PTM-driven mechanisms of cell signaling

This MIRA/R35 application is designed to replace our NIGMS R01 GM147310-01 and a recent NIGMS R01application that was scored on first submission but rejected based on not being able to have two simultaneousNIGMS R01s. Following NIGMS advice I have consolidated these projects into this MIRA application. Ouroverarching goal is to uncover fundamental cell signaling mechanisms that can be therapeutically targeted indisease. All projects began with PTM discovery approaches and have now grown into multi-pronged efforts thatspan molecular biology/biochemistry structural biology and drug development. The first project addresses theregulation 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 inTNK1 convert it into an oncogenic driver yet its normal day job function and mechanism of regulation are stillpoorly understood. We also developed the first TNK1 small molecule inhibitor TP5801 which has passed INDhurdles 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 ofTP5801. Our work on TNK1 takes advantage of cell-based and in vitro biochemical systems recently developedin the lab and also extends to mouse models in which we have CRISPR-engineered constitutively active (bydisrupting 14-3-3 binding) or kinase-dead tnk1 alleles. Together our preliminary data point to a role for TNK1 insensing poly-ubiquitin and instigating inflammatory signaling and also help explain how genomicrearrangements aberrantly activate the kinase. The second project addresses key gaps in our understandingaggrephagy a form of autophagy that occurs in nutrient replete conditions and rids cells of toxic proteinaggregates that could otherwise cause degenerative proteopathies (e.g. ALS). Specifically we focus on theearliest stepshow the lipid scramblase ATG9A is recruited to sites of aggrephagy referred to as ubiquitin-richcondensates and engages with autophagy/aggrephagy machinery to encapsulate and degrade proteinaggregates. 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 signalingthat could explain why persistent pathological inflammation is a hallmark of proteopathy diseases. A third relatedproject extends our work on aggrephagy to understand how disruption of ATG9A-mediated aggrephagysensitizes cells to nucleic-acid sensing pathways that activate interferon (IFN) signaling. Together theseaggrephagy-focused projects uncover fundamental mechanisms and links to inflammation that could beexploited for new approaches to manipulate IFN signaling in disease.