缺氧状态下丝裂原活化蛋白激酶磷酸酶1对缺氧诱导因子1α和p300在体外结合的影响

目的探讨丝裂原活化的蛋白激酶磷酸酶1（MKP-1）对缺氧诱导因子1α（HIF-1α）和p300在体外结合的影响。方法采用DNA重组技术构建含HIF-1α反式激活域（TAD）的原核表达载体并进行原核表达;利用谷胱甘肽-琼脂糖珠子纯化原核表达的谷胱甘肽S转移酶（GST）融合蛋白;利用脂质体将含MKP-1的正义真核表达载体和空载体转入SGC7901细胞;借助Pull-down试验和Western印迹检测MKP-1或PD98059对HIF-1d与协同激活因子p300在体外结合的影响;Western印迹检测MKP-1对p300表达的影响。结果（1）成功构建了含HIF-1αTAD的原核表达载体,使含HIF-1αTAD的GST融合蛋白在BL-21大肠杆菌中得到表达。（2）利用谷胱甘肽-琼脂糖珠子纯化得到了含HIF-1αTAD的GST融合蛋白。（3）缺氧12h后,GST融合蛋白HIF-1αTAD从PD98059处理细胞裂解液中洗脱的p300量低于从二甲基亚砜（DMSO）处理细胞和SGC7901细胞裂解液中洗脱的p300。（4）分别将MKP-1正义真核表达载体和pcDNA3．1空载体瞬时转入SGC7901细胞;缺氧12h后,GST融合蛋白从MKP-1正义真核表达载体转染细胞（SGC7901-MKP-1）裂解液中洗脱的p300的量低于从空载体转染细胞（SGC7901-空载体）和未转染细胞（SGC7901）裂解液中洗脱的p300。（5）缺氧12h后,p300的表达量在SGC7901-MKP-1、SGC7901-空载体和SGC79013组细胞中无变化。结论MKP-1抑制HIF-1α和p300在体外结合。

Inhibition of the in vitro interaction between hypoxia inducible factor 1alpha and p300 by mitogen-activated protein kinase phosphatase 1

OBJECTIVE: To investigate the effects of mitogen-activated protein kinase phosphatase-1 (MKP-1) on the interaction between hypoxia-inducible factor (HIF)-1alpha and coactivator p300. METHODS: Prokaryotic expression vector PGEX-4T1 was constructed based on DNA recombination technology. GST fusion proteins were purified by glutathione-agarose beads and transfected into Eschericha coli BL-21. Effects of MKP-1 or PD98059 on in vitro interaction between HIF-1alpha and p300 was determined by Pull-down assay coupled with Western bloting. Effects of MKP-1 on the p300 expression level was detected using Western bloting. RESULTS: (1) GST fusion proteins were expressed in BL-21 Escherichia coli and purified proteins were obtained. (2) Less amount of p300 was pulled down by GST fusion protein from SGC7901-MKP-1 cells than from control cells after 12 hours of exposure to hypoxia. (3) 12 hours after hypoxia, Less amount of p300 was pulled down by GST fusion protein from PD98059 treated cells than from DMSO treated cells or SGC7901 cells. (4) There were no significant differences in p300 expression levels between recombinant MKP-1 transfected cells and control cells after 12 hours of exposure to hypoxia. CONCLUSION: MKP-1 inhibits the in vitro interaction between HIF-1alpha and p300.