FRET analysis of protein tyrosine kinase c-Src activation mediated via aryl hydrocarbon receptor.
FRET analysis of protein tyrosine kinase c-Src activation mediated via aryl hydrocarbon receptor.
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通过芳基烃受体介导的蛋白酪氨酸激酶C-SRC激活的FRET分析。
DOI：
10.1016/j.bbagen.2010.11.007
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发表时间：
2011-04
影响因子：
3
通讯作者：
Vogel, Christoph Franz Adam
中科院分区：
文献类型：
--
作者：
Dong, Bin;Cheng, Wei;Li, Wen;Zheng, Jie;Wu, Dalei;Matsumura, Fumio;Vogel, Christoph Franz Adam
蛋白质酪氨酸激酶c-Src（c-Src激酶）在暴露于环境污染物2，3，7，8-四氯二苯并-对-二恶英（TCDD）诱导下的激活已在多种细胞类型中显示。大多数先前的工作使用Western印迹分析来检测激活c-Src激酶的Tyr 416残基上的磷酸化。在这里，我们比较了C-Src酪氨酸磷酸化的结果，通过芳烃受体（AhR）依赖的机制，从蛋白质印迹分析与荧光共振能量转移（FRET）测定检测C-Src激活后，用TCDD激活AhR在两种不同的人类细胞类型的治疗。Western blot分析显示TCDD在HepG 2和MCF-10A细胞中对c-Src的磷酸化具有时间依赖性。从FRET分析的数据可视化和量化的c-Src激酶的激活诱导的TCDD在两种细胞类型的活细胞。在TCDD处理后5 min，FRET效率下降了20%，并继续下降，直到实验结束，TCDD处理后25 min。PP 2是一种c-Src特异性抑制剂，可抑制TCDD和表皮生长因子（EGF）诱导的c-Src活化。相反，AhR拮抗剂3 '-甲氧基-4'-硝基黄酮（MNF）仅阻断TCDD诱导的c-Src活化，而不阻断EGF诱导的c-Src活化。目前的研究表明，使用FRET分析可以在活细胞中观察到c-Src通过EGF和AhR信号通路的早期激活，这与蛋白质印迹分析一致。FRET检测提供了一个有用的工具，可视化和定量c-Src激酶激活通过AhR在活细胞中。
Activation of the protein tyrosine kinase c-Src (c-Src kinase) induced by the exposure to the environmental pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has been shown in various cell types. Most previous works used Western blot analysis to detect the phosphorylation on the Tyr416 residue, which activates c-Src kinase. Here we compared results of c-Src tyrosine phosphorylation via aryl hydrocarbon receptor (AhR)-dependent mechanisms from Western blot analysis with fluorescent resonance energy transfer (FRET) assay detecting c-Src activation after treatment with TCDD to activate AhR in two different human cell types. Western blot analyses show time-dependent phosphorylation of c-Src by TCDD in HepG2 and MCF-10A cells. Data from FRET assay visualized and quantified activation of c-Src kinase induced by TCDD in living cells of both cell types. The FRET efficiency decreased by 20%, 5 min after TCDD treatment and continued decreasing until the end of the experiment, 25 min after TCDD treatment. PP2, a c-Src specific inhibitor, suppressed both TCDD- and epidermal growth factor- (EGF) induced c-Src activation. In contrast, the AhR antagonist 3’-methoxy-4’nitroflavone (MNF) blocked only TCDD- but not EGF-induced activation of c-Src. The current study shows that early activation of c-Src via EGF and AhR signaling pathways can be visualized in living cells using FRET assay which is in line with Western blot analysis. The FRET assay provides a useful tool to visualize and quantify c-Src kinase activation via AhR in living cells.
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DOI：
10.1073/pnas.0701764104
发表时间：
2007-05-22
影响因子：
11.1
作者：
Fritsche, Ellen;Schaefer, Claudia;Krutmann, Jean
通讯作者：
Krutmann, Jean
影响因子：
4.8
作者：
Vogel, CFA;Sciullo, E;Matsumura, F
通讯作者：
Matsumura, F
影响因子：
3.6
作者：
Blankenship, A;Matsumura, F
通讯作者：
Matsumura, F
影响因子：
3.6
作者：
Liu, Xueqing;Jefcoate, Colin
通讯作者：
Jefcoate, Colin
影响因子：
--
作者：
Vogel, Christoph F. A.;Sciullo, Eric;Matsumura, Fumio
通讯作者：
Matsumura, Fumio