Fortilin binds Ca2+ and blocks Ca2+-dependent apoptosis in vivo.
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 mM 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.
Graidist
Potchanapond
P
Department of Biomedical Sciences, Faculty of Medicine, Prince of Songkla University, Hat-Yai, Songkhla, Thailand, 90110.
Yazawa
Michio
M
Tonganunt
Moltira
M
Nakatomi
Akiko
A
Lin
Curtis Chun-Jen
CC
Chang
Jui-Yoa
JY
Phongdara
Amornrat
A
Fujise
Ken
K
D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D014408:Biomarkers, Tumor / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002118:Calcium / Q000037:antagonists & inhibitors* / Q000378:metabolism* / Q000502:physiology; D002413:Cations, Divalent / Q000378:metabolism; D002460:Cell Line; D015536:Down-Regulation / Q000502:physiology; D007424:Intracellular Fluid / Q000737:chemistry / Q000378:metabolism; D051379:Mice; D011485:Protein Binding / Q000502:physiology; D017433:Protein Structure, Secondary / Q000502:physiology; D015398:Signal Transduction / Q000502:physiology; D000090324:Tumor Protein, Translationally-Controlled 1
D014408:Biomarkers, Tumor; D002413:Cations, Divalent; D000090324:Tumor Protein, Translationally-Controlled 1; D002118:Calcium
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