-
[show abstract]
[hide abstract]
ABSTRACT: Angiogenesis-the growth of new blood vessels from preexisting vessels-is an important physiological process and is considered to play a key role in tumor growth and metastasis. We identified the immunoglobulin-containing and proline-rich receptor-1 (IGPR-1, also called TMIGD2) gene as a novel cell adhesion receptor that is expressed in various human organs and tissues, mainly in cells with epithelium and endothelium origins. IGPR-1 regulates cellular morphology, homophilic cell aggregation, and cell-cell interaction. IGPR-1 activity also modulates actin stress fiber formation and focal adhesion and reduces cell migration. Silencing of expression of IGPR-1 by small interfering RNA (siRNA) and by ectopic overexpression in endothelial cells showed that IGPR-1 regulates capillary tube formation in vitro, and B16F melanoma cells engineered to express IGPR-1 displayed extensive angiogenesis in the mouse Matrigel angiogenesis model. Moreover, IGPR-1, through its proline-rich cytoplasmic domain, associates with multiple Src homology 3 (SH3)-containing signaling proteins, including SH3 protein interacting with Nck (SPIN90/WISH), bullous pemphigoid antigen-1, and calcium channel β2. Silencing of expression of SPIN90/WISH by siRNA in endothelial cells showed that SPIN90/WISH is required for capillary tube formation. These features of IGPR-1 suggest that IGPR-1 is a novel receptor that plays an important role in cell-cell interaction, cell migration, and angiogenesis.
Molecular biology of the cell 03/2012; 23(9):1646-56. · 5.98 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The internalization and degradation of vascular endothelial growth factor receptor 2 (VEGFR-2), a potent angiogenic receptor tyrosine kinase, is a central mechanism for the regulation of the coordinated action of VEGF in angiogenesis. Here, we show that VEGFR-2 is ubiquitinated in response to VEGF, and Lys 48-linked polyubiquitination controls its degradation via the 26S proteosome. The degradation and ubiquitination of VEGFR-2 is controlled by its PEST domain, and the phosphorylation of Ser1188/Ser1191 is required for the ubiquitination of VEGFR-2. F-box-containing β-Trcp1 ubiquitin E3 ligase is recruited to S1188/S1191 VEGFR-2 and mediates the ubiquitination and degradation of VEGFR-2. The PEST domain also controls the activation of p38 mitogen-activated protein kinase (MAPK) through phospho-Y1173. The activation of p38 stabilizes VEGFR-2, and its inactivation accelerates VEGFR-2 downregulation. The VEGFR-2-mediated activation of p38 is established through the protein kinase A (PKA)/MKK6 pathway. PKA is recruited to VEGFR-2 through AKAP1/AKAP149, and its phosphorylation requires Y1173 of VEGFR-2. The study has identified a unique mechanism in which VEGFR-2 stability and degradation is modulated. The PEST domain acts as a dual modulator of VEGFR-2; the phosphorylation of S1188/S1191 controls ubiquitination and degradation via β-Trcp1, where the phosphorylation of Y1173 through PKA/p38 MAPK controls the stability of VEGFR-2.
Molecular and cellular biology 03/2011; 31(10):2010-25. · 6.06 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Activation of phospholipase Cγ1 (PLCγ1) by vascular endothelial growth factor receptor (VEGFR)-2 is necessary for proliferation and tube formation of endothelial cells in vitro. Previous work has demonstrated that Casitas B-lineage lymphoma (c-Cbl) promotes ubiquitination of PLCγ1 and suppression of its tyrosine phosphorylation. This study was designed to evaluate the importance of PLCγ1 and c-Cbl in experimental choroidal neovascularization (CNV).
The role of PLCγ1 was studied in three models of angiogenesis: the endothelial cell culture system, the chorioallantoic membrane (CAM) assay, and the laser-induced CNV model. Endothelial cells were analyzed for the role of PLCγ1 in promoting tube formation. CAMs were incubated with pharmacologic agents that either inhibit or stimulate PLCγ1. CNV was induced in wild-type and c-Cbl-knockout mice, and the progression of CNV was evaluated by fluorescein angiography.
Activation of PLCγ1 was necessary for tube formation of endothelial cells. PLCγ1 stimulation increased the growth of blood vessels and conversely, PLCγ1 inhibition decreased the growth of blood vessels in the CAM model. CNV lesions in the c-Cbl-knockout mice were significantly greater in number, more confluent, and increased in size with time, compared with those in the control wild-type mice.
The data show that PLCγ1 plays an important role in angiogenesis. Loss of c-Cbl results in enhanced CNV in the eye. The study also shows that c-Cbl plays an important role in ocular angiogenesis, suggesting that modulation of c-Cbl activity or inhibition of PLCγ1 would be a compelling target for antiangiogenesis therapy.
Investigative ophthalmology & visual science 12/2010; 51(12):6803-9. · 3.43 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Sex differences in liver gene expression are dictated by sex differences in circulating GH profiles. Presently, the pituitary hormone dependence of mouse liver gene expression was investigated on a global scale to discover sex-specific early GH response genes that could contribute to sex-specific regulation of downstream GH targets and to ascertain whether intrinsic sex differences characterize hepatic responses to plasma GH stimulation. Global RNA expression analysis identified two distinct classes of sex-specific mouse liver genes: genes subject to positive regulation (class I) and genes subject to negative regulation by pituitary hormones (class II). Genes activated or repressed in hypophysectomized (Hypox) mouse liver within 30-90 min of GH pulse treatment at a physiological dose were identified as putative direct targets of GH action (early response genes). Intrinsic sex differences in the GH responsiveness of a subset of these early response genes were observed. Notably, 45 male-specific genes, including five encoding transcriptional regulators that may mediate downstream sex-specific transcriptional responses, were induced by GH within 30 min in Hypox male but not Hypox female mouse liver. The early GH response genes were enriched in 29 male-specific targets of the transcription factor myocyte enhancer factor 2, whose activation in hepatic stellate cells is associated with liver fibrosis leading to hepatocellular carcinoma, a male-predominant disease. Thus, the rapid activation by GH pulses of certain sex-specific genes is modulated by intrinsic sex-specific factors, which may be associated with prior hormone exposure (epigenetic mechanisms) or genetic factors that are pituitary-independent, and could contribute to sex differences in predisposition to liver cancer or other hepatic patho-physiologies.
Molecular Endocrinology 02/2010; 24(3):667-78. · 4.54 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The transcriptional repressor Bcl6 is a male-specific rat liver gene product and one of 24 early GH-response genes encoding DNA-binding proteins. Presently, the sex specificity of Bcl6 was shown to emerge at puberty, when hepatic Bcl6 mRNA was induced in males and repressed in females by the female plasma GH profile. Hepatic Bcl6 mRNA was increased to near-normal male levels in hypophysectomized females and was extinguished in intact males given a continuous GH infusion (female-like GH pattern). Bcl6 was also repressed in adult male somatostatin-deficient mice, where plasma GH profiles are female like. Hepatic Bcl6 RNA was rapidly down-regulated by GH pulse treatment, both in hypophysectomized male rats and in primary rat hepatocytes. Bcl6 was substantially induced in female mice deficient in hepatic signal transducer and activator of transcription (STAT)5a/STAT5b, suggesting that these STAT transcriptional mediators of GH signaling repress Bcl6. Indeed, STAT5 was bound to Bcl6 STAT5-binding region-B, previously associated with Bcl6 repression, in both male and female liver chromatin. STAT5 also bound to Bcl6 region-A in male chromatin but only during a plasma GH pulse. Analysis of primary transcripts (heterogeneous nuclear RNA) across the Bcl6 gene revealed a novel mechanism of GH-dependent sex specificity, with two apparent blocks in Bcl6 transcription elongation seen in female liver and in continuous GH-treated male liver, one early in intron 4 and one in exon 5, which together reduced transcription beyond exon 5 more than 300-fold. Finally, Bcl6 was bound to a subset of STAT5-binding sites in male liver chromatin, including a Socs2 STAT5-binding site where Bcl6 binding increased substantially between plasma GH pulses, i.e. when STAT5 binding was low. Bcl6 and STAT5 binding are thus inversely coordinated by the endogenous pulses of pituitary GH release, suggesting this male-specific transcriptional repressor modulates hepatic GH signaling to select STAT5 target genes.
Molecular Endocrinology 10/2009; 23(11):1914-26. · 4.54 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Vascular endothelial growth factor receptor-1/fms-related tyrosine kinase 1 (VEGFR-1/FLT1) is expressed as a membrane-bound receptor tyrosine kinase and as an alternatively spliced soluble protein (sVEGFR-1) containing the 1-6 IgG-like domain of its ectodomain. sVEGFR-1 is known as a naturally occurring inhibitor of angiogenesis and as a surrogate marker for cancer progression; it is also linked to pregnancy-induced hypertension called preeclampsia and to avascularity of normal cornea. It remains an open question whether alternative mRNA splicing is the only mechanism by which sVEGFR-1 is generated. In this study, we show that in leukemic cancer cells, PlGF and VEGF-A both induce tyrosine phosphorylation of VEGFR-1 and render it susceptible to ectodomain shedding, resulting in the generation of sVEGFR-1 and an intracellular cytoplasmic fragment. Activation of protein kinase C and tumor necrosis factor-alpha-converting enzyme family metalloproteases are critically required for the occurrence of sVEGFR-1. Following the removal of the ectodomain, the remnant of VEGFR-1 remains attached to the membrane, and the activity of gamma-secretase/presenilin is required for its release from the cell membrane. We propose that sVEGFR-1 produced via ectodomain shedding plays a prominent role in the VEGF receptor system by antagonizing VEGF receptor signaling by acting as a dominant-negative form and/or forming a nonsignaling dimerizing complex with VEGF receptors.
Cancer Research 04/2009; 69(6):2607-14. · 7.86 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Vascular endothelial growth factor receptor-2 (VEGFR-2) signaling is an obligate requirement for normal development and pathological angiogenesis such as cancer and age-related macular degeneration. Although autophosphorylation of tyrosine 1173 (Y1173) of VEGFR-2 is considered a focal point for its angiogenic signal relay, however, the mechanism of phosphorylation of Y1173, signaling proteins that are recruited to this residue and their role in angiogenesis is not fully understood.
In this study we demonstrate that c-Src kinase directly through its Src homology 2 (SH2) domain and indirectly via c-Cbl binds to phospho-Y1057 of VEGFR-2. Activation of c-Src kinase by a positive feedback mechanism phosphorylates VEGFR-2 at multi-docking site, Y1173. c-Src also catalyzes tyrosine phosphorylation of IQGAP1 and acts as an adaptor to bridge IQGAP1 to VEGFR-2. In turn, IQGAP1 activates b-Raf and mediates proliferation of endothelial cells. Silencing expression of IQGAP1 and b-Raf revealed that their activity is essential for VEGF to stimulate angiogenesis in an in vivo angiogenesis model of chicken chorioallantoic membrane (CAM).
Angiogenesis contributes to the pathology of numerous human diseases ranging from cancer to age-related macular degeneration. Determining molecular mechanism of tyrosine phosphorylation of VEGFR-2 and identification of molecules that are relaying its angiogenic signaling may identify novel targets for therapeutic intervention against angiogenesis-associated diseases. Our study shows that recruitment and activation of c-Src by VEGFR-2 plays a pivotal role in relaying angiogenic signaling of VEGFR-2; it phosphorylates VEGFR-2 at Y1173, facilitates association and activation of IQGAP1 and other signaling proteins to VEGFR-2. IQGAP1-dependent signaling, in part, is critically required for endothelial cell proliferation, a key step in angiogenesis. Thus, Y1057 of VEGFR-2 serves to regulate VEGFR-2 function in a combinatorial manner by supporting both diversity of recruitment of angiogenic signaling proteins to VEGFR-2, and its ability to promote angiogenesis.
PLoS ONE 02/2008; 3(12):e3848. · 4.09 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Activation of phospholipase Cgamma1 (PLCgamma1) by vascular endothelial growth factor receptor-2 (VEGFR-2) in endothelial cells in part is responsible for angiogenesis in vivo. The cellular mechanisms exerting negative control over PLCgamma1 activation, however, remain unaddressed. Here by using in vitro and in vivo binding assays, we show that the Casitas B-lineage lymphoma (c-Cbl) E3 ubiquitin ligase constitutively associates with PLCgamma1 via its C-terminal domain and conditionally interacts with VEGFR-2 via the N-terminal/TKB domain. Site-directed mutagenesis of VEGFR-2 showed that full activation of c-Cbl requires its direct association with phospho-tyrosines 1052 and 1057 of VEGFR-2 via its TKB domain and indirect association with phospho-tyrosine 1173 of VEGFR-2 via PLCgamma1. The tertiary complex formation between VEGFR-2, PLCgamma1 and c-Cbl selectively promotes ubiquitylation and suppression of tyrosine phosphorylation of PLCgamma1 by a proteolysis-independent mechanism. Further analysis showed that association of c-Cbl with VEGFR-2 does not impact ubiquitylation, down-regulation, or tyrosine phosphorylation of VEGFR-2. Silencing of c-Cbl by siRNA revealed that endogenous c-Cbl plays an inhibitory role in angiogenesis. Our data demonstrate that corecruitment of c-Cbl and PLCgamma1 to VEGFR-2 serves as a mechanism to fine-tune the angiogenic signal relay of VEGFR-2.
Proceedings of the National Academy of Sciences 04/2007; 104(13):5413-8. · 9.68 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Activation loop tyrosine autophosphorylation is an essential requirement for full kinase activation of receptor tyrosine kinases (RTKs). However, mechanisms involved are not fully understood. In general, kinase domains of RTKs are folded into two main lobes, NH2- and COOH-terminal lobes. The COOH-terminal lobe of vascular endothelial growth factor receptor-2 (VEGFR-2) is folded into seven alpha-helices (alphaD-alphaI). In the studies presented here we demonstrate that leucine residues of helix I (alphaI) regulate tyrosine autophosphorylation and phosphotransferase activity of VEGFR-2. The presence of leucines 1158, 1161, and 1162 are essential for tyrosine autophosphorylation and kinase activation of VEGFR-2 and are involved in helix-helix packing via hydrophobic interactions. The presence of leucine 1158 is critical for kinase activation of VEGFR-2 and appears to interact with alphaE, alphaF, alphaH, and beta7. The analogous residue, leucine 957 on platelet-derived growth factor receptor-beta and leucine 910 on colony stimulating factor-1R are also found to be critical for tyrosine autophosphorylation of these receptors. Leucines 1161 and 1162 are also involved in helix-helix packing but they play a less critical role in VEGFR-2 activation. Thus, we conclude that leucine motif-mediated helix-helix interactions are critical for kinase regulation of type III RTKs. This mechanism is likely to be shared with other kinases and might provide a basis for the design of a novel class of tyrosine kinase inhibitors.
Journal of Biological Chemistry 04/2006; 281(13):8620-7. · 4.77 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: VEGFR-1 is a kinase-defective receptor tyrosine kinase (RTK) and negatively modulates angiogenesis by acting as a decoy receptor. The decoy characteristic of VEGFR-1 is required for normal development and angiogenesis. To date, there is no molecular explanation for this unusual characteristic of VEGFR-1. Here we show that the molecular mechanisms underlying the decoy characteristic of VEGFR-1 is linked to the replacement of a highly conserved amino acid residue in the activation loop. This amino acid is highly conserved among all the type III RTKs and corresponds to aspartic acid, but in VEGFR-1 it is substituted to asparagine. Mutation of asparagine (Asn(1050)) within the activation loop to aspartic acid promoted enhanced ligand-dependent tyrosine autophosphorylation and kinase activation in vivo and in vitro. The mutant VEGFR-1 (Asp(1050)) promoted endothelial cell proliferation but not tubulogenesis. It also displayed an oncogenic phenotype as its expression in fibroblast cells elicited transformation and colony growth. Furthermore, mutation of the invariable aspartic acid to asparagine in VEGFR-2 lowered the autophosphorylation of activation loop tyrosines 1052 and 1057. We propose that the conserved aspartic acid in the activation loop favors the transphosphorylation of the activation loop tyrosines, and its absence renders RTK to a less potent enzyme by disfavoring transphosphorylation of activation loop tyrosines.
Journal of Biological Chemistry 02/2006; 281(2):867-75. · 4.77 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Activation of vascular endothelial growth factor receptor-1 and -2 (VEGFR-1 and VEGFR-2) plays a critical role in vasculogenesis and angiogenesis. However, the mechanism by which activation of VEGFRs elicits these cellular events is not fully understood. We recently constructed chimeric receptors containing the extracellular domain of human CSF-1R/c-fms fused with the entire transmembrane and cytoplasmic domains of murine VEGFR-1 and VEGFR-2. Selective activation of chimeric VEGFR-2, but not chimeric VEGFR-1, stimulated endothelial cell growth, migration, and differentiation. Stimulation of cells coexpressing chimeric VEGFR-1 and VEGFR-2 suppressed VEGFR-2-mediated endothelial cell growth. Site-directed mutagenesis demonstrated that tyrosines 799 and 1173 are required for VEGFR-2-mediated endothelial cell growth and activation of PI3 kinase. Further site-directed mutagenesis demonstrated that tyrosine 1212, located in the carboxyl tail of VEGFR-2, is required for the ligand-dependent autophosphorylation of the receptor and its ability to activate signaling proteins. Collectively, our results suggest that activation of VEGFR-1 and VEGFR-2 differentially regulates endothelial cell function and angiogenesis. Second, activation of VEGFR-2 is associated with many endothelial cell functions, including cell proliferation, migration, and differentiation. Third, activation of PI3 kinase by VEGFR-2 regulates endothelial cell proliferation.
Annals of the New York Academy of Sciences 01/2006; 995(1):200 - 207. · 3.15 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Vascular endothelial growth factor receptor-2 (VEGFR-2/Flk-1) is a receptor tyrosine kinase (RTK) whose activation regulates angiogenesis. The regulatory mechanisms that attenuate VEGFR-2 signal relay are largely unknown. Our study shows that VEGFR-2 promotes phosphorylation of c-Cbl, but activation, ubiquitylation, and down-regulation of VEGFR-2 are not influenced by c-Cbl activity. A structure-function analysis of VEGFR-2 and pharmacological approach revealed that down-regulation of VEGFR-2 is mediated by a distinct mechanism involving PKC. A tyrosine mutant VEGFR-2, defective in PLC-gamma1 activation underwent down-regulation efficiently in response to ligand stimulation, suggesting that activation of classical PKCs are not involved in VEGFR-2 down-regulation. Further studies showed that the ectodomain of VEGFR-2 is dispensable for PKC-dependent down-regulation. Progressive deletion of the carboxyl-terminal domain showed that at least 39 amino acids within the carboxyl-terminal domain, immediately C-terminal to the kinase domain, is required for efficient PKC-mediated down-regulation of VEGFR-2. Mutation of serine sites at 1188 and 1191, within this 39 amino acid region, compromised the ability of VEGFR-2 to undergo efficient ligand-dependent down-regulation. Altogether the results show that the regulatory mechanisms involved in the attenuation of VEGFR-2 activation is mediated by nonclassical PKCs and the presence of serine sites in the carboxyl terminal of VEGFR-2.
Molecular Biology of the Cell 05/2005; 16(4):2106-18. · 4.94 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: VEGFR-1 is devoid of ligand-dependent tyrosine autophosphorylation and its activation is not associated with proliferation of endothelial cells. The molecular mechanism responsible for this characteristic of VEGFR-1 is not known. In this study, we show that VEGFR-1 is devoid of ligand-dependent downregulation and failed to stimulate intracellular calcium release, cell migration and angiogenesis in vitro. To understand the molecular mechanisms responsible for the poor tyrosine autophosphorylation of VEGFR-1, we have either deleted the carboxyl terminus of VEGFR-1 or exchanged it with the carboxyl terminus of VEGFR-2. The deletion of carboxyl terminus of VEGFR-1 did not reverse its defective ligand-dependent autophosphorylation. The carboxyl terminus-swapped VEGFR-1, however, displayed ligand-dependent autophosphorylation, downregulation and also conveyed strong mitogenic responses. Thus, the carboxyl tail of VEGFR-1 restrains the ligand-dependent kinase activation and downregulation of VEGFR-1 and its ability to convey the angiogenic responses in endothelial cells.
Oncogene 08/2004; 23(32):5523-31. · 6.37 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Vascular endothelial growth factor receptor-2 (VEGFR-2/FLK-1) is a receptor tyrosine kinase whose activation stimulates angiogenesis. We recently generated a chimeric VEGFR-2 in which the extracellular domain of VEGFR-2 was replaced with the extracellular domain of human colony stimulating factor-1 receptor and expressed in endothelial cells. To study the contribution of the carboxyl terminus to activation of VEGFR-2, we created a panel of truncated receptors in which the carboxyl terminus of VEGFR-2 was progressively deleted. Removal of the entire carboxyl terminus eliminated activation of VEGFR-2, its ability to activate signaling proteins, and its ability to stimulate cell proliferation. The carboxyl terminus-deleted VEGFR-2 exhibited impaired ligand-dependent down-regulation and inhibited the activation of wild-type receptor in a dominant-negative fashion. Furthermore, introducing the carboxyl terminus of another receptor, i.e., VEGFR-1, restored the ligand-dependent activation of the carboxyl terminus-deleted VEGFR-2 and its ability to stimulate cell proliferation. Our findings suggest that the carboxyl terminus of VEGFR-2 plays a critical role in VEGFR-2 activation, its ability to activate signaling proteins, and its ability to induce biological responses. The presence of at least 57 amino acids at the carboxyl terminus of VEGFR-2 are required for VEGFR-2 activation. Thus, we propose that the carboxyl terminus is required for activation of VEGFR-2, and absence of the carboxyl terminus renders VEGFR-2 inactive.
Journal of Biological Chemistry 02/2004; 279(1):735-42. · 4.77 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Activation of vascular endothelial growth factor receptor-1 and -2 (VEGFR-1 and VEGFR-2) plays a critical role in vasculogenesis and angiogenesis. However, the mechanism by which activation of VEGFRs elicits these cellular events is not fully understood. We recently constructed chimeric receptors containing the extracellular domain of human CSF-1R/c-fms fused with the entire transmembrane and cytoplasmic domains of murine VEGFR-1 and VEGFR-2. Selective activation of chimeric VEGFR-2, but not chimeric VEGFR-1, stimulated endothelial cell growth, migration, and differentiation. Stimulation of cells coexpressing chimeric VEGFR-1 and VEGFR-2 suppressed VEGFR-2-mediated endothelial cell growth. Site-directed mutagenesis demonstrated that tyrosines 799 and 1173 are required for VEGFR-2-mediated endothelial cell growth and activation of PI3 kinase. Further site-directed mutagenesis demonstrated that tyrosine 1212, located in the carboxyl tail of VEGFR-2, is required for the ligand-dependent autophosphorylation of the receptor and its ability to activate signaling proteins. Collectively, our results suggest that activation of VEGFR-1 and VEGFR-2 differentially regulates endothelial cell function and angiogenesis. Second, activation of VEGFR-2 is associated with many endothelial cell functions, including cell proliferation, migration, and differentiation. Third, activation of PI3 kinase by VEGFR-2 regulates endothelial cell proliferation.
Annals of the New York Academy of Sciences 06/2003; 995:200-7. · 3.15 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Vascular endothelial growth factor-mediated angiogenic signal transduction relay is achieved by coordinated induction of endothelial cell proliferation, migration, and differentiation. These complex cellular processes are most likely controlled by activation of both cooperative and antagonistic signals by vascular endothelial growth factor receptors (VEGFRs). Here, we investigated the contribution of tyrosine-phosphorylated residues of VEGFR-2/fetal liver kinase-1 to endothelial cell proliferation and differentiation and activation of signaling proteins. Mutation of tyrosine 1006 of VEGFR-2 to phenylalanine severely impaired the ability of this receptor to stimulate endothelial cell differentiation and tubulogenesis. Paradoxically, the mutant receptor stimulated endothelial cell proliferation far better than the wild-type receptor. Further analysis showed that tyrosine 1006 is responsible for phospholipase Cgamma1 (PLCgamma1) activation and intracellular calcium release in endothelial cells. Activation of PLCgamma1 was selectively mediated by tyrosine 1006. Mutation of tyrosines 799, 820, 949, 994, 1080, 1173, and 1221 had no measurable effect on the ability of VEGFR-2 to stimulate PLCgamma1 activation. Association of VEGFR-2 with PLCgamma1 was mainly established between tyrosine 1006 and the C-terminal SH2 domain of PLCgamma1 in vitro and in vivo. Taken together, the results indicate that phosphorylation of tyrosine 1006 is essential for VEGFR-2-mediated PLCgamma1 activation, calcium flux, and cell differentiation. More importantly, VEGFR-2-mediated endothelial cell proliferation is inversely correlated with the ability of VEGFR-2 to associate with and activate PLCgamma1.
Journal of Biological Chemistry 06/2003; 278(18):16347-55. · 4.77 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Vascular endothelial growth factor receptor (VEGFR)-2 plays a critical role in vasculogenesis during embryonic development and pathological angiogenesis, but little is known about the molecular mechanisms governing its functions. Here we investigated the role of tyrosine 1212 on mouse VEGFR-2 autophosphorylation and its signal transduction relay in endothelial cells. Mutation of tyrosine 1212 on VEGFR-2 to phenylalanine severely impaired the ligand-dependent autophosphorylation of VEGFR-2 and its ability to associate with and activate Src. This mutation also reduced the VEGFR-2 ability to phosphorylate phospholipase Cgamma1 and mitogen-activated protein kinase (MAPK). Unlike mutation of tyrosine 1212 to phenylalanine, replacement of tyrosine 1212 with glutamic acid preserved the ligand-dependent activation of VEGFR-2 and activation of VEGFR-2-associated signaling proteins including Src, phospholipase Cgamma1, and MAPK. Further analysis showed that Src activation is not required for activation of VEGFR-2, since cells co-expressing wild type receptor with kinase dead Src or wild type Src displayed no apparent effect in the ligand-dependent autophosphorylation of VEGFR-2. Similarly, expression of wild type VEGFR-2 in fibroblast (SYF) cells obtained from the triple knockout Src family kinases showed normal ligand-dependent autophosphorylation. Collectively, these results suggest that phosphorylation of tyrosine 1212 of VEGFR-2 plays a crucial role in the activation of VEGFR-2 and subsequently VEGFR-2-mediated angiogenesis.
Journal of Biological Chemistry 08/2002; 277(30):27081-7. · 4.77 Impact Factor
