Robust co-regulation of tyrosine phosphorylation sites on proteins reveals novel protein interactions.

TitleRobust co-regulation of tyrosine phosphorylation sites on proteins reveals novel protein interactions.
Publication TypeJournal Article
Year of Publication2012
AuthorsNaegle, KM, White, FM, Lauffenburger, DA, Yaffe, MB
JournalMol Biosyst
Volume8
Issue10
Pagination2771-82
Date Published2012 Oct
ISSN1742-2051
KeywordsChromatography, Liquid, Data Mining, Epithelial Cells, Female, Humans, Immunohistochemistry, Immunoprecipitation, Ligands, LIM Domain Proteins, Mammary Glands, Human, Microfilament Proteins, Phosphoproteins, Phosphorylation, Primary Cell Culture, Protein Binding, Protein Interaction Mapping, Receptor, Epidermal Growth Factor, Signal Transduction, Tandem Mass Spectrometry, Tyrosine
Abstract

Cell signaling networks propagate information from extracellular cues via dynamic modulation of protein-protein interactions in a context-dependent manner. Networks based on receptor tyrosine kinases (RTKs), for example, phosphorylate intracellular proteins in response to extracellular ligands, resulting in dynamic protein-protein interactions that drive phenotypic changes. Most commonly used methods for discovering these protein-protein interactions, however, are optimized for detecting stable, longer-lived complexes, rather than the type of transient interactions that are essential components of dynamic signaling networks such as those mediated by RTKs. Substrate phosphorylation downstream of RTK activation modifies substrate activity and induces phospho-specific binding interactions, resulting in the formation of large transient macromolecular signaling complexes. Since protein complex formation should follow the trajectory of events that drive it, we reasoned that mining phosphoproteomic datasets for highly similar dynamic behavior of measured phosphorylation sites on different proteins could be used to predict novel, transient protein-protein interactions that had not been previously identified. We applied this method to explore signaling events downstream of EGFR stimulation. Our computational analysis of robustly co-regulated phosphorylation sites, based on multiple clustering analysis of quantitative time-resolved mass-spectrometry phosphoproteomic data, not only identified known sitewise-specific recruitment of proteins to EGFR, but also predicted novel, a priori interactions. A particularly intriguing prediction of EGFR interaction with the cytoskeleton-associated protein PDLIM1 was verified within cells using co-immunoprecipitation and in situ proximity ligation assays. Our approach thus offers a new way to discover protein-protein interactions in a dynamic context- and phosphorylation site-specific manner.

DOI10.1039/c2mb25200g
Alternate JournalMol Biosyst
PubMed ID22851037
PubMed Central IDPMC3501258
Grant ListP50 GM068762 / GM / NIGMS NIH HHS / United States
P50-GM68762 / GM / NIGMS NIH HHS / United States
R01 CA096504 / CA / NCI NIH HHS / United States
R01 ES-015339 / ES / NIEHS NIH HHS / United States
R01 ES015339 / ES / NIEHS NIH HHS / United States
R01-CA96504 / CA / NCI NIH HHS / United States
U54 CA112967 / CA / NCI NIH HHS / United States
U54-CA112967 / CA / NCI NIH HHS / United States