Elucidating the Mechanism of Angiopoeitin-mediated Tie2 Signalling

Abstract

This abstract is for the thesis entitled 'Elucidating the Mechanism ofAngiopoeitin-mediated Tie2 Signalling' submitted for the Degree of Doctorof Philosophy in 2012 to the Faculty of Life Sciences at the University ofManchester by Alengo Nyamay'Antu.Research on angiogenesis has been focused on developing antiangiogenictherapies to target endothelial cell-specific signalling pathways,as a mean to limit tumour outgrowth and metastasis. One of the maintargets is the endothelial cell-specific Tie2 receptor and its ligands theangiopoietins, which controls the later stages of angiogenesis. Althoughthe angiopoietin/Tie2 signalling pathways have been well characterized,the molecular mechanism by which the ligands regulate Tie2 activityremains unclear. To address this question, we determined whether theactivation mechanism of Tie2 is induced by dimerisation alone, or whethersubsequent relative rotation of the kinase domain is required. Here weemployed a coiled-coiled based protein engineering approach to identifythe relative orientations of the kinase domains that are optimal for Tie2activation. By replacing the extracellular domain of Tie2 with the dimericparallel coiled-coil motif Put3cc, we generated ligand-independenthomodimers of the kinase domains Put3cc-Tie2 I-VII that have distinctorientations. We show that dimerisation is sufficient to induce Tie2activation and downstream activation of Akt, and that varying the interfaceof the kinase domain in Tie2 dimers can increase its catalytic efficiency. Inaddition we examined for the presence of potential dimerisation within thetransmembrane and intracellular domain of Tie2. We show that the KDand potentially the TM contain dimerisation motifs that stabilise Tie2 in theinactive and active conformations. In addition, we show that deletion of thepotential coiled-coil motif in the JM does not disrupt dimerisation butdecreases the catalytic efficiency of Tie2. Finally, we propose that theactivation mechanism of Tie2 may be similar to the previously describedasymmetric dimer formation of EGFR and FGFR receptors.

Date of Award	1 Aug 2013
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Christoph Ballestrem (Supervisor) & Stephen High (Supervisor)