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

J Biol Chem. 2001 Mar 23;276(12):9230-8. doi: 10.1074/jbc.M009853200. Epub 2000 Nov 13.


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.

MeSH terms

  • Adaptor Proteins, Signal Transducing*
  • Amino Acid Sequence
  • Apoptosis Regulatory Proteins
  • Apoptosis*
  • Base Sequence
  • Caenorhabditis elegans Proteins*
  • Calcium-Binding Proteins / chemistry
  • Calcium-Binding Proteins / genetics*
  • Carrier Proteins / chemistry
  • Carrier Proteins / genetics*
  • Cell Line
  • Chromosome Mapping
  • Cloning, Molecular
  • DNA, Complementary
  • Helminth Proteins / chemistry
  • Helminth Proteins / genetics*
  • Humans
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • NLR Proteins
  • Protein Isoforms / genetics*
  • RNA, Messenger / genetics
  • Recombinant Fusion Proteins / metabolism
  • Sequence Homology, Amino Acid


  • Adaptor Proteins, Signal Transducing
  • Apoptosis Regulatory Proteins
  • Caenorhabditis elegans Proteins
  • Calcium-Binding Proteins
  • Carrier Proteins
  • Ced-4 protein, C elegans
  • DNA, Complementary
  • Helminth Proteins
  • NLR Proteins
  • NLRP1 protein, human
  • Protein Isoforms
  • RNA, Messenger
  • Recombinant Fusion Proteins

Associated data

  • RefSeq/NP_055737