Screening for PTB domain binding partners and ligand specificity using proteome-derived NPXY peptide arrays.
Modular interaction domains that recognize peptide motifs in target proteins can impart selectivity in signaling pathways. Phosphotyrosine binding (PTB) domains are components of cytoplasmic docking proteins that bind cell surface receptors through NPXY motifs. We have employed a library of human proteome-derived NXXY sequences to explore PTB domain specificity and function. SPOTS peptide arrays were used to create a comprehensive matrix of receptor motifs that were probed with a set of 10 diverse PTB domains. This approach confirmed that individual PTB domains have selective and distinct recognition properties and provided a means to explore over 2,500 potential PTB domain-NXXY interactions. The results correlated well with previously known associations between full-length proteins and predicted novel interactions, as well as consensus binding data for specific PTB domains. Using the Ret, MuSK, and ErbB2 receptor tyrosine kinases, we show that interactions of these receptors with PTB domains predicted to bind by the NXXY arrays do occur in cells. Proteome-based peptide arrays can therefore identify networks of receptor interactions with scaffold proteins that may be physiologically relevant.
Smith
Matthew J
MJ
Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada M5G 1X5.
Hardy
W Rod
WR
Murphy
James M
JM
Jones
Nina
N
Pawson
Tony
T
D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001665:Binding Sites; D002460:Cell Line; D016000:Cluster Analysis; D030562:Databases, Protein; D006801:Humans; D008024:Ligands; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000378:metabolism; D009687:Nuclear Proteins / Q000378:metabolism; D010750:Phosphoproteins; D010766:Phosphorylation; D040081:Protein Array Analysis / Q000379:methods*; D025941:Protein Interaction Mapping; D017434:Protein Structure, Tertiary; D020543:Proteome / Q000378:metabolism*; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics* / Q000378:metabolism; D055765:Shc Signaling Adaptor Proteins; D015398:Signal Transduction; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1
C091042:APBB1 protein, human; D048868:Adaptor Proteins, Signal Transducing; C115089:DOK2 protein, human; D008024:Ligands; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D010750:Phosphoproteins; D020543:Proteome; C528024:SHC1 protein, human; D055765:Shc Signaling Adaptor Proteins; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D020794:Receptor Protein-Tyrosine Kinases