Fortilin binds and stabilizes MEF2C, activates it through phosphorylation, and drives transcription of the cell structural and survival protein CTNNA3

<h2>Abstract</h2> (words: 246): Fortilin, a 172 amino acid modulator protein that positively regulates survival and growth pathways, is one of the most abundantly expressed proteins in the heart, and its loss leads to lethal heart failure. While fortilin binds and protects catenin alpha-3 (CTNNA3)—a cardiomyocyte structural and survival protein—against degradation, its role in the transcriptional regulation of CTNNA3 has remained unknown. Here, we report that fortilin also promotes CTNNA3 transcription. Mechanistically, we found that fortilin specifically bound to the N-terminal region (amino acids 1–85) of myocyte enhancer factor 2C (MEF2C)—a transcription factor that drives CTNNA3 expression—but not to MEF2A, MEF2B, or MEF2D, under the experimental conditions tested and as shown by microscale thermophoresis, proximity ligation assay, and in vitro and in vivo co-immunoprecipitation western blot analyses. Molecular docking and site-directed mutagenesis identified a critical binding interface involving Asp²⁵ of fortilin, whereby an Asp²⁵-to-alanine mutation markedly weakened binding to MEF2C. Additionally, fortilin protected MEF2C against ubiquitination and proteasomal degradation and promoted MEF2C serine 59 phosphorylation, a modification essential for its transcriptional activity, both in a binding-dependent manner. Loss of fortilin significantly impaired MEF2C binding to nuclear DNA, reduced CTNNA3 promoter-driven luciferase activity in a MEF2C-dependent fashion, and lowered RNA polymerase II occupancy on the CTNNA3 locus. These data suggest that fortilin is a previously unrecognized transcriptional co-factor of MEF2C. By stabilizing MEF2C and promoting its activating phosphorylation, fortilin enhances the transcription of CTNNA3 while simultaneously stabilizing CTNNA3 protein, thereby sustaining CTNNA3 expression and supporting myocardial structural integrity.