期刊
JOURNAL OF BIOLOGICAL CHEMISTRY
卷 275, 期 12, 页码 8461-8468出版社
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.275.12.8461
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资金
- NCI NIH HHS [CA-34556] Funding Source: Medline
- NHLBI NIH HHS [2T32HL07517] Funding Source: Medline
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 Il-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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