Expression and Functional Analysis of Apaf-1 Isoforms

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 addi-tional 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 per-formed 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 iso-form, Apaf-1LN, containing the NH 2 -terminal insert, but lacking the additional WD-40 repeat. Functional analy-sis 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 pro-caspase-9, and formation of active Apaf-1 oligomers. These experiments demonstrate the expression of mul-tiple Apaf-1 isoforms and show that only those contain-ing the additional WD-40 repeat bind and activate pro-caspase-9 in response to cytochrome c and dATP.