[TRF1的丝氨酸219磷酸化在细胞周期调控中的重要作用]

[Important role of Ser 219 phosphorylation of TRF1 in regulation of cell cycle].

作者信息

Xu Yi-yi, Lan Jian-ping, Zhu Yuan-yuan

机构信息

The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou 310003, China.

出版信息

Zhejiang Da Xue Xue Bao Yi Xue Ban. 2007 Jul;36(4):325-30. doi: 10.3785/j.issn.1008-9292.2007.04.003.

DOI:10.3785/j.issn.1008-9292.2007.04.003

PMID:17717821

Abstract

OBJECTIVE

To investigate the role of Ser 219 phosphorylation of TRF1 (telomere repeat binding factor 1) in regulation of cell cycle.

METHODS

The mimicking phosphorylation mutant (TRF1S219D-GFP) and the non-phosphorylatable mutant (TRF1S219A-GFP) were constructed; the mutant genes and corresponding proteins were checked by sequencing and Western blot, respectively. Immunofluorescence staining was performed to detect the localization of mutants in HeLa cells. Cell cycle was analyzed by flow cytometry and ATM level was evaluated by immunoblotting.

RESULTS

The mutant genes were verified by direct sequencing and protein expression of GFP-tagged mutants was confirmed by immunoblotting.TRF1S219A-GFP and TRF1S219D-GFP were both localized in telomere of HeLa cells. Moreover, overexpression of TRF1-GFP or TRF1S219A-GFP resulted in an accumulation of HeLa cells in G2/M (P<0.05). The protein level of ATM was increased when overexpression the wide type or mutants.

CONCLUSION

The Ser 219 phosphorylation of TRF1 by ATM could result in cell cycle arrest in G2/M, which is related to overexpression of TRF1.

摘要

目的

研究端粒重复结合因子1（TRF1）的丝氨酸219磷酸化在细胞周期调控中的作用。

方法

构建模拟磷酸化突变体（TRF1S219D-GFP）和不可磷酸化突变体（TRF1S219A-GFP）；分别通过测序和蛋白质印迹法检测突变基因和相应蛋白质。进行免疫荧光染色以检测突变体在HeLa细胞中的定位。通过流式细胞术分析细胞周期，并通过免疫印迹法评估ATM水平。

结果

通过直接测序验证了突变基因，通过蛋白质印迹法确认了GFP标记突变体的蛋白质表达。TRF1S219A-GFP和TRF1S219D-GFP均定位于HeLa细胞的端粒。此外，TRF1-GFP或TRF1S219A-GFP的过表达导致HeLa细胞在G2/M期积累（P<0.05）。过表达野生型或突变体时，ATM的蛋白质水平升高。