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高等学校化学研究 ›› 2011, Vol. 27 ›› Issue (6): 988-991.

• Articles • 上一篇    下一篇

Serine659 in ClC-2—Target Site for Phosphorylation by MAPK

ZHAO Jing1, ZHENG Ya-juan2, LI Gui-rong2, CHEN Jie2, YU Qian1* and XIN Hua1*   

  1. 1. China-Japan Union Hospital of Jilin University, Changchun 130031, P. R. China;
    2. The Second Hospital of Jilin University, Changchun 130041, P. R. China
  • 收稿日期:2011-08-17 修回日期:2011-10-09 出版日期:2011-11-25 发布日期:2011-11-07
  • 通讯作者: YU Qian;XIN Hua E-mail:yuq_zrly@yahoo.com.cn; xinhua7254@yahoo.com.cn
  • 基金资助:

    Supported by the National Natural Science Foundation of China(No.30973274).

Serine659 in ClC-2—Target Site for Phosphorylation by MAPK

ZHAO Jing1, ZHENG Ya-juan2, LI Gui-rong2, CHEN Jie2, YU Qian1* and XIN Hua1*   

  1. 1. China-Japan Union Hospital of Jilin University, Changchun 130031, P. R. China;
    2. The Second Hospital of Jilin University, Changchun 130041, P. R. China
  • Received:2011-08-17 Revised:2011-10-09 Online:2011-11-25 Published:2011-11-07
  • Contact: YU Qian;XIN Hua E-mail:yuq_zrly@yahoo.com.cn; xinhua7254@yahoo.com.cn
  • Supported by:

    Supported by the National Natural Science Foundation of China(No.30973274).

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摘要： In order to further investigate the role of ClC-2(ClC=chloride-ion channel) played in the regulation of cell proliferation and differentiation, the capablity of ClC-2 phosphorylation catalyzed by mitogen-activated protein kinase(MAPK) was studied. A mutation of 659Ser to Ala(S659A) of the rabbit ClC-2 cDNA in the consensus sequence of MAPK phosphorylation was introduced by overlap extension polymerase chain reaction(PCR). Recombinant vectors pGEX-4T-1/ClC-2-2CT and pGEX-4T-1/ClC-2CT(S659A) were constructed. They were transformed to E. coli BL21, expressed by isopropy-β-D-thiogalactoside(IPTG) induction, the recombinant proteins were subjected to purification by glutathione sepharose 4B affinity chromatography. In vitro phosphorylation of the fusion proteins catalyzed by MAPK was performed. The results show that fusion protein GST/ClC-2CT(wild type) can be phosphorylated by MAPK, and this phosphorylation can be restrained by the inhibitor p42/44MAPK, PD98095; while the phosphorylation level of fusion protein GST/ClC-2CT(S659A)(mutant) was significantly reduced. Therefore, ClC-2 can be phosphorylated by MAPK and the target site of the phosphorylation is most likely the 659Ser residue.

关键词: ClC-2 channel, Fusion protein, Mitogen-activated protein kinase(MAPK), In vitro phosphorylation

Abstract: In order to further investigate the role of ClC-2(ClC=chloride-ion channel) played in the regulation of cell proliferation and differentiation, the capablity of ClC-2 phosphorylation catalyzed by mitogen-activated protein kinase(MAPK) was studied. A mutation of 659Ser to Ala(S659A) of the rabbit ClC-2 cDNA in the consensus sequence of MAPK phosphorylation was introduced by overlap extension polymerase chain reaction(PCR). Recombinant vectors pGEX-4T-1/ClC-2-2CT and pGEX-4T-1/ClC-2CT(S659A) were constructed. They were transformed to E. coli BL21, expressed by isopropy-β-D-thiogalactoside(IPTG) induction, the recombinant proteins were subjected to purification by glutathione sepharose 4B affinity chromatography. In vitro phosphorylation of the fusion proteins catalyzed by MAPK was performed. The results show that fusion protein GST/ClC-2CT(wild type) can be phosphorylated by MAPK, and this phosphorylation can be restrained by the inhibitor p42/44MAPK, PD98095; while the phosphorylation level of fusion protein GST/ClC-2CT(S659A)(mutant) was significantly reduced. Therefore, ClC-2 can be phosphorylated by MAPK and the target site of the phosphorylation is most likely the 659Ser residue.

Key words: ClC-2 channel, Fusion protein, Mitogen-activated protein kinase(MAPK), In vitro phosphorylation