MUTATIONAL ANALYSIS OF THE INTERACTING CELL-DEATH REGULATORS CED-9 AND CED-4

The genes ced-3, ced-4 and ced-9 are central components in the cell de ath pathway of the nematode C, elegans, Ced-9, which functions to inhi bit cell death, is homologous to the Bcl-2 family of mammalian anti-ap optotic genes, The ced-3 gene encodes a protein homologous to the casp ases, a family of cysteine proteases involved in the execution of prog rammed cell death, It has recently been demonstrated that CED-4, an in ducer of apoptosis for which no mammalian equivalent has been reported , can interact with CED-9 and Bcl-x(L). Here we confirm that CED-9 and CED-4 interact and using a series of deletion mutants, demonstrate th at only short N-terminal deletions are tolerated in each molecule with out loss-of-interaction, Two loss-of-function point mutations in diffe rent regions of CED-4 also lead to a significant loss of interaction s uggesting further that the relevant interaction domains are not short linear sequences, but rather, are formed by more complex structural de terminants in each molecule, Furthermore, we demonstrate that CED-4 no t only interacts with Bcl-X-L but also with its homologue, Bcl-2, and that the unstructured loop region present in Bcl-x(L) and Bcl-2 can re gulate the CED-4 interaction, Lastly, we show that a BH3 peptide that can inhibit Bcl-2 family interactions also inhibits the interaction be tween Bcl-x(L) and CED-4.