title: Expression and functional analysis of Apaf-1 isoforms. Extra Wd-40 repeat is required for cytochrome c binding and regulated activation of procaspase-9.
abstract: Apaf-1 is an important apoptotic signaling molecule that can activate procaspase-9 in a cytochrome c/dATP-dependent fashion. Alternative splicing can create an NH(2)-terminal 11-amino acid insert between the caspase recruitment domain and ATPase domains or an additional COOH-terminal WD-40 repeat. Recently, several Apaf-1 isoforms have been identified in tumor cell lines, but their expression in tissues and ability to activate procaspase-9 remain poorly characterized. We performed analysis of normal tissue mRNAs to examine the relative expression of the Apaf-1 forms and identified Apaf-1XL, containing both the NH(2)-terminal and COOH-terminal inserts, as the major RNA form expressed in all tissues tested. We also identified another expressed isoform, Apaf-1LN, containing the NH(2)-terminal insert, but lacking the additional WD-40 repeat. Functional analysis of all identified Apaf-1 isoforms demonstrated that only those with the additional WD-40 repeat activated procaspase 9 in vitro in response to cytochrome c and dATP, while the NH(2)-terminal insert was not required for this activity. Consistent with this result, in vitro binding assays demonstrated that the additional WD-40 repeat was also required for binding of cytochrome c, subsequent Apaf-1 self-association, binding to procaspase-9, and formation of active Apaf-1 oligomers. These experiments demonstrate the expression of multiple Apaf-1 isoforms and show that only those containing the additional WD-40 repeat bind and activate procaspase-9 in response to cytochrome c and dATP.
authors.affiliation: Department of Pathology, Comprehensive Cancer Center and Cellular and Molecular Biology Program, University of Michigan Medical School, Ann Arbor, Michigan 48109, USA.
authors.forename: M A
authors.initials: MA
authors.lastname: Benedict
authors.forename: Y
authors.initials: Y
authors.lastname: Hu
authors.forename: N
authors.initials: N
authors.lastname: Inohara
authors.forename: G
authors.initials: G
authors.lastname: Núñez
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D017398:Alternative Splicing; D017209:Apoptosis / Q000502:physiology; D053477:Apoptotic Protease-Activating Factor 1; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism*; D003574:Cytochrome c Group / Q000378:metabolism*; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000378:metabolism*; D015801:Helminth Proteins; D006801:Humans; D011485:Protein Binding; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D020449:Repetitive Sequences, Amino Acid
chemical_list: C506592:APAF1 protein, human; D053477:Apoptotic Protease-Activating Factor 1; D003574:Cytochrome c Group; D004792:Enzyme Precursors; D015801:Helminth Proteins; D020033:Protein Isoforms; D011506:Proteins; D011994:Recombinant Proteins; C400683:che-2 protein, C elegans; D000255:Adenosine Triphosphate; C506433:CASP9 protein, human; D053453:Caspase 9; D020169:Caspases