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Cell. 2019 Oct 3;179(2):543-560.e26. doi: 10.1016/j.cell.2019.09.008.

Oncogenic Mutations Rewire Signaling Pathways by Switching Protein Recruitment to Phosphotyrosine Sites.

Author information

1
Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Faculty of Health and Medical Sciences, Blegdamsvej 3b, DK-2200 Copenhagen, Denmark; Department of Biomedical Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark. Electronic address: alicia.lundby@sund.ku.dk.
2
Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Faculty of Health and Medical Sciences, Blegdamsvej 3b, DK-2200 Copenhagen, Denmark.
3
Biotech Research and Innovation Centre (BRIC), Faculty of Health and Medical Sciences, University of Copenhagen, 2200 Copenhagen, Denmark.
4
Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Faculty of Health and Medical Sciences, Blegdamsvej 3b, DK-2200 Copenhagen, Denmark; Novo Nordisk A/S, Novo Nordisk Park, DK-2760 Maaloev, Denmark.
5
Department of Biomedical Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
6
Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Faculty of Health and Medical Sciences, Blegdamsvej 3b, DK-2200 Copenhagen, Denmark. Electronic address: jesper.olsen@cpr.ku.dk.

Abstract

Tyrosine phosphorylation regulates multi-layered signaling networks with broad implications in (patho)physiology, but high-throughput methods for functional annotation of phosphotyrosine sites are lacking. To decipher phosphotyrosine signaling directly in tissue samples, we developed a mass-spectrometry-based interaction proteomics approach. We measured the in vivo EGF-dependent signaling network in lung tissue quantifying >1,000 phosphotyrosine sites. To assign function to all EGF-regulated sites, we determined their recruited protein signaling complexes in lung tissue by interaction proteomics. We demonstrated how mutations near tyrosine residues introduce molecular switches that rewire cancer signaling networks, and we revealed oncogenic properties of such a lung cancer EGFR mutant. To demonstrate the scalability of the approach, we performed >1,000 phosphopeptide pulldowns and analyzed them by rapid mass spectrometric analysis, revealing tissue-specific differences in interactors. Our approach is a general strategy for functional annotation of phosphorylation sites in tissues, enabling in-depth mechanistic insights into oncogenic rewiring of signaling networks.

KEYWORDS:

EGFR; SH2; cancer signaling; interaction proteomics; mass spectrometry; peptide pulldown; phosphopeptides; phosphoproteomics; phosphotyrosine; zebrafish

PMID:
31585087
DOI:
10.1016/j.cell.2019.09.008

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