Motif Decomposition of the Phosphotyrosine Proteome Reveals a New N-terminal Binding Motif for SHIP2

Martin Lee Miller,Stefan Hanke,Anders M⊘Rkeberg Hinsby,Carsten Friis,S⊘Ren Brunak,Matthias Mann,Nikolaj Blom

doi:10.1074/mcp.m700241-mcp200

Abstract

Advances in mass spectrometry-based proteomics have yielded a substantial mapping of the tyrosine phosphoproteome and thus provided an important step toward a systematic analysis of intracellular signaling networks in higher eukaryotes. In this study we decomposed an uncharacterized proteomics data set of 481 unique phosphotyrosine (Tyr(P)) peptides by sequence similarity to known ligands of the Src homology 2 (SH2) and the phosphotyrosine binding (PTB) domains. From 20 clusters we extracted 16 known and four new interaction motifs. Using quantitative mass spectrometry we pulled down Tyr(P)-specific binding partners for peptides corresponding to the extracted motifs. We confirmed numerous previously known interaction motifs and found 15 new interactions mediated by phosphosites not previously known to bind SH2 or PTB. Remarkably, a novel hydrophobic N-terminal motif ((L/V/I)(L/V/I)pY) was identified and validated as a binding motif for the SH2 domain-containing inositol phosphatase SHIP2. Our decomposition of the in vivo Tyr(P) proteome furthermore suggests that two-thirds of the Tyr(P) sites mediate interaction, whereas the remaining third govern processes such as enzyme activation and nucleic acid binding.

Highlights

Advances in mass spectrometry-based proteomics have yielded a substantial mapping of the tyrosine phosphoproteome and provided an important step toward a systematic analysis of intracellular signaling networks in higher eukaryotes
We estimate that one-third of the in vivo Tyr(P) sites are not directly involved in interaction via domains such as Src homology 2 (SH2) and phosphotyrosine binding (PTB) but rather are sites that could alter the catalytic activity of enzymes or modulate the DNA binding affinity of e.g. transcription factors
A New Clustering Approach for Motif Extraction and Classification—To discover new interaction motifs in large aligned peptide data sets, we developed a method that partitions the sequences based on similarity with proteins previously known to be involved in interaction

Summary

Introduction

Advances in mass spectrometry-based proteomics have yielded a substantial mapping of the tyrosine phosphoproteome and provided an important step toward a systematic analysis of intracellular signaling networks in higher eukaryotes. In this study we decomposed an uncharacterized proteomics data set of 481 unique phosphotyrosine (Tyr(P)) peptides by sequence similarity to known ligands of the Src homology 2 (SH2) and the phosphotyrosine binding (PTB) domains. “Linear motifs” (unstructured sequence recognition patches with conserved residues at specific positions [3]) that direct Tyr(P)-dependent interaction have traditionally been studied using degenerate oriented peptide libraries. Such studies revealed that PTB and SH2 domains have preference for specific amino acids N- and C-terminal to the Tyr(P) residue, respectively [4, 5]. The above mentioned methods are in silico approaches and do not combine prediction with experimental validation

Results

Discussion

Conclusion