Abstract
Here we describe a novel method for studying the protein "interactome" in primary human cells and apply this method to investigate the effect of posttranslational protein modifications (PTMs) on the protein's functions. We created a novel "biomimetic microsystem platform" (Bio-MSP) to isolate the protein complexes in primary cells by covalently attaching purified His-tagged proteins to a solid microscale support. Using this Bio-MSP, we have analyzed the interactomes of unphosphorylated and phosphomimetic end-binding protein-3 (EB3) in endothelial cells. Pathway analysis of these interactomes demonstrated the novel role of EB3 phosphorylation at serine 162 in regulating the protein's function. We showed that phosphorylation "switches" the EB3 biological network to modulate cellular processes such as cell-to-cell adhesion whereas dephosphorylation of this site promotes cell proliferation. This novel technique provides a useful tool to study the role of PTMs or single point mutations in activating distinct signal transduction networks and thereby the biological function of the protein in health and disease.