A Bio-orthogonal Approach for Identifying Direct Kinase Substrates and their phosphorylation sites

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With this award, the Chemistry of Life Processes Program in the Chemistry Division is funding Dr. Kavita Shah from Purdue University. The investigator is developing a high-throughput chemical tool to identify and validate the direct targets of a class of enzymes called kinases. Kinases are key cell regulators that perform phosphorylation reactions with proteins in the cell. They have an important role in biological outcomes that support each tissue's unique physiology. It is estimated that ~33,000 proteins are phosphorylated by kinases in a cell. At present, it is a formidable endeavor to identify each of the 518 human kinases. This project is developing special probes, with exquisite sensitivity for specifically tagging and isolating kinase targets. This method is widely applicable and could be used to identify and validate the targets of any desired kinase in cells. This project allows graduate students, undergraduate students, and postdoctoral fellows to acquire highly interdisciplinary training. The scientific areas of training include chemical synthesis, protein engineering, and enzymatic assays. This project is integrated into an outreach program which consists of an interdisciplinary Chemical Biology course. This course is designed for Chemistry majors and graduate students. It also comprises part of a research training program for under-represented undergraduates recruited through specific programs at Purdue University. The Chemical Biology course is also used as a professional development opportunity for high school teachers. The overall goal is to increase STEM education and retention for high school students, under-represented minorities, and graduate students.The goal of this proposal is to develop a high-throughput bio-orthogonal chemical approach that enables rapid identification of low abundance kinase substrates and their phosphorylation sites on a proteome-wide scale. A "catch and release" strategy, in combination with an innovative chemical genetic approach, is used to specifically label with a unique phospho-tag, the substrates of any desired kinase of interest. The exquisite sensitivity of this approach stems from the phospho-tag, which will be used to selectively isolate substrates from the whole proteome, without the need for any other separation techniques. These substrates are validated in a high throughput screen using CRISPR-engineered cell lines and orthogonal inhibitors. A few chosen substrates are used as cues to uncover the mechanisms by which these kinases regulate various cellular processes, potentially leading to the discovery of novel pathways and networks. A rapid and comprehensive analysis of a desired kinase's substrates and their phosphorylation sites is crucial for dissecting the contribution of the kinase in activating distinct signaling cascades.