Proximity-dependent biotinylation to elucidate the interactome of TNK2 non-receptor tyrosine kinase

Non-receptor tyrosine kinases represent an important class of signaling molecules which are involved in driving diverse cellular pathways. Although, the large majority have been well-studied in terms of their protein-binding partners, the interactomes of some of the key non-receptor tyrosine kinases such as TNK2 (also known as activated Cdc42-associated kinase 1 or ACK1) have not been systematically investigated. Aberrant expression and hyperphosphorylation of TNK2 has been implicated in a number of cancers. However, the exact proteins and cellular events that mediate phenotypic changes downstream of TNK2 are unclear. Biological systems that employ proximity-dependent biotinylation methods, such as BioID, are being increasingly used to map protein-protein interactions as they provide increased sensitivity in discovering interaction partners. In this study, we employed BioID coupled to the biotinylation site identification technology (BioSITe) method that we recently developed to perform molecular mapping of intracellular proteins associated with TNK2. We also employed stable isotope labeling with amino acids in cell culture (SILAC) to quantitatively explore the interactome of TNK2. By performing a controlled comparative analysis between full-length TNK2 and its truncated counterpart, we were not only able to confidently identify site-level biotinylation of previously well-established TNK2 binders and substrates such as NCK1, NCK2, CTTN, STAT3, but also discover several novel TNK2 interacting partners. We validated TNK2 interaction with one of the novel TNK2 interacting protein, clathrin interactor 1 (CLINT1), using immunoblot analysis. Overall, this work reveals the power of the BioSITe method coupled to BioID and highlights several molecules that warrant further exploration to assess their functional significance in TNK2-mediated signaling.