Figure 2

Survivin-ΔEx3 functions as an adaptor. (A) A predicted model for survivin-ΔEx3 in complex with active caspase-3. Caspase-3 catalytic domain (upper panel) is composed of a large (blue) and a small (green) subunit, and binds to survivin-ΔEx3 BIR domain (red). The substrate-binding pocket is shown with an arrow. This model is based on the existing interaction between active caspase-3 and the XIAP BIR2 domain (lower panel). (B) Survivin-ΔEx3 interacts directly through its BIR domain with active caspase-3. Upper, schematic representation of the domain organisation of the recombinant proteins. Green lines indicate caspase-3-binding proteins, while red does not. Lower, bound caspase-3 is analysed by Western blot with an anti-active caspase-3 polyclonal antibody. A Coomassie blue-stained gel shows the expression level of the different mutants. (C) Survivin-ΔEx3 binds to Bcl-2 in vivo, through its BH2 domain. Immunoprecipitations were performed in HeLa transfected with indicated plasmids. (D) Bcl-2 precipitates active caspase-3 in the presence of survivin-ΔEx3. Survivin-ΔEx3-expressing HeLa cells were treated with TNFα (10 ng ml⁻¹) plus cycloheximide (1 μg ml⁻¹) for 2 h. Immunoprecipitations were performed as previously, and were analysed with anti-active caspase-3 antibody. (E) Survivin-ΔEx3 is an essential bridge between Bcl-2 and active caspase-3. HeLa cells were pre-infected with either empty lentivirus (V) or lentivirus stably expressing RNAi against all isoforms of survivin (KO). Apoptosis and immunoprecipitation were performed as described previously.