The Problem Protein kinases are master regulators of signal transduction within the cell, controlling cellular processes by substrate-specific phosphorylation of proteins. Phosphorylation is a major post-translational modification (PTM) affecting approximately 30% of cellular proteins. Consequently, kinases are integral to many key cell fate decisions such as cell profliferation and migration, differentiation, and metabolism. Unsurprisingly, protein kinases are preferentially mutated in various cancers, and kinase activity can be used as a correlative biomarker of chemotherapeutic response. Hence, sensitive and accurate quantitation of kinase activity is critical for both preventative and curative purposes. However, current tools to image kinase activity in situ depend on intensity-based measurements that can be limited by spectral bleed-through and photobleaching.
The Approach The Parker laboratory has developed a novel in silico pipeline to design efficient and specific kinase substrates, enabling multiplexed detection of various clinically-indicated PTMs. We have previously shown these artificial peptide biosensors enable readout of kinase-specific phosphorylation activity within minutes of cell exposure. Fluorescence lifetime imaging microscopy (FLIM) can detect the fluorophore lifetime shifts of these biosensors upon phosphorylation, and is not confunded by photobleaching or autofluorescence. My current work leverages the multiplexing capabilities of our FLIM probes to study cancer cell response to extracellular matrix-mediated signaling in a physiologically relevant 3D context. I am studying the effect of a 3D microenvironment on cell phenotype and drug resistance to clinically-relevant kinase inhibitors.
Project Funding NIH R33 Diversity Supplement (CA217780)
Relevant Publications & Presentations
1. Jena, S., Bastidas, O., Pratt, E.D., Allendorf, S., Cizubu, B., and Parker, L.L. (2019). Developing and characterizing flim probes to detect subcellular tyrosine kinase activity. In 15th US human proteome organization (US-HUPO) annual conference.