Fortilin binds Ca2+ and blocks Ca2+-dependent apoptosis in vivo

Potchanapond Graidist, Michio Yazawa, Moltira Tonganunt, Akiko Nakatomi, Curtis Chun Jen Lin, Jui Yoa Chang, Amornrat Phongdara, Ken Fujise

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51 Scopus citations

Abstract

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 wildtype 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.

Original languageEnglish (US)
Pages (from-to)181-191
Number of pages11
JournalBiochemical Journal
Volume408
Issue number2
DOIs
StatePublished - Dec 1 2007

Keywords

  • Apoptosis
  • Ca
  • Cell death
  • Fortilin
  • Thapsigargin

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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    Graidist, P., Yazawa, M., Tonganunt, M., Nakatomi, A., Lin, C. C. J., Chang, J. Y., Phongdara, A., & Fujise, K. (2007). Fortilin binds Ca2+ and blocks Ca2+-dependent apoptosis in vivo. Biochemical Journal, 408(2), 181-191. https://doi.org/10.1042/BJ20070679