A Unique Autophosphorylation Site on Tie2/Tek Mediates Dok-R Phosphotyrosine Binding Domain Binding and Function

Abstract

Tie2/Tek is an endothelial cell receptor tyrosine kinase that induces signal transduction pathways involved in cell migration upon angiopoietin-1 (Ang1) stimulation. To address the importance of the various tyrosine residues of Tie2 in signal transduction, we generated a series of Tie2 mutants and examined their signaling properties. Using this approach in conjunction with a phosphorylation state-specific antibody, we identified tyrosine residue 1106 on Tie2 as an Ang1-dependent autophosphorylation site that mediates binding and phosphorylation of the downstream-of-kinase-related (Dok-R) docking protein. This tyrosine residue is contained within a unique interaction motif for the phosphotyrosine binding domain of Dok-R, and the pleckstrin homology domain of Dok-R further contributes to Tie2 binding in a phosphatidylinositol 3′-kinase-dependent manner. Introduction of a Tie2 mutant lacking tyrosine residue 1106 into endothelial cells interferes with Dok-R phosphorylation in response to Ang1. Furthermore, this mutant is unable to restore the migration potential of endothelial cells derived from mice lacking Tie2. Together, these findings demonstrate that tyrosine residue 1106 on Tie2 is critical for coupling downstream cell migration signal transduction pathways with Ang1 stimulation in endothelial cells.

ACKNOWLEDGMENTS

We gratefully acknowledge Urban Deutsch (Max-Planck Institut, Bad Nauheim, Germany) for providing the Tie2^wt/wt and Tie2^Δsp/Δsp endothelial cell lines. We also thank Gisele Knowles for FACS analysis and Jamie Jones for technical assistance.

This work was supported by grants from the Canadian Institute for Health Research (CIHR) and National Cancer Institute of Canada (NCIC). S.C. is the recipient of an Ontario Graduate Scholarship in Science and Technology. Z.M. and C.S. are supported by studentships from the CIHR and the CIHR—Heart and Stroke, respectively. J.T. is supported by an NCIC studentship. R.S.K. is supported by grants from the CIHR. D.J.D. is a CIHR Scientist and is a member of the Heart and Stroke/Richard Lewar Centre of Excellence, University of Toronto, Toronto, Canada.