A 14‐3‐3‐mediated mechanism of regulation for the ubiquitin‐sensing kinase TNK1

Thirty -eight negative kinase 1 (TNK1) is a poorly characterized member of the ACK family of non-receptor tyrosine kinases. Previous studies suggest a role for TNK1 in oncogenic transformation; however, the biological function, regulation and substrates of TNK1 remain unknown. We previously discovered that the phospho-binding protein 14-3-3 binds to a phospho-serine (S502) within a C-terminal proline-rich domain of TNK1, sequestering TNK1 in the cytosol and inhibiting its kinase activity. Conversely, the release of TNK1 from 14-3-3 activates TNK1 and increases its oncogenic activity1 . However, how 14-3-3 controls TNK1 localization and activity is still not understood. We recently found that the release of TNK1 from 14-3-3 allows TNK1 to interact with ubiquitin-rich cytosolic condensates, where TNK1 is fully active. Furthermore, we found that the interaction between TNK1 and ubiquitin-rich condensates requires an unusual ubiquitin association (UBA) domain on the TNK1 C-terminus that binds with high affinity to K63-linked poly-ubiquitin chains. Thus, 14-3-3 acts as a toggle to turn on and off the ubiquitin-binding ability of TNK1, which in turn controls TNK1 oncogenic activation. Our current data suggest that this occurs through a two-step mechanism. First, the release of TNK1 from 14-3-3 activates the TNK1 kinase domain, which leads to an autophosphorylation within the TNK1 UBA domain. Second, this autophosphorylation enhances the affinity of the UBA domain for poly-ubiquitin, which promotes the clustering of active TNK1 at ubiquitin-rich condensates where TNK1 phosphorylates downstream substrates. 1. Chan and Egbert et al. TNK1 is a ubiquitin-binding and 14-3-3 regulated kinase that can be targeted to block tumor growth. Nat Commun 12, 5337 (2021).