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 p...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 ki-nase（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.展开更多
基金Supported by the National Natural Science Foundation of China(No.30973274)
文摘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 ki-nase（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.
