PTM-driven mechanisms of cell signaling

Utah State Higher Education System--University Of Utah, Salt Lake City UT

Investigators

Abstract

PROJECT SUMMARY This MIRA/R35 application is designed to replace our NIGMS R01 GM147310-01 and a recent NIGMS R01 application that was scored on first submission but rejected based on not being able to have two simultaneous NIGMS R01s. Following NIGMS advice, I have consolidated these projects into this MIRA application. Our overarching goal is to uncover fundamental cell signaling mechanisms that can be therapeutically targeted in disease. All projects began with PTM discovery approaches and have now grown into multi-pronged efforts that span molecular biology/biochemistry, structural biology, and drug development. The first project addresses the regulation and function of kinases in the understudied ‘dark kinome’, with current focus on an unusual ubiquitin- binding non-receptor tyrosine kinase called TNK1 (currently funded by GM147310-01). C-terminal truncations in TNK1 convert it into an oncogenic driver, yet it’s normal “day job” function and mechanism of regulation are still poorly understood. We also developed the first TNK1 small molecule inhibitor, TP5801, which has passed IND hurdles for phase-I trials but lacks clear clinical direction due to our still nascent understanding of TNK1 biology. Thus, our research addresses critical gaps in TNK1 function and regulation that will inform the clinical path of TP5801. Our work on TNK1 takes advantage of cell-based and in vitro biochemical systems recently developed in the lab, and also extends to mouse models in which we have CRISPR-engineered constitutively active (by disrupting 14-3-3 binding) or kinase-dead tnk1 alleles. Together, our preliminary data point to a role for TNK1 in sensing poly-ubiquitin and instigating inflammatory signaling, and also help explain how genomic rearrangements aberrantly activate the kinase. The second project addresses key gaps in our understanding aggrephagy, a form of autophagy that occurs in nutrient replete conditions and rids cells of toxic protein aggregates that could otherwise cause degenerative proteopathies (e.g., ALS). Specifically, we focus on the earliest steps—how the lipid scramblase ATG9A is recruited to sites of aggrephagy, referred to as “ubiquitin-rich condensates”, and engages with autophagy/aggrephagy machinery to encapsulate and degrade protein aggregates. This project also addresses how ubiquitin-rich condensates can act as platforms to assemble pro- inflammatory (and other) signaling complexes, providing a link between aggrephagy and inflammatory signaling that could explain why persistent, pathological inflammation is a hallmark of proteopathy diseases. A third related project extends our work on aggrephagy to understand how disruption of ATG9A-mediated aggrephagy sensitizes cells to nucleic-acid sensing pathways that activate interferon (IFN) signaling. Together these aggrephagy-focused projects uncover fundamental mechanisms and links to inflammation that could be exploited for new approaches to manipulate IFN signaling in disease.