Abstract
We report the deduced amino acid sequences of two alternately spliced isoforms, 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 (Mg(2+)-binding site). Like Nod1, but different from Apaf-1, DEFCAP contains a putative regulatory domain containing multiple leucine-rich repeats (LRR). However, a distinguishing feature of the primary sequence of DEFCAP is that DEFCAP contains at its NH(2) terminus a pyrin-like motif and a proline-rich sequence, possibly involved in protein-protein interactions with Src homology domain 3-containing proteins. By using in vitro coimmunoprecipitation experiments, both long and short isoforms were capable of strongly interacting with caspase-2 and exhibited a weaker interaction with caspase-9. Transient overexpression of full-length DEFCAP-L, but not DEFCAP-S, in breast adenocarcinoma cells MCF7 resulted in significant levels of apoptosis. In vitro death assays with transient overexpression of deletion constructs of both isoforms using beta-galactosidase as a reporter gene in MCF7 cells suggest the following: 1) the nucleotide binding domain may act as a negative regulator of the killing activity of DEFCAP; 2) the LRR/CARD represents a putative constitutively active inducer of apoptosis; 3) the killing activity of LRR/CARD is inhibitable by benzyloxycarbonyl-Val-Ala-Asp (OMe)-fluoromethyl ketone and to a lesser extent by Asp-Glu-Val-Asp (OMe)-fluoromethyl ketone; and 4) the CARD is critical for killing activity of DEFCAP. These results suggest that DEFCAP is a novel member of the mammalian Ced-4 family of proteins capable of inducing apoptosis, and understanding its regulation may elucidate the complex nature of the mammalian apoptosis-promoting machinery.
Highlights
Apoptosis is the genetically determined cell suicide program resulting in distinct biochemical and morphological features
In vitro death assays with transient overexpression of deletion constructs of both isoforms using -galactosidase as a reporter gene in MCF7 cells suggest the following: 1) the nucleotide binding domain may act as a negative regulator of the killing activity of DEFCAP; 2) the leucine-rich repeats (LRR)/caspase recruitment domain (CARD) represents a putative constitutively active inducer of apoptosis; 3) the killing activity of LRR/CARD is inhibitable by benzyloxycarbonyl-ValAla-Asp (OMe)-fluoromethyl ketone and to a lesser extent by Asp-Glu-Val-Asp (OMe)-fluoromethyl ketone; and 4) the CARD is critical for killing activity of DEFCAP. These results suggest that DEFCAP is a novel member of the mammalian Ced-4 family of proteins capable of inducing apoptosis, and understanding its regulation may elucidate the complex nature of the mammalian apoptosis-promoting machinery
Cloning of DEFCAP-L from a K562 cDNA Library and Chromosomal Assignment—By homology search using BLASTP for CARD-containing proteins, we identified clone KIAA0926 (GenBankTM accession number NP_055737), which was kindly provided by the Kazusa DNA Research Institute [21]
Summary
Apoptosis (programmed cell death) is the genetically determined cell suicide program resulting in distinct biochemical and morphological features. 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 site).
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