Abstract 3988: Mst1 Physically Interacts With and Phosphorylates Foxo1, Thereby Stimulating Cell Protective Mechanisms of Foxo1 in the Heart
Mst1 (mammalian sterile 20-like kinase 1) regulates growth and death of cardiomyocytes (CMs) through phosphorylation of multiple substrates, thereby playing an important role in mediating cardiac dysfunction. Mst1 interacts with the FoxO family transcription factors, critical regulators of cell growth and death. We hypothesized that FoxO1 modulates the function of Mst1 in CMs. Co-immunoprecipitation assays showed that Mst1 physically interacts with FoxO1. In vitro kinase assays showed that Mst1 phosphorylates FoxO1. Overexpression of Mst1 induced nuclear translocation of FoxO1 in CMs, as determined by immunostaining. However, Mst1 failed to induce nuclear translocation of a FoxO1 mutant, in which Ser209/215/231/232 at known Mst1 phosphorylation sites are replaced with Ala. Although Mst1 significantly increased apoptosis in CMs, co-expresed FoxO1 significantly reduced Mst1-induced CM apoptosis, and shRNA-mediated knock-down of FoxO1 exacerbated Mst1-induced CM apoptosis (TUNEL-positive nuclei: LacZ=1.3±0.2%, Mst1=13±3%*, Foxo1=9.5±1%*, Mst1+FoxO1=6.4±0.4%*#, Mst1+shFoxO1=23±3%*#, *p<0.05 vs LacZ, #p<0.05 vs Mst1, n=6). Co-overexpression of Mst1 and FoxO1 decreased mRNA expression of FasL and Bim, while it upregulated expression of catalase mRNA, and the ability of FoxO1 to stimulate FasL transcription was attenuated by Mst1. These results suggest that Mst1 stimulates the anti-apoptotic actions and inhibits the pro-apoptotic actions of FoxO1. In order to examine the functional significance of the Mst1/FoxO1 interaction in vivo, transgenic mice (Tg) in which both FoxO1 and Mst1 were overexpressed in the heart were generated. Co-overexpression of FoxO1 significantly improved the left ventricular dysfunction observed in Tg with overexpression of Mst1 alone (ejection fraction: Wild type (WT) = 72±1%, FoxO1-Tg =71±2%, Mst1-Tg=47±3%*, Mst1-FoxO1 bigenic mice= 58±6%*#, *p<0.05 vs WT, #p<0.05 vs Mst1, n=4). In summary, Mst1 induces nuclear translocation of FoxO1 through phosphorylation, thereby inhibiting the pro-apoptotic function of FoxO1 and/or stimulating the cell protective function of FoxO1. Conversely, the presence of FoxO1 negatively affects the function of Mst1, possibly by stimulating a cell protective mechanism.
This research has received full or partial funding support from the American Heart Association, National Center.