Thirty‐eight‐negative kinase 1 (TNK1) is a poorly characterized non‐receptor tyrosine kinase (NRTK) first isolated from umbilical cord blood. TNK1 differs from most NRTKs in that it possesses a functional ubiquitin association (UBA) domain at its C‐terminus. We previously observed that the TNK1 UBA domain interacts with a variety of poly‐ubiquitin chains, is essential for full TNK1 activation, and facilitates the clustering of active TNK1 into ubiquitin‐rich condensates.1 Paradoxically, a genetic inversion that truncates the TNK1 C‐terminus (including the UBA domain) in human lymphoma hyperactivates the kinase and coverts it into an oncogenic driver. Our current research focuses on how deletion of the UBA, which is important for native TNK1 function, helps activate the kinase. Here we show that immediately adjacent to the UBA domain on TNK1 is an inhibitory 14‐3‐3 binding site, which is also truncated in lymphoma. Furthermore, point mutations that disrupt the 14‐3‐3 binding site are sufficient to convert TNK1 into a hyperactive oncogenic kinase. In addition, we show that although truncation of the UBA perturbs the normal function of TNK1, it also stabilizes TNK1 protein levels. Thus, the lymphoma‐associated truncations in TNK1 activate and stabilize the kinase, resulting in a highly expressed and active TNK1. Finally, we show that truncation of the 14‐3‐3 binding site and UBA domain of TNK1 is sufficient to transform pro‐B cells to growth factor‐independence and grow tumors in vivo. Thus, our data explain how genetic inversions convert TNK1 into an oncogenic driver in human cancers. 1. Chan, TY., Egbert, C.M., Maxson, J.E. 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). https://doi.org/10.1038/s41467-021-25622-3
Thirty-eight-negative kinase 1 (TNK1) is a poorly characterized member of the ACK family of non-receptor tyrosine kinases, which we recently identified as a driver of cell survival in a subset of primary hematological malignancies. However, the biological function of TNK1 is not well understood. We also found that TNK1 is unusual among kinases for the presence of a functional ubiquitin association (UBA) domain on its C-terminus. We discovered that the TNK1 UBA domain binds to poly-ubiquitin with high affinity and has no apparent preference for ubiquitin chain length or linkage type. Interestingly, the UBA domain is important for TNK1 function, given that deletion of the UBA domain (TNK1 ΔUBA) alters its localization and phospho-substrate network, while also weakening the oncogenic activity of TNK1 in cell transformation assays. Based on these data, we hypothesized that the UBA domain tethers TNK1 to its substrates. To test this hypothesis, we performed quantitative phospho-tyrosine proteomics using murine pro-B cells transformed with either TNK1 full length or TNK1 ΔUBA and identified TANK-binding kinase 1 (TBK1) as a putative UBA-dependent TNK1 substrate. TBK1 is a serine/threonine kinase involved in the regulation of inflammation, autophagy, and NF-ĸB signaling. During the selective autophagic degradation of misfolded proteins (aggrephagy), TBK1 phosphorylates p62 at S403 to increase its affinity to ubiquitin, and thereby increases p62-mediated recruitment of misfolded proteins into the condensate. Unchecked growth of ubiquitin condensates can overwhelm the size capacity of autophagosomes, potentially causing misfolded proteins to become proteotoxic aggregates. Our preliminary data suggest a model in which active TNK1 accumulates at ubiquitin condensates to phosphorylate and inhibit TBK1. Based on these data, we propose that TNK1 acts as a kinase sensor of poly-ubiquitin to control the TBK1-mediated growth of ubiquitin condensates. Citation Format: Emmalee Kohler, Tania Lopez-Palacios, Tsz-Yin Chan, Christina Egbert, Jacob Truman, Spencer Ashworth, Alec Vaughan, Joshua Andersen, D. Madhusanka. Determining the biological function of TNK1. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5029.
A20 is an anti-inflammatory protein with a dual ubiquitin-editing activity. Through its N-terminal ovarian tumor (OTU) domain, A20 negatively regulates NF-ĸB by removing the K63-linked ubiquitin chains on RIP1 that act as platforms for NF-ĸB activation downstream of tumor necrosis factor alpha (TNFα). On the other hand, A20 also catalyzes the addition of K48-linked ubiquitin chains on RIP1 via its C-terminal zinc finger domain 4 (ZnF4) to promote its proteasomal degradation. Through both mechanisms, A20 is a means of negative feedback on TNFα-induced NF-ĸB activity. Importantly, inactivation of A20 is frequently found in B-cell lymphomas, where loss-of-function A20 truncations promote cell proliferation and upregulation of NF-ĸB signaling. Among the post-translational modifications (PTMs) reported in A20, there are several phosphorylation events in its C-terminus, including within the ZnF4 domain. Besides its known E3 ubiquitin ligase activity, previous reports show that the C-terminal region of A20 is important for ubiquitin binding, having preference for K63-linked poly-ubiquitin. However, a little is known about the effects of phosphorylation at this region on A20 activity as well as its consequences in downstream signaling. Our current work suggests that phosphorylation in the C-terminus of A20 inhibits its E3 ubiquitin ligase activity and disrupts its interaction with ubiquitin, which may also affect its deubiquitinase activity by preventing its recruitment to ubiquitinated proteins in the TNFα receptor (TNFR) complex. We also propose that the C-terminal region is important for the formation of the A20 ubiquitin-editing complex, necessary to downregulate NF-ĸB-activated inflammatory signals. Based on these observations, we hypothesize that not only gene-inactivating mutations, but also post-translational mechanisms, such as phosphorylation, inhibit A20 to promote cancer development. Our research is now focused on understanding the upstream signaling that regulates A20 phosphorylation. Citation Format: Tania Lopez Palacios, Tsz-Yin Chan, Christina Egbert, Jacob Truman, Spencer Ashworth, Alec Vaughan, Joshua L. Andersen. The regulation of inflammatory signaling in cancer via A20 phosphorylation. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5028.
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 (http://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.
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).
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