Phosphotyrosine interactome of the ErbB-receptor kinase family

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Waltraud X Schulze¹, Lei Deng¹ & Matthias Mann¹

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Synopsis

A large-scale peptide-protein interaction assay using quantitative proteomics reveals the entire phosphotyrosine-dependent interactome of the ErbB family

One of the main interests in systems biology is to understand how cells process information. In signaling pathways, ligands bind cell surface receptors and this information is passed on via intracellular phosphorylation events. An important subset of these signal-dependent phosphorylation events creates new protein-protein interaction sites, which serve to recruit effector molecules to activated receptors. Mechanistically, signal transduction events in all organisms are characterized by modification-dependent protein-protein interactions between domains and extended peptide motifs. Consensus peptide sequences recognized by different protein domains have been studied using oriented peptide libraries, peptide arrays or phage display. Activated tyrosine kinase receptors and their interactions with specific downstream signaling molecules have been investigated by immunoprecipitation and detection of selected proteins by Western blotting. However, most of these methods lack specificity for modification-dependent interactions and only a few of them are unbiased or can be performed at a proteomic scale.

In the past, detection of protein-protein interactions has been a balance between specificity (background reduction) and affinity (detection of weak interactions). Recently, the introduction of stable isotope labeling to distinguish specific from unspecific interaction partners has enabled detection of weak binders in the presence of background proteins. We have developed a proteomic screen for peptide motif-based interactions, which we have optimized here for high-throughput analysis. Using synthetic peptide pairs in phosphorylated and unphosphorylated form, pull-down experiments are performed on normal and SILAC (Stable Isotope Labeling by Amino acids in Cell culture)-labeled cell lysates. This approach allows us to identify specific binding partners of the phosphorylated bait peptides by mass spectrometry, even in the presence of a large excess of background binders, which are recognized by exhibiting a one-to-one ratio between labeled and unlabeled state. Here, we use this improved pull-down approach to profile the entire phosphotyrosine interactome of the ErbB-receptor family.

The relatively high throughput of our screen allowed a systematic study of all tyrosine residues of the ErbB-receptor family without making prior assumptions about whether a site might be phosphorylated in vivo or what the interaction partner might be. We analyzed all 89 cytosolic tyrosine residues in the four receptors EGFR, ErbB2, ErbB3 and ErbB4. Not surprisingly, 49 out of the 89 investigated tyrosine residues did not have an interaction partner to their phosphorylated form. Most of the tyrosine residues without interaction partners were located in and around the kinase domain, whereas the residues that interacted with specific partners accumulated at the C-terminal regions of the receptors. Strikingly, all binding partners with a significant ratio in our assay had either an SH2 or a PTB domain and we did not find any evidence for false-positive interactions.

The distribution of interaction partners of the different members of the ErbB-receptor family shows clear differences between individual receptors, and also a large overlap. EGFR is the family member with most interaction partners and the highest percentage of tyrosine residues with more than one binding partner. ErbB3 is characterized by a large number of binding sites for phosphatidylinositol-3-kinase (PI3K), while ErbB2 has only few interaction partners with Shc as the most frequent one. ErbB4 and EGFR have a variety of phosphotyrosines that bind Grb2, or Grb2 and Shc. The EGFR and ErbB4 have a greater diversity of interaction partners than ErbB2 and ErbB3.

To correlate our findings with in vivo phosphorylation of the receptor, we directly analyzed the kinetics of in vivo phosphorylation of several tyrosine residues upon EGF stimulation by mass spectrometry. Phosphopeptides of immunoprecipitated EGFR were analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) after different times of stimulation with EGF. In this experiment, we identified novel EGFR phosphorylation sites at Y801 and Y998. Our results show that different phosphorylation sites have different interaction partners and show different phosphorylation dynamics after stimulation. This may reflect distinct temporal roles in signaling. Interestingly, these differences in phosphorylation dynamics correlate with the interaction partners identified for each of these sites. The autophosphorylation sites at Y1110, Y1172 and Y1197 display rapid phosphorylation with a maximum after 4 min and they have the same set of interaction partners (Grb2 and Shc). The novel phosphorylation site at Y998 is increasingly phosphorylated over 15 min, and interacts with STAT5, PTP-2c, Shc, Crk, and the phosphotyrosine kinase Src or Csk. In contrast, the phosphorylation site at Y869 and Y801 showed distinct phosphorylation patterns but had no interaction partners.

Our studies show that different receptors in the ErbB family clearly differ in their preferred interaction partners indicating distinct roles in signaling. The results are consistent with the idea that EGFR and ErbB4 function as 'control centers' and sites of integration for signaling processes involving stimulation with their ligands, EGF and neuregulin, respectively. This idea is supported by findings of overexpression studies, in which EGFR dimerizes preferably with ErbB2 and ErbB3, while ErbB4 dimerizes with ErbB2 and ErbB3, but to a lesser extent with EGFR. In this respect, it should be kept in mind that ErbB2 has a nonfunctional extracellular ligand binding domain and is a common dimerization partner of other members of the ErbB family. In contrast, ErbB3 has an inactive kinase domain; thus, transphosphorylation in different heterodimers may lead to phosphorylation of different sites in ErbB3. Furthermore, the striking difference in interaction partners of kinase-inactive ErbB3 compared to the other family members may indicate a specific role in signaling to the PI3K pathway as part of heterodimers with EGFR, ErbB4 or ErbB2 in different cell types or differentiation stages. In conclusion, we have shown that the peptide-protein interaction screen can be performed in a large-scale fashion and that it is indeed a suitable method to obtain a broad picture of shared and distinct interaction partners of a whole protein family.

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