Directed Network Wiring Identifies a Key Protein Interaction in Embryonic Stem Cell Differentiation

•Defining roles of individual interactions in complex networks is challenging•Protein design tailors Grb2 SH2 into “pY-clamps” specific for single pY-ligands•Rewiring the Grb2 PPI network using synthetic Grb2 comprising pY-clamps•Identified Ptpn11 pY580 as a key Grb2 SH2 ligand in stem cell fate specification

SummaryCell signaling depends on dynamic protein-protein interaction (PPI) networks, often assembled through modular domains each interacting with multiple peptide motifs. This complexity raises a conceptual challenge, namely to define whether a particular cellular response requires assembly of the complete PPI network of interest or can be driven by a specific interaction. To address this issue, we designed variants of the Grb2 SH2 domain (“pY-clamps”) whose specificity is highly biased toward a single phosphotyrosine (pY) motif among many potential pYXNX Grb2-binding sites. Surprisingly, directing Grb2 predominantly to a single pY site of the Ptpn11/Shp2 phosphatase, but not other sites tested, was sufficient for differentiation of the essential primitive endoderm lineage from embryonic stem cells. Our data suggest that discrete connections within complex PPI networks can underpin regulation of particular biological events. We propose that this directed wiring approach will be of general utility in functionally annotating specific PPIs.

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