A Novel Phosphopeptide Microarray Based Interactome Map in Breast Cancer Cells Reveals Phosphoprotein-GRB2 Cell Signaling Networks

<div><p>The architecture of cellular proteins connected to form signaling pathways in response to internal and external cues is much more complex than a group of simple protein-protein interactions. Post translational modifications on proteins (e.g., phosphorylation of serine, threonine and tyrosine residues on proteins) initiate many downstream signaling events leading to protein-protein interactions and subsequent activation of signaling cascades leading to cell proliferation, cell differentiation and cell death. As evidenced by a rapidly expanding mass spectrometry database demonstrating protein phosphorylation at specific motifs, there is currently a large gap in understanding the functional significance of phosphoproteins with respect to their specific protein connections in the signaling cascades. A comprehensive map that interconnects phospho-motifs in pathways will enable identification of nodal protein interactions that are sensitive signatures indicating a disease phenotype from the physiological hemostasis and provide clues into control of disease. Using a novel phosphopeptide microarray technology, we have mapped endogenous tyrosine-phosphoproteome interaction networks in breast cancer cells mediated by signaling adaptor protein GRB2, which transduces cellular responses downstream of several RTKs through the Ras-ERK signaling cascade. We have identified several previously reported motif specific interactions and novel interactions. The peptide microarray data indicate that various phospho-motifs on a single protein are differentially regulated in various cell types and shows global downregulation of phosphoprotein interactions specifically in cells with metastatic potential. The study has revealed novel phosphoprotein mediated signaling networks, which warrants further detailed analysis of the nodes of protein-protein interaction to uncover their biomarker or therapeutic potential.</p>