Overview
The overarching goal of our lab is to understand molecular mechanisms of tyrosine kinase and tyrosine phosphatase signaling. We focus on these proteins both because of their biomedical importance – many are dysregulated in diseases and are the targets of therapeutics – and because they are fascinating model systems to study interaction specificity, allostery, evolution, and drug resistance. Our lab aims to address the following big-picture questions:
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What is the full complement of interactions in the cell that a phosphotyrosine signaling protein participates in? This information will help define the topologies of signaling pathways, clarify the biological roles of those proteins, and identify new signaling nodes that can be targeted by therapeutics.
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What is the molecular basis for the interactions and regulation of phosphotyrosine signaling proteins? A molecular description of specificity and allostery will help explain functional differences between related proteins, predict uncharacterized interactions, and design new interventions.
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How do mutations in phosphotyrosine signaling proteins alter their biochemical and cellular activities, thereby leading to diseases or drug resistance? The question of mutational effects on protein structure, function, and signaling resonates throughout many projects in the lab.
Although we often use classical biochemical, biophysical, and cell biology techniques to tackle these questions, we are drawn to new chemical tools and high-throughput biochemical methods, which enable analyses that were not previously possible. These approaches are yielding exciting new mechanistic insights into phosphotyrosine signaling proteins.
Understanding sequence recognition
Phosphotyrosine signaling proteins phosphorylate, dephosphorylate, and interact with other proteins in a sequence-dependent manner. We have developed a high-throughput platform to profile the sequence specificity of signaling proteins using genetically-encoded peptide libraries and next-generation DNA sequencing. This method allows us to compare the activities of thousands of substrate or ligand peptides simultaneously.
We are using this platform to predict tyrosine kinase and phosphatase substrates, pinpoint the molecular determinants of sequence recognition, and dissect the effects of disease-associated mutations in phosphotyrosine signaling enzymes and at tyrosine phosphorylation sites. Using these deep profiles of kinase and phosphatase sequence recognition, we ultimately aim to design selective inhibitors and engineer signaling pathways for basic mechanistic studies and therapeutic applications.
Using our high-throughput platform, we can dissect subtle specificity differences between closely-related kinases (left panel). We have identified peptides that are highly selective substrates for individual tyrosine kinases and may serve as templates for the design of selective inhibitors (right panel).
Mapping signaling interactions
An outstanding challenge in understanding phosphotyrosine signaling is to map the connectivity between individual proteins in these pathways. Specifically, it remains difficult to identify true physiological substrates and regulatory partners of tyrosine kinases and phosphatases. In addition to peptide screening to map interactions driven by sequence specificity, we are taking two approaches to tackle this problem:
We are mapping the “interactomes” of phosphotyrosine signaling enzymes using proximity-labeling proteomics. We anticipate that this method will help explain the distinct signaling profiles and phenotypes associated with mutant enzymes found in human diseases.
Guided by structures, we are engineering phosphotyrosine signaling enzymes to incorporate photo-crosslinkers that can be used to trap and identify native cellular interaction partners.
Dissecting regulatory mechanisms
Our understanding of cell signaling processes is guided by our knowledge of how signaling proteins are switched on and off through post-translational modifications and binding interactions. For many tyrosine kinases, we have a thorough understanding of what input signals regulate enzyme activity and the molecular mechanisms by which activity is modulated. By contrast, there is a dearth of such knowledge for the vast majority of tyrosine phosphatases. We have started to fill this gap in by discovering and characterizing specific allosteric regulatory interactions for tyrosine phosphatases. We are also pursuing two new strategies to fill this void (below). Using these two approaches, we hope to understand when and where tyrosine phosphatases are dynamically regulated as well as how this regulation is achieved at the molecular level.
We are designing small molecule probes that covalently label tyrosine phosphatases. Our long-term goal is to use these molecules to track changes in phosphatase activity in cells using mass spectrometry proteomics
We are developing selection assays to conduct deep mutational scanning and related multiplexed assays of variant effects. These experiments will allow us to gauge the functional effects of thousands of protein variants in a high-throughput fashion and map resistance mutation profiles for inhibitors of signaling proteins. They are revealing new structural and mechanistic insights into the allosteric regulation of signaling enzymes.
