Open AccessProximity-Dependent Biotinylation to Elucidate the Interactome of TNK2 Nonreceptor Tyrosine Kinase
Author(s) -
Raiha Tahir,
Anil K. Madugundu,
Savita Udainiya,
Jevon Cutler,
Santosh Renuse,
Li Wang,
Nicole A Pearson,
Christopher J. Mitchell,
Nupam P. Mahajan,
Akhilesh Pandey,
Xinyan Wu
Publication year - 2021
Publication title -
journal of proteome research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.644
H-Index - 161
eISSN - 1535-3907
pISSN - 1535-3893
DOI - 10.1021/acs.jproteome.1c00551
Subject(s) - biotinylation , interactome , immunoprecipitation , computational biology , protein–protein interaction , biology , microbiology and biotechnology , proteomics , kinase , biochemistry , genetics , cell culture , gene
Nonreceptor tyrosine kinases (NRTKs) represent an important class of signaling molecules driving diverse cellular pathways. Aberrant expression and hyperphosphorylation of TNK2, an NRTK, have been implicated in multiple 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 stable isotope labeling with amino acids in cell culture and BioID coupled to the biotinylation site identification technology (BioSITe) method that we recently developed to quantitatively explore the interactome of TNK2. By performing a controlled comparative analysis between full-length TNK2 and its truncated counterpart, we were able to not only identify site-level biotinylation of previously well-established TNK2 binders and substrates including NCK1, NCK2, CTTN, and STAT3, but also discover several novel TNK2 interacting partners. We also performed co-immunoprecipitation and immunofluorescence analysis to validate the interaction between TNK2 and CLINT1, a novel TNK2 interacting protein. 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.