KDR Stimulates Endothelial Cell Migration through Heterotrimeric G Protein Gq/11-mediated Activation of a Small GTPase RhoA
2002; Elsevier BV; Volume: 277; Issue: 48 Linguagem: Inglês
10.1074/jbc.m206133200
ISSN1083-351X
AutoresHuiyan Zeng, Dezheng Zhao, Debabrata Mukhopadhyay,
Tópico(s)Angiogenesis and VEGF in Cancer
ResumoVascular permeability factor/vascular endothelial growth factor (VPF/VEGF) functions by activating two receptor tyrosine kinases, Flt-1 (VEGFR-1) and KDR (VEGFR-2), both of which are selectively expressed on the primary vascular endothelium. KDR is responsible for VPF/VEGF-stimulated endothelial cell (EC) proliferation and migration, whereas Flt-1 down-modulates KDR-mediated EC proliferation. Flt-1 mediates down-regulation of EC proliferation through pertussis toxin-sensitive G proteins, βγ subunits, small GTPase CDC42, and partly by Rac-1. However, the molecular mechanism by which KDR mediates EC migration is not clear yet. Here we show for the first time that activation of RhoA and Rac1 is fully and partially required for KDR-mediated human umbilical vein endothelial cell (HUVEC) migration, respectively, and that CDC42, however, is not involved. Furthermore, overexpression of the RhoA dominant negative mutant RhoA-19N does not affect VPF/VEGF-stimulated KDR phosphorylation, intracellular Ca2+ mobilization, and mitogen-activated protein kinase phosphorylation. Utilizing the receptor chimeras (EGDR and EGLT) in which the extracellular domain of the epidermal growth factor receptor (EGFR) was fused to the transmembrane domain and the intracellular domains of KDR and Flt-1, respectively, we demonstrate that RhoA activation is mediated by EGDR, not by EGLT, and that EGDR mediates activation of Rac1, not CDC42. Furthermore, the EGDR-mediated RhoA and Rac1 activation is regulated by G proteins Gq/11, Gβγ, and phospholipase C independent of phosphatidylinositol 3-kinase and intracellular Ca2+ mobilization. Interestingly, the RhoA activation can be partially inhibited by overexpression of Rac1–17N, but overexpression of RhoA-19N has no effect on Rac1 activation. Finally, Gq/11 and Gβγ subunits are also required for VPF/VEGF-stimulated HUVEC migration. Taken together, our results indicate that KDR stimulates endothelial cell migration through a heterotrimeric G protein Gq/11 and Gβγ-mediated RhoA pathway. Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) functions by activating two receptor tyrosine kinases, Flt-1 (VEGFR-1) and KDR (VEGFR-2), both of which are selectively expressed on the primary vascular endothelium. KDR is responsible for VPF/VEGF-stimulated endothelial cell (EC) proliferation and migration, whereas Flt-1 down-modulates KDR-mediated EC proliferation. Flt-1 mediates down-regulation of EC proliferation through pertussis toxin-sensitive G proteins, βγ subunits, small GTPase CDC42, and partly by Rac-1. However, the molecular mechanism by which KDR mediates EC migration is not clear yet. Here we show for the first time that activation of RhoA and Rac1 is fully and partially required for KDR-mediated human umbilical vein endothelial cell (HUVEC) migration, respectively, and that CDC42, however, is not involved. Furthermore, overexpression of the RhoA dominant negative mutant RhoA-19N does not affect VPF/VEGF-stimulated KDR phosphorylation, intracellular Ca2+ mobilization, and mitogen-activated protein kinase phosphorylation. Utilizing the receptor chimeras (EGDR and EGLT) in which the extracellular domain of the epidermal growth factor receptor (EGFR) was fused to the transmembrane domain and the intracellular domains of KDR and Flt-1, respectively, we demonstrate that RhoA activation is mediated by EGDR, not by EGLT, and that EGDR mediates activation of Rac1, not CDC42. Furthermore, the EGDR-mediated RhoA and Rac1 activation is regulated by G proteins Gq/11, Gβγ, and phospholipase C independent of phosphatidylinositol 3-kinase and intracellular Ca2+ mobilization. Interestingly, the RhoA activation can be partially inhibited by overexpression of Rac1–17N, but overexpression of RhoA-19N has no effect on Rac1 activation. Finally, Gq/11 and Gβγ subunits are also required for VPF/VEGF-stimulated HUVEC migration. Taken together, our results indicate that KDR stimulates endothelial cell migration through a heterotrimeric G protein Gq/11 and Gβγ-mediated RhoA pathway. VPF/VEGF 1The abbreviations used for: VPF/VEGF, vascular permeability factor/vascular endothelial growth factor; VEGFR, VEGF receptor; EC, endothelial cell; PLC, phospholipase C; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; HUVEC, human umbilical vein endothelial cells; EGF, epidermal growth factor; EGFR, EGF receptor; EGDR, fusion protein with the extracellular domain of the EGFR and the transmembrane/intracellular domain of KDR; EGLT, fusion protein with the extracellular domain of the EGFR and the transmembrane/intracellular domain of Flt-1; FITC, fluorescein isothiocyanate; GST, glutathione S-transferase; TRBD, GST Rhotekin binding domain; PI3K, phosphatidylinositol 3-kinase; PBS, phosphate-buffered saline; BPAEC, bovine pulmonary artery endothelial cell; GEF, guanine nucleotide exchange factor; GDI, guanine nucleotide dissociation inhibitor; CRIB, Cdc42/Rac-interactive domain. is a multifunctional cytokine that is required for tumorigenesis as well as vasculogenesis (1Barleon B. 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Dvorak H.F. Mukhopadhyay D. J. Biol. Chem. 2001; 276: 26969-26979Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar). It is also indicated that Flt-1 mediated down-regulation of endothelial cell proliferation through pertussis toxin-sensitive G proteins, Gbγ subunits, small GTPase CDC42, and partly by Rac-1 (11Zeng H. Dvorak H.F. Mukhopadhyay D. J. Biol. Chem. 2001; 276: 26969-26979Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar, 12Zeng H. Zhao D. Mukhopadhyay D. J. Biol. Chem. 2002; 277: 4003-4009Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). A variety of evidence indicates that the activity of phospholipase C (PLC) is required for both EC proliferation and migration, and intracellular Ca2+ mobilization and MAPK phosphorylation are required for VPF/VEGF-stimulated proliferation of human umbilical vein endothelial cells (HUVEC) but not migration (11Zeng H. Dvorak H.F. Mukhopadhyay D. J. Biol. Chem. 2001; 276: 26969-26979Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar,13Zeng H. Sanyal S. Mukhopadhyay D. J. Biol. Chem. 2001; 276: 32714-32719Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). However the molecular mechanism by which VPF/VEGF stimulates HUVEC migration is not clear. The Rho family of the small GTPase superfamily has been shown to play an important role in cell growth, migration, transformation, and gene expression (14Aspenstrom P. Curr. Opin. Cell Biol. 1999; 11: 95-102Crossref PubMed Scopus (286) Google Scholar). The Rho family includes Rho (RhoA, RhoB, RhoC, RhoE, and RhoG), Rac (Rac1, Rac2, Rac3, and RhoG), CDC42 (CDC42Hs, G25K, and TC10), Rnd (RhoE/Rnd3, Rnd1/Rho6, and Rnd2/Rh07), RhoD, and TTF (14Aspenstrom P. Curr. Opin. Cell Biol. 1999; 11: 95-102Crossref PubMed Scopus (286) Google Scholar). Among them, RhoA, Rac1, and CDC42 are the most extensively studied members of this family. RhoA primarily induces the formation of stress fibers, whereas Rac1 and CDC42 promote the formation of lamellipodia and filopodia, respectively, when they are expressed in cells (15Hall A. Science. 1998; 279: 509-514Crossref PubMed Scopus (5220) Google Scholar, 16Machesky L.M. Insall R.H. J. Cell Biol. 1999; 146: 267-272Crossref PubMed Scopus (214) Google Scholar). It was reported that VPF/VEGF induces actin-based mobility (17Rousseau S. Houle F. Huot J. Trends Cardiovasc. Med. 2000; 10: 321-327Crossref PubMed Scopus (114) Google Scholar), suggesting that Rho family proteins might be involved in this response. Recently, we reported that CDC42 and Rac1 are required and partially required for Flt-1-mediated antiproliferation activity, respectively. However, whether RhoA family members play any role in VPF/VEGF-stimulated HUVEC migration is not known. In this study, we show that overexpression of dominant negative RhoA (RhoA-19N) and Rac1 (Rac1–17N) inhibits and partially inhibits VPF/VEGF-stimulated HUVEC migration, respectively. However, overexpression of dominant negative CDC42 (CDC42–17N) has no effect. Overexpression of RhoA-19N has no effect on intracellular Ca2+ mobilization and MAPK phosphorylation in VPF/VEGF-stimulated HUVECs. Recently, we reported (12Zeng H. Zhao D. Mukhopadhyay D. J. Biol. Chem. 2002; 277: 4003-4009Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar) that CDC42 and Rac1 were directly activated in VPF/VEGF-stimulated HUVECs and that the CDC42 and Rac1 activation could be mediated by Flt-1. Here, we show that RhoA is also activated in VPF/VEGF-stimulated HUVECs. Using our receptor chimeras EGDR and EGLT, in which the N-terminal domains of KDR and Flt-1, respectively, were replaced with the N-terminal domain of epidermal growth factor receptor (EGFR), we further show that the activation of RhoA is mediated by EGDR, not EGLT. EGDR also mediates Rac1 activation but not CDC42 activation. Furthermore, we show that an EGDR mutant (EGDR(Y951F)) lacking the migrating activity of EGDR also lost the ability to induce Rho activity; however, this mutant could partially activate Rac1 (13Zeng H. Sanyal S. Mukhopadhyay D. J. Biol. Chem. 2001; 276: 32714-32719Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). Moreover, EGDR-mediated activation of RhoA and Rac1, respectively, requires PLC activity but not PI3K activity or intracellular calcium mobilization. However, different pathways are required for Rac1-dependent and -independent RhoA activation. Finally, we show that Gq/11 proteins and Gβγ subunits are required for KDR-mediated RhoA and Rac1 activation and for VPF/VEGF-stimulated HUVEC migration. Recombinant VPF/VEGF was obtained from R&D Systems (Minneapolis, MN). EGM-MV Bullet kit, trypsin-EDTA, and trypsin neutralization solution were obtained from Clonetics (San Diego, CA). Vitrogen 100 was purchased from Collagen Biomaterials (Palo Alto, CA). Mouse monoclonal antibodies against the KDR C-terminal domain and a rabbit polyclonal antibody against Gq/11, Gi, βARK, Rho, and Rac1 were obtained from Santa Cruz Biotechnology. Anti-phosphotyrosine antibody was obtained from Upstate Biotechnology (Lake Placid, NY). Anti-phospho-p42/p44 MAPK antibodies were obtained from New England Biolabs (Beverly, MA). Transwell plate inserts were from Fisher Scientific. CyQuant, Fura-2 AM, and Pluronic F-127 were obtained from Molecular Probes (Eugene, Oregon). Primary human umbilical vein endothelial cells were obtained from Clonetics (San Diego, CA). Cells were grown on plates coated with 30 μg/ml vitrogen in the EGM-MV Bullet kit (5% FBS in endothelial basal medium with 12 μg/ml bovine brain extract, 1 μg/ml hydrocortisone, 1 μl/ml GA-1000, and EGF). HUVECs traduced with EGDR or EGLT were grown in the same medium without EGF. HUVECs (passages 3 or 4) that were ∼80% confluent were used for most experiments. Cells were serum-starved in 0.1% FBS in endothelial basal medium for 24 h prior to treatment. CDC42–17N, Rac1–17N, and RhoA-19N were kindly provided by Margaret M. Chou (University of Pennsylvania). The fragments encoding the genes were subcloned to a retroviral vector pMMP (11Zeng H. Dvorak H.F. Mukhopadhyay D. J. Biol. Chem. 2001; 276: 26969-26979Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar). The hβARK1 (495) minigene was cloned by reverse transcription PCR from HUVEC RNA and subcloned to a retrovirus vector (18Zhao D. Keates A.C. Kuhnt-Moore S. Moyer M.P. Kelly C.P. Pothoulakis C. J. Biol. Chem. 2001; 276: 44464-44471Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). Retrovirus preparation and HUVEC infection with retrovirus were carried out as described (11Zeng H. Dvorak H.F. Mukhopadhyay D. J. Biol. Chem. 2001; 276: 26969-26979Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar, 13Zeng H. Sanyal S. Mukhopadhyay D. J. Biol. Chem. 2001; 276: 32714-32719Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). A 3′-end FITC-labeled phosphorothioated Gαq/11 antisense oligonucleotide (ODN-Gq/11; 5′-CCATGCGGTTCTCATTGTCTG-3′) and a 3′-end FITC-labeled phosphorothioated random oligonucleotide (ODN-RD; 5′-CCCTTATTTACTACTTTCGC-3′) (19Sanchez-Blazquez P. Garzon J. J. Pharmacol. Exp. Ther. 1998; 285: 820-827PubMed Google Scholar) were synthesized by Genemed Synthesis (Genemed Synthesis, South San Francisco, CA). Transfection was carried out as described (20Paik J.H. Chae S. Lee M.J. Thangada S. Hla T. J. Biol. Chem. 2001; 276: 11830-11837Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar). Assays were carried out as described (11Zeng H. Dvorak H.F. Mukhopadhyay D. J. Biol. Chem. 2001; 276: 26969-26979Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar,13Zeng H. Sanyal S. Mukhopadhyay D. J. Biol. Chem. 2001; 276: 32714-32719Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). Serum-starved HUVECs (infected with retrovirus or transfected with antisense oligonucleotides) were detached from tissue culture plates as described (11Zeng H. Dvorak H.F. Mukhopadhyay D. J. Biol. Chem. 2001; 276: 26969-26979Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar, 13Zeng H. Sanyal S. Mukhopadhyay D. J. Biol. Chem. 2001; 276: 32714-32719Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar), washed twice with endothelial basal medium containing 0.1% FBS, seeded (1 × 105 cells per well) into the transwells coated with vitrogen (30 μg/ml), and the transwells were inserted into a 24-well plate containing 1 ml of the same medium. Cells over a range from 3 × 103 to 1 × 105 cells per well were seeded in a 96-well plate for the standard curve. Cells were incubated at 37 °C for 1 h to allow the cells to attach, then VPF/VEGF was added at a final concentration of 10 ng/ml. After incubation for an additional 2 h, cells remaining on the upper surface of the transwell filter membrane were wiped off with a cotton tip. The whole transwell membrane was cut out and placed in an individual well of the 96-well plate that contained the cells for the standard curve. 200 μl of Cyquant DNA stain was added to each well containing cells or membrane, and the plate was kept at 4 °C overnight. After warming to room temperature, stained cells were counted in a spectrofluorometer (SpectraFluor; TECAN) with Delta Soft 3 software. Data are expressed as the mean ± S.D. of quadruplicate values. All experiments were repeated at least three times. Serum-starved HUVECs transduced with LacZ or Rho-19N were treated with 10 ng/ml VPF/VEGF for different time intervals as indicated. Cell lysates were either subjected to Western blot analysis using an antibody specific for active forms of MAPK/ERK kinase or immunoprecipitated with an KDR antibody followed by immunoblotting with a phospho-tyrosine antibody as described previously (11Zeng H. Dvorak H.F. Mukhopadhyay D. J. Biol. Chem. 2001; 276: 26969-26979Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar, 12Zeng H. Zhao D. Mukhopadhyay D. J. Biol. Chem. 2002; 277: 4003-4009Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Serum-starved HUVECs transfected with LacZ or RhoA-19N were loaded with Fura-2 AM and stimulated with 10 ng/ml VPF/VEGF. An assay was carried out as described (11Zeng H. Dvorak H.F. Mukhopadhyay D. J. Biol. Chem. 2001; 276: 26969-26979Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar, 13Zeng H. Sanyal S. Mukhopadhyay D. J. Biol. Chem. 2001; 276: 32714-32719Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). All experiments were repeated at least three times. The RhoA activity assay was modified from that used by Ren et al. (21Ren X.D. Kiosses W.B. Schwartz M.A. EMBO J. 1999; 18: 578-585Crossref PubMed Scopus (1365) Google Scholar, 22Petrova T.V. Makinen T. Alitalo K. Exp. Cell Res. 1999; 253: 117-130Crossref PubMed Scopus (241) Google Scholar, 23English J. Pearson G. Wilsbacher J. Swantek J. Karandikar M. Xu S. Cobb M.H. Exp. Cell Res. 1999; 253: 255-270Crossref PubMed Scopus (377) Google Scholar). The glutathione S-transferase (GST)-Rhotekin Rho binding domain (TRBD) fusion protein was kindly provided by Dr. Martin Schwartz (Scripps Institute). Bacteria were grown to an optical density of 0.8 at a wavelength of 600 nm and induced with 1 mm of isopropylthiogalactoside (IPTG) for 3 h. Bacteria were aliquoted per 50 ml and collected by centrifugation at 5000 rpm for 20 min and frozen at −80 °C. To prepare the GST·TRBD beads, each aliquot of 50 ml of frozen bacteria was resuspended in 2 ml of cold PBS followed by the addition of 20 μl of 1 m dithiothreitol, 20 μl of 0.2 m phenylmethylsulfonyl fluoride, and 40 μl of lysozyme (50 μg/ml). After incubation on ice for 30 min, 225 μl of 10% Triton X-100, 22.5 μl of 1 m MgCl2, 22.5 μl of DNase I (2,000 kilo-units/ml) were added. After another 30 min incubation on ice, the bacteria were centrifuged at 10,000 rpm for 5 min. The supernatant was incubated with 200 μl of glutathione-coupled Sepharose 4B beads (Amersham Biosciences), which were washed with bead-washing buffer (PBS with 10 mmdithiothreitol and 1% Triton X-100) three times. After incubation at 4 °C for 45 min, beads were washed three times with bead-washing buffer and resuspended in the bead-washing buffer to give out 50% bead slur. For 24 h, serum-starved HUVECs with or without infection with retrovirus or transfected with antisense oligonucleotides were stimulated with 10 ng/ml VPF/VEGF or EGF for different time intervals. Stimulation was stopped by the addition of ice-cold PBS. Cells were washed three times with PBS and lysed with lysis buffer (150 mm NaCl, 0.8 mm MgCl2, 5 mm EGTA, 1% IGEPAL, 50 mm HEPES, pH 7.5, 1 μm phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, and 10 μg/ml aprotinin). Cell lysates were centrifuged at 14,000 rpm for 3 min. The supernatant was incubated with 50 μl of GST·TRBD beads at 4 °C for 45 min. Proteins bound to beads were washed trice with AP wash buffer (50 mm Tris-HCl, pH 7.2, 1% Triton X-100, 150 mm NaCl, 10 mm MgCl2, 1 μm phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin and 10 μg/ml aprotinin) and analyzed by SDS-PAGE. For inhibitor experiments, different concentrations of inhibitors as indicated were added 5 min before EGF treatment. All experiments were repeated at least three times. CDC and Rac1 assays were carried out as described previously (12Zeng H. Zhao D. Mukhopadhyay D. J. Biol. Chem. 2002; 277: 4003-4009Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Basely, these were the same as the RhoA assay described above, except that GST·Pak·CRIB beads, not GST·TRBD beads, were used. Recently, we have shown (12Zeng H. Zhao D. Mukhopadhyay D. J. Biol. Chem. 2002; 277: 4003-4009Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar) that dominant negative mutants of RhoA, Rac1, and Cdc42 (RhoA-19N, Rac1–17N, and CDC42–17N) that block their endogenous functions have different effects on VPF/VEGF-stimulated HUVEC proliferation. CDC42–17N and Rac1–17N completely and partially inhibit Flt-1-mediated anti-proliferative activity, respectively, whereas RhoA-19N has no effect. In this study we further examined whether the dominant negative mutants CDC42–17N, RhoA-19N, and Rac1–17N have any effects on VPF/VEGF-stimulated HUVEC migration. CDC42–17N, Rho-19N, and Rac1–17N were overexpressed in HUVECs as described previously (11Zeng H. Dvorak H.F. Mukhopadhyay D. J. Biol. Chem. 2001; 276: 26969-26979Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar, 12Zeng H. Zhao D. Mukhopadhyay D. J. Biol. Chem. 2002; 277: 4003-4009Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 13Zeng H. Sanyal S. Mukhopadhyay D. J. Biol. Chem. 2001; 276: 32714-32719Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar), with a retrovirus system that demonstrates almost 100% infection yield in HUVECs. Fig.1 a shows that the expression levels of the dominant negative mutants CDC42–17N, Rac1–17N, and RhoA-19N are much higher than those of their corresponding endogenous proteins. HUVECs transduced with CDC42–17N, RhoA-19N, and Rac1–17N were serum starved and subjected to migration assay as described elsewhere (11Zeng H. Dvorak H.F. Mukhopadhyay D. J. Biol. Chem. 2001; 276: 26969-26979Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar, 13Zeng H. Sanyal S. Mukhopadhyay D. J. Biol. Chem. 2001; 276: 32714-32719Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). The results show that overexpression of RhoA-19N almost completely inhibits VPF/VEGF-stimulated HUVEC migration. Overexpression of Rac1–17N inhibits VPF/VEGF-stimulated HUVEC migration by ∼50%, whereas overexpression of CDC42–17N has no effect (Fig. 1 b). Fig. 1 a also clearly shows that the ratios of endogenous G proteins (CDC42, Rac1, and RhoA) to their corresponding overexpressed dominant negative mutants (CDC42–17N, Rac1–17N, and RhoA-19N) are similar to each other. Furthermore, overexpression of CDC42–17N and Rac1–17N was shown to inhibit the Flt-1-mediated signaling pathway in HUVECs stimulated with VPF/VEGF (12Zeng H. Zhao D. Mukhopadhyay D. J. Biol. Chem. 2002; 277: 4003-4009Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Therefore, the partial inhibition of Rac1–17N on VPF/VEGF-stimulated HUVEC migration is not due to the lesser expression of Rac1–17N, and the functional CDC42–17N has no effect on this response. These data indicate that three major members of the Rho family small GTPases differentially regulate VPF/VEGF-stimulated HUVEC migration. It is known that VPF/VEGF stimulates KDR phosphorylation, intracellular Ca2+mobilization, and MAPK phosphorylation in the EC (22,23). Therefore, we first examined whether RhoA mediates KDR phosphorylation. Serum-starved HUVECs transduced with LacZ- or RhoA-19N-expressing viruses were stimulated with VPF/VEGF for 1, 5, and 10 min. Cellular extracts were immunoprecipitated with an antibody against KDR and immunoblotted with an antibody against phosphotyrosine. The results show that RhoA-19N has no effect on KDR phosphorylation (Fig.2 a). To examine whether RhoA mediates VPF/VEGF-induced intracellular Ca2+ mobilization, serum-starved HUVECs transduced with LacZ or RhoA-19N were stimulated with VPF/VEGF, and the intracellular calcium mobilization was monitored. The data show that overexpression of RhoA-19N has no effect on VPF/VEGF-induced calcium response (Fig. 2 b). Similarly, serum-starved HUVECs transduced with LacZ or RhoA-19N-expressing viruses were stimulated with VPF/VEGF for 1, 5, and 10 min. Cellular extracts were subjected to immunoblot analysis using an antibody specifically against phosphorylated MAPK. Fig. 2 c clearly shows that MAPK was phosphorylated to the same level in HUVECs transduced with LacZ or RhoA-19N. Recently, we showed that VPF/VEGF activates CDC42 and Rac1 in HUVECs (12Zeng H. Zhao D. Mukhopadhyay D. J. Biol. Chem. 2002; 277: 4003-4009Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Because RhoA-19N blocks VPF/VEGF-induced HUVEC migration (Fig. 1), we examined whether RhoA can also be activated in HUVECs stimulated by VPF/VEGF. The activity of RhoA was measured by a pull-down assay using a GST·TRBD fusion protein that binds only to the GTP-bound form of RhoA (21Ren X.D. Kiosses W.B. Schwartz M.A. EMBO J. 1999; 18: 578-585Crossref PubMed Scopus (1365) Google Scholar). Serum-starved HUVECs were stimulated with VPF/VEGF for different lengths of time as indicated. Cellular extracts were incubated with freshly prepared GST·TRBD beads. Proteins bound to the beads were subjected to Western blot analysis using the antibodies against RhoA. Fig. 3 shows that RhoA is activated as early as 0.5 min and remains high at 5 min after VPF/VEGF treatment. To systematically study the roles of Rho family GTPases in VPF/VEGF signaling pathways, we used the recently developed receptor chimeras EGDR and EGLT in which the N-terminal domain of KDR or Flt-1, respectively, was replaced with that of EGFR to dissect the signaling pathways mediated by KDR or Flt-1 (11Zeng H. Dvorak H.F. Mukhopadhyay D. J. Biol. Chem. 2001; 276: 26969-26979Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar). It was previously shown that HUVECs were not responsive to EGF treatment in the experimental conditions used (11Zeng H. Dvorak H.F. Mukhopadhyay D. J. Biol. Chem. 2001; 276: 26969-26979Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar, 12Zeng H. Zhao D. Mukhopadhyay D. J. Biol. Chem. 2002; 277: 4003-4009Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). As expected, in the HUVECs transduced with LacZ-expressing viruses, EGF did not activate RhoA (Fig.4 a, top panel). When serum-starved HUVECs transduced with EGDR- or EGLT-expressing viruses were stimulated with EGF for different time intervals, EGDR mediated RhoA activation in a similar time course to that in VPF/VEGF-stimulated HUVECs (Fig. 4 a,middle panel). In contrast, stimulation of EGLT did not induce RhoA activation (Fig. 4 a, bottom panel). Because both CDC42 and Rac1 are activated in EGF-stimulated HUVECs transduced with EGLT (12Zeng H. Zhao D. Mukhopadhyay D. J. Biol. Chem. 2002; 277: 4003-4009Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar), we examined whether EGDR mediates CDC42 and Rac1 activation. A pull-down assay using the GST·Pak·CRIB fusion protein, which binds only to the GTP-bound forms of CDC42 and Rac1, was used to measure the activity of CDC42 and Rac1 (24Bagrodia S. Taylor S.J. Creasy C.L. Chernoff J. Cerione R.A. J. Biol. Chem. 1995; 270: 22731-22737Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar, 12Zeng H. Zhao D. Mukhopadhyay D. J. Biol. Chem. 2002; 277: 4003-4009Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). HUVECs transduced with LacZ or EGDR were serum starved and then stimulated with 10 ng/ml EGF for different time intervals as indicated. Cellular extracts were incubated with freshly prepared GST·Pak·CRIB beads. Proteins bound to the beads were subjected to immunoblot analysis using the antibodies against CDC42 or Rac1, respectively. Figs. 4 b shows that Rac1 was activated in EGDR/HUVECs stimulated with EGF for 0.5 and 1 min. At 5 min of EGF stimulation, no Rac1 activation was detected. This EGDR-mediated Rac1 activation is in a similar time course to that in VPF/VEGF-stimulated HUVECs (12Zeng H. Zhao D. Mukhopadhyay D. J. Biol. Chem. 2002; 277: 4003-4009Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). However, Fig. 4 c shows that EGDR cannot mediate CDC42 activation. Recently, we demonstrated that the tyrosine 951 residue of KDR, which is partially required for KDR phosphorylation, is essential for VPF/VEGF-induced migration but not proliferation (13Zeng H. Sanyal S. Mukhopadhyay D. J. Biol
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