Proximity-Dependent Biotinylation to Elucidate the Interactome of TNK2 Nonreceptor Tyrosine Kinase.

Raiha Tahir, Anil K Madugundu, Savita Udainiya, Jevon A Cutler, Santosh Renuse, Li Wang, Nicole A Pearson, Christopher J Mitchell, Nupam Mahajan, Akhilesh Pandey, Xinyan Wu

Journal: Journal of proteome research 2021;20(9):4566-4577

PMID: 34428048

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Abstract

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.

Address: Biochemistry, Cellular and Molecular Biology Graduate Program, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, United States.; Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, United States.; Ginkgo Bioworks, Boston, Massachusetts 02210, United States.; McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, United States.; Institute of Bioinformatics, International Technology Park, Bangalore 560066, Karnataka, India.; Manipal Academy of Higher Education (MAHE), Manipal 576104, Karnataka, India.; Center for Molecular Medicine, National Institute of Mental Health and Neurosciences (NIMHANS), Hosur Road, Bangalore 560029, Karnataka, India.; Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minnesota 55905, United States.; Pre-Doctoral Training Program in Human Genetics, McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, United States.; Center for Individualized Medicine, Mayo Clinic, Rochester, Minnesota 55905, United States.; Siteman Cancer Center, Washington University, St. Louis, Missouri 63110, United States.; Departments of Pathology and Oncology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, United States.; Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Rochester, Minnesota 55905, United States.

DOI: 10.1021/acs.jproteome.1c00551