E1B 19,000-MOLECULAR-WEIGHT PROTEIN INTERACTS WITH AND INHIBITS CED-4-DEPENDENT, FLICE-MEDIATED APOPTOSIS

Genetic studies of the nematode Caenorhabditis (C. elegans) have ident ified several important components of the cell death pathway, most not ably CED-3, CED-4, and CED-9. CED-4 directly interacts with the Bcl-2 homologue CED-9 (or the mammalian Bcl-2 family member Bcl-x(L)) and th e caspase CED-3 (or the mammalian caspases ICE and FLICE). This trimol ecular complex of CED-4, CED-3, and CED-9 is functional in that CED-9 inhibits CED-4 from activating CED-3 and thereby inhibits apoptosis in heterologous systems. The E1B 19,000-molecular weight protein (E1B 19 K) is a potent apoptosis inhibitor and the adenovirus homologue of Bcl -2-related apoptosis inhibitors. Since E1B 19K and Bcl-X-L, have funct ional similarity, we determined if E1B 19K interacts with CED-4 and re gulates CED-4-dependent caspase activation, Binding analysis indicated that E1B 19K interacts with CED-4 in a Saccharomyces cerevisiae two-h ybrid assay, in vitro, and in mammalian cell lysates. The subcellular localization pattern of CED-4 was dramatically changed by E1B 19K, sup porting the theory of a functional interaction between CED-4 and E1B 1 9K. Whereas expression of CED-4 alone could not induce cell death, coe xpression of CED-4 and FLICE augmented cell death induction by FLICE, which was blocked by expression of E1B 19K. Even though E1B 19K did no t prevent FLICE-induced apoptosis, it did inhibit CED-4-dependent, FLI CE-mediated apoptosis, which suggested that CED-4 was required for E1B 19K to block FLICE activation. Thus, E1B 19K functions through intera cting with CED-4, and presumably a mammalian homologue of CED-4, to in hibit caspase activation and apoptosis.