Effects of phosphorylation sites of FoxO1 on protein synthesis and proteolysis in rat skeletal muscle cells exposure to hypoxia

Abstract

Objective To investigate the effects of different phosphorylation sites of FoxO1 on protein synthesis and proteolysis in rat skeletal muscle L6 cells under the condition of hypoxia. Methods Adenoviral vectors (FoxO1, FoxO1T24A/D, FoxO1S250A/D, and FoxO1AS313A/D) were constructed, and then used to infect the differentiated rat skeletal muscle cells respectively with appropriate multiples of infection (MOI). These L6 cells were corresponding named to 8 groups: EGFP, rFoxO1, T24A, T24D, S250A, S250D, S313A and S313D, and then each transfection group was divided into normoxic group (C) and hypoxic group (H). Adenovirus transfection was observed using a fluorescence confocal microscope. Puromycin-binding peptides, ubiquitin labeling protein, synthesis and decomposition-related proteins were detected using Western blotting. Results Compared with the C-EGFP group, the expression levels of puromycin-binding polypeptide (1.38±0.06), mTOR (1.54±0.21) and p-mTOR (1.57±0.34) were significantly increased in the C-rFoxO1 group (all P 0.05). Conclusion FoxO1 can improve protein synthesis by enhancing the activity of mTOR, but this process is strictly regulated by hypoxia. Under hypoxic condition, FoxO1 inhibits protein synthesis via phosphorylation sites Thr24 and Ser250.

Keywords

• foxo1
• phosphorylation site
• protein synthesis
• protein proteolysis
• sunset