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J Biol Chem, Vol. 275, Issue 12, 8461-8468, March 24, 2000

Expression and Functional Analysis of Apaf-1 Isoforms
EXTRA WD-40 REPEAT IS REQUIRED FOR CYTOCHROME c BINDING AND REGULATED ACTIVATION OF PROCASPASE-9^*

Mary A. Benedict, Yuanming Hu^§, Naohiro Inohara, and Gabriel Núñez^¶

From the Department of Pathology, Comprehensive Cancer Center and Cellular and Molecular Biology Program, University of Michigan Medical School, Ann Arbor, Michigan 48109

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 NH₂-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 NH₂-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 NH₂-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 NH₂-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.

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^* This work was supported in part by National Institutes of Health Grant CA-64556 (to G. N.) and U. S. Army Medical Research Command Grant DAMD196-609.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Supported by a pre-doctoral fellowship from the U. S. Army Medical Research Command.

^§ Supported by National Institutes of Health Postdoctoral Training Grant 2T32HL07517.

^¶ Recipient of Research Career and Development Award CA-64421 from the National Institutes of Health. To whom correspondence should be addressed. Tel.: 734-764-8514; Fax: 734-647-9654; E-mail: Gabriel.Nunez@umich.edu.

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Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.

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