October 16, 2023
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12:00 PM
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1:00 PM
Kinase assay tool development using mass spectrometry and fluorescence lifetime imaging
Laurie Parker, PhD
Professor, Department of Biochemistry, Molecular Biology and Biophysics
Associate Dean for Undergraduate Education, College of Biological Sciences
University of Minnesota
Co-sponsored by Morgridge Institute for Research and the UW SPIE/Optica Chapter
Abstract:
Protein phosphorylation is a crucial post-translational modification in all cells, carried out by kinase enzymes and reversed by phosphatase enzymes. It is regulated by a broad range of factors including protein-ligand and protein-protein interactions, scaffolding, and subcellular localization. Dysregulation of kinase activity leads to cellular abnormalities and disease, and thus kinases are a key target for drug discovery. Most assays to detect kinase activity are either endpoint-based and/or require lysis of cells (which loses contextual information), or require genetically encoded sensors in engineered cellular systems. We have used phosphoproteomics and AI-based structural modeling to determine substrate preferences and design novel synthetic, kinase substrate sequences. We have also linked fluorophore-labeled versions to cell penetrating peptides, delivering peptide-based biosensors to live cells and mapping more than one subcellular kinase activity at a time via fluorescence lifetime imaging microscopy. Overall, we show how synthetic peptides can be used in vitro and in cells to detect dynamic kinase activity to better evaluate inhibitor pharmacology and/or understand target kinase biology in a more biologically-relevant system than in recombinant assays.
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