A mechanism of regulation for the ubiquitin-sensing kinase TNK1.

TNK1 is a poorly understood non-receptor tyrosine kinase that by mutation can be converted into an oncogenic driver. While little is known about the normal function of TNK1, it possesses a C-terminal ubiquitin binding domain (UBA) that interacts with high affinity to multiple poly-ubiquitin linkages. To our knowledge, this ability to directly interact with poly-ubiquitin makes TNK1 unique across the human kinome. Our recent data suggest that the UBA domain of TNK1 homes the kinase to phase-separated condensates of poly-ubiquitinated proteins where TNK1 becomes fully active. We hypothesize that the interaction between the TNK1 UBA and poly-ubiquitin at these ubiquitin-rich condensates tethers the kinase to its substrates, which are involved in condensate biology and cell survival signaling. These findings led us to question how the interaction between TNK1 and ubiquitin is regulated. We have focused on a phosphorylation at Y661 within the TNK1 UBA domain, which has been identified in high throughput PTM mass spectrometry (phosphosite.org) but is of unknown function. Here we show that intrinsic TNK1 kinase activity is required for phosphorylation of Y661, suggesting that it may be an autophosphorylation. Furthermore, our preliminary data suggest that a phosphomimic mutation at Y661 leads to an increase in ubiquitin binding, suggesting that this phosphorylation may enhance the affinity of the TNK1 UBA for ubiquitin. Together, our data suggest a feed-forward mechanism of TNK1 regulation in which activation of TNK1 kinase activity primes the UBA for binding ubiquitin via phosphorylation at Y661. In turn, the UBA domain helps cluster active TNK1 at ubiquitin-rich condensates where TNK1 is brought into proximity to its substrates.