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Development of the cardiovascular system is critically dependent on the ability of endothelial cells (ECs) to reorganize their intracellular actin architecture to facilitate migration, adhesion, and morphogenesis. Nck family cytoskeletal adaptors function as key mediators of actin dynamics in numerous cell types, though their role in EC biology remains largely unexplored. Here, we demonstrate an essential requirement for Nck within ECs. Mouse embryos lacking endothelial Nck1/2 expression develop extensive angiogenic defects that result in lethality at about embryonic day 10. Mutant embryos show immature vascular networks, with decreased vessel branching, aberrant perivascular cell recruitment, and reduced cardiac trabeculation. Strikingly, embryos deficient in endothelial Nck also fail to undergo the endothelial-to-mesenchymal transition (EnMT) required for cardiac valve morphogenesis, with loss of Nck disrupting expression of major EnMT markers, as well as suppressing mesenchymal outgrowth. Furthermore, we show that Nck-null ECs are unable to migrate downstream of vascular endothelial growth factor and angiopoietin-1, and they exhibit profound perturbations in cytoskeletal patterning, with disorganized cellular projections, impaired focal adhesion turnover, and disrupted actin-based signaling. Our collective findings thereby reveal a crucial role for Nck as a master regulator within the endothelium to control actin cytoskeleton organization, vascular network remodeling, and EnMT during cardiovascular development. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]