distribution of viable cells was assessed by cell-cycle analysis. (E) A549 cells expressing Flag-Nup107 (457–618) were treated or not with cisplatin (20 μM, 24 h) and the phosphorylation of Chk1 (serine 317) was assayed by protein gel blotting.
