Figure 5. Dimerized caspase‐2 is ubiquitylated in a TRAF2‐dependent manner at K15, K152, and K153, which in turn promotes further TRAF2 binding in a positive feedback loop.

• A
  Casp2pro BiFC cells were treated with 20 μM cisplatin for 24 h in the presence of 10 μM Q‐VD(OMe)‐OPh, followed by GFP‐Trap IP and IB with anti‐ubiquitin or anti‐GFP antibody.
• B
  HeLa cells were treated with 20 μM cisplatin for 24 h in the presence of 10 μM Q‐VD(OMe)‐OPh. Lysates were denatured/renatured and immunoprecipitated with anti‐caspase‐2 antibody or control IgG, followed by IB with anti‐ubiquitin or anti‐caspase‐2 antibody.
• C
  HeLa cells were transfected with TRAF2 siRNA for 24 h, then transfected with Casp2pro‐mVenus for 48 h, followed by GFP‐Trap IP and IB.
• D
  Casp2(C320A)‐mVenus was co‐expressed with the indicated TRAF2 constructs and then pulled down with GFP‐Trap and analyzed by IB.
• E
  In vitro ubiquitylation of recombinant Casp2‐Flag by Myc‐TRAF2 (wild type or ΔRING) purified from HEK293T cells.
• F, G
  Casp2pro‐mVenus wild type and indicated lysine mutants were expressed for 24 h in HEK293T cells, followed by GFP‐Trap IP and IB.
• H
  HEK293T cells were transfected with Casp2(C320A)‐mVenus (wild type or K15/152/153R (3KR) mutant) constructs for 48 h, followed by GFP‐Trap IP and IB.
• I
  HeLa cells were transfected with Casp2pro‐mVenus for 48 h and lysed. Recombinant MBP‐TRAF2 or MBP control proteins were incubated in the lysate for 1 h, followed by amylose pulldown and IB to detect caspase‐2 binding.
• J
  In vitro ubiquitylation was performed as in (E), with recombinant Casp2‐Myc protein and Flag‐TRAF2 (wild type or ΔRING) purified from HEK293T cells. After 3‐h incubation at 37°C (Ub reaction (+)) or on ice (No Ub reaction), the reaction was incubated with anti‐Flag beads. Immunoprecipitated and unbound fractions were analyzed by IB.

Source data are available online for this figure.