Identification and characterization of downstream signaling partners of the endothelial cell-specific receptor tyrosine kinase, Tek/Tie-2

grantor: University of TorontoTek/Tie-2 is an endothelial cell-specific receptor tyrosine kinase that plays a pivotal role in physiological and pathological angiogenesis. A family of ligands known as the angiopoietins has recently been shown to signal through Tek although little is known about the molecules that are involved in Tek-mediated signal transduction pathways. The goal of this study was to determine exactly how Tek is eliciting its signals in endothelial cells through the identification of substrates and docking molecules that bind the activated receptor. In order to circumvent the requirement for a ligand to specifically activate the receptor, we have used the yeast two-hybrid system to identify molecules that interact with phosphorylated Tek. Using this approach, we were able to demonstrate that a number of signaling molecules could associate with Tek, including the adaptor molecules Grb2, Grb7 and Grb14, the tyrosine phosphatase Shp2, the p85 regulatory subunit of phosphatidylinositol 3' kinase and one novel docking protein known as Dok-R. Mapping of the binding sites of these molecules on Tek has revealed the presence of a multi-functional docking site on Tek that is required for binding of Grb2 and Grb7 and for tyrosine phosphorylation of Grb7 and p85. Activation of Tek by Angiopoietin-1 results in tyrosine phosphorylation of p85 as well as stimulation of endothelial cell migration and survival pathways that are partly dependent on phosphatidylinositol 3' kinase activity. These findings support the role of Tek signaling in sprouting angiogenesis and endothelial cell survival. Dok-R contains numerous sites of tyrosine phosphorylation that mediate binding to RasGAP and Nck. Interestingly, overexpression of Dok-R downstream of the epidermal growth factor receptor can attenuate mitogen activated protein kinase activation and 'Dokr' maps to a locus that is rearranged in a number of lymphoproliferative disorders. These results suggest that Dok-R may be required for negative regulation of signal transduction. Further characterization of these downstream signaling molecules should prove useful in elucidating the role of Tek within the endothelial cell lineage, and this will ultimately contribute to our understanding of vascular development in both the embryo and the adult, as well as in pathological situations such as tumour-mediated angiogenesis.Ph.D