New Molecular Probes For Protein Kinases

The long-term objective of this project is to merge molecular tool development with functional studies of kinases that are of biological interest. To date, we’ve developed new chemical tools for modulating kinase regulatory interactions, global conformational state, and interactomes, which we’ve used to understand how differentially modulating these parameters affects the intracellular functions of protein kinases. During the previous funding period, we demonstrated that new and unexpected mechanistic discoveries can be made on even the most well characterized kinases through the application of new molecular analysis tools. We found that the phosphotransferase activity of the tyrosine kinase Src is regulated by its N-terminal SH4 domain, which was previously believed to only serve as a membrane tether. In this proposal, we will leverage many of the same methodologies what we used to stusy Src in order obtain a detailed molecular understanding of Lck, which is a Src-Family Kinase (SFK) that plays a specialized role in T cell receptor (TCR) signaling. SFKs that contain identical domain architectures as Lck cannot replicate Lck’s role in TCR signaling and our preliminary results suggest that Lck’s regulatory module is unique. Our goal is to comprehensively profile the regulatory properties of Lck and to determine what makes it unique amongst the SFKs. In three Aims, spanning enzymology, biochemistry, cell biology, and chemoproteomics, we propose to study the intrinsic and extrinsic regulation of Lck and the functional consequences of manipulating its regulatory apparatus during TCR signaling. In addition, we describe a new technology for profiling the regulatory and conformational states of kinases during TCR signaling.