Fortilin binds Ca2+ and blocks Ca2+-dependent apoptosisin vivo

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 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.

The Translational Controlled Tumour Protein TCTP: Biological Functions and Regulation

“…This result was consolidated and further refined in a very recent study (Lucas et al 2014). TCTP/fortilin has been reported to act as Ca 2+ -scavenger, and this was proposed as a yet another mechanism of its antiapoptotic activity (Graidist et al 2007). Indeed, overexpression of TCTP has been shown to protect cells against Ca 2+ -dependent apoptosis induced by thapsigargin (Graidist et al 2007;Bommer et al 2010), a reagent that inhibits the Ca 2+ -pump of the ER membrane, resulting in a significant increase in cytosolic Ca 2+ -levels.…”

“…Na-K-ATPase C-terminus Na-K-ATPase inhibition Jung et al (2004), Kim et al (2008a) Sorting nexin 6 (SNX6) Na-K-ATPase activation Yoon et al (2006) Ca 2+ N-and C-terminus Ca 2+ -scavenging Feng et al (2007a), Graidist et al (2007), Kim et al (2000) Vitamin-D3 1. Cooperation with other anti-apoptotic proteins.…”

“…Although TCTP does not have a canonical Ca 2+ -binding site , its Ca 2+ -binding activity has been confirmed by several laboratories using a range of methods (Feng et al 2007a;Graidist et al 2007;Kim et al 2000;Lucas et al 2014;Arcuri et al 2004Arcuri et al , 2005Sanchez et al 1997). However, the different approaches used to map the Ca 2+ -binding site at the TCTP protein yielded different results (Feng et al 2007a;Graidist et al 2007;Kim et al 2000). Feng et al demonstrated that it is a weak binding site (Feng et al 2007a); they provided the NMR structure of human TCTP (Fig.…”

“…TCTP/fortilin has been reported to act as Ca 2+ -scavenger, and this was proposed as a yet another mechanism of its antiapoptotic activity (Graidist et al 2007). Indeed, overexpression of TCTP has been shown to protect cells against Ca 2+ -dependent apoptosis induced by thapsigargin (Graidist et al 2007;Bommer et al 2010), a reagent that inhibits the Ca 2+ -pump of the ER membrane, resulting in a significant increase in cytosolic Ca 2+ -levels. The notion of TCTP acting as a Ca 2+ -scavenger, or more generally as a 'buffer-like' molecule for Ca 2+ , is consistent with its high abundance and with the fact that its binding site is rather weak (Lucas et al 2014).…”

Roles of the translationally controlled tumour protein (TCTP) and the double-stranded RNA-dependent protein kinase, PKR, in cellular stress responses

“…Our laboratory has characterized the microtubule-stabilizing activity of TCTP and mapped its tubulin-binding domain (Gachet et al, 1999). Ca þ þ -binding activity has been reported for TCTP by several laboratories; however, there are conflicting reports on the precise localization of the Ca þ þ -binding site (Kim et al, 2000;Feng et al, 2007;Graidist et al, 2007).…”