Comprehensive binary interaction mapping of SH2 domains via fluorescence polarization reveals novel functional diversification of ErbB receptors

PLoS One. 2012;7(9):e44471. doi: 10.1371/journal.pone.0044471. Epub 2012 Sep 4.

Abstract

First-generation interaction maps of Src homology 2 (SH2) domains with receptor tyrosine kinase (RTK) phosphosites have previously been generated using protein microarray (PM) technologies. Here, we developed a large-scale fluorescence polarization (FP) methodology that was able to characterize interactions between SH2 domains and ErbB receptor phosphosites with higher fidelity and sensitivity than was previously achieved with PMs. We used the FP assay to query the interaction of synthetic phosphopeptides corresponding to 89 ErbB receptor intracellular tyrosine sites against 93 human SH2 domains and 2 phosphotyrosine binding (PTB) domains. From 358,944 polarization measurements, the affinities for 1,405 unique biological interactions were determined, 83% of which are novel. In contrast to data from previous reports, our analyses suggested that ErbB2 was not more promiscuous than the other ErbB receptors. Our results showed that each receptor displays unique preferences in the affinity and location of recruited SH2 domains that may contribute to differences in downstream signaling potential. ErbB1 was enriched versus the other receptors for recruitment of domains from RAS GEFs whereas ErbB2 was enriched for recruitment of domains from tyrosine and phosphatidyl inositol phosphatases. ErbB3, the kinase inactive ErbB receptor family member, was predictably enriched for recruitment of domains from phosphatidyl inositol kinases and surprisingly, was enriched for recruitment of domains from tyrosine kinases, cytoskeletal regulatory proteins, and RHO GEFs but depleted for recruitment of domains from phosphatidyl inositol phosphatases. Many novel interactions were also observed with phosphopeptides corresponding to ErbB receptor tyrosines not previously reported to be phosphorylated by mass spectrometry, suggesting the existence of many biologically relevant RTK sites that may be phosphorylated but below the detection threshold of standard mass spectrometry procedures. This dataset represents a rich source of testable hypotheses regarding the biological mechanisms of ErbB receptors.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Chromatography, Gel
  • ErbB Receptors / genetics
  • ErbB Receptors / metabolism*
  • Fluorescence Polarization / methods*
  • Phosphopeptides / isolation & purification
  • Phosphopeptides / metabolism
  • Phosphorylation
  • Protein Array Analysis
  • Protein Interaction Mapping / methods*
  • Receptor, ErbB-2 / genetics
  • Receptor, ErbB-2 / metabolism*
  • Surface Plasmon Resonance
  • src Homology Domains / genetics*

Substances

  • Phosphopeptides
  • EGFR protein, human
  • ErbB Receptors
  • Receptor, ErbB-2

Grant support

This work was supported by awards from the Cancer Research Foundation and The Illinois Department of Health. Additional support was provided by National Institutes of Health Postdoctoral Cardiovascular Training Grant (JB), NIH Cancer Biology Postdoctoral Training Grant (CPC), NIH Cancer Biology Pre-doctoral Training grant (KKL), NIH Genetics and Regulation Pre-doctoral Training Grant (RJH), and NIH Systems Biology of Oxygen Predoctoral Training Grant (MFC). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.