EGF诱导的Src信号动力学的实时光学成像研究
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摘要
蛋白质酪氨酸磷酸化对诸多基本细胞过程起着关键的调控作用，包括细胞生长、粘附、增殖等。因为蛋白质酪氨酸激酶（Protein Tyrosine Kinases, PTKs）与蛋白质酪氨酸磷脂酶（Protein Tyrosine Phosphatases, PTPs）的相互拮抗，所以一个特定蛋白质的酪氨酸磷酸化水平是PTKs和PTPs共同作用的结果。最近研究发现酪氨酸磷酸化过程受双氧水(Hydrogen Peroxide, H_2O_2)介导的氧化还原过程调节。证据显示H_2O_2可以通过氧化PTPs活性中心的半胱氨酸残基而抑制其活性。目前逐渐形成有关H_2O_2在生长因子诱导的酪氨酸磷酸化信号中的作用的假说：PTKs的激活不足以提高蛋白质的酪氨酸磷酸化稳态水平，同时也需要内源性H_2O_2介导的PTPs抑制作用的参与。虽然H_2O_2参与酪氨酸磷酸化信号通路的分子机制已经被广为接受，但是H_2O_2是以何种方式调节酪氨酸磷酸化信号动力学过程及其特点仍不清楚，尤其是缺乏活细胞内的实时动态调节信息；此外，在外源性H_2O_2诱导的氧化应激中，细胞内谷胱甘肽（Glutathione, GSH）氧化还原电势与酪氨酸磷酸化动力学之间的关系至今未见报道。
本研究使用基于荧光蛋白的荧光能量共振转移（Fluorescence Resonance EnergyTransfer, FRET）探针Src探针在活细胞水平对上表皮生长因子（Epidermal GrowthFactor, EGF）诱导的和Src激酶介导的酪氨酸磷酸化过程（简称Src信号）进行了定量研究，发现内源性H_2O_2通过抑制PTPs活性，正向地调节Src信号的峰值强度和持续时间。为了在单个细胞内对Src信号动力学与外源性H_2O_2诱导的细胞GSH氧化还原电势变化动力学进行同步研究，我们发展了基于mVenus/mKOκ FRET对的Src探针和基于单个荧光蛋白的Grx1-roGFP2探针的双分子成像方法，在单个细胞中，实现了Src信号和氧化还原电势信号动力学变化的实时同步成像。研究显示，在外源性H_2O_2诱导下，细胞内GSH氧化还原体系参与负调节EGF诱导的Src信号。
主要研究结果如下：
1）活细胞内，在EGF刺激下，利用Src光学探针，我们得到了Src信号动力学。
结果显示Src信号动力学曲线与用生化方法得到的EGF诱导的细胞外信号调节激酶（Extracellular signal-regulated kinase, ERK）信号动力学曲线类似，通过借鉴用来描述ERK动力学的数学模型和参数，我们引入三个简化的参数来定量描述Src信号动力学：信号峰值、信号持续时间和积分信号强度。并建立了这三个参数与Src活化程度和PTPs活性之间的关联。
2）在活细胞内，发现内源性H_2O_2通过抑制PTPs活性来调节Src的信号峰值和持续时间。通过细胞内表达H_2O_2相关调节基因Rac1-N17和Prx1-Y197F，降低内源H_2O_2水平，结果显示Src的信号峰值和持续时间均大大降低。而PTPs特异抑制剂vanadate能够消除此影响。
3）结果提示EGF诱导的内源性H_2O_2必须局部产生，这样可以避免触发细胞抗氧化防御机制。动力学数据显示这个防御机制不利于Src介导的酪氨酸磷酸化信号。
4）发展了基于黄色荧光蛋白mVenus和橙色荧光蛋白mKOκ的新FRET对，建立了基于mVenus/mKOκ (简称YO) FRET对和单荧光蛋白探针Grx1-roGFP2的双比率实时同步成像新方法。实验结果显示，在进行多色荧光信号双比率实时成像时，无需特殊的算法处理，即可获得双生物分子信号互不干扰的动态信息。
5）设计了基于YO FRET对的Src激酶探针：YO-Src。在单细胞内，对YO-Src和Grx1-roGFP2探针进行同步成像，结果显示，外源H_2O_2对EGF诱导的Src信号起负向调节作用。
本论文，以可视化、定量化的实验方法，在活细胞水平，显示了EGF诱导的Src信号动力学。阐明了内源性和外源性H_2O_2在Src信号动力学中的作用特点，同时增进了对信号分子H_2O_2在细胞中重要作用的理解。本论文建立的荧光信号定量表征分子事件的研究方法，也适用于其它信号途径中分子事件的研究；建立的双比率成像方法，可以广泛地应用到细胞信号转导途径双分子事件时空特性描述中。
Reversible protein tyrosine phosphorylation controls a wide range of fundamentalcellular processes, including cell growth, adhesion, and proliferation. Because proteintyrosine kinases (PTKs) are counteracted by protein tyrosine phosphatases (PTPs), thetyrosine phosphorylation level of a given protein is the result of the concerted action of therelated PTKs and PTPs. Recently, accumulating evidence reveals a critical role ofhydrogen peroxide (H_2O_2)-dependent modification in regulating tyrosine phosphorylationsignaling that is induced by growth factors such as epidermal growth factor (EGF) andplatelet-derived growth factor (PDGF). H_2O_2potentially inhibits activity of PTPs throughoxidizing a cysteine in the active site of PTPs to create a cysteine-sulfenic derivative. Thecurrent paradigm proposes that activation of PTKs is insufficient for elevating thesteady-state level of protein tyrosine phosphorylation and that H_2O_2-mediated inhibition ofPTPs is also required. However, this proposal lacks real-time dynamic evidence in livingcells. Furthermore, the temporal kinetic correlation of tyrosine phosphorylation signlingand GSH redox potential under H_2O_2exposure is also unclear.
In this dissertation, we used a genetically encoded Src kinase-specific biosensor basedon fluorescence resonance energy transfer (FRET) to image the kinetics of theSrc-mediated tyrosine phosphorylation signaling (Src signaling) induced by epidermalgrowth factor (EGF). Through straightforward quantitative analyses method thatcharacterized the signaling kinetics, we demonstrated that H_2O_2modulated both theamplitude and the duration of the signal by inhibiting PTPs’ activity. Furthermore, weestablished a novel combination of FP biosensors for dual-parameter ratiometric imaging,consisting of a new fluorescence resonance energy transfer pair mVenus (yellowFP)/mKOκ (orange FP)-based (abbreviated as YO) biosensor and a single FP-based biosensor. By using this dual-parameter ratiometric imaging approach, we achievedsimultaneous imaging of Src signaling and glutathione (GSH) redox potential in a singlecell. The results showed that the GSH redox system negatively regulate EGF-induced Srcsignaling upon exogenous H_2O_2stimulation. The main results are shown as following:
1) In living cells, we obtained kinetics of EGF-induced Src signaling using Srckinase biosensor. Given that the kinetic profile of Src signaling resembles that of ERK(Extracellular signal-regulated kinase) signaling, we introduced three simplifiedparameter to quantatively describe Src signaling, namely, peak amplitude of signaling(PAS), duration of signaling (DS) and integral signaling strength (ISS). We furthercorrelated those parameter with Src and PTPs’ activity.
2) We found that H_2O_2modulated both the amplitude and the duration of the signalby inhibiting PTPs’ activity under EGF simulation. By down-regualting endogenous H_2O_2via expressing Rac1-N17and Prx1-Y197F protein, results showed that PAS and DS ofSrc signaling are substantially reduced. PTPs specific inhibitor vanadate is able to abolishthis effect.
3) Our results suggested that in the context of EGF-induced signal transductionpathway, EGF-induced H_2O_2has to be locally generated. Otherwise, it triggersantioxidant defenses, leading to attenuation of EGF signal transduction.
4) We developed a novel combination of FP biosensors for dual-parameterratiometric imaging, consisting of a new FRET pair mVenus (yellow FP)/mKOκ (orangeFP)-based (abbreviated as YO) biosensor and a single FP-based biosensor. Resultsshowed that this combination of FP biosensor does not require intensity correcton inliving cell imaging.
5) We designed a Src specific biosensor based on YO FRET pair (named YO-Src).By using this dual-parameter ratiometric imaging approach, we achieved simultaneousimaging of Src signaling and glutathione (GSH) redox potential in a single cell, providing direct evidence that epidermal growth factor (EGF)-induced Src signaling was negativelyregulated by H_2O_2via its effect on GSH-based redox system.
Collectively, we quantitatively visualize the EGF-induced Src signaling in living cells.And we elucidate the effect of endogenous and exogenous H_2O_2on Src signaling. Theresults promote understanding of H_2O_2, which is considered the important signalmolecule. Furthermore, our research approach, which analyzes the FRET-biosensorderived kinetic data with mathematical parameters, can be applied to molecular event inother signal transduction. The dual-molecule ratiometric imaging approach can beextensively applied for the elucidation of the interplay of two molecular events in singleliving cells.
本研究使用基于荧光蛋白的荧光能量共振转移（Fluorescence Resonance EnergyTransfer, FRET）探针Src探针在活细胞水平对上表皮生长因子（Epidermal GrowthFactor, EGF）诱导的和Src激酶介导的酪氨酸磷酸化过程（简称Src信号）进行了定量研究，发现内源性H_2O_2通过抑制PTPs活性，正向地调节Src信号的峰值强度和持续时间。为了在单个细胞内对Src信号动力学与外源性H_2O_2诱导的细胞GSH氧化还原电势变化动力学进行同步研究，我们发展了基于mVenus/mKOκ FRET对的Src探针和基于单个荧光蛋白的Grx1-roGFP2探针的双分子成像方法，在单个细胞中，实现了Src信号和氧化还原电势信号动力学变化的实时同步成像。研究显示，在外源性H_2O_2诱导下，细胞内GSH氧化还原体系参与负调节EGF诱导的Src信号。
主要研究结果如下：
1）活细胞内，在EGF刺激下，利用Src光学探针，我们得到了Src信号动力学。
结果显示Src信号动力学曲线与用生化方法得到的EGF诱导的细胞外信号调节激酶（Extracellular signal-regulated kinase, ERK）信号动力学曲线类似，通过借鉴用来描述ERK动力学的数学模型和参数，我们引入三个简化的参数来定量描述Src信号动力学：信号峰值、信号持续时间和积分信号强度。并建立了这三个参数与Src活化程度和PTPs活性之间的关联。
2）在活细胞内，发现内源性H_2O_2通过抑制PTPs活性来调节Src的信号峰值和持续时间。通过细胞内表达H_2O_2相关调节基因Rac1-N17和Prx1-Y197F，降低内源H_2O_2水平，结果显示Src的信号峰值和持续时间均大大降低。而PTPs特异抑制剂vanadate能够消除此影响。
3）结果提示EGF诱导的内源性H_2O_2必须局部产生，这样可以避免触发细胞抗氧化防御机制。动力学数据显示这个防御机制不利于Src介导的酪氨酸磷酸化信号。
4）发展了基于黄色荧光蛋白mVenus和橙色荧光蛋白mKOκ的新FRET对，建立了基于mVenus/mKOκ (简称YO) FRET对和单荧光蛋白探针Grx1-roGFP2的双比率实时同步成像新方法。实验结果显示，在进行多色荧光信号双比率实时成像时，无需特殊的算法处理，即可获得双生物分子信号互不干扰的动态信息。
5）设计了基于YO FRET对的Src激酶探针：YO-Src。在单细胞内，对YO-Src和Grx1-roGFP2探针进行同步成像，结果显示，外源H_2O_2对EGF诱导的Src信号起负向调节作用。
本论文，以可视化、定量化的实验方法，在活细胞水平，显示了EGF诱导的Src信号动力学。阐明了内源性和外源性H_2O_2在Src信号动力学中的作用特点，同时增进了对信号分子H_2O_2在细胞中重要作用的理解。本论文建立的荧光信号定量表征分子事件的研究方法，也适用于其它信号途径中分子事件的研究；建立的双比率成像方法，可以广泛地应用到细胞信号转导途径双分子事件时空特性描述中。
Reversible protein tyrosine phosphorylation controls a wide range of fundamentalcellular processes, including cell growth, adhesion, and proliferation. Because proteintyrosine kinases (PTKs) are counteracted by protein tyrosine phosphatases (PTPs), thetyrosine phosphorylation level of a given protein is the result of the concerted action of therelated PTKs and PTPs. Recently, accumulating evidence reveals a critical role ofhydrogen peroxide (H_2O_2)-dependent modification in regulating tyrosine phosphorylationsignaling that is induced by growth factors such as epidermal growth factor (EGF) andplatelet-derived growth factor (PDGF). H_2O_2potentially inhibits activity of PTPs throughoxidizing a cysteine in the active site of PTPs to create a cysteine-sulfenic derivative. Thecurrent paradigm proposes that activation of PTKs is insufficient for elevating thesteady-state level of protein tyrosine phosphorylation and that H_2O_2-mediated inhibition ofPTPs is also required. However, this proposal lacks real-time dynamic evidence in livingcells. Furthermore, the temporal kinetic correlation of tyrosine phosphorylation signlingand GSH redox potential under H_2O_2exposure is also unclear.
In this dissertation, we used a genetically encoded Src kinase-specific biosensor basedon fluorescence resonance energy transfer (FRET) to image the kinetics of theSrc-mediated tyrosine phosphorylation signaling (Src signaling) induced by epidermalgrowth factor (EGF). Through straightforward quantitative analyses method thatcharacterized the signaling kinetics, we demonstrated that H_2O_2modulated both theamplitude and the duration of the signal by inhibiting PTPs’ activity. Furthermore, weestablished a novel combination of FP biosensors for dual-parameter ratiometric imaging,consisting of a new fluorescence resonance energy transfer pair mVenus (yellowFP)/mKOκ (orange FP)-based (abbreviated as YO) biosensor and a single FP-based biosensor. By using this dual-parameter ratiometric imaging approach, we achievedsimultaneous imaging of Src signaling and glutathione (GSH) redox potential in a singlecell. The results showed that the GSH redox system negatively regulate EGF-induced Srcsignaling upon exogenous H_2O_2stimulation. The main results are shown as following:
1) In living cells, we obtained kinetics of EGF-induced Src signaling using Srckinase biosensor. Given that the kinetic profile of Src signaling resembles that of ERK(Extracellular signal-regulated kinase) signaling, we introduced three simplifiedparameter to quantatively describe Src signaling, namely, peak amplitude of signaling(PAS), duration of signaling (DS) and integral signaling strength (ISS). We furthercorrelated those parameter with Src and PTPs’ activity.
2) We found that H_2O_2modulated both the amplitude and the duration of the signalby inhibiting PTPs’ activity under EGF simulation. By down-regualting endogenous H_2O_2via expressing Rac1-N17and Prx1-Y197F protein, results showed that PAS and DS ofSrc signaling are substantially reduced. PTPs specific inhibitor vanadate is able to abolishthis effect.
3) Our results suggested that in the context of EGF-induced signal transductionpathway, EGF-induced H_2O_2has to be locally generated. Otherwise, it triggersantioxidant defenses, leading to attenuation of EGF signal transduction.
4) We developed a novel combination of FP biosensors for dual-parameterratiometric imaging, consisting of a new FRET pair mVenus (yellow FP)/mKOκ (orangeFP)-based (abbreviated as YO) biosensor and a single FP-based biosensor. Resultsshowed that this combination of FP biosensor does not require intensity correcton inliving cell imaging.
5) We designed a Src specific biosensor based on YO FRET pair (named YO-Src).By using this dual-parameter ratiometric imaging approach, we achieved simultaneousimaging of Src signaling and glutathione (GSH) redox potential in a single cell, providing direct evidence that epidermal growth factor (EGF)-induced Src signaling was negativelyregulated by H_2O_2via its effect on GSH-based redox system.
Collectively, we quantitatively visualize the EGF-induced Src signaling in living cells.And we elucidate the effect of endogenous and exogenous H_2O_2on Src signaling. Theresults promote understanding of H_2O_2, which is considered the important signalmolecule. Furthermore, our research approach, which analyzes the FRET-biosensorderived kinetic data with mathematical parameters, can be applied to molecular event inother signal transduction. The dual-molecule ratiometric imaging approach can beextensively applied for the elucidation of the interplay of two molecular events in singleliving cells.
引文
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