Interactome Mapping Uncovers a General Role for Numb in Protein Kinase Regulation

Ran Wei,Tomonori Kaneko,Xuguang Liu,Huadong Liu,Lei Li,Courtney Voss,Eric Liu,Ningning He,Shawn S.-C Li

doi:10.1074/mcp.ra117.000114

Abstract

Cellular functions are frequently regulated by protein-protein interactions involving the binding of a modular domain in one protein to a specific peptide sequence in another. This mechanism may be explored to identify binding partners for proteins harboring a peptide-recognition domain. Here we report a proteomic strategy combining peptide and protein microarray screening with biochemical and cellular assays to identify modular domain-mediated protein-protein interactions in a systematic manner. We applied this strategy to Numb, a multi-functional protein containing a phosphotyrosine-binding (PTB) domain. Through the screening of a protein microarray, we identified >100 protein kinases, including both Tyr and Ser/Thr kinases, that could potentially interact with the Numb PTB domain, suggesting a general role for Numb in regulating kinase function. The putative interactions between Numb and several tyrosine kinases were subsequently validated by GST pull-down and/or co-immunoprecipitation assays. Furthermore, using the Oriented Peptide Array Library approach, we defined the specificity of the Numb PTB domain which, in turn, allowed us to predict binding partners for Numb at the genome level. The combination of the protein microarray screening with computer-aided prediction produced the most expansive interactome for Numb to date, implicating Numb in regulating phosphorylation signaling through protein kinases and phosphatases. Not only does the data generated from this study provide an important resource for hypothesis-driven research to further define the function of Numb, the proteomic strategy described herein may be employed to uncover the interactome for other peptide-recognition domains whose consensus motifs are known or can be determined.

Highlights

Protein-protein interactions (PPIs)1 play a pivotal role in all essential cellular processes, and dysregulation of PPI may lead to diseases such as cancer [1]
Identification of Modular Domain-binding Proteins by Complementary Peptide and Protein Array Screening—We have developed an integrative approach combining protein and peptide array screening with computer-aided ligand prediction to systematically identify the PPI network mediated by modular domains
We evaluated this approach using the Numb phosphotyrosine binding (PTB) domain

Summary

MATERIALS AND METHODS

Reagents and Antibodies—The SKBR3, Hela and HCC78 cell lines were obtained from American Tissue Culture Collection. Far-Western Blotting of Peptide Array—The peptide spot array membrane was blocked with 3% BSA in TBST buffer (0.1 M Tris-HCl, pH 7.4, 150 mM NaCl, and 0.1% Tween 20) for 1 h at room temperature, with gentle rocking. GST-fusion proteins were added directly into the blocking buffer to reach a final concentration of 1 ␮g/ml and incubated with the peptide array membrane for 1 h at room temperature, with gentle shaking. Immunoprecipitation, GST Pulldown and Western Blotting—Cultured cells were lysed in ice-cold lysis buffer (1% Triton X-100, 50 mM Tris-pH 7.2, 150 mM NaCl, 2 mM MgCl2, 0.1 mM EDTA, 0.1 mM EGTA, 0.5 mM DTT, 10 mM NaF) containing complete protease inhibitors (Roche, Basel, Switzerland). The scoring matrix was imported to SMALI and used to search the UniProt protein database [37] to generate a ranked list of potential Numb PTB domain binding peptides/proteins

RESULTS

SMALI Score

PGLDNPAYVSS TGFSNMSFEDF

DISCUSSION