ABSTRACT
ErbB2 signaling pathways are linked to breast cancer formation, growth, and aggression; therefore, understanding the behavior of proteins associated with these pathways as well as regulatory factors that influence ErbB2 function is essential. MEMO1 is a redox active protein that is shown to associate with phosphorylated ErbB2 and mediate cell motility. We have developed a fluorescence polarization assay to probe the interaction between MEMO1 and an ErbB2-derived peptide containing a phosphorylated tyrosine residue. This interaction is shown to be pH-dependent and stronger with longer peptides as would be expected for protein-protein interactions. We have quantitatively mapped the binding interface of MEMO1 to the peptide using the fluorescence polarization assay and molecular dynamics simulations. We have confirmed that phosphorylation of the peptide is essential for binding and through mutagenesis have identified residues that contribute to favorable interactions. Our results highlight the importance of the protein-protein interactions of MEMO1 that complement the oxidase activity. In the future, these studies will provide a method for screening for selective modulators of MEMO1, which will allow for additional biological investigations.
