Abstract 1883 14-3-3 inhibits TNK1-mediated signaling by blocking its ability to undergo liquid-liquid phase separation

TNK1 is a poorly understood non-receptor tyrosine kinase that has been implicated as an oncogene and mediator of inflammation. TNK1 has unique features for a kinase, including a C-terminal ubiquitin-association (UBA) domain. The UBA domain is linked to the kinase via an intrinsically disordered proline-rich region (PRR) that we have shown has a phosphorylated docking site for the adapter protein 14-3-3. Loss of 14-3-3 binding to TNK1, either through naturally occurring PRR truncations in lymphoma or phospho-null point mutations within the PRR, make TNK1 highly active and oncogenic. However, the mechanism by which 14-3-3 regulates TNK1 is still unknown. Our preliminary data show that loss of 14-3-3 binding allows TNK1 to form kinase-active condensates in cells. These TNK1 condensates show liquid-like properties, including recovery after photobleaching and condensate fusion/fission. Although the TNK1 UBA domain is not required for condensate formation, it ultimately determines where these TNK1 condensates reside, with UBA-intact forms of TNK1 localizing to ubiquitin-rich sites within the cytosol and UBA-deleted forms of TNK1 forming untethered cytosolic condensates. Our preliminary data suggest that TNK1 is able to sequester substrates into these condensates to form signaling complexes. Together, our data support a model in which 14-3-3 acts as a brake on TNK1 activity by inhibiting its ability to phase separate, likely by reducing the flexibility and multivalency of the TNK1 PRR. This work was supported by the National Institutes of Health (NIGMS R01 GM147310-01).