EphB4 Cellular Kinase Activity Assayed Using an Enzymatic Protein Interaction System

• DOI: 10.1089/adt.2012.490
• Publication Date: 2013-04-04T14:04:10Z
• Authors: Tom Wehrman; Mimi Nguyen; Wei Feng; Benjamin Bader
• Keywords: Enzyme Activation; 0303 health sciences; 03 medical and health sciences; Drug Discovery; Protein Interaction Mapping; Receptor, EphB4; Enzyme Assays; High-Throughput Screening Assays; Substrate Specificity; 3. Good health

Receptor tyrosine kinases (RTKs) are important players in various cellular processes, including proliferation, migration, metabolism, and neuronal development. EphB4 RTK is essential for the development of a functional arterial-venous network in embryonic and adult neoangiogenesis. To develop novel inhibitors of EphB4 that might have applications in severe diseases like cancer and retinopathies, assays need to be in place that resemble, in a most physiological fashion, the activation and downstream function of the kinase. In addition, such assays need to be amenable to high-throughput screening to serve efficiently the modern drug discovery processes in the pharmaceutical industry. The authors have developed an enzyme fragment complementation assay that measures the interaction of a downstream docking protein to the activated and phosphorylated full-length EphB4 kinase in cells. The assay is specific, robust, and amenable to miniaturization and high-throughput screening. It covers most steps in the activation process of EphB4, including ligand binding, autophosphorylation, and docking of a downstream interactor. This assay format can be transferred to other RTKs and adds an important cell-based kinase assay option to researchers in the field.