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

Jie JIA; Pengyu FU; Rongxin ZHU; Chunyan XU; Lijing GONG

doi:10.16016/j.1000-5404.202003281

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

Basic Medicine | 更新时间：2026-01-09

- Effects of phosphorylation sites of FoxO1 on protein synthesis and proteolysis in rat skeletal muscle cells exposure to hypoxia
- Journal of Third Military Medical University Vol. 42, Issue 15, Pages: 1519-1530(2020)
- 作者机构：
  
  1.100084 北京, 北京体育大学：中国运动与健康研究院
  2.100084 北京, 北京体育大学：运动人体科学学院
  3.200030 上海, 上海体育科学研究所
- 作者简介：
- 基金信息：
  
  the Fundamental Research Fund for the Central Universities of China(2019PT003);the General Program of National Natural Science Foundation of China(31771317)
- DOI：10.16016/j.1000-5404.202003281
  CLC： R322.74;R329.26;R341
- Received：22 March 2020，
  
  Revised：2020-05-15，
  
  Published：15 August 2020
- 稿件说明：
移动端阅览
Jie JIA, Pengyu FU, Rongxin ZHU, et al. Effects of phosphorylation sites of FoxO1 on protein synthesis and proteolysis in rat skeletal muscle cells exposure to hypoxia[J]. Journal of Third Military Medical University, 2020, 42(15): 1519-1530.
DOI：

Jie JIA, Pengyu FU, Rongxin ZHU, et al. Effects of phosphorylation sites of FoxO1 on protein synthesis and proteolysis in rat skeletal muscle cells exposure to hypoxia[J]. Journal of Third Military Medical University, 2020, 42(15): 1519-1530. DOI： 10.16016/j.1000-5404.202003281.

摘要

目的

探究FoxO1不同磷酸化位点在低氧暴露下大鼠L6成肌细胞蛋白质合成和分解中的调节作用。

方法

构建FoxO1过表达、FoxO1

T24A/D

、FoxO1

S250A/D

、FoxO1

S313A/D

位点的质粒，使用腺病毒作为载体，分别感染分化后的大鼠L6骨骼肌细胞6 h，将细胞分别命名为rFoxO1、T24A、T24D、S250A、S250D、S313A、S313D，并对应分为7个转染组，以转染空白对照质粒作为对照组(EGFP)，随后将各转染组再分为常氧组(normoxic group，C)和低氧组(hypoxic group，H)。采用荧光共聚焦显微镜观察腺病毒转染情况；采用Western blot对嘌呤霉素(puromycin)结合多肽进行分析，并对泛素(ubiquitin)标记蛋白和分解相关蛋白表达进行检测。

结果

与C-EGFP组相比，C-rFoxO1组puromycin结合多肽(1.38±0.06)、mTOR(1.54±0.21)和p-mTOR(1.57±0.34)表达显著增加(

0.05)。与C-EGFP组相比，C-T24D组mTOR(1.

33±0.35)和p-mTOR(1.60±0.44)表达显著上升(

0.01)；与C-T24D组相比，H-T24D组mTOR表达显著降低(

0.01)。与C-EGFP组相比，C-S250A组蛋白质积累显著上升(

0.01)，与C-S250A组相比，H-S250A组蛋白质积累显著下降(

0.05)。与EGFP组相比，S313A和S313D组蛋白质积累差异无统计学意义(

0.05)。

结论

FoxO1通过上调mTOR活性来增加蛋白质合成，但受低氧调控；低氧下FoxO1经Thr24和Ser250两个位点磷酸化来抑制机体中蛋白质合成。

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

FoxO1

T24A/D

FoxO1

S250A/D

and FoxO1

AS313A/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

0.05). The levels of mTOR (1.33±0.35) and p-mTOR (1.60 ± 0.44) were also elevated in the C-T24D group than the C-EGFP group (

0.01). The level of mTOR in the H-T24D group was notably reduced when compared with the C-T24D group (

0.01). Protein accumulation was remarkably increased in the C-S250A group than the C-EGFP group

(

0.01)

and was statistically decreased in the H-S250A group than the C-S250A group (

0.05). But no such differences were seen in the protein accumulation in the S313A and S313D groups when compared with that in the C-EGFP group (

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.

关键词

Keywords

references

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