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

Area of Science:

• Molecular Biology
• Proteomics
• Cancer Signaling

Background:

• Tyrosine phosphorylation is crucial for cellular signaling in health and disease.
• Existing methods lack high-throughput functional annotation of phosphotyrosine sites in tissues.
• Understanding signaling networks is vital for deciphering (patho)physiology.

Purpose of the Study:

• To develop a mass spectrometry-based interaction proteomics approach for functional annotation of phosphotyrosine sites directly in tissue samples.
• To decipher the in vivo epidermal growth factor (EGF)-dependent signaling network in lung tissue.
• To assign function to EGF-regulated phosphotyrosine sites by identifying their recruited protein signaling complexes.

Main Methods:

• Developed and applied a mass-spectrometry-based interaction proteomics strategy.
• Quantified over 1,000 phosphotyrosine sites in EGF-stimulated lung tissue.
• Performed over 1,000 phosphopeptide pulldowns followed by rapid mass spectrometric analysis.

Main Results:

• Successfully mapped the in vivo EGF-dependent signaling network in lung tissue, identifying over 1,000 phosphotyrosine sites.
• Determined the protein signaling complexes recruited by EGF-regulated phosphotyrosine sites.
• Demonstrated that mutations near tyrosine residues can rewire cancer signaling networks, identifying oncogenic properties of a lung cancer EGFR mutant.
• Revealed tissue-specific differences in protein interactors through scalable phosphopeptide pulldowns.

Conclusions:

• The developed approach provides a general strategy for functional annotation of phosphorylation sites in tissues.
• Enables in-depth mechanistic insights into the oncogenic rewiring of signaling networks.
• Facilitates the study of tyrosine phosphorylation in diverse physiological and pathological contexts.