Dataset Information

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

ABSTRACT: 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 important 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 have been implicated in a number of cancers, although the exact proteins and cellular events that mediate phenotypic changes downstream of TNK2 are unclear. Biological systems that employ proximity-dependent protein labeling methods, such as biotinylation identification (BioID), are being increasingly used to map protein-protein interactomes as they provide increased sensitivity in finding interaction partners. In the present study, we employ BioID coupled to a Biotinylation Site Identification Technology (BioSITe) method we recently developed to perform molecular mapping of intracellular protein interactors 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, but also several novel binders of TNK2 that may help explain its role in oncogenic signaling.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Breast, Breast Cancer Cell Line

DISEASE(S): Breast Ductal Carcinoma

SUBMITTER: Akhilesh Pandey

LAB HEAD: Akhilesh Pandey

PROVIDER: PXD010474 | Pride | 2021-08-25

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	RT_TNK2_BioSITe_SILAC_REP1.raw	Raw
	RT_TNK2_BioSITe_SILAC_REP2.raw	Raw
	RT_TNK2_BioSITe_SILAC_REP3.pdResult	Other
	RT_TNK2_BioSITe_SILAC_REP3.raw	Raw
	TNK2_BioSITe-SILAC_REP1-REP3.mzid	Mzid

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Publications

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

Tahir Raiha R Madugundu Anil K AK Udainiya Savita S Cutler Jevon A JA Renuse Santosh S Wang Li L Pearson Nicole A NA Mitchell Christopher J CJ Mahajan Nupam N Pandey Akhilesh A Wu Xinyan X

Journal of proteome research 20210824 9

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 th ...[more]

PMID: 34428048

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Project description:Background: Cell surface proteins perform critical functions related to immune response, signal transduction, cell-cell interactions, and cell migration. Expression of specific cell surface proteins can determine cell-type identity, and can be altered in diseases including infections, cancer and genetic disorders. Identification of the cell surface proteome remains a challenge despite the development of several enrichment methods exploiting their biochemical and biophysical properties. Methods: We report a novel method for enrichment of proteins localized to the cell surface. We developed surface Biotinylation Site Identification Technology (sBioSITe) by adapting our previously published method for direct identification of biotinylated peptides. The primary amine groups of lysines on cell surface proteins in live PC3 cells in culture were first labeled with biotin with subsequent enrichment of biotinylated peptides with anti-biotin antibodies. The enriched peptides containing biotin were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Biotinylated peptides identified in triplicate were mapped to proteins. Results: By direct detection of biotinylated lysines using sBioSITe, we identified 5,851 peptides biotinylated on the cell surface derived from 1,409 proteins. 533 of these proteins have been previously shown or predicted to be localized to the cell surface, or secreted into the extracellular space. Importantly, several of the identified markers have known associations with prostate cancer and metastasis including CD59, epithelial growth factor receptor (EGFR), 4F2 cell-surface antigen heavy chain (SLC3A2) and Adhesion G protein-coupled receptor E5 (ADGRE5) thus highlighting the robustness of this method. Conclusions: Detection of biotinylation sites on cell surface proteins modified in vitro provides a reliable method for the identification of cell surface proteins. This method is an excellent tool that complements existing methods for the analysis of surface expressed proteins in qualitative as well as comparative studies.