title: Molecular cloning and characterization of DEFCAP-L and -S, two isoforms of a novel member of the mammalian Ced-4 family of apoptosis proteins.
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.
authors.affiliation: Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan 48109, USA.
authors.forename: T
authors.initials: T
authors.lastname: Hlaing
authors.forename: R F
authors.initials: RF
authors.lastname: Guo
authors.forename: K A
authors.initials: KA
authors.lastname: Dilley
authors.forename: J M
authors.initials: JM
authors.lastname: Loussia
authors.forename: T A
authors.initials: TA
authors.lastname: Morrish
authors.forename: M M
authors.initials: MM
authors.lastname: Shi
authors.forename: C
authors.initials: C
authors.lastname: Vincenz
authors.forename: P A
authors.initials: PA
authors.lastname: Ward
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins; D001483:Base Sequence; D029742:Caenorhabditis elegans Proteins; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000235:genetics*; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics*; D002460:Cell Line; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D018076:DNA, Complementary; D015801:Helminth Proteins / Q000737:chemistry / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D000070576:NLR Proteins; D020033:Protein Isoforms / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D051017:Apoptosis Regulatory Proteins; D029742:Caenorhabditis elegans Proteins; D002135:Calcium-Binding Proteins; D002352:Carrier Proteins; C089224:Ced-4 protein, C elegans; D018076:DNA, Complementary; D015801:Helminth Proteins; D000070576:NLR Proteins; C427903:NLRP1 protein, human; D020033:Protein Isoforms; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins