Real-time Optical Imaging Of EGF-induced Src Signal Kinetics

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 （H₂O₂）-dependent modification in regulating tyrosine phosphorylationsignaling that is induced by growth factors such as epidermal growth factor （EGF） andplatelet-derived growth factor （PDGF）. H₂O₂potentially 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 H₂O₂-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 H₂O₂exposure 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 H₂O₂modulated 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 H₂O₂stimulation. 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 H₂O₂modulated both the amplitude and the duration of the signalby inhibiting PTPs’ activity under EGF simulation. By down-regualting endogenous H₂O₂via 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 H₂O₂has 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 H₂O₂via 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 H₂O₂on Src signaling. Theresults promote understanding of H₂O₂, 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.