Fortilin, a 172-amino-acid polypeptide present both in the cytosol and nucleus, possesses potent anti-apoptotic activity. Although fortilin is known to bind Ca2+, the biochemistry and biological significance of such an interaction remains unknown. In the present study we report that fortilin must bind Ca2+ in order to protect cells against Ca2+-dependent apoptosis. Using a standard Ca2+-overlay assay, we first validated that full-length fortilin binds Ca2+ and showed that the N-terminus (amino acids 1–72) is required for its Ca2+-binding. We then used flow dialysis and CD spectropolarimetry assays to demonstrate that fortilin binds Ca2+ with a dissociation constant (Kd) of approx. 10 μM and that the binding of fortilin to Ca2+ induces a significant change in the secondary structure of fortilin. In order to evaluate the impact of the binding of fortilin to Ca2+in vivo, we measured intracellular Ca2+ levels upon thapsigargin challenge and found that the lack of fortilin in the cell results in the exaggerated elevation of intracellular Ca2+ in the cell. We then tested various point mutants of fortilin for their Ca2+ binding and identified fortilin(E58A/E60A) to be a double-point mutant of fortilin lacking the ability of Ca2+-binding. We then found that wild-type fortilin, but not fortilin(E58A/E60A), protected cells against thapsigargin-induced apoptosis, suggesting that the binding of fortilin to Ca2+ is required for fortilin to protect cells against Ca2+-dependent apoptosis. Together, these results suggest that fortilin is an intracellular Ca2+ scavenger, protecting cells against Ca2+-dependent apoptosis by binding and sequestering Ca2+ from the downstream Ca2+-dependent apoptotic pathways.
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December 2007
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Research Article|
November 14 2007
Fortilin binds Ca2+ and blocks Ca2+-dependent apoptosis in vivo
Potchanapond Graidist;
Potchanapond Graidist
*Department of Biomedical Sciences, Faculty of Medicine, Prince of Songkla University, Hat-Yai, Songkhla, Thailand, 90110
†Brown Foundation Institute of Molecular Medicine for the Prevention of Human Diseases, Houston, TX 77030, U.S.A.
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Michio Yazawa;
Michio Yazawa
‡Faculty of Advanced Life Science, Division of Cellular Life Science, Hokkaido University, Sapporo, Japan, 060-0810
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Moltira Tonganunt;
Moltira Tonganunt
†Brown Foundation Institute of Molecular Medicine for the Prevention of Human Diseases, Houston, TX 77030, U.S.A.
§Center for Genomics and Bioinformatics Research, Faculty of Science, Prince of Songkla University, Hat-Yai, Songkhla, Thailand, 90112
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Akiko Nakatomi;
Akiko Nakatomi
‡Faculty of Advanced Life Science, Division of Cellular Life Science, Hokkaido University, Sapporo, Japan, 060-0810
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Curtis Chun-Jen Lin;
Curtis Chun-Jen Lin
†Brown Foundation Institute of Molecular Medicine for the Prevention of Human Diseases, Houston, TX 77030, U.S.A.
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Jui-Yoa Chang;
Jui-Yoa Chang
†Brown Foundation Institute of Molecular Medicine for the Prevention of Human Diseases, Houston, TX 77030, U.S.A.
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Amornrat Phongdara;
Amornrat Phongdara
§Center for Genomics and Bioinformatics Research, Faculty of Science, Prince of Songkla University, Hat-Yai, Songkhla, Thailand, 90112
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Ken Fujise
Ken Fujise
1
†Brown Foundation Institute of Molecular Medicine for the Prevention of Human Diseases, Houston, TX 77030, U.S.A.
∥Division of Cardiology, Department of Internal Medicine, Medical School, University of Texas Health Science Center at Houston, Houston, TX 77030, U.S.A.
¶St. Luke's Episcopal Hospital, Houston, TX 77030, U.S.A.
1To whom correspondence should be addressed (email Kenichi.Fujise@uth.tmc.edu).
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Publisher: Portland Press Ltd
Received:
May 22 2007
Revision Received:
July 30 2007
Accepted:
August 17 2007
Accepted Manuscript online:
August 17 2007
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© The Authors Journal compilation © 2007 Biochemical Society
2007
Biochem J (2007) 408 (2): 181–191.
Article history
Received:
May 22 2007
Revision Received:
July 30 2007
Accepted:
August 17 2007
Accepted Manuscript online:
August 17 2007
Citation
Potchanapond Graidist, Michio Yazawa, Moltira Tonganunt, Akiko Nakatomi, Curtis Chun-Jen Lin, Jui-Yoa Chang, Amornrat Phongdara, Ken Fujise; Fortilin binds Ca2+ and blocks Ca2+-dependent apoptosis in vivo. Biochem J 1 December 2007; 408 (2): 181–191. doi: https://doi.org/10.1042/BJ20070679
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