Expression and Characterization of the Receptor Tyrosine Kinase Tie2

Abstract

Nearly one‐third of human kinases are implicated in disease, and this enzyme class has proved to be an enormously successful drug target for combating many diseases. Kinases are key regulators in signaling pathways responsible for such processes as growth, proliferation, and angiogenesis. Tie2 is an endothelium‐specific receptor tyrosine kinase (RTK) responsible for angiogenesis and vasculature maintenance. Mutations in this enzyme can cause venous malformations for which no current therapies are available, and Tie2 has been proposed to be a target for anticancer therapies. However, the molecular mechanisms of Tie2 activity are not well understood. Here, we developed a robust purification strategy using heterologous expression in insect cells, optimizing expression and purification with a post‐infection incubation time of 72 hours and utilization of affinity chromatography. We have created autophosphorylation assays to establish catalytic rates of wild‐type Tie2 and mutants implicated in disease. We hypothesize that mutant Tie2 will have faster rates of autophosphorylation, likely through stabilization of the active conformation of the protein. These studies in kinase activity can improve our understanding of the catalytic features of wild‐type and mutant Tie2, and can inform the development of therapeutic targets for vascular disease.Support or Funding InformationNIHThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.