Protein phosphorylation plays an essential role in orchestrating the myriad aspects of cell function. Kinases, which phosphorylate proteins, are encoded by 2% of the human genome and are critically involved in diverse conditions such as cancer, metabolic disorders and neurodegeneration. Current scientific tools for detecting cellular phosphorylation are limited, because most tools examine this process at a single time point, or in cell lysates, or are restricted to single kinases. Tools that enable detection of phosphorylation continuously in real time within living cells and are adaptable to multiple kinases would be a significantly improved method. Lucidicor Inc. has developed such a method. The technology, PhosFluorTM, is an extensively engineered protein that fluoresces robustly only when phosphorylated. To our knowledge, this is the only fluorescent protein to date with this property. By engineering substrate recognition sites into PhosFluor, this molecule can be converted into a biosensor for the activity of virtually any protein kinase. Using PhosFluor, we have already created and validated biosensors for Protein Kinase A (PKA), cyclin- dependent kinases (cdks), and Src-family kinases. A 700% change in the fluorescence of PhosFluor biosensors is observed when cells are stimulated physiologically. By contrast, dual-color fluorescent protein biosensors that utilize FRET (Förster Resonance Energy Transfer) exhibit a small 25-30% change in overall fluorescence during a physiological stimulus. The higher responsiveness of PhosFluor greatly optimizes fluorescence detection and signal/noise, which are essential for consistently accurate measurements within cells. We envision that the utility of PhosFluor will be greatly enhanced if it is integrated with other methods. Specifically, integration of PhosFluor with advanced microscopy would allow precise visualization and measurement of the spatio-temporal action of kinases. Integration with viral transduction would permit analyses of kinase activity in diverse cell types, which in turn has potential in diagnostics or drug discovery. To accomplish these goals, the following Aims are proposed: #1-Determine the limits of correlation between the phosphorylation state and fluorescence of PhosFluor; #2-Demonstrate that PhosFluor can be developed into a broad set of biosensors for detecting phosphorylation; and #3-Develop viral transduction method for introducing PhosFluor. biosensors into cells. In Phase II, we plan to extend the utility of our biosensors to neurons, primary cells, and human specimens. PhosFluor's low background and high signal/noise lends itself to high throughput applications, such as the capture of information in microscopy-based high content screening. Collectively, this will enable creation of a kinome toolbox that can be used by basic and clinical investigators, and the pharmaceutical industry.
Public Health Relevance Statement: Public Health Relevance Statement Protein phosphorylation is a key biochemical reaction that underlies serious medical conditions such as cancer, dementia and metabolic disorders. Our technology is capable of monitoring protein phosphorylation continually in living cells. The goal of this application is to further develop our technology to facilitate accurate measurements in a wide range of cells, which in turn, accelerates research into disease mechanisms, or supports patient care as a companion diagnostic.
Project Terms: Animal Model; base; Basic Science; Biochemical Reaction; Biosensor; Cell Line; Cell physiology; cell type; Cells; clinical application; Clinical Investigator; Collection; Color; companion diagnostics; Cyclic AMP-Dependent Protein Kinases; Cyclin-Dependent Kinases; Dementia; design; Detection; Diagnosis; Diagnostic; Directed Molecular Evolution; Disease; drug discovery; Drug Industry; Engineering; Exhibits; Experimental Animal Model; Family; Flow Cytometry; Fluorescence; Fluorescence Microscopy; Fluorescence Resonance Energy Transfer; Gene Expression; Goals; Green Fluorescent Proteins; Human; Human Genome; Imagery; improved; Investigation; Knowledge; Malignant Neoplasms; MAP Kinase Gene; Measurement; Medical; Metabolic Diseases; Methods; Microscopy; Monitor; Nerve Degeneration; Neurons; Noise; novel; outcome forecast; Patient Care; Phase; Phospho-Specific Antibodies; Phosphorylation; Phosphotransferases; Physiological; Play; Process; Property; Protein Engineering; Protein Kinase; Proteins; Proxy; public health relevance; red fluorescent protein; Regulation; Research; Resolution; Role; Safety; screening; sensor; Signal Transduction; Site; spatiotemporal; Specimen; src-Family Kinases; Stimulus; System; Technology; Testing; Therapeutic; Time; tool; transduction efficiency; Transfection; Viral; virtual
