TNK1 negatively regulates TBK1 at ubiquitin condensates.

Thirty-eight-negative kinase 1 (TNK1) is a poorly understood member of the ACK family of non-receptor tyrosine kinases. TNK1 has been linked to oncogenic activity. However, the biological function of TNK1 remains unclear. Recently, we discovered the presence of a functional ubiquitin association (UBA) domain on the C-terminus of TNK1, which is an unusual feature on a kinase. Previously, we demonstrated that the TNK1 UBA domain binds to poly-ubiquitin with high affinity and with no preference for length or linkage type (1). Interestingly, deletion of the UBA domain (TNK1 ΔUBA) affects TNK1 intracellular localization and decreases its phospho-substrates network. Based on these data, we hypothesized that the UBA domain homes TNK1 to substrates. To test this hypothesis, we used quantitative phospho-tyrosine proteomics in pro-B cells transformed with either TNK1 or TNK1 ΔUBA to identify UBA-dependent TNK1 substrates. Among the putative UBA-dependent substrates, we identified phospho-Y235 of TANK-binding kinase 1 (TBK1). TBK1 is an important serine/threonine protein kinase involved in the regulation of inflammation, autophagy, and NF-kB signaling. Through confocal microscopy, we demonstrated a colocalization between TNK1 and TBK1 at p62-positive phase-separated clusters of ubiquitin-referred to here as ubiquitin condensates. We found that TNK1 localizes to ubiquitin condensates in a UBA-dependent manner. Furthermore, our data suggest that TBK1 is inhibited by phosphorylation at Y235, as phospho-null mutation at Y235 (Y235F) significantly increases phosphorylation of the TBK1 substrates p62 and OPTN. We also found that CRISPR/Cas9 deletion of TNK1 increased TBK1 activity and phosphorylation of p62 and OPTN. These data suggest that TNK1 negatively regulates TBK1 activity. Intriguingly, we also identified an enrichment of other putative UBA-dependent TNK1 substrates in the NF-kB signaling pathway, suggesting that the UBA domain tethers TNK1 to multiple substrates in this pathway, perhaps as a means to dampen NF-kB signaling. Our current work focuses on the biological impact of these phosphorylations on NF-kB activity, inflammatory signaling, and autophagy. 1. Chan, TY., Egbert, C.M. 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).