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.