Abstract Tyrosine non-receptor kinase 1 (TNK1) is a poorly characterized member of the ACK family of non-receptor tyrosine kinases. We identified TNK1 as a mediator of cell survival in subsets of primary hematological malignancies. Previous studies have indicated a role for TNK1 in driving oncogenic potential; however, the function, regulation, and substrates of TNK1 still remain unknown. We identified an interaction between TNK1 and 14-3-3 that is promoted by a MARK-mediated phosphorylation at S502 on TNK1. 14-3-3 binding sequesters TNK1 to the cytosol. Upon the release of 14-3-3 binding, TNK1 moves to perinuclear puncta and is in a highly active state. This highly active form of TNK1 is able to drive growth factor independent proliferation of lymphoid cells in culture and in mice more potently than TNK1 WT. A unique feature of TNK1 is its C-terminal ubiquitin association domain (UBA) domain. In vitro ubiquitin pulldowns indicate a high affinity between the TNK1 UBA and polyubiquitin of varying linkages, with dissociation constants in the low nanomolar range. Mutation of residues within ubiquitin-binding interfaces of the UBA and ubiquitin leads to a reduction of ubiquitin association and a marked shift of TNK1 from perinuclear puncta to a diffuse cytosolic localization. Importantly, we show that loss of ubiquitin binding reduces TNK1 activity and oncogenic signaling. Taken together, our data uncovers a 14-3-3 and UBA-mediated mechanism of TNK1 regulation. Citation Format: Christina Marie Egbert, Tsz Yin Chan, Logan Larsen, Eranga Roshan Balasooriya, Kristina Kohler, Jeremy Tsang, Madison Frey, Julia Maxson, David Huang, Kenneth A. Christensen, James Moody, Jeffrey W. Tyner, Joshua L. Andersen. The ubiquitin-binding non-receptor tyrosine kinase TNK1 is regulated by a MARK-mediated interaction with 14-3-3 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2306.