Novel fluorescent biosensors to continuously visualize real-time protein phosphorylation in live cells

Project SummaryProtein phosphorylation plays an essential role in orchestrating the myriad aspects of cell functionKinaseswhich phosphorylate proteinsare encoded byof the human genome and are critically involved in diverse conditions such as cancermetabolic disorders and neurodegenerationCurrent scientific tools for detecting cellular phosphorylation are limitedbecause most tools examine this process at a single time pointor in cell lysatesor are restricted to single kinasesTools that enable detection of phosphorylation continuously in real time within living cells and are adaptable to multiple kinases would be a significantly improved methodLucidicor Inchas developed such a methodThe technologyPhosFluorTMis an extensively engineered protein that fluoresces robustly only when phosphorylatedTo our knowledgethis is the only fluorescent protein to date with this propertyBy engineering substrate recognition sites into PhosFluorthis molecule can be converted into a biosensor for the activity of virtually any protein kinaseUsing PhosFluorwe have already created and validated biosensors for Protein Kinase APKAcyclindependent kinasescdksand Src family kinasesAchange in the fluorescence of PhosFluor biosensors is observed when cells are stimulated physiologicallyBy contrastdual color fluorescent protein biosensors that utilize FRETF rster Resonance Energy Transferexhibit a smallchange in overall fluorescence during a physiological stimulusThe higher responsiveness of PhosFluor greatly optimizes fluorescence detection and signal noisewhich are essential for consistently accurate measurements within cellsWe envision that the utility of PhosFluor will be greatly enhanced if it is integrated with other methodsSpecificallyintegration of PhosFluor with advanced microscopy would allow precise visualization and measurement of the spatio temporal action of kinasesIntegration with viral transduction would permit analyses of kinase activity in diverse cell typeswhich in turn has potential in diagnostics or drug discoveryTo accomplish these goalsthe following Aims are proposedDetermine the limits of correlation between the phosphorylation state and fluorescence of PhosFluorDemonstrate that PhosFluor can be developed into a broad set of biosensors for detecting phosphorylationandDevelop viral transduction method for introducing PhosFluorbiosensors into cellsIn Phase IIwe plan to extend the utility of our biosensors to neuronsprimary cellsand human specimensPhosFluorandapos s low background and high signal noise lends itself to high throughput applicationssuch as the capture of information in microscopy based high content screeningCollectivelythis will enable creation of a kinome toolbox that can be used by basic and clinical investigatorsand the pharmaceutical industry Public Health Relevance Statement Protein phosphorylation is a key biochemical reaction that underlies serious medical conditions such as cancerdementia and metabolic disordersOur technology is capable of monitoring protein phosphorylation continually in living cellsThe goal of this application is to further develop our technology to facilitate accurate measurements in a wide range of cellswhich in turnaccelerates research into disease mechanismsor supports patient care as a companion diagnostic