Global Methods for Discovering and Characterizing Regulatory Mechanisms of Multi-Domain Protein Kinase

Abstract

Perturbations to cellular phosphorylation levels are highly correlated with a variety of disease states. Because protein kinases are the enzymes responsible for protein phosphorylation, they play a central role in maintaining homeostatic phosphorylation levels, and as such have become attractive drug targets. Consequently, the regulatory mechanisms that govern protein kinase activity have been studied for decades. Roughly half of protein kinases have at least one protein domain in addition to their catalytic kinase domain and in many cases these domains serve as “regulatory domains” by making physical contacts with surfaces on the catalytic domain, disrupting the alignment of catalytically necessary residues. While the intramolecular regulatory mechanisms of many kinases have been delineated, there are many layers of regulation that lack definition. Specifically, a collaborative effort between the Maly and Fowler labs (University of Washington Department of Genome Sciences) revealed new putative regulatory surfaces on the catalytic domain of the long-studied Src kinase. One central hypothesis of this work is that there are similar but distinct regulatory surfaces on other members of Src Family of Kinases (SFKs), which give rise to differences in kinase substrate specificity, localization, temporal control over kinase activity, and overall mechanisms of regulation. Given the involvement of the SFKs Lck and Fyn in T-cell development and mature thymocyte signaling, we would like to better understand how these regulatory surfaces contribute to productive T cell receptor (TCR) signaling, which has yet to be systematically explored. Therefore, we identified putative inter- and intramolecular regulatory surfaces on Lck—the most centrally involved SFK in TCR signaling—using a series of saturation mutagenesis Deep Mutational Scans (DMS) in yeast. In addition to revealing fundamental information about the roles of Lck in mediating healthy TCR signaling, the methods described herein are general, and can be applied to study any protein of interest.