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

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.