Molecular cloning and characterization of DEFCAP-L and -S, two isoforms ofa novel member of the mammalian Ced-4 family of apoptosis proteins

We report the deduced amino acid sequences of two alternately spliced isofo rms, designated DEFCAP-L and -S, that differ in 44 amino acids and encode a novel member of the mammalian Ced-4 family of apoptosis proteins. Similar to the other mammalian Ced-4 proteins (Apaf-1 and Nod1), DEFCAP contains a caspase recruitment domain (CARD) and a putative nucleotide binding domain, signified by a consensus Walker's A box (P-loop) and B box (Mg2+-binding s ite). Like Nod1, but different from Apaf-1, DEFCAP contains a putative regu latory domain containing multiple leucine-rich repeats (I;RR). However, a d istinguishing feature of the primary sequence of DEFCAP is that DEFCAP cont ains at its NH,terminus a pyrin-like motif and a proline-rich sequence, pos sibly involved in protein-protein interactions with Src homology domain 3-c ontaining proteins. By using in vitro coimmunoprecipitation experiments, bo th long and short isoforms were capable of strongly interacting with caspas e-2 and exhibited a weaker interaction with caspase-9, Transient overexpres sion of full-length DEFCAP-L, but not DEFCAP-S, in breast adenocarcinoma ce lls MCF7 resulted in significant levels of apoptosis, lie. vitro death assa ys with transient overexpression of deletion constructs of both isoforms us ing P-galactosidase as a reporter gene in MCF7 cells suggest the following: 1) the nucleotide binding domain may act as a negative regulator of the ki lling activity of DEFCAP; 2) the LRR/CARD represents a putative constitutiv ely active inducer of apoptosis; 3) the killing activity of LRR/CARD is inh ibitable by benzyloxycarbonyl-Val-Ala-Asp (OMe)-fluoromethyl ketone and to a lesser extent by Asp-Glu-Val-Asp (OMe)-fluoromethyl ketone; and 4) the CA RD is critical for killing activity of DEFCAP; These results suggest that D EFCAP is a novel member of the mammalian Ced-4 family of proteins capable o f inducing apoptosis, and understanding its regulation may elucidate the co mplex nature of the mammalian apoptosis-promoting machinery.