Development of a functional cardiovascular system is dependent on the regulated proliferation, migration, and differentiation of endothelial cells in two discrete processes known as vasculogenesis and angiogenesis (47
). Vasculogenesis occurs principally during embryonic development to establish the early vessel network, which is subsequently remodeled through angiogenesis. Here, new capillaries arise from preexisting larger vessels to give rise to a more complex vascular network with a hierarchy of both large and small vessels. Periendothelial support cells are then recruited to the nascent vessels to surround the endothelial tubes and stabilize the vessel (9
Cellular events in vascular development are controlled by molecular signal transduction pathways that are often mediated by cell surface growth factor receptors known as receptor tyrosine kinases. A number of these receptors, including those from the vascular endothelial growth factor (VEGF) receptor and TIE receptor subfamilies, have been identified on the surfaces of endothelial cells (65
). Such receptors are membrane-spanning proteins comprising an extracellular ligand binding domain and an intracellular catalytic tyrosine kinase domain, followed by a carboxy-terminal tail. Ligand-mediated receptor oligomerization triggers activation of the kinase and autophosphorylation at a specific set of tyrosine residues, which serve as docking sites for intracellular signaling molecules containing Src homology 2 (SH2) or phosphotyrosine binding (PTB) domains (45
). Functional differences between the VEGF and TIE receptors within the endothelial cell lineage may be explained in part by the unique series of signaling molecules associated with each receptor (55
Growth factors acting on the vascular endothelium presently include five members of the VEGF family and four members of the angiopoietin family (65
). Although the VEGF family possesses overlapping receptor specificity, the angiopoietins isolated to date appear to bind exclusively to the Tie2/Tek receptor tyrosine kinase and the ligand for the closely related Tie1 receptor remains elusive. Interestingly, these ligands can dynamically regulate receptor activation as angiopoietin-1 (Ang1) and Ang4 stimulate tyrosine phosphorylation of the receptor while Ang2 and Ang3 can inhibit this phosphorylation in certain cellular contexts (10
). Identification of a family of natural agonists and competitive antagonists for Tie2 implies that there is exquisite control over the signal transduction pathways mediated by this receptor.
Coordinated expression of the angiopoietins and Tie2 is required for the angiogenic remodeling and vessel stabilization processes that occur subsequent to the initial vasculogenic actions of VEGF receptors 1 and 2. Gene-targeting studies have revealed that mice deficient in Tie2 or Tie2 kinase activity do not undergo sufficient sprouting and remodeling of the primary capillary plexus, leading to incomplete development of the heart and head regions (13
). There is also a dramatic reduction in the number of endothelial cells in these mice (13
), owing to impaired survival of the endothelium in the absence of Tie2 (25
). Disruption of the Tie2 agonistic ligand, Ang1, results in embryonic lethality, with defects in angiogenesis that are strikingly similar to those seen upon disruption of Tie2 (54
). Interestingly, however, the defects observed in these mice are less severe than those observed in mice lacking Tie2, implicating the additional angiopoietins in Tie2 function. Transgenic overexpression of Ang2 in endothelial cells results in vascular defects that resemble those seen in the absence of Ang1 or Tie2 (35
), demonstrating that Ang2 can potentially regulate Ang1 function in vivo by antagonizing the effects of Ang1 on Tie2.
Underdevelopment of the vasculature in mice lacking Tie2 signaling pathways has been attributed to defects in both sprouting angiogenesis and intussusceptive microvascular growth (44
). During sprouting angiogenesis, activated endothelial cells secrete matrix-degrading proteinases and migrate through the basement membrane into the surrounding tissue. A role for Tie2 signaling in endothelial cell migration is supported by numerous studies showing that activation of Tie2 by Ang1 results in the stimulation of cell motility, including sprout and tubule formation (19
), and that a modified form of Ang1 known as Ang1* synergizes with VEGF during sprouting angiogenesis in vivo (1
). Ang2 can also stimulate tyrosine phosphorylation of Tie2 in several endothelial cell types, leading to tubule formation on fibrin and collagen matrices (38
), and Ang2 augments fibroblast growth factor 2-induced chemotaxis (38
). Endothelial cell migration in response to Ang1 is contingent upon changes in the intracellular architecture of the cell, which may depend in part on the activity of phosphatidylinositol (PI) 3′ kinase. Upon Ang1 stimulation, the SH2 domain-containing p85 subunit of PI 3′ kinase is recruited to Tie2 via tyrosine residue 1100 in the carboxy-terminal tail of the receptor, leading to activation of the enzyme (24
). Addition of PI 3′ kinase inhibitors in cell motility assays blocks Ang1-stimulated migration of both endothelial and nonendothelial cells expressing Tie2 (15
) as well as Ang2-stimulated chemotaxis of endothelial cells (38
). Interestingly, however, inhibition of PI 3′ kinase activity can only partially inhibit the chemotactic effect of Ang1 on endothelial cells (24
), thereby implying that additional Tie2 binding partners may also contribute to Ang1-mediated endothelial cell migration.
Phosphorylation of Tie2 further results in its association with a docking protein related to downstream of kinase (Dok), known as Dok-R (also referred to as p56Dok2
and FRIP) (22
). Five Dok family proteins that are characterized by an amino-terminal pleckstrin homology (PH) domain, a central PTB domain, and a carboxy-terminal region rich in tyrosine and proline residues have been isolated (5
). Recruitment of Dok-R to the activated Tie2 receptor via its PTB domain results in the phosphorylation of Dok-R on multiple tyrosine residues. This phosphorylation establishes binding sites for the Ras GTPase-activating protein and the adaptor protein Nck, both of which have been implicated in cell motility and actin rearrangement (16
). An equivalent Nck binding site on Dok (also known as p62Dok1
) mediates cell migration in response to insulin (41
), and overexpression of a mutant form of Dok lacking the carboxy-terminal region (which includes the Nck binding site) inhibits the migration potential of metastatic melanoma cells (21
). We have recently demonstrated that the expression of Dok-R can potentiate Ang1-induced cell migration (36
). This effect is dependent on the association of Dok-R with an Nck/p21 activated kinase (Pak) complex, and the localization of Pak with Tie2 at the membrane surface is required for Pak kinase activation (36
). Enzyme activation is essential for Pak function (20
), which underscores the importance of recruiting this molecular complex to the phosphorylated Tie2 receptor to mediate Ang1-stimulated cell motility.
Despite intense investigation into the signal transduction pathways initiated by Tie2, it is presently not well understood which tyrosine residues are phosphorylated on the activated receptor to bridge interactions with downstream signaling molecules. A recent mass spectrometry-based approach to identify phosphorylation sites on the autophosphorylated Tie2 intracellular domain revealed tyrosine residue 1106 but not 1100 as an autophosphorylation site (39
). Using mutant forms of Tie2 in conjunction with a phosphorylation-state-specific antibody, we have identified tyrosine residue 1106 on Tie2 as an Ang1-dependent autophosphorylation site that is required to mediate the binding and phosphorylation of Dok-R. Binding of Dok-R to Tie2 is mediated through a unique PTB domain binding motif flanking tyrosine residue 1106, and the PH domain of Dok-R also contributes to Tie2 binding in a PI 3′-kinase-dependent manner. Mutation in tyrosine residue 1106 cannot restore the migratory potential of endothelial cells derived from Tie2-null mice, thereby demonstrating the importance of this tyrosine residue in linking downstream cell migration signal transduction pathways with Ang1 stimulation in endothelial cells.