Abstract 1307 Oncogenic mutations in the ubiquitin-binding kinase TNK1 reveal its function and mechanism of regulation

Thirty-eight negative kinase-1 (TNK1) is an understudied non-receptor tyrosine kinase with no established function or mechanism of regulation. We identified TNK1 as the primary kinase driver of cell survival in subsets of patient blood cancer samples1. In addition, rare genomic rearrangements that C-terminally truncate TNK1 convert the kinase into a potent oncogenic driver, hinting at critical regulatory domains within the TNK1 C-terminus. One unusual feature at the TNK1 C-terminus is a ubiquitin-association (UBA) domain that we found binds with high affinity to multiple poly-ubiquitin linkages. The TNK1 UBA domain shows an atypical five-helix structure with two ubiquitin-binding interfaces, which likely increase the avidity of TNK1 for poly-ubiquitin. We found that TNK1 relies on its UBA domain to localize to ubiquitin-rich condensates in the cytosol where it becomes active. To our knowledge, this feature of TNK1-a bona-fide ubiquitin-association domain involved in kinase activity-makes TNK1 unique across the human kinome. We also discovered that within the C-terminus and adjacent to the TNK1 UBA domain is a phosphorylation site (Ser502) that triggers TNK1 binding to the adapter protein 14-3-3. Our data suggest that this interaction with 14-3-3 inhibits TNK1 activity by preventing the inclusion of TNK1 in biomolecular condensates where TNK1 is otherwise active. We are currently using in vitro biochemical systems to understand the biophysical mechanism by which 14-3-3 regulates TNK1 condensate formation. Lastly, using proteomics to identify TNK1 substrates, we found that mutations that mimic the patient-associated TNK1 C-terminal truncations have a dual effect on TNK1 activity/function: they hyperactivate the kinase due to loss of 14-3-3 binding and also alter its substrate profile due to loss of the UBA domain. Together, our data support a model in which the UBA domain tethers the kinase to its native 'day job' substrates (via interaction with poly-ubiquitin), whereas loss of the UBA skews the TNK1 substrate profile toward pro-growth/oncogenic signaling. 1. Chan TY*, Egbert CM*, Maxson JE, Siddiqui A, Larsen LJ, Kohler K, Balasooriya ER, Pennington KL, Tsang TM, Frey M, Soderblom EJ, Geng H, Müschen M, Forostyan TV, Free S, Mercenne G, Banks CJ, Valdoz J, Whatcott CJ, Foulks JM, Bearss DJ, O'Hare T, Huang DCS, Christensen KA, Moody J, Warner SL, Tyner JW, Andersen JL. (2021) TNK1 is a ubiquitin-binding and 14-3-3-regulated kinase that can be targeted to block tumor growth. Nature Communications, Sept 9;12(1):5337, PMID: 34504101, *co-first authors. This study was supported by an American Cancer Society Research Scholar grant (133550-RSG-19-006- 01-CCG) and an NIH/NIGMS R01 (GM147310-01) to JLA.