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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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:Ubash3b, also known as suppressor of T-cell receptor signaling or Sts-1, is an ill-studied atypical tyrosine phosphatase with ubiquitin binding ability. In our previous study, we hypothesized that Ubash3b plays an inhibitory role in BCR-ABL signaling through binding and dephosphorylating BCR-ABL and its interactors. The Hantschel lab recently solved the crystal structures of the p210 PH and DH domains, which are absent in the p190 variant, and demonstrated that loss-of-function mutations in the PH domain altered BCR-ABL localization, thereby reducing the interaction between Ubash3b and p2104. Taken together, this suggests differential subcellular localization of Ubash3b as a mechanism by which it interacts more strongly with p201 as compared to p190. To better understand the global impact Ubash3b has on p210, its direct kinase substrates and proteins in its phosphotyrosine signaling network, we have taken an integrative approach by combining global phosphotyrosine profiling, proximity-dependent biotinylation (BioID) and total protein analysis to investigate p210 signaling upon Ubash3b knockdown (KD). The BioID system was used to characterize Ubash3b function in p210 signaling by examining its interactome. Importantly, in all of our BioID experiments, we employed a newly technique that we have recently developed, Biotinylation Site Identification Technology (BioSITe), which directly identifies biotinylated peptides thereby increasing the reliability of the identified interactors. Here, we additionally used short hairpin RNA (shRNA) interference and generated Ubash3b knock-down (KD) and non-targeting control shRNA lines in Ba/F3 BirA*-p210 cells. Ubash3b expression was reduced >90 % in the KD cells and had a substantial effect on global tyrosine phosphorylation and on the interactome of p210. Of the 1,421 unique tyrosine phosphorylation sites identified from 830 proteins, 379 sites (from 286 proteins) exhibited a substantial increase (ÃƒÂ¢Ã¢â‚¬Â°Ã‚Â¥2-fold) in tyrosine phosphorylation upon Ubash3b KD cells compared to control cells. To date, the interactome of Ubash3b has not been extensively investigated, however, some examination of Ubash3b in the context p210 signaling has been undertaken. We designed constructs of C-terminal BirA* tagged full length Ubash3b and a deletion mutant lacking the UBA and SH3 domains leaving only the phosphatase domain tethered to BirA*. A comparative analysis of the core interactors of p210 from previous studies and Ubash3b interactome from the current study revealed 36 proteins that interact with both p210 and Ubash3b.

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.

Dataset Information

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

Publications

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

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