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. 1999 Jan 5;96(1):145–150. doi: 10.1073/pnas.96.1.145

Figure 4.

Figure 4

Direct interaction of CED-4 with drICE and requirement of ATP binding for caspase activation. (A) Schematic representation of CED-4 and its mutants. The amino acid sequence in CED-4 is numbered. Single-letter abbreviations for the amino acid residues are as follows; I, Ile; K, Lys; N, Asn; R, Arg. (B) CED-4 and its mutants were transfected into S2 cells, and their expressions were confirmed by Western blot (data not shown). CED-4 (K165R) suppressed wild-type CED-4-induced caspase activity in S2 cells. Caspase activities were measured and represented as mean ± SD. (C) ATPase inhibitor FSBA blocked CED-4-dependent caspase activity in S2 cells. Cytoplasmic lysates expressing CED-4 were preincubated with various amounts of FSBA, with or without 1 mM ATP at 37°C for 30 min. Caspase activities were then measured (mean ± SD). (D) CED-4 and CED-4 (K165R) coimmunoprecipitate with drICE. His6-tagged drICEmt (C211A) was cotransfected with ced-4 or ced-4 (K165R) into S2 cells, and cell lysates were prepared 24 h after heat shock, immunoprecipitation, and separation by 12.5% SDS/PAGE (see Materials and Methods). Expression of drICE, CED-4, or CED-4 (K165R) in the lysates used for immunoprecipitation experiments is shown (Lower).