The p53 K120E and p53 R280A mutants are impaired for transcription-independent cell death. A, Western blot analysis of Saos-2 cells stably transfected with temperature-sensitive wild type p53 and the p53 K120E and R280A temperature-sensitive mutants for p53-induced proteins p21 and MDM2 and caspase-cleaved products of MDM2 (asterisk), p85 PARP, and caspase 3 after temperature shift of cells from 39 °C (mutant conformation p53) to 32 °C (wild type conformation p53). B, colony formation assay of Saos-2 cells transfected with an expression vector encoding either mitochondrially targeted wt p53 (L-p53 wt) or mutant proteins (L-p53 K120E and L-p53 R280A) compared with parental vector after selection in G418. C, percentage of Saos-2 cells viable after transfection with wild type L-p53, L-p53 K120E, or L-p53 R280A after selection in G418. Cell viability was determined by the GUAVA ViaCount assay. The graph shows the average number of viable cells and S.D. of three independent experiments.

Mutation of glutamic acids 24 and 25 within an electrostatic region of BAK diminishes binding of BAK to p53. A, surface diagram of p53 depicting electropositive (blue) and electronegative (red) areas within p53. Lysine 120, arginine 248, and arginine 280 together form an electropositive area on p53 (lower part of the molecule). B, surface diagram of BAK depicting electropositive (blue) and electronegative (red) regions. Glutamic acids 24 and 25 and aspartic acid 160 together form an electronegative area on BAK (lower part of the molecule). C, binding of in vitro translated p53 to ³⁵S-labeled in vitro translated Bcl-xl and BAX (left panel). In the middle panel, in vitro translated p53 was incubated with ³⁵S-labeled in vitro translated wild type BAK or BAK mutated at glutamic acids 24/25, 32, and 48 to alanine. After interaction, bound complexes were immunoprecipitated with a p53-specific antibody, washed extensively in 0.5% CHAPS buffer, separated by SDS-PAGE, and subjected to autoradiography (middle panel). In the right panel in vitro translated p53 was incubated with the BAK mutants indicated (E24A/E25A and D160A) in 1% CHAPS binding buffer and washed in 1% CHAPS binding buffer. Input lanes contain 1% of ³⁵S-labeled proteins used in the binding reaction. D, molecular model of the interaction of the p53 DBD with full-length BAK. The backbone of the p53 DBD is on the left, colored in yellow, green, orange, and red. The p53 H2 helix is highlighted in red, whereas the L1 loop is highlighted in green. Critical p53 residues in the H2 helix and the L1 and L3 loop involved in the interaction with BAK are shown as ball and stick. The backbone of BAK is colored in green and blue, and BAK residues 25 and 160 are denoted.

BAK-null MEFs are markedly impaired for p53-dependent apoptosis. A, TUNEL analysis of apoptosis in MEFs with wt p53 (columns 1 and 2) or from p53-null littermates (columns 5 and 6) or the BAK-null mouse (columns 3 and 4) infected with retrovirus encoding adenovirus E1A (E1A) or viral vector alone (vector) and treated with adriamycin (0.5 μg/ml) for 24 h. Data depicted are the average of three independent experiments, with S.E. B, Western blot analysis of p53, BAK, E1A, and β-actin control in lysates isolated from the samples depicted in A, treated or untreated with adriamycin (ADR). C, Saos-2-tsp53 and H1299-Tet-On-p53 cells were transfected with BAK-specific siRNA. Saos-2-tsp53 cells were temperature-shifted for 24 h from 39 °C (mutant conformation) to 32 °C (wild type conformation) to induce p53. D, H1299-Tet-On-p53 cells were treated for 3 h with 0.75 μg/μl doxycycline followed by a 20-h treatment with 0.5 μg/μl adriamycin. p53 expression, actin expression, siRNA-mediated knockdown of BAK, and appearance of the caspase-cleaved products of PARP (p85 PARP) and caspase 3 were monitored by Western blot analysis of whole cell lysates. NTC, non-targeting control.

Mutations within the L1 and L3 loop of p53 affect oligomerization of BAK by p53. A, ribbon diagram of the p53 DNA binding domain, showing the proximity of the L1 and L3 loops to the H2 helix. B, BAK oligomerization assays using 20 μg of mitochondria purified from H1299 cells incubated with 0.5 μg of wt p53 or the p53 L1 loop mutants denoted, in the backbone of the core DNA binding domain of p53 (102-292) isolated away from GST using PreScission protease digestion. After incubation and cross-linking with BMH, BAK monomers and oligomers were detected by immunoblotting (IB) using a BAK-specific antibody. Arrows indicate BAK oligomers; open arrowheads indicate BAK monomers; the asterisk indicates a common intramolecular cross-link in BAK. To assess the integrity of purified mitochondria, levels of the mitochondrial (mito) proteins cytochrome c (cyto c), Grp75, Hsp60, and the nuclear protein proliferating cell nuclear antigen (PCNA) were assessed by immunoblotting (right panel). To ensure equal input of recombinant proteins, 0.5 μg of protein were subjected to SDS-PAGE and Coomassie staining (bottom panel). C, BAK oligomerization assays using 20 μg of mitochondria purified from H1299 cells incubated with 0.5 μg of wt p53 or the p53 L3 loop mutant R248Q, in the backbone of the core DNA binding domain of p53 (102-292). After incubation and cross-linking with BMH, BAK monomers and oligomers were detected by immunoblotting using a BAK-specific antibody. To ensure equal input of recombinant proteins, 0.5 μg of protein were subjected to SDS-PAGE and Coomassie staining (bottom panel). D, mitochondria purified from H1299 cells were incubated with 3 μg of wt or mutant recombinant p53 (102-292) proteins for 45 min and spun down, and the supernatant was assayed for released cytochrome c by immunoblotting. As a positive control for cytochrome c release, 3 mm CaCl₂ was added to purified mitochondria. Equal input of recombinant proteins was assessed by SDS-PAGE and Coomassie staining (bottom panel).

Mutations within the H2 α-helix of p53 and Cep-1 impair BAK oligomerization. A, alignment of the p53 H2 helix with the BH3 domains of Bid, BAK, and Bad. Conserved residues are boxed. Residues critical for classical BH3 functions are underlined in the Bid BH3 domain. B, BAK oligomerization assays using 20 μg of mitochondria purified from H1299 cells incubated with 0.5 μg of wt p53 or the p53 mutants denoted in the backbone of GST-p53 (160-318). After incubation and cross-linking with BMH, BAK monomers and oligomers were detected by immunoblotting (IB) using a BAK-specific antibody (left and middle panels). Arrows indicate BAK oligomers; open arrowheads indicate BAK monomers; asterisk indicates a common intramolecular cross-link in BAK detected after the use of BMH. To assess the integrity of purified mitochondria, levels of the mitochondrial (mito) proteins cytochrome c (cyto c), Grp75, Hsp60, and the nuclear protein proliferating cell nuclear antigen (PCNA) were assessed by immunoblotting (right panel). To ensure equal input of recombinant proteins, proteins were subjected to SDS-PAGE and Coomassie staining (bottom panels). C, BAK oligomerization assays using the GST fusion proteins depicted in the backbone of the core DNA binding domain of p53 (102-292), isolated away from GST by digestion with PreScission protease. Controls for the purity of mitochondria are depicted in the right panel. D, alignment of p53 and Cep-1 H2 α-helix. Conserved residues are indicated. Incubation of 0.5 μg of GST-Cep-1 wt and mutant proteins with 20 μg of mitochondria isolated from H1299 cells. Residues (405 and 406) within the Cep-1 α-helix were mutated to alanine. Bak monomers and oligomers were detected by immunoblotting using a Bak-specific antibody. The purity of mitochondrial preparations was detected by immunoblotting using antisera to the proteins indicated (right panel). Input of recombinant proteins was assessed by subjecting proteins to SDS-PAGE and Coomassie staining (bottom panel); although considerably more R405A was used in this experiment, the impairment of this mutant in BAK oligomerization is evident.

Oligomerization of BAK by the isolated p53 DNA binding domain (102-292), C. elegans p53 Cep-1 (220-420), and tumor-derived mutants of p53. A, 20 μg of mitochondria purified from H1299 cells were incubated with 0.25, 0.5, and 1.0 μg of recombinant GST, GST-p53 (102-292), and p53 DNA binding domain alone (102-292). The purity of mitochondrial preparations was detected by immunoblotting (IB) with antisera to controls, cytochrome c (cyto c), GRP75, and Hsp60 (mitochondrial proteins) as well as proliferating cell nuclear antigen (cytosolic/nuclear protein) (right panel). To ensure equal input of recombinant proteins, 0.25, 0.5, and 1.0 μg of protein were subjected to SDS-PAGE and Coomassie staining (bottom panel). B, the indicated concentrations of recombinant GST-p53 and GST-Cep-1 (0.25, 0.5, and 1.0 μg) were incubated with 20 μg of mitochondria purified from H1299 cells and subsequently cross-linked with BMH. BAK oligomers after BMH cross linking are depicted in the left panel; the purity of mitochondrial preparations was confirmed by immunoblotting as described in A. Equal input of recombinant proteins was assessed by SDS-PAGE and Coomassie staining (bottom panel). C, mitochondria purified from H1299 cells were incubated with 1 or 4 μg of wt or mutant recombinant GST-p53 (160-318) proteins for 30 min and subsequently cross-linked with BMH. BAK monomers and oligomers were detected by immunoblotting using a BAK-specific antibody. Arrows indicate BAK oligomers; open arrowheads indicate BAK monomers, and the asterisk indicates an intramolecular cross-link seen with BAK monomers treated with BMH. The purity of mitochondrial preparations was detected by immunoblotting with antisera to controls (right panel). Equal input of recombinant proteins was assessed by SDS-PAGE and Coomassie staining (bottom panel). The slight mobility shift evident in the R175H protein reflects usage of a stop codon within the GST vector rather than in the p53 coding sequence. D, mitochondria purified from H1299 cells were incubated with 3 μg of wt or mutant recombinant GST-p53 (160-318) proteins for 45 min. As a positive control for cytochrome c release, 3 mm CaCl₂ was added to purified mitochondria. Cytochrome c release from mitochondria was detected by immunoblotting using an antibody specific for cytochrome c. Equal input of recombinant proteins was assessed by SDS-PAGE and Coomassie staining (bottom panel). E, Saos-2 cells stably transfected with vector or p53 mutated at codons 175, 273, or 277 were treated with camptothecin for 5 h. In parallel, Saos-2 cells stably transfected with temperature-sensitive p53 were temperature-shifted from 39 °C (mutant conformation p53) to 32 °C (wild type conformation p53). 300 μg of whole cell lysate was immunoprecipitated (IP) with a BAK-specific antibody (BAK-NT, Upstate). Immunoprecipitated complexes were captured with protein G-agarose beads and subjected to SDS-PAGE and Western blotting with a p53 specific antibody (Ab-6, EMD, Biosciences). In the bottom panel whole cell lysates of cells expressing mutant p53 were subjected to Western blotting with a p53-specific antibody (Ab-6, EMD Biosciences) and an actin specific antibody (Ac-15, Sigma) to confirm equal protein loading. F, colony formation assay of Saos-2 cells transfected with an expression vector encoding either mitochondrially targeted wt p53 (L-p53 wt) or mutant proteins (L-p53 R175H, L-p53 R273H, and L-p53 C277F), compared with parental vector following selection in G418.