TNK1 is a targetable JAK-independent driver of STAT signaling and inflammation

Deregulated inflammatory signaling via STAT family transcription factors, particularly STAT1, underlies a variety of immune-related diseases, including inflammatory bowel disease. Whereas activation of STATs by JAKs via canonical receptor-driven JAK-STAT signaling is well understood, little is known about JAK-independent mechanisms of STAT activation. Here, we identify the understudied nonreceptor tyrosine kinase TNK1 as a therapeutically targetable, JAK-independent activator of STAT signaling. Using a multiomics approach, we mapped a network of TNK1 substrates associated with protein condensates and proinflammatory signaling, including STAT1. We found that TNK1, but not its sister kinase ACK1, directly phosphorylates STATs at well described STAT-activating JAK sites. In cells, TNK1-mediated STAT1 phosphorylation and activation occurs independently of JAKs. Imaging and interactomics data suggest that TNK1 interacts with STAT1 in cytosolic condensates, which likely compartmentalize TNK1-substrate interactions. We show that an intrinsically disordered proline-rich region in TNK1, which includes a 14-3-3 docking phosphorylation site, is required for the formation of kinase-active TNK1 condensates and STAT1 phosphorylation. Mutations within the proline-rich region that eliminate 14-3-3 binding increase formation of TNK1 condensates, suggesting a model in which 14-3-3 acts as a clamp that constrains the flexible PRR to inhibit condensate formation and STAT1 activation. Finally, we show that TNK1 is a targetable driver of STAT1-mediated inflammation in the gut as inhibition of TNK1 reduces active STAT1 in the colon and ameliorates colitis symptoms in mice.