Vascular Endothelial Growth Factor-dependent Down-regulation of Flk-1/KDR Involves Cbl-mediated Ubiquitination

Ligand-stimulated degradation of receptor tyrosine kinase (RTK) is an important regulatory step of signal transduction. The vascular endothelial growth factor (VEGF) receptor Flk-1/KDR is responsible for the VEGF-stimulated nitric oxide (NO) production from endothelial cells. Cellular mechanisms mediating the negative regulation of Flk-1 signaling in endothelial cells have not been investigated. Here we show that Flk-1 is rapidly down-regulated following VEGF stimulation of bovine aortic endothelial cells (BAECs). Consequently, VEGF pretreatment of endothelial cells prevents any further stimulation of Flk-1, resulting in decreased NO production from subsequent VEGF challenges. Ubiquitination of RTKs targets them for degradation; we demonstrate that activation of Flk-1 by VEGF leads to its polyubiquitination in BAECs. Furthermore, VEGF stimulation of BAECs or COS-7 cells transiently transfected with Flk-1 results in the phosphorylation of the ubiquitin ligase Cbl, the enhanced association of Cbl with Flk-1, and the relocalization of Cbl to vesicular structures in BAECs. Overexpression of Cbl in COS-7 cells enhances VEGF-induced ubiquitination of Flk-1, whereas a Cbl mutant lacking the ubiquitin ligase RING finger domain, 70Z/3-Cbl, does not. Moreover, expression of Cbl in contrast to 70Z/3-Cbl inhibits the Flk-1-dependent activation of eNOS and, thus, NO release. In BAEC overexpressing Cbl, the degradation of Flk-1 upon VEGF stimulation is accelerated compared with cells transfected with a control vector (green fluorescent protein). Our findings demonstrate that Flk-1 is rapidly down-regulated following sustained VEGF stimulation and identify Cbl as a negative regulator of Flk-1 signaling to eNOS. Cbl thus plays a role in the regulation of VEGF signaling by mediating the stimulated ubiquitination and, consequently, degradation of Flk-1 in endothelial cells. Ligand-stimulated degradation of receptor tyrosine kinase (RTK) is an important regulatory step of signal transduction. The vascular endothelial growth factor (VEGF) receptor Flk-1/KDR is responsible for the VEGF-stimulated nitric oxide (NO) production from endothelial cells. Cellular mechanisms mediating the negative regulation of Flk-1 signaling in endothelial cells have not been investigated. Here we show that Flk-1 is rapidly down-regulated following VEGF stimulation of bovine aortic endothelial cells (BAECs). Consequently, VEGF pretreatment of endothelial cells prevents any further stimulation of Flk-1, resulting in decreased NO production from subsequent VEGF challenges. Ubiquitination of RTKs targets them for degradation; we demonstrate that activation of Flk-1 by VEGF leads to its polyubiquitination in BAECs. Furthermore, VEGF stimulation of BAECs or COS-7 cells transiently transfected with Flk-1 results in the phosphorylation of the ubiquitin ligase Cbl, the enhanced association of Cbl with Flk-1, and the relocalization of Cbl to vesicular structures in BAECs. Overexpression of Cbl in COS-7 cells enhances VEGF-induced ubiquitination of Flk-1, whereas a Cbl mutant lacking the ubiquitin ligase RING finger domain, 70Z/3-Cbl, does not. Moreover, expression of Cbl in contrast to 70Z/3-Cbl inhibits the Flk-1-dependent activation of eNOS and, thus, NO release. In BAEC overexpressing Cbl, the degradation of Flk-1 upon VEGF stimulation is accelerated compared with cells transfected with a control vector (green fluorescent protein). Our findings demonstrate that Flk-1 is rapidly down-regulated following sustained VEGF stimulation and identify Cbl as a negative regulator of Flk-1 signaling to eNOS. Cbl thus plays a role in the regulation of VEGF signaling by mediating the stimulated ubiquitination and, consequently, degradation of Flk-1 in endothelial cells. Vascular endothelial growth factor (VEGF) 1The abbreviations used are: VEGF, vascular endothelial growth factor; NO, nitric oxide; PI3-kinase, phosphatidylinositol 3-kinase; eNOS, endothelial NO synthase; RTK, receptor tyrosine kinase; EGFR, epidermal growth factor receptor; PDGFR, platelet-derived growth factor receptor; BAEC, bovine aortic endothelial cells; HA, hemagglutinin A; PBS, phosphate-buffered saline; β-Gal, β-galactosidase; GFP, green fluorescent protein. is a multifunctional cytokine essential for vascular development and maintenance (1Matsumoto T. Claesson-Welsh L. Sci STKE 2001. 2001; 112 (stke.sciencemag.org): RE21Google Scholar). VEGF activates specific receptor tyrosine kinases, namely Flt-1 (VEGF receptor 1), Flk-1 (VEGF receptor 2, KDR), and Flt-4 (VEGF receptor 3) (2de Vries C. Escobedo J.A. Ueno H. 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Chem. 2000; 275: 5096-5103Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar, 15Kroll J. Waltenberger J. J. Biol. Chem. 1997; 272: 32521-32527Abstract Full Text Full Text PDF PubMed Scopus (389) Google Scholar, 16D'Angelo G. Martini J.F. Iiri T. Fantl W.J. Martial J. Weiner R.I. Mol. Endocrinol. 1999; 13: 692-704Crossref PubMed Google Scholar, 17Ratcliffe K.E. Tao Q. Yavuz B. Stoletov K.V. Spring S.C. Terman B.I. Oncogene. 2002; 21: 6307-6316Crossref PubMed Scopus (15) Google Scholar). Substantial amounts of data describe the receptorial components necessary for VEGF-mediated activation of Flk-1; however, little is known about factors mediating down-regulation or negative regulation of Flk-1 signaling. One of the mechanisms by which growth factor responses are negatively regulated is through ligand-stimulated degradation of the receptors. Several RTKs, such as the epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor (PDGFR), and the colony-stimulating factor-1 (CSF-1), are actively degraded following sustained exposure to their respective ligands. Degradation of RTKs is mainly initiated by the addition of ubiquitin moieties following receptor engagement, which has been show to target them to the lysosomal/proteasomal machinery for degradation (18Ettenberg S.A. Rubinstein Y.R. Banerjee P. Nau M.M. Keane M.M. Lipkowitz S. Mol. Cell. Biol. Res. Commun. 1999; 2: 111-118Crossref PubMed Scopus (46) Google Scholar, 19Miyake S. Lupher Jr., M.L. Druker B. Band H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7927-7932Crossref PubMed Scopus (237) Google Scholar, 20Kallies A. Rosenbauer F. Scheller M. Knobeloch K.P. Horak I. Blood. 2002; 99: 3213-3219Crossref PubMed Scopus (14) Google Scholar, 21Thien C.B. Langdon W.Y. Nat. Rev. Mol. Cell Biol. 2001; 2: 294-307Crossref PubMed Scopus (523) Google Scholar). Ubiquitination of substrates requires three concerted steps for the conjugation of ubiquitin molecules to a substrate protein. Initially, the ubiquitin-activating enzyme (E1) forms a thiol ester bond with the carboxy-terminal glycine of ubiquitin. Then the ubiquitin molecule is transferred to an ubiquitin-conjugating enzyme (also known as ubiquitin-carrier enzyme or E2), again involving the carboxyl terminus of ubiquitin. The final step involves an ubiquitin ligase (E3) enzyme that complexes with the substrate and catalyzes the transfer from E2 to the ϵ-amino group of a lysine residue on the target protein (22Weissman A.M. Nat. Rev. Mol. Cell Biol. 2001; 2: 169-178Crossref PubMed Scopus (1262) Google Scholar). The multi-adaptor protein Cbl has been shown to possess E3 ligase activity and, thus, has been implicated in the ubiquitination of activated EGFR, PDGFR, and other RTKs (19Miyake S. Lupher Jr., M.L. Druker B. Band H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7927-7932Crossref PubMed Scopus (237) Google Scholar, 23Levkowitz G. Waterman H. Zamir E. Kam Z. Oved S. Langdon W.Y. Beguinot L. Geiger B. Yarden Y. Genes Dev. 1998; 12: 3663-3674Crossref PubMed Scopus (720) Google Scholar). The demonstration of a prominent and inducible association of Cbl with the EGFR coupled to the observation that Cbl overexpression induced EGFR and PDGFR ubiquitination confirmed Cbl as a regulatory component of RTK function. The Flk-1 receptor has been shown to internalize upon VEGF stimulation (24Dougher M. Terman B.I. Oncogene. 1999; 18: 1619-1627Crossref PubMed Scopus (165) Google Scholar). However, the regulation of VEGF-dependent down-regulation of this important and essential component of vascular integrity has never been investigated. In this study, we demonstrate that sustained VEGF stimulation of endothelial cells results in Flk-1 protein down-regulation. This results in impaired eNOS activation and NO release during subsequent VEGF challenges. Consistent with the VEGF-stimulated degradation of Flk-1, we demonstrate that VEGF stimulates ubiquitination of Flk-1 and that Cbl mediates this effect through its ubiquitin ligase activity. Finally, we also demonstrate that Cbl negatively regulates Flk-1 signaling and VEGF-induced NO release. Our results identify Cbl as a novel component in the negative regulation of VEGF signaling. Cell Culture—Bovine aortic endothelial cells (BAECs) were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (HyClone, Logan, UT), 2.0 mm l-glutamine, 100 units/ml penicillin, and 100 COS-7 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum 2 mm l-glutamine, 100 units/ml penicillin, and 100 were used and human VEGF from the of the of and used for cell stimulation this and bovine eNOS human Flk-1 and were D. Gratton J.P. McCabe T.J. Fontana J. Fujio Y. Walsh K. Franke T.F. Papapetropoulos A. Sessa W.C. Nature. 1999; 399: 597-601Crossref PubMed Scopus (2239) Google Scholar, J. D. Y. McCabe T.J. N. T. Sessa W.C. Res. 2002; PubMed Scopus Google Scholar). Cbl and the mutant were from A. and from S. COS-7 cells were transfected in or and transfected BAECs were transfected in as P. G. D. J. 2000; PubMed Scopus Google Scholar). The expression used for the of as as for control in BAEC Cell were following and protein expression confirmed of cell by and were serum for and for the of the of NO in by as W.C. G. J. A. J. S. K.M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). were in is to NO, which by a with in a NO COS-7 cells were also for the of cells were stimulated with VEGF for 2 and NO as and Cbl and ubiquitin were from from and were from Cell and from BAECs were with following VEGF cells were in some by to the cells were with a mm mm mm mm mm mm mm and a proteins were by for and the were for 2 with the of a and for an The complexes were by three with in and by a and as J.P. M. Y. D. Walsh K. Sessa W.C. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). by a Ubiquitination cells were transfected with a of of for Flk-1, Cbl, and and VEGF-stimulated cells were in were for in mm mm mm mm mm mm mm mm and a and were as were cultured on cells were stimulated for with VEGF were with and for in were with and with in for cells were with and for with the in bovine serum in and were following of and were a VEGF VEGF to down-regulation of has been shown for several VEGF-dependent down-regulation of Flk-1 signaling has not been investigated. VEGF-dependent of Flk-1 receptor signaling in endothelial we the effect of VEGF pretreatment on subsequent VEGF NO release from bovine aortic endothelial cells that VEGF pretreatment in a of VEGF-stimulated NO The and any subsequent VEGF-stimulated NO production from BAEC demonstrate the of this VEGF or were used as for NO production pretreatment with VEGF not stimulated NO release by These results that the responses of BAECs to VEGF are specific to the down-regulation of the Flk-1 further the VEGF of NO we the of a VEGF pretreatment on Akt phosphorylation and on eNOS phosphorylation Ser1179 stimulated by a VEGF stimulation the pretreatment with VEGF any further increase in Akt and eNOS phosphorylation following a VEGF stimulation of cells. fetal bovine activation of Akt not by VEGF pretreatment not in Flk-1 protein were and VEGF pretreatment down-regulated the expression of Flk-1, which responsible for the impaired to further VEGF VEGF-dependent of Flk-1 in further the VEGF-dependent down-regulation of Flk-1 shown in we by on cell from the effect of a VEGF on Flk-1 protein cells were in VEGF stimulation induced a in Flk-1 protein results were cells were in that in of Flk-1 following stimulation for the down-regulation This of stimulation and 2, and of BAEC for with of VEGF that this effect and In the of used as a control for protein were not by VEGF These results that VEGF stimulation specific degradation of Flk-1, which to the VEGF-dependent eNOS activation following a VEGF pretreatment of endothelial cells. VEGF Ubiquitination of ubiquitination of proteins targets them for the the of VEGF to the addition of ubiquitin moieties on the activated Flk-1 BAECs were stimulated with of VEGF which is to Flk-1 its degradation to a ubiquitin on the Flk-1 that Flk-1 is in to VEGF of BAECs The that the ubiquitination machinery and of ubiquitin moieties to Flk-1, the form of Flk-1 is as a of the with that Flk-1 is rapidly and transiently tyrosine in to VEGF and that ubiquitination follows the VEGF-induced of Flk-1 VEGF Cbl and to The ubiquitin ligase Cbl has been shown to down-regulation of several the of VEGF to Cbl and its association to Flk-1 upon in the activation of Flk-1 in a rapid and transient phosphorylation of Flk-1 the BAEC were with Cbl, and the tyrosine phosphorylation of Cbl and its association with Flk-1 were of the Cbl confirmed that VEGF stimulation of BAEC to a rapid and sustained phosphorylation of Cbl on tyrosine Furthermore, with that VEGF stimulation of BAEC the of Flk-1 in the Cbl the VEGF-dependent association of Cbl with Flk-1, we transiently COS-7 cells with for Flk-1 and or Cbl Overexpression of Cbl in COS-7 cells in of Cbl with the transfected Flk-1 receptor and Moreover, in COS-7 VEGF stimulation in enhanced association of Cbl with Flk-1 2 and Cbl Flk-1 were on COS-7 cells overexpressing Flk-1 and Cbl, that the specific for the of Flk-1 2 and results that stimulation of Flk-1 activates Cbl and also its to the activated Flk-1 Cbl Ubiquitination of demonstrate that Cbl has the to ubiquitination of Flk-1, we the of Cbl overexpression on thus transfected COS-7 cells with Flk-1 and ubiquitin in the or of Cbl or the mutant form 70Z/3-Cbl has been shown to an form of Cbl that of the ubiquitin ligase RING C.B. Langdon W.Y. Nat. Rev. Mol. Cell Biol. 2001; 2: 294-307Crossref PubMed Scopus (523) Google Scholar). The addition of on Flk-1 in transfected COS-7 cell for VEGF stimulation of COS-7 cells in of in the Flk-1 The is again as a a as Flk-1 is in to VEGF overexpression of the mutant 70Z/3-Cbl not VEGF-induced ubiquitination of the transfected Flk-1 Moreover, the addition of in specific for Flk-1, in Flk-1 from cells transfected with and Cbl These findings demonstrate that Cbl Flk-1 ubiquitination and that the RING finger of Cbl is necessary for this Cbl Overexpression Flk-1-dependent the effect of Cbl on VEGF-mediated NO we thus transfected COS-7 cells with Flk-1 and eNOS expression and the effect of overexpression of Cbl and 70Z/3-Cbl on VEGF-dependent NO release oxide in the as by specific for in of of of NO in the were from COS-7 cells eNOS as compared with cells not D. Gratton J.P. McCabe T.J. Fontana J. Fujio Y. Walsh K. Franke T.F. Papapetropoulos A. Sessa W.C. Nature. 1999; 399: 597-601Crossref PubMed Scopus (2239) Google Scholar). of Flk-1 with eNOS in the of VEGF NO production compared with eNOS with This that Flk-1 activation has the to NO release in transfected COS-7 cells of Cbl with Flk-1 and eNOS the Flk-1-dependent NO release from VEGF-stimulated cells 2 and In Cbl not any on eNOS and This that the of Cbl on NO release are specific to Flk-1-dependent and not on eNOS activity Finally, 70Z/3-Cbl in contrast to Cbl, not NO release. 70Z/3-Cbl a however, not to increase NO release induced by stimulation of Flk-1 2 and These results show that Cbl negatively Flk-1-dependent eNOS activation and that the RING finger is essential for the of Cbl on NO VEGF-dependent Degradation of Flk-1 in BAEC the regulatory role of Cbl in endothelial cell we transiently transfected BAEC with expression that for Cbl or as a in BAEC are however, results that BAEC transfected with Cbl a overexpression of Cbl compared with control cells transfected with vector VEGF stimulation of BAEC overexpressing Cbl in a rapid of Flk-1 degradation compared with cells. of Flk-1 in or BAECs were BAECs overexpressing Cbl and of VEGF a in the receptor compared with cells that a in of VEGF These results a role for Cbl in VEGF-dependent degradation of Flk-1 in endothelial cells. VEGF of of Cbl to we of Flk-1 and Cbl by in and BAECs following VEGF in Flk-1 is prominent in the as structures as as the of cells. Cbl in control a and In VEGF-stimulated Flk-1 and the VEGF stimulation of BAEC a relocalization of Cbl vesicular in with cells were with a serum as as a not The results show the Flk-1 is rapidly down-regulated following sustained VEGF stimulation of endothelial cells. VEGF stimulation Cbl activation and enhances its association to 3) Cbl is responsible for the VEGF-stimulated ubiquitination and degradation of thus that negative regulation of the Flk-1 receptor is a novel for down-regulation of VEGF-stimulated NO production from endothelial cells. oxide release from endothelial cells is in of VEGF as in eNOS NO release has been shown to essential for VEGF-mediated in vascular and endothelial cell M.J. Claesson-Welsh L. Pharmacol. Sci. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, D. T. A. Y. J. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus Google Scholar, M. D. G. Fujio Y. Walsh K. Sessa W.C. Res. 2000; PubMed Scopus Google Scholar). regulation of VEGF NO is Our that VEGF pretreatment of BAEC prevents any further activation of the signaling from a VEGF Our results that VEGF-dependent degradation of Flk-1 is in this of cells to VEGF The degradation of Flk-1 actively in the regulation of VEGF responses in endothelial cells through the rapid of VEGF-stimulated eNOS activation and NO release and One also that rapid degradation of Flk-1 also in the regulation of other components of VEGF signaling. However, the on of VEGF such as endothelial cell migration, proliferation, and have not been investigated. Ubiquitination of RTKs is known to target them to the and C.B. Langdon W.Y. Nat. Rev. Mol. Cell Biol. 2001; 2: 294-307Crossref PubMed Scopus (523) Google Scholar). with findings we demonstrate following VEGF Flk-1 is in BAEC and Flk-1 transfected COS-7 cells and of RTKs, has as a that the of proteins that are regulated or in to the (22Weissman A.M. Nat. Rev. Mol. Cell Biol. 2001; 2: 169-178Crossref PubMed Scopus (1262) Google Scholar). show that polyubiquitination is in the regulation of Flk-1 receptor Our results also demonstrate that Cbl is tyrosine and recruited to Flk-1 following VEGF stimulation of BAEC Cbl has been as a multi-adaptor protein in the of signaling phosphorylation of Cbl upon stimulation results in its association to the Src homology 2 proteins such as the of PI3-kinase, the factor and the and G. T. S. S. K. Druker B. S. L. Band H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, C. I. A. J. 1997; Google Scholar, N. I. Band H. J. Biol. 2002; Google Scholar, Oncogene. 1997; PubMed Scopus Google Scholar). Furthermore, Cbl a that is in Src homology The demonstration of an of Cbl in the negative regulation of RTKs from of that that a of of the Cbl for signaling J. Science. PubMed Scopus Google Scholar, PubMed Google Scholar). that Cbl as an ubiquitin ligase that E2 to a receptor M. T. A. M. W.C. H. A. R. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). Our results show that the association of Cbl with Flk-1 is enhanced in to VEGF stimulation of BAEC and in transfected COS-7 cells. This association of Cbl to activated RTKs is mediated by the of However, we that a protein as an in the of Cbl to the proteins to in in for the RING finger of Cbl to transfer the ubiquitin group the target we demonstrate that overexpression of Cbl enhances ubiquitination and degradation of Flk-1 and that a Cbl mutant lacking the RING finger domain, 70Z/3-Cbl, Flk-1 ubiquitination M. T. A. M. W.C. H. A. R. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). Our also that Cbl regulates VEGF-stimulated NO release from cells. The of Cbl with Flk-1 and eNOS inhibits VEGF-stimulated NO release Furthermore, the mutant form of Cbl, 70Z/3-Cbl, to NO release and, to have a negative effect on Cbl by NO release as as VEGF-stimulated Flk-1 ubiquitination and These results are in with that forms of Cbl, such as and 70Z/3-Cbl, to enhanced signaling from RTKs and, in some to and C.B. Langdon W.Y. Oncogene. 1997; 15: PubMed Scopus Google Scholar, C.B. Langdon W.Y. EMBO J. 13: PubMed Scopus Google Scholar, S. Lupher Jr., M.L. Langdon W.Y. Band H. Mol. Cell. Biol. 1997; PubMed Google Scholar). The VEGF-stimulated association of Cbl with Flk-1 is and the of Flk-1 we demonstrate that overexpression of Cbl in BAEC VEGF-stimulated degradation of This in endothelial Cbl mediates Flk-1 degradation and that the of expression of Cbl Flk-1 responses by receptor following stimulation of cells. In results that Cbl expression does however, of the Flk-1 protein upon stimulation of cells; Cbl the of its The Cbl on RTKs is as some show that receptor ubiquitination in and other that this the the G. Waterman H. Zamir E. Kam Z. Oved S. Langdon W.Y. Beguinot L. Geiger B. Yarden Y. Genes Dev. 1998; 12: 3663-3674Crossref PubMed Scopus (720) Google Scholar, G. J. H. J. J. Cell Sci. 2001; PubMed Google Scholar). Moreover, data show following a VEGF stimulation of endothelial Cbl is in structures This to in with that Cbl with the receptor and G. J. H. J. J. Cell Sci. 2001; PubMed Google Scholar). Our results show that negative regulation of Flk-1 by Cbl results in impaired NO release and that ubiquitination of Flk-1 through Cbl is in the of the to rapid VEGF signaling. Cbl has been shown to activated in to endothelial cells to and also in the activation of following Y. H. S. S. S. J. Cell 2002; PubMed Scopus Google Scholar, H. S. Wang Y. A. S. Biochem. Biophys. Res. Commun. 2002; PubMed Scopus Google Scholar). that Cbl as an molecule as as an E3 is to that on endothelial cells results in decreased Flk-1 protein In that polyubiquitination of the VEGF Flk-1, by the ubiquitin ligase Cbl, mediates VEGF-dependent degradation of that to important in endothelial cells such as the of VEGF-mediated NO release and enhanced Flk-1 demonstrate the of this novel of control of Flk-1 signaling in other of are to for for with for and for and of the

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