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 (K_d) 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.