A negative regulatory role for Y1111 on the Tie-2 RTK.

Tie2 is a receptor tyrosine kinase (RTK) essential for aspects of both normal and pathological angiogenesis. Understanding how this receptor is regulated is important for development of therapeutic angiogenic agents. Evidence suggests the C-terminal tail of the receptor plays a negative regulatory role in Tie2 signaling and function. Here we investigated the role of a specific C-tail residue, Y1111, in Tie2 signaling by generating a number of receptor point mutants. We found that mutation of this site to phenylalanine (Y1111F) results in an increase in receptor phosphorylation and kinase activity, as well as increased downstream signaling. Furthermore, mutation of Y1111 to the highly charged aspartate (Y1111D) or glutamate (Y1111E) results in even more dramatic increase in receptor phosphorylation and activity. Limited protease digestion studies indicate that these mutations may alter receptor conformation and potentially relieve negative inhibition imparted by the C-tail of Tie2. These studies suggest that Y1111 plays a key role in negative regulation of Tie2 activity and they provide important insight into molecular mechanisms behind the intrinsic ability of this RTK to regulate its own activity.

Chronic systemic delivery of angiopoietin-2 reveals a possible independent angiogenic effect.

Angiopoietin-2 has been implicated in the angiogenic response; however, this response has been tied to the expression of VEGF, and an independent angiogenic role has yet to be described. In this report, we detail the generation of transgenic mice that conditionally express angiopoietin-2 in the liver, resulting in sustained increases in circulating levels. These animals survive gestation and present with several vascular abnormalities, including an increase in the diameter of myocardial coronary vessels and a reduction in the density of endocardial vessels. In the lung, prominent increases in vessel diameter were observed. These vascular remodeling changes occurred in the absence of any apparent increase in VEGF expression. Our results illustrate that chronic systemic delivery of angiopoietin-2 induces angiogenesis in the absence of increased VEGF expression and that angiopoietin-2 promotes myocardial coronary vessel remodeling.

Molecular mechanisms in lymphangiogenesis: model systems and implications in human disease.

The basic science and development of therapies targeting the blood vascular system has enjoyed much focus due to the knowledge of the molecular mechanisms behind its development and roles in disease. However, the closely associated lymphatic system, while also being responsible for a number of serious and debilitating diseases, has not garnered as much attention due to the lack of specific molecular markers, thereby limiting this field to no more than descriptive analysis. Within the past decade, great strides have been taken to identify a number of molecular signatures unique to the lymphatic system. To this end, the timeline for lymphatic development has now been redefined at the molecular level, and diseases associated with lymphatics now have a molecular basis. With this knowledge, the current modes of treatment for disease such as lymphedema, lymphangiomas, and metastatic progression can now be augmented with potential molecular therapies that have currently been tested in a number of animal models. Much like the therapeutics that have been associated with vasculogenesis and angiogenesis, manipulation of the molecular pathways that define lymphatic development may lead to better clinical outcomes associated with developmental defects and disease.

Dok-R plays a pivotal role in angiopoietin-1-dependent cell migration through recruitment and activation of Pak.

Tek/Tie-2 is an endothelial cell (EC)-specific receptor tyrosine kinase that plays a critical role in angiogenesis via its regulation by the angiopoietin family of growth factor ligands. Angiopoietin-1 (Ang1) can promote EC migration; however, the signaling mechanisms underlying this process remain elusive. Here we demonstrate that Dok-R/Dok-2 can associate with Tek in ECs following Ang1 stimulation, resulting in tyrosine phosphorylation of Dok-R and the subsequent recruitment of Nck and the p21-activating kinase (Pak/Pak1) to the activated receptor. Ang1-mediated migration is increased upon Dok-R overexpression and this requires a functional Nck binding site on Dok-R. Localization of this Dok-R-Nck-Pak complex to the activated Tek receptor at the cellular membrane is coincident with activation of Pak kinase. The ability of Dok-R to bind Nck is required for maximal activation of Pak and overexpression of Pak results in increased Ang1-mediated cell motility. Our study outlines a novel signaling pathway underlying Ang1-driven cell migration that involves Dok-R and its recruitment of Nck and the subsequent activation of Pak.

Multiple ErbB-2/Neu Phosphorylation Sites Mediate Transformation through Distinct Effector Proteins.

Amplification of the type I receptor tyrosine kinase ErbB-2 (HER2/Neu) is observed in 20-30% of human mammary carcinomas, correlating with a poor clinical prognosis. We have previously demonstrated that four (Tyr(1144), Tyr(1201), Tyr(1226/1227), or Tyr(1253)) of the five known Neu/ErbB-2 autophosphorylation sites can independently mediate transforming signals. The transforming potential of at least two of these autophosphorylation sites (Tyr(1144) and Tyr(1226/1227)) has been further correlated with their ability to associate with Grb2 and Shc adapter proteins, respectively. To confirm the specificity of these interactions, we have created a series of second site mutants in these phosphorylation sites. The results showed that Grb2 recruitment to site 1144 is absolutely required for transforming signal from this autophosphorylation site, whereas association of Shc-mediated transformation is dependent on conservation of the NPXY motif spanning Tyr(1227). A stretch of amino acid identity around tyrosines 1201 (ENPEYLTP)and 1253 (ENPEYLDL) exists, and mutation of key residues within this motif reveals distinct requirements for an intact protein tyrosine-binding protein (NPXY). We show that DOK-R, a protein tyrosine-binding site-containing protein implicated in Ras signaling, interacts with Neu/ErbB-2 at Tyr(1253) as do two unidentified proteins, p150 and p34, the latter correlating with transformation. Together these data argue that ErbB-2/Neu is capable of mediating transformation through distinct effector pathways.

Rescue of the early vascular defects in Tek/Tie2 null mice reveals an essential survival function.

Disruption of the signaling pathways mediated by the receptor tyrosine kinase Tek/Tie2 has shown that this receptor plays a pivotal role in vascularization of the developing embryo. In this report, we have utilized the tetracycline-responsive binary transgenic system to overcome the early lethal cardiovascular defects associated with the tekDeltasp null allele in order to investigate the role of Tek in later stages of vessel growth. We show for the first time in vivo that synchronized loss of tek expression correlates with rapid endothelial cell apoptosis in hemorrhagic regions of the embryo, demonstrating an ongoing requirement for Tek-mediated signal transduction in vascular maintenance.

Tie receptors: new modulators of angiogenic and lymphangiogenic responses.

Angiogenesis is required for normal embryonic vascular development and aberrant angiogenesis contributes to several diseases, including cancer, diabetes and tissue ischaemia. What are the molecular mechanisms that regulate this important process? The Tie family of receptors and their ligands, the angiopoietins, are beginning to provide insight into how vessels make decisions such as whether to grow or regress--processes that are important not only during development but throughout an organism's life.

Biological action of angiopoietin-2 in a fibrin matrix model of angiogenesis is associated with activation of Tie2.

The endothelial cell (EC) specific tyrosine kinase receptor, Tie2, interacts with at least two ligands, angiopoietin-1 (Ang1) and angiopoietin-2 (Ang2). Ang1 stimulates Tie2 receptor autophosphorylation, while Ang2 has been reported to inhibit Ang1-induced Tie2 receptor autophosphorylation. We studied the effects of Ang1 and Ang2 in an in vitro model of angiogenesis. Human ECs (HUVEC), cultured on 3-D fibrin matrices, were treated with conditioned media (CM) from stably transfected cells expressing human Ang1 or Ang2, or with purified recombinant proteins. EC tube formation was measured as a differentiation index (DI), calculated as the ratio of total tube length over residual of EC monolayer. CM from Ang1 overexpressing A10 SMC or HEK293T cells induced profound HUVEC differentiation, resulting in the formation of extensive capillary-like tubes within 48 h (DI: 24.58+/-5.91 and 19.13+/-7.86, respectively) vs. control (DI: 2.73+/-1.68 and 2.15+/-1.45, respectively, both P<0.001). Interestingly, CM from two independent cell lines overexpressing Ang2 also produced a significant increase in EC differentiation (DI: 9.22+/-3.00 and 9.72+/-4.84, both P<0.005 vs. control) although the degree of angiogenesis was significantly less then that seen with Ang1. Addition of Ang1* (a genetically engineered variant of naturally occurring Ang1) or Ang2 also resulted in dose dependent increases in DI, which were blocked by an excess of soluble Tie2 receptor (20 microg/ml). Both Ang1* and Ang2 induced modest increases in [3H]thymidine incorporation into HUVECs (20 and 26%, respectively), which were inhibited by excess soluble Tie2. Although Ang2 was unable to induce significant Tie2 receptor phosphorylation during a 5-min exposure, a 24-h pretreatment with Ang2, followed by brief re-exposure, produced Tie2 phosphorylation in HUVEC comparable to that produced by Ang1*. These results demonstrate for the first time that Ang2 may have a direct role in stimulating Tie2 receptor signaling and inducing in vitro angiogenesis. Our findings suggest that the physiological role of Ang2 is more complex than previously recognized: acting alternately to promote or blunt Tie2 receptor signaling in endothelial cells, depending on local conditions.

VEGF-C signaling pathways through VEGFR-2 and VEGFR-3 in vasculoangiogenesis and hematopoiesis.

Signaling by vascular endothelial growth factors (VEGFs) through VEGF receptors (VEGFRs) plays important roles in vascular development and hematopoiesis. The authors analyzed the function of VEGF-C signaling through both VEGFR-2 and VEGFR-3 in vasculoangiogenesis and hematopoiesis using a coculture of para-aortic splanchnopleural mesoderm (P-Sp) explants from mouse embryos with stromal cells (OP9). Vasculogenesis and angiogenesis were evaluated by the extent of vascular bed and network formation, respectively. Addition of VEGF-C to the P-Sp culture enhanced vascular bed formation and suppressed definitive hematopoiesis. Both vascular bed and network formations were completely suppressed by addition of soluble VEGFR-1-Fc competitor protein. Formation of vascular beds but not networks could be rescued by VEGF-C in the presence of the competitor, while both were rescued by VEGF-A. VEGFR-3-deficient embryos show the abnormal vasculature and severe anemia. Consistent with these in vivo findings, vascular bed formation in the P-Sp from the VEGFR-3-deficient embryos was enhanced to that in wild-type or heterozygous embryos, and hematopoiesis was severely suppressed. When VEGFR-3-Fc chimeric protein was added to trap endogenous VEGF-C in the P-Sp culture of the VEGFR-3-deficient embryos, vascular bed formation was suppressed and hematopoiesis was partially rescued. These results demonstrate that because VEGF-C signaling through VEGFR-2 works synergistically with VEGF-A, the binding of VEGF-C to VEGFR-3 consequently regulates VEGFR-2 signaling. In VEGFR-3-deficient embryos, an excess of VEGF-C signals through VEGFR-2 induced the disturbance of vasculogenesis and hematopoiesis during embryogenesis. This indicates that elaborated control through VEGFR-3 signaling is critical in vasculoangiogenesis and hematopoiesis. (Blood. 2000;96:3793-3800)

The SH2 inositol 5-phosphatase Ship1 is recruited in an SH2-dependent manner to the erythropoietin receptor.

Ship1 (SH2 inositol 5-phosphatase 1) has been shown to be a target of tyrosine phosphorylation downstream of cytokine and immunoregulatory receptors. In addition to its catalytic activity on phosphatidylinositol substrates, it can serve as an adaptor protein in binding Shc and Grb2. Erythropoietin (EPO), the primary regulator of erythropoiesis, has been shown to activate the tyrosine phosphorylation of Shc, resulting in recruitment of Grb2. However, the mechanism by which the erythropoietin receptor (EPO-R) recruits Shc remains unknown. EPO activates the tyrosine phosphorylation of Ship1, resulting in the interdependent recruitment of Shc and Grb2. Ship1 is recruited to the EPO-R in an SH2-dependent manner. Utilizing a panel of EPO-R deletion and tyrosine mutants, we have discovered remarkable redundancy in Ship1 recruitment. EPO-R Tyr(401) appears to be a major site of Ship1 binding; however, Tyr(429) and Tyr(431) can also serve to recruit Ship1. In addition, we have shown that EPO stimulates the formation of a ternary complex consisting of Ship1, Shc, and Grb2. Ship1 may modulate several discrete signal transduction pathways. EPO-dependent activation of ERK1/2 and protein kinase B (PKB)/Akt was examined utilizing a panel of EPO-R deletion mutants. Activation of ERK1/2 was observed in EPO-RDelta99, which retains only the most proximal tyrosine, Tyr(343). In contrast, EPO-dependent PKB activation was observed in EPO-RDelta43, but not in EPO-RDelta99. It appears that EPO-dependent PKB activation is downstream of a region that indirectly couples to phosphatidylinositol 3-kinase.

Tek/Tie2 signaling: new and old partners.

A common property amongst angiogenic ligands such as the vascular endothelial growth factors and the angiopoietins is that they can elicit multiple responses depending upon the context of their expression and the presence of other growth factors. Study of the signal transduction pathways initiated by these growth factors provides insight into the molecular and cellular mechanisms that regulate vessel assembly. Key components of signal transduction cascades can then be used as potential targets in angiogenic therapies. This commentary reviews the recent advances into understanding the molecular signaling pathways mediated through the angiopoietin receptor, Tek/Tie2, as well as their effect on the regulation of distinct cellular aspects of angiogenesis.

A murine model of hereditary hemorrhagic telangiectasia.

Endoglin (CD105), an accessory protein of the TGF-beta receptor superfamily, is highly expressed on endothelial cells. Hereditary hemorrhagic telangiectasia type 1 (HHT1) is associated with mutations in the Endoglin gene, leading to haploinsufficiency. To generate a disease model and ascertain the role of endoglin in development, we generated mice lacking 1 or both copies of the gene. Endoglin null embryos die at gestational day 10.0-10.5 due to defects in vessel and heart development. Vessel formation appears normal until hemorrhage occurs in yolk sacs and embryos. The primitive vascular plexus of the yolk sac fails to mature into defined vessels, and vascular channels dilate and rupture. Internal bleeding is seen in the peritoneal cavity, implying fragile vessels. Heart development is arrested at day 9.0, and the atrioventricular canal endocardium fails to undergo mesenchymal transformation and cushion-tissue formation. These data suggest that endoglin is critical for both angiogenesis and heart valve formation. Some heterozygotes, either with an inbred 129/Ola or mixed C57BL/6-129/Ola background, show signs of HHT, such as telangiectases or recurrent nosebleeds. In this murine model of HHT, it appears that epigenetic factors and modifier genes, some of which are present in 129/Ola, contribute to disease heterogeneity.

Identification of Tek/Tie2 binding partners. Binding to a multifunctional docking site mediates cell survival and migration.

The Tek/Tie2 receptor tyrosine kinase plays a pivotal role in vascular and hematopoietic development. To study the signal transduction pathways that are mediated by this receptor, we have used the yeast two-hybrid system to identify signaling molecules that associate with the phosphorylated Tek receptor. Using this approach, we demonstrate that five molecules, Grb2, Grb7, Grb14, Shp2, and the p85 subunit of phosphatidylinositol 3-kinase can interact with Tek in a phosphotyrosine-dependent manner through their SH2 domains. Mapping of the binding sites of these molecules on Tek reveals the presence of a multisubstrate docking site in the carboxyl tail of Tek (Tyr(1100)). Mutation of this site abrogates binding of Grb2 and Grb7 to Tek in vivo, and this site is required for tyrosine phosphorylation of Grb7 and p85 in vivo. Furthermore, stimulation of Tek-expressing cells with Angiopoietin-1 results in phosphorylation of both Tek and p85 and in activation of endothelial cell migration and survival pathways that are dependent in part on phosphatidylinositol 3-kinase. Taken together, these results demonstrate that Angiopoietin-1-induced signaling from the Tek receptor is mediated by a multifunctional docking site that is responsible for activation of both cell migration and cell survival pathways.

Recruitment of Dok-R to the EGF receptor through its PTB domain is required for attenuation of Erk MAP kinase activation.

Dok (for downstream of tyrosine kinases) proteins are a newly identified family of docking molecules that are characterized by the presence of an amino-terminal pleckstrin homology (PH) domain, a central putative phosphotyrosine-binding (PTB) domain and numerous potential sites of tyrosine phosphorylation [1] [2] [3] [4] [5] [6]. Here, we explore the potential role of the Dok family member Dok-R (also known as p56(Dok2) or FRIP) in signaling pathways mediated by the epidermal growth factor (EGF) receptor. An intact PTB domain in Dok-R was critical for its association with two PTB-binding consensus sites on the EGF receptor and the PH domain further contributed to stable in vivo binding and tyrosine phosphorylation of Dok-R. Multiple sites on Dok-R were tyrosine-phosphorylated following EGF stimulation; phosphorylated Tyr276 and Tyr304 are proposed to dock the tandem Src homology 2 (SH2) domains of the p21(Ras) GTPase-activating protein rasGAP and Tyr351 mediates an association with the SH2 domain of the adapter protein Nck. Interestingly, we have found that Dok-R could attenuate EGF-stimulated mitogen-activated protein (MAP) kinase activation independently of its association with rasGAP. Together, these results suggest that Dok-R has an important role downstream of growth factor receptors as a potential negative regulator of signal transduction.

SHIP is a negative regulator of growth factor receptor-mediated PKB/Akt activation and myeloid cell survival.

SHIP is an inositol 5' phosphatase that hydrolyzes the PI3'K product PI(3,4,5)P3. We show that SHIP-deficient mice exhibit dramatic chronic hyperplasia of myeloid cells resulting in splenomegaly, lymphadenopathy, and myeloid infiltration of vital organs. Neutrophils and bone marrow-derived mast cells from SHIP-/- mice are less susceptible to programmed cell death induced by various apoptotic stimuli or by growth factor withdrawal. Engagement of IL3-R and GM-CSF-R in these cells leads to increased and prolonged PI3'K-dependent PI(3,4,5)P3 accumulation and PKB activation. These data indicate that SHIP is a negative regulator of growth factor-mediated PKB activation and myeloid cell survival.

Critical role of the TIE2 endothelial cell receptor in the development of definitive hematopoiesis.

We have investigated the function of TIE2/TEK receptor tyrosine kinase in the development of definitive hematopoiesis. In the vitelline artery at 9.5 days postcoitum (d.p.c.), TIE2+ hematopoietic cells aggregated and adhered to TIE2+ endothelial cells. Soluble TIE2-Fc chimeric protein inhibited the development of hematopoiesis and angiogenesis in the para-aortic splanchnopleural mesoderm (P-Sp) explant culture, and TIE2-deficient mice showed severely impaired definitive hematopoiesis. An in vitro study revealed that Angiopoietin-1 but not Angiopoietin-2 promoted the adhesion to fibronectin (FN) through integrins in TIE2-transfected cells and primary TIE2+ cells sorted from 9.5 d.p.c. P-Sp. Adhesion of TIE2+ cells induced by Angiopoietin-1 enhanced the proliferation of hematopoietic progenitor cells.

Cardiovascular failure in mouse embryos deficient in VEGF receptor-3.

Vascular endothelial growth factor (VEGF) is a key regulator of blood vessel development in embryos and angiogenesis in adult tissues. Unlike VEGF, the related VEGF-C stimulates the growth of lymphatic vessels through its specific lymphatic endothelial receptor VEGFR-3. Here it is shown that targeted inactivation of the gene encoding VEGFR-3 resulted in defective blood vessel development in early mouse embryos. Vasculogenesis and angiogenesis occurred, but large vessels became abnormally organized with defective lumens, leading to fluid accumulation in the pericardial cavity and cardiovascular failure at embryonic day 9.5. Thus, VEGFR-3 has an essential role in the development of the embryonic cardiovascular system before the emergence of the lymphatic vessels.

The Tek/Tie2 receptor signals through a novel Dok-related docking protein, Dok-R.

Tek/Tie2 is an endothelial cell-specific receptor tyrosine kinase that has been shown to play a role in vascular development of the mouse. Targeted mutagenesis of both Tek and its agonistic ligand, Angiopoietin-1, result in embryonic lethality, demonstrating that the signal transduction pathway(s) mediated by this receptor are crucial for normal embryonic development. In an attempt to identify downstream signaling partners of the Tek receptor, we have used the yeast two-hybrid system to identify phosphotyrosine-dependent interactions. Using this approach, we have identified a novel docking molecule called Dok-R, which has sequence and structural homology to p62dok and IRS-3. Mapping of the phosphotyrosine-interaction domain within Dok-R shows that Dok-R interacts with Tek through a PTB domain. Dok-R is coexpressed with Tek in a number of endothelial cell lines. We show that coexpression of Dok-R with activated Tek results in tyrosine phosphorylation of Dok-R and that rasGAP and Nck coimmunoprecipitate with phosphorylated Dok-R. Furthermore, Dok-R is constitutively bound to Crk presumably through the proline rich tail of Dok-R. The cloning of Dok-R represents the first downstream substrate of the activated Tek receptor, and suggests that Tek can signal through a multitude of pathways.