摘要
皮层蛋白(cortactin)是一种癌基因蛋白,是酪氨酸磷酸化激酶Src的作用底物,到目前为止,皮层蛋白受磷酸化调控的机制还不清楚。本研究用荧光共振能量转移(FRET)分析皮层蛋白受磷酸化调控后的蛋白质结构变化,推测皮层蛋白受磷酸化调控的可能机制。采用蓝绿色荧光蛋白(CFP)和黄色荧光蛋白(YFP)作为配对,设计和构建基于皮层蛋白羧基端SH3功能的蛋白质传感器,转染NIH/3T3细胞,将细胞以过钒酸盐(PV)和PP2处理,人为诱导或抑制细胞蛋白磷酸化,比较皮层蛋白的磷酸化和非磷酸化状态FRET的不同;进一步观察皮层蛋白磷酸化位点突变后FRET的变化,以此判断皮层蛋白的磷酸化作用后结构的变化。所设计的生物传感器供体和受体工作良好。FRET分析显示,在PP2处理后,FRET能量变化明显较对照组增高,但用PV处理后,FRET下降。皮层蛋白的磷酸化位点突变后,产生的FRET明显较野生型皮层蛋白产生的FRET为弱。因此推测,皮层蛋白在磷酸化后其分子结构处于"展开"状态,并可能以此增强和其他细胞蛋白的相互作用。
Oncoprotein cortactin is a substrate of tyrosine phosphorylation kinase Src. So far it is still not fully understood about the mechanism of phosphorylation of cortactin. The purpose of this study is to probe the conformation change of cortactin in response to phosphorylation. Bio-sensor of cortactin using cyan fluorescence protein （CFP） and yellow fluorescent protein （YFP） was first designed. Cortactin DNA was inserted into plasmid MGIN and transfected NIH/3T3 cells. Expression was examined and analyzed by fluorescence resonance energy transfer （FRET）. Cells were then treated with pervanadate （PV） and PP2. FRET change was recorded before and after treatment. Meanwhile, F-mutant cortactin was in place of wild type cortactin in the biosensor to observe the change of FRET after cell treatment. Biosensor of cortactin worked effectly in cells. The FRET was increased after PP2 treatment of cells and decreased after PV treatment in comparison with the controls. When wild type cortactin was replaced by F-mutant one in the sensor, FRET was attenuated compared to the wild type one. Thus, cortactin molecule might be changed to an ＇unfolded＇ conformation after phosphorylation and more available for cell proteins to interact.
出处
《细胞生物学杂志》
CSCD
2009年第5期689-693,共5页
Chinese Journal of Cell Biology
基金
国家自然科学基金(No.30771126)
教育部回国留学人员启动基金(2008-890)
江苏省自然科学基金(No.BK2006058)资助项目~~
