This is a new F32 application to develop and apply chemical rescue technology to the functional analysis of protein kinases. Protein kinases regulate virtually every aspect of cellular physiology in health and disease through phosphorylation of their substrates, typically on Tyr and Ser/Thr residues. Despite intense efforts even the most ?well-studied? protein kinases remain incompletely understood. Methods to study protein kinase signaling in cellular contexts are urgently needed, but unfortunately, few methods exist for activating specific kinases in living cells. The techniques that are available for this purpose require extensive engineering, which raises concerns about preservation of natural regulatory mechanisms and has likely limited their widespread application. Previously, our lab developed chemical rescue as a method for rapidly activating protein Tyr kinases in living cells. A single point mutant is generated within the catalytic site, which renders the kinase catalytically inactive without disturbing its structure or regulation. A small molecule is then added which complements this mutation and rapidly switches the kinase back on in a reversible manner. This technique is conceptually simple and has been proven useful in dissecting cell signaling of the protein Tyr kinases Csk, Src, and Abl. However, due to the historical difficulty with generating point mutants in mammalian cells, chemical rescue has not yet been widely applied. Additionally, a chemical rescue technique has not been developed for Ser/Thr kinases, which make up approximately 80% of the human kinome. This proposal seeks to overcome both of these limitations. By using CRISPR/Cas9 gene editing to introduce the required point mutations in model cell lines, we will use chemical rescue to identify substrates and cell signaling pathways of two enigmatic Tyr kinases.
In Aim 1, ALK, a receptor Tyr kinase commonly mutated in cancer but with unclear physiological function will be analyzed in its wild-type and oncogenic forms to identify its cellular targets.
In Aim 2, ZAP70, a nonreceptor Tyr kinase essential for T-cell activation but with very few known substrates will be investigated. For these two aims, we will use an unbiased mass spectrometry based phosphoproteomic approach to identify substrate proteins and activated signaling pathways.
In Aim 3, we will develop proof-of-concept for chemical rescue of Ser/Thr kinases using the model protein Akt1. Collectively, this work will open new doors to investigating kinase signaling and advance our understanding of three key enzymes in biomedical research. In addition, this proposal builds on the chemical biology background of the PI and promises to greatly broaden the PI's skills in enzymology, mass spectrometry, and cell biology. It is anticipated that the training plan outlined here will position the PI for success in an independent academic research career.
Protein kinases are key regulators of human physiology and are frequently misregulated in a variety of disease states. There are very few robust methods for activating specific kinases in cellular contexts, and consequently, the substrates and signaling pathways of the vast majority of human kinases remain unknown. The goal of this work is to develop chemical rescue, a technique for kinase activation, to study cell signaling through both of the major classes of protein kinases in order to advance our understanding of human physiology and pathophysiology.
