PROJECT SUMMARY/ABSTRACT Alterations in kinase signaling underlie many of the most devastating human diseases, including degenerative disease, autoimmunity and cancer. Thus, not surprisingly, kinases are the second most targeted group of drug targets (next to G-protein coupled receptors). Yet despite their importance in disease, only about 8% of kinases are targets of FDA approved drugs and roughly a quarter of the 634 kinases in the human kinome is still considered ‘understudied’, leaving over 100 kinases untapped as potential therapeutic targets and without clear biological functions. This proposal focuses on TNK1, a poorly understood member of the ACK kinase family of non-receptor tyrosine kinases (NRTKs). Our recently published data (Nat. Comm. 2021) uncovered the first mechanism of regulation and the unusual presence of a ubiquitin-association (UBA) domain on this kinase. Critical gaps relating to this mechanism and the still unknown cellular function of TNK1 are addressed in this proposal. Our long-term goal is to discover mechanisms of cell growth and survival that can be therapeutically targeted in disease. The overall objectives of this proposal are to establish the first detailed mechanism and function of this understudied kinase. The central hypothesis is that the binding of the TNK1 UBA domain to clusters of poly-ubiquitin at protein aggregate condensates acts as a form of induced proximity to oligomerize and activate TNK1 (aim 1). We also hypothesize that the interaction of 14-3-3 with phospho-Ser502 of TNK1 inhibits TNK1 oligomerization and conceals the UBA domain, thereby sequestering TNK1 away from ubiquitin in an inactive state (aim 2). Finally, we posit that TNK1 senses the accumulation of poly-ubiquitin to phosphorylate substrates that promote the lysosomal degradation of condensates (aim 3). The proposal is significant because it fills a basic gap in our understanding of TNK1, provides a framework to understand how mutations activate TNK1 in disease, and perhaps most importantly will inform pharmacological strategies that take advantage of our recently developed TNK1 inhibitor to target TNK1 in disease. The proposal is innovative because it addresses a novel mechanism of kinase activation through direct interaction with poly- ubiquitin, thereby establishing condensates as organizing platforms for kinase signaling. In addition, this proposal lays the first foundation to target TNK1 in disease.