The Adaptor Protein Shb Binds to Tyrosine 1175 in Vascular Endothelial Growth Factor (VEGF) Receptor-2 and Regulates VEGF-dependent Cellular Migration

Previous studies have shown that the adaptor protein Shb is involved in receptor tyrosine kinase signaling. In this study, we demonstrate that Shb is phosphorylated in an Src-dependent manner upon vascular endothelial growth factor (VEGF) stimulation using porcine aortic endothelial cells expressing the human VEGF receptor 2 (VEGFR-2) (KDR). In co-immunoprecipitation experiments, we could detect an interaction between Shb and the VEGFR-2 in human telomerase-immortalized microvascular endothelial cells. Furthermore, in a glutathione S-transferase pull-down assay, the Src homology 2 domain of Shb was shown to interact with phosphorylated tyrosine 1175 in the C-terminal tail of VEGFR-2. VEGF-induced Shb phosphorylation was lost in porcine aortic endothelial cells expressing a chimeric murine VEGFR-2 (Flk-1) with a mutation at the corresponding position. Shb expression was specifically decreased by 80%, in a transient manner, by using the short interfering RNA technique. Reduced Shb expression led to a loss of stimulation of phosphatidylinositol 3-kinase, phosphorylation of focal adhesion kinase at tyrosine 576, the generation of focal adhesions, and stress fiber formation in response to VEGF. Furthermore, we show that VEGF-induced migration is inhibited in Shb short interfering RNA-treated cells. Our data demonstrate that Shb is important for VEGF signaling in endothelial cells. This is achieved by Shb binding to tyrosine 1175 in the VEGFR-2, which regulates VEGF-induced formation of focal adhesions and cell migration, of which the latter occurs in a phosphatidylinositol 3-kinase-dependent manner. Previous studies have shown that the adaptor protein Shb is involved in receptor tyrosine kinase signaling. In this study, we demonstrate that Shb is phosphorylated in an Src-dependent manner upon vascular endothelial growth factor (VEGF) stimulation using porcine aortic endothelial cells expressing the human VEGF receptor 2 (VEGFR-2) (KDR). In co-immunoprecipitation experiments, we could detect an interaction between Shb and the VEGFR-2 in human telomerase-immortalized microvascular endothelial cells. Furthermore, in a glutathione S-transferase pull-down assay, the Src homology 2 domain of Shb was shown to interact with phosphorylated tyrosine 1175 in the C-terminal tail of VEGFR-2. VEGF-induced Shb phosphorylation was lost in porcine aortic endothelial cells expressing a chimeric murine VEGFR-2 (Flk-1) with a mutation at the corresponding position. Shb expression was specifically decreased by 80%, in a transient manner, by using the short interfering RNA technique. Reduced Shb expression led to a loss of stimulation of phosphatidylinositol 3-kinase, phosphorylation of focal adhesion kinase at tyrosine 576, the generation of focal adhesions, and stress fiber formation in response to VEGF. Furthermore, we show that VEGF-induced migration is inhibited in Shb short interfering RNA-treated cells. Our data demonstrate that Shb is important for VEGF signaling in endothelial cells. This is achieved by Shb binding to tyrosine 1175 in the VEGFR-2, which regulates VEGF-induced formation of focal adhesions and cell migration, of which the latter occurs in a phosphatidylinositol 3-kinase-dependent manner. The adaptor protein Shb has been shown previously to be involved in platelet-derived growth factor (PDGF), 1The abbreviations used are: PDGF, platelet-derived growth factor; VEGF, vascular endothelial growth factor; VEGFR-2, vascular endothelial growth factor receptor 2; FAK, focal adhesion kinase; FGF, fibroblast growth factor; PI, phosphatidylinositol; PAE, porcine aortic endothelial; TIME, telomerase-immortalized microvascular endothelial; GST, glutathione S-transferase; SH2, Src homology 2; siRNA, short interfering RNA; IL2, interleukin 2; IL2R, IL2 receptor; PTB, phosphotyrosine binding; MAPK, mitogen-activated protein kinase; PBS, phosphate-buffered saline; BSA, bovine serum albumin; FCS, fetal calf serum; PLC-γ, phospholipase C-γ; CSF, colony-stimulating factor; EGF, epidermal growth factor; TBS, Tris-buffered saline; FGFR-1, fibroblast growth factor receptor 1; PDGFR, platelet-derived growth factor receptor; FACS, fluorescence-activated cell sorter. fibroblast growth factor (FGF), T cell, and interleukin (IL) 2-receptor signaling (1Hooshmand-Rad R. Lu L. Heldin C. H. Claesson-Welsh L. Welsh M. Exp. Cell Res. 2000; 257: 245-254Crossref PubMed Scopus (20) Google Scholar, 2Cross M. J. Lu L. Magnusson P. Nyqvist D. Holmqvist K. Welsh M. Claesson-Welsh L. Mol. Biol. Cell. 2002; 8: 2881-2893Crossref Google Scholar, 3Lindholm C. K. Gylfe E. Zhang W. Samelson L. E. Welsh M. J. Biol. Chem. 1999; 274: 28050-28057Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 4Lindholm C. K. Biochem. Biophys. Res. Commun. 2002; 296: 929-936Crossref PubMed Scopus (18) Google Scholar). Shb interacts directly with these receptors, initiating a specific cellular response depending on the growth factor stimulus. In this study we have investigated the role of Shb in vascular endothelial growth factor receptor 2 (VEGFR-2) signaling. Shb consists of a C-terminal Src homology 2 (SH2) domain, a central phosphotyrosine binding (PTB) domain, four central putative tyrosine phosphorylation sites, and a proline-rich N terminus (5Welsh M. Mares J. Karlsson T. Lavergne C. Breant B. Claesson-Welsh L. Oncogene. 1994; 9: 19-27PubMed Google Scholar, 6Karlsson T. Songyang Z. Landgren E. Lavergne C. DiFiore P. P. Anafi M. Pawson T. Cantley L. C. Claesson-Welsh L. Welsh M. Oncogene. 1995; 10: 475-483Google Scholar, 7Lindholm C. K. Frantz J. D. Shoelson S. E. Welsh M. Biochem. Biophys. Res. Commun. 2000; 278: 537-543Crossref PubMed Scopus (25) Google Scholar). VEGF is a potent angiogenic factor that binds to VEGFR-2 (Flk-1/KDR) present on endothelial cells, evoking an intracellular signaling cascade leading to a number of physiological responses (8Matsumoto T. Claesson-Welsh L. Science's STKE. 2001; http: //www. stke. org/cgi/content/full/OCₛigtrans;2001/112/re21PubMed Google Scholar, 9Cross M. J. Dixelius J. Matsumoto T. Claesson-Welsh L. Trends Biochem. Sci. 2003; 28: 488-494Abstract Full Text Full Text PDF PubMed Scopus (519) Google Scholar). Gene inactivation of VEGFR-2 in mice leads to embryonal death at day 8. 5-9. 5, because of lack of endothelial cells (10Shalaby F. Rossant J. Yamaguchi P. Gertsenstein M. Wu X. F. Breitman M. L. Schuh A. C. Nature. 1995; 376: 62-66Crossref PubMed Scopus (3371) Google Scholar). We have shown previously that Shb plays a number of important roles in endothelial cells. It can bind directly to the FGFR-1, via its SH2 domain, and regulate mitogen-activated protein kinase (MAPK) activation and mitogenicity (2Cross M. J. Lu L. Magnusson P. Nyqvist D. Holmqvist K. Welsh M. Claesson-Welsh L. Mol. Biol. Cell. 2002; 8: 2881-2893Crossref Google Scholar). It can also bind directly to focal adhesion kinase (FAK), via its PTB domain, and regulate cell spreading (11Holmqvist K. Cross M. Riley D. Welsh M. Cell. Signal. 2003; 15: 171-179Crossref PubMed Scopus (28) Google Scholar) and tubular morphogenesis (12Lu L. Holmqvist K. Cross M. Welsh M. Cell Growth Differ. 2002; 13: 141-148PubMed Google Scholar). Activation of VEGFR-2 is known to lead to cytoskeletal reorganization and increased cellular migration. This process has been shown to require the activation of phosphatidylinositol 3-kinase (PI 3-kinase) (8Matsumoto T. Claesson-Welsh L. Science's STKE. 2001; http: //www. stke. org/cgi/content/full/OCₛigtrans;2001/112/re21PubMed Google Scholar, 9Cross M. J. Dixelius J. Matsumoto T. Claesson-Welsh L. Trends Biochem. Sci. 2003; 28: 488-494Abstract Full Text Full Text PDF PubMed Scopus (519) Google Scholar). In addition, knockout experiments have demonstrated the importance of the cytoplasmic tyrosine kinase FAK for cellular migration (13Ilic D. Furuta Y. Kanazawa S. Takeda N. Sobue K. Nakatsuji N. Nomora S. Fujimoto J. Okada M. Yamamoto T. Nature. 1995; 377: 539-544Crossref PubMed Scopus (1591) Google Scholar). In this study, we demonstrate that Shb is phosphorylated and binds directly to tyrosine 1175 upon VEGF stimulation in PAE/VEGFR-2 cells. By use of the siRNA technique, we find that Shb is required for VEGF-mediated PI 3-kinase activity, stress fiber formation, and cellular migration. Taken together, the data indicate that Shb plays a critical role in VEGFR-2-mediated signaling. Materials—Phosphate-buffered saline (PBS) for cell culture, bovine serum albumin (BSA), and gelatin were purchased from Sigma. Ham's F-12 medium, trypsin, Opti-MEM medium, Lipofectin® reagent, and fetal calf serum were from The was from were from were purchased from was from and and were from and were purchased from human was purchased from human was purchased from The Src kinase was purchased from The Shb has been previously T. Welsh M. Oncogene. 13: Google Scholar). The was from FAK and were purchased from phosphorylated tyrosine 1175 in the VEGFR-2 and was by Cell The was from The was from was from was from and was purchased from cells expressing chimeric VEGFR-2 domain of human with the and cytoplasmic of murine VEGFR-2 have been N. K. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). siRNA were from was from VEGFR-2 corresponding to phosphorylated and and were by at the was from was purchased from Cell cells and cells expressing the human 2 J. Claesson-Welsh L. M. Heldin J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar) were on in Ham's F-12 with at in cells expressing a chimeric VEGFR-2 the domain of human with the and the cytoplasmic of murine VEGFR-2 (Flk-1) and cells expressing chimeric VEGFR-2 with a mutation at tyrosine in the cytoplasmic domain were and cells N. K. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). human microvascular endothelial cells E. C. T. M. Exp. Cell Res. 2002; PubMed Scopus Google Scholar) were on gelatin in in endothelial cell FCS, epidermal growth factor VEGF growth and at in The cells were in FCS, of growth to the and cells were on and in Ham's F-12 with stimulation were with VEGF for were with and in of 2 and for on to The were at for at The were with and with Shb on for 2 a with protein The was in and in Tris-buffered saline The were in for and by The were which were in in The were with in BSA, for 2 by in The were with the in and the by the were in and at for of and corresponding to the SH2 domain and Shb with the SH2 domain were (5Welsh M. Mares J. Karlsson T. Lavergne C. Breant B. Claesson-Welsh L. Oncogene. 1994; 9: 19-27PubMed Google Scholar, M. Songyang Z. Frantz J. D. T. Karlsson T. M. Cantley L. C. Shoelson S. E. Oncogene. PubMed Scopus Google Scholar). PAE/VEGFR-2 cells were on and to and with VEGF for and in from cells were at at for was and with of protein SH2, to and phosphotyrosine was to a of were for at and corresponding to tyrosine 1175 in the VEGFR-2 were and used in pull-down experiments to the interaction at and The were in and in was and was The were and for phosphotyrosine for the siRNA were by using the The used were and The been to the and was to a of in 2 PAE/VEGFR-2 cells were in in Ham's F-12 with The cells were the day using Lipofectin® of Opti-MEM was with of and of Opti-MEM was with of siRNA The were and for at of Opti-MEM was and the was to cells The was to Ham's F-12 with and the cells were in and for Shb and expression by the cells were in Ham's F-12 with The day the cells were with VEGF for for The cells were in and in and on a PAE/VEGFR-2 cells were with and the cells were and with Ham's F-12 with The were using a PI were with siRNA the cells were for which were for with VEGF The cells were in and were and the were using a phosphotyrosine The were to the PI 3-kinase (1Hooshmand-Rad R. Lu L. Heldin C. H. Claesson-Welsh L. Welsh M. Exp. Cell Res. 2000; 257: 245-254Crossref PubMed Scopus (20) Google Scholar). of PI is shown of and on of the were by to protein and PAE/VEGFR-2 cells were in in Ham's F-12 with The the cells were with siRNA using Lipofectin® The cells were using Ham's F-12 with the the cells were with VEGF for The cells were in PBS, in in for and using at for The were and were by for with was for The cells were was and the were The were using the in a the cells were in and on an the cells were for to stimulation with VEGF for and The cells were in and the cells were in reagent, by with in and by with the were in and using a cells were with siRNA The cells were in Ham's F-12 with for the cells and on a in a of at the cells were in with and in The cells, on the of the were and the was on a using cells were using The of the migration were of response in Shb upon VEGF studies have demonstrated that the adaptor protein Shb is phosphorylated in response to a number of J. T. Holmqvist K. Lu L. R. Welsh M. Claesson-Welsh L. 2000; PubMed Google growth factor T. K. Welsh M. Cell Growth Differ. 9: Google (1Hooshmand-Rad R. Lu L. Heldin C. H. Claesson-Welsh L. Welsh M. Exp. Cell Res. 2000; 257: 245-254Crossref PubMed Scopus (20) Google and IL2 C. K. Biochem. Biophys. Res. Commun. 2002; 296: 929-936Crossref PubMed Scopus (18) Google Scholar). The of this study was to Shb has a role in VEGF signaling. this we cells with the human VEGFR-2 J. Claesson-Welsh L. M. Heldin J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar). of VEGFR-2 in these cells was by of PAE/VEGFR-2 cells with VEGF in a phosphorylated protein of which to the VEGFR-2 PAE/VEGFR-2 cells were with VEGF, and the phosphorylation of Shb was Shb was phosphorylated in response to VEGF stimulation at phosphorylated of corresponding to the VEGFR-2, was with demonstrate an interaction between Shb and the VEGFR-2, we telomerase-immortalized human microvascular endothelial cells. cells were with VEGF, with and with Shb The demonstrated that Shb with the VEGFR-2 that Shb and VEGFR-2 interact in a manner. VEGF-induced Shb have shown previously that Shb phosphorylation is Src-dependent (11Holmqvist K. Cross M. Riley D. Welsh M. Cell. Signal. 2003; 15: 171-179Crossref PubMed Scopus (28) Google Scholar). the role of Src in VEGF-induced Shb phosphorylation was investigated by use of the Src kinase PAE/VEGFR-2 cells were with stimulation with VEGF. The cells were and with Shb and was VEGF-induced Shb phosphorylation was by with Furthermore, of Src the of Shb with a protein at because the of with the of Shb in the The was also with an phosphorylated tyrosine 1175 of the VEGFR-2, which that Shb and phosphorylated VEGFR-2 of cell from the that directly the phosphorylation of the VEGFR-2 Our data show that Src kinase is for Shb phosphorylation for the of Shb and VEGFR-2 in PAE/VEGFR-2 cells. Shb to 1175 in the Shb adaptor protein SH2 domain has been shown previously to bind to the T. Songyang Z. Landgren E. Lavergne C. DiFiore P. P. Anafi M. Pawson T. Cantley L. C. Claesson-Welsh L. Welsh M. Oncogene. 1995; 10: 475-483Google Scholar). This to tyrosine in the (2Cross M. J. Lu L. Magnusson P. Nyqvist D. Holmqvist K. Welsh M. Claesson-Welsh L. Mol. Biol. Cell. 2002; 8: 2881-2893Crossref Google Scholar) and tyrosine in the (1Hooshmand-Rad R. Lu L. Heldin C. H. Claesson-Welsh L. Welsh M. Exp. Cell Res. 2000; 257: 245-254Crossref PubMed Scopus (20) Google Scholar). In the human VEGFR-2 tyrosine 1175 has the that be a this PAE/VEGFR-2 cells were with VEGF, and with the SH2 domain of Shb Shb with a of the SH2 The binding was with the of phosphotyrosine phosphorylated at the tyrosine 1175 in the VEGFR-2 The that the Shb SH2 domain to tyrosine 1175 in the VEGFR-2 in a manner. Furthermore, Shb with an SH2 domain also to bind to the VEGFR-2, this was the role of tyrosine 1175 in Shb we cells expressing a chimeric receptor of the domain of the human colony-stimulating factor receptor and the intracellular domain of the murine VEGFR-2 (Flk-1) C. N. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). The tyrosine 1175 in the human VEGFR-2 is and to tyrosine in the murine VEGFR-2 We also a murine receptor in which tyrosine was to Activation of the chimeric with in receptor phosphorylation of Shb phosphorylation that activation of the chimeric receptor in Shb activation of the receptor Shb phosphorylation of the of of intracellular signaling that the receptor could a activation of MAPK, was to the phosphorylation of FAK at tyrosine Taken together, these that Shb binds to the in the VEGFR-2 Gene by siRNA Shb the physiological role of Shb in VEGFR-2 we to siRNA using a RNA We of the Shb and and a PAE/VEGFR-2 cells were with a and was which a of at Furthermore, Shb protein expression was specifically decreased upon with the siRNA in a transient By using the we to Shb expression by and with a a of Shb The was also for in to the of the Shb Our data show transient of Shb protein expression in PAE/VEGFR-2 cells using the siRNA technique. VEGF-induced of FAK in siRNA to the of Shb protein intracellular signaling were Shb has been shown to bind and regulate FAK phosphorylation (11Holmqvist K. Cross M. Riley D. Welsh M. Cell. Signal. 2003; 15: 171-179Crossref PubMed Scopus (28) Google we investigated the phosphorylation of FAK these We that VEGF-induced phosphorylation of FAK tyrosine was in the siRNA cells by In in the and in the cells, VEGF increased FAK tyrosine phosphorylation by increased FAK phosphorylation in cells, that in the siRNA cells, FAK tyrosine could be The was and with an FAK, which We also the of Shb expression on VEGFR-2, phospholipase and that Shb expression with the siRNA VEGFR-2 phosphorylation and the activation of and The data in the of VEGFR-2 Shb regulates FAK Shb for PI to FAK, PLC-γ, and MAPK, was of to the role of Shb in VEGF-induced PI 3-kinase because was shown previously T. Songyang Z. Landgren E. Lavergne C. DiFiore P. P. Anafi M. Pawson T. Cantley L. C. Claesson-Welsh L. Welsh M. Oncogene. 1995; 10: 475-483Google Scholar) that the domain of the of PI 3-kinase could in with proline-rich of with siRNA to PI 3-kinase in response to VEGF the the Shb with an the of PI 3-kinase, for an between Shb and PI 3-kinase was Shb is required for PI 3-kinase activation in response to VEGF, for a between Shb and PI 3-kinase was Reduced Shb to of and in to VEGF to use PAE/VEGFR-2 cells to study the role of Shb in VEGF-induced stress fiber The PAE/VEGFR-2 cells were with VEGF, and for using and of stimulation with VEGF stress fiber formation in cells and in cells. in the siRNA cells, VEGF to stress fiber formation and of Shb expression a of the protein The cells were also for a for focal The and cells increased number of focal adhesions and stimulation was short to an of VEGF on the formation of focal adhesions In to increased of focal adhesions, these were and The cells, were to to VEGF the with increased formation of focal adhesions Our data that Shb is important for VEGF-induced formation of stress and focal VEGF-induced by siRNA of PAE/VEGFR-2 the physiological role of Shb in the VEGFR-2 we to study cellular PAE/VEGFR-2 cells were with siRNA for and on a in a with VEGF and lead to an in migration in and cells In the siRNA cells, VEGF to could a This that Shb is required for VEGFR-2-mediated migration in endothelial cells. the role of PI 3-kinase in VEGF-induced cell migration, PAE/VEGFR-2 cell migration was in the of the PI 3-kinase VEGF-induced cell migration was inhibited in the of this of a role of PI 3-kinase in the In this study we have the role of the adaptor protein Shb in VEGF signaling. Shb has been shown previously to in and T cell and signaling. In addition, we have shown that Shb plays an important role in signaling in endothelial cells (2Cross M. J. Lu L. Magnusson P. Nyqvist D. Holmqvist K. Welsh M. Claesson-Welsh L. Mol. Biol. Cell. 2002; 8: 2881-2893Crossref Google Scholar, K. Cross M. Riley D. Welsh M. Cell. Signal. 2003; 15: 171-179Crossref PubMed Scopus (28) Google Scholar, L. Holmqvist K. Cross M. Welsh M. Cell Growth Differ. 2002; 13: 141-148PubMed Google Scholar). VEGF is a potent of in endothelial cells, we were in the role of Shb in VEGFR-2 Our data show that stimulation of PAE/VEGFR-2 cells with VEGF leads to an between VEGFR-2 and Shb and increased Shb with an Src kinase the VEGF-induced Shb phosphorylation is data were with to signaling in endothelial cells, Shb phosphorylation was Src-dependent (11Holmqvist K. Cross M. Riley D. Welsh M. Cell. Signal. 2003; 15: 171-179Crossref PubMed Scopus (28) Google Scholar). It has been shown J. Biochem. 2002; PubMed Scopus Google Scholar) that Src is with the VEGFR-2, the role of Src in VEGF signaling Taken together, that Src Shb in response to a number of We also an between Shb and the VEGFR-2 in the human cells, a microvascular endothelial cell that Shb is in response to VEGFR-2 phosphorylation in a number of endothelial cells. Our data the tyrosine 1175 in the VEGFR-2 a binding of the Shb SH2 The Shb SH2 domain has been shown previously to bind tyrosine in the (2Cross M. J. Lu L. Magnusson P. Nyqvist D. Holmqvist K. Welsh M. Claesson-Welsh L. Mol. Biol. Cell. 2002; 8: 2881-2893Crossref Google tyrosine in the (1Hooshmand-Rad R. Lu L. Heldin C. H. Claesson-Welsh L. Welsh M. Exp. Cell Res. 2000; 257: 245-254Crossref PubMed Scopus (20) Google tyrosine in the and the in the T cell receptor M. Songyang Z. Frantz J. D. T. Karlsson T. M. Cantley L. C. Shoelson S. E. Oncogene. PubMed Scopus Google Scholar). of cells expressing the chimeric (Flk-1) tyrosine 1175 the binding for in the FGFR-1, Shb and can bind to the at tyrosine (2Cross M. J. Lu L. Magnusson P. Nyqvist D. Holmqvist K. Welsh M. Claesson-Welsh L. Mol. Biol. Cell. 2002; 8: 2881-2893Crossref Google Scholar, M. D. R. F. W. M. M. J. Mol. Cell. Biol. 10: Google Scholar). In the of the VEGFR-2, has been previously to bind to tyrosine 1175 T. Yamaguchi S. K. M. J. 2001; PubMed Scopus Google Scholar). in the of the angiogenic tyrosine kinase VEGFR-2 and FGFR-1, Shb and have the to bind to the (2Cross M. J. Lu L. Magnusson P. Nyqvist D. Holmqvist K. Welsh M. Claesson-Welsh L. Mol. Biol. Cell. 2002; 8: 2881-2893Crossref Google Scholar, Biochem. Biophys. Res. Commun. PubMed Scopus Google Scholar). In addition, Shb and can with C. K. Gylfe E. Zhang W. Samelson L. E. Welsh M. J. Biol. Chem. 1999; 274: 28050-28057Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). that Shb and for the binding in (2Cross M. J. Lu L. Magnusson P. Nyqvist D. Holmqvist K. Welsh M. Claesson-Welsh L. Mol. Biol. Cell. 2002; 8: 2881-2893Crossref Google Scholar) in the VEGFR-2, shown in the siRNA phosphorylation was upon Shb adaptor has also been shown to interact with tyrosine 1175 in the VEGFR-2 J. Biochem. J. 2000; PubMed Scopus Google Scholar). This that this interacts with signaling the with the (2Cross M. J. Lu L. Magnusson P. Nyqvist D. Holmqvist K. Welsh M. Claesson-Welsh L. Mol. Biol. Cell. 2002; 8: 2881-2893Crossref Google Shb to regulate in response to VEGFR-2 shown in the cells and in the Shb siRNA we that Shb is to intracellular signaling from receptor tyrosine This could be by the of in signaling. This adaptor protein is required for a process that Shb (2Cross M. J. Lu L. Magnusson P. Nyqvist D. Holmqvist K. Welsh M. Claesson-Welsh L. Mol. Biol. Cell. 2002; 8: 2881-2893Crossref Google Scholar). by using and cells, we could detect activation in response to VEGF that the VEGFR-2 to of and In to the binding of the Shb SH2 domain protein to tyrosine 1175 in the VEGFR-2, we also an interaction between the protein with the SH2 domain and phosphorylated VEGFR-2. The of this interaction because phosphotyrosine inhibited this because this protein also the proline-rich domain of in to the PTB domain, is that the interaction with the VEGFR-2 is and is by with Shb and the VEGFR-2. We have used the siRNA to specifically Shb expression in PAE/VEGFR-2 cells. siRNA was in S. S. E. Nature. PubMed Scopus Google Scholar). Shb using Shb siRNA in a loss of PI 3-kinase activation in response to VEGF. Furthermore, the murine VEGFR-2 PI 3-kinase activation in response to VEGF N. K. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, C. N. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). binding to important for the PI 3-kinase It is this is We have previously that the domain of PI 3-kinase can interact in with the proline-rich of Shb have for a between Shb and PI the could be via FAK Claesson-Welsh L. Exp. Cell Res. 2001; PubMed Scopus Google Scholar, P. L. F. F. R. B. M. D. E. F. R. F. D. E. Cell. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). The of FAK could to this because we that FAK tyrosine phosphorylation was in the cells with Shb upon VEGF in FAK activation was in response to activation of the murine VEGFR-2 (Flk-1) chimeric the of Shb on FAK phosphorylation these is and to the of Shb on PI 3-kinase FAK is phosphorylated in by Src at a number of and of tyrosine 576, in the FAK kinase domain, the of FAK Mol. Cell. Biol. 1995; Google Scholar). is that Shb is required for VEGF-mediated activation of FAK by Src to tyrosine we have shown previously (11Holmqvist K. Cross M. Riley D. Welsh M. Cell. Signal. 2003; 15: 171-179Crossref PubMed Scopus (28) Google Scholar) that the PTB domain of Shb can bind directly to FAK and regulate its in response to activation in endothelial cells. Taken together, that Shb regulates FAK in response to and VEGFR-2 The of FAK tyrosine phosphorylation by the Shb to have because these cells to to VEGF stimulation with an increased formation of focal VEGF has been shown previously to the formation of focal adhesions J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google an that occurs a Y. S. M. S. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). Taken a data indicate FAK in response to VEGF upon Shb PI 3-kinase activation has been shown to be important for cell migration in response to a number of VEGF M. J. Dixelius J. Matsumoto T. Claesson-Welsh L. Trends Biochem. Sci. 2003; 28: 488-494Abstract Full Text Full Text PDF PubMed Scopus (519) Google Scholar). This the of which regulate stress and Trends Cell Biol. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). of stress fiber formation Shb siRNA stress in response to VEGF. We have shown previously that stimulation of endothelial cells Shb leads to an in stress fiber formation and the formation of (12Lu L. Holmqvist K. Cross M. Welsh M. Cell Growth Differ. 2002; 13: 141-148PubMed Google that Shb plays an important role in the This could have for cell migration, because Shb expression by siRNA also the of endothelial cells to in response to VEGF. the importance of the PI 3-kinase in this by the of the PI 3-kinase FAK could be of because cells from mice and spreading (13Ilic D. Furuta Y. Kanazawa S. Takeda N. Sobue K. Nakatsuji N. Nomora S. Fujimoto J. Okada M. Yamamoto T. Nature. 1995; 377: 539-544Crossref PubMed Scopus (1591) Google Scholar, J. D. Mol. Cell. Biol. 1999; PubMed Scopus Google Scholar). Furthermore, FAK is known to be critical for the focal adhesion and required for migration we directly an of FAK in VEGF-induced migration Oncogene. 2000; PubMed Scopus Google Scholar). by PI 3-kinase and FAK activation in Shb could cellular migration. cell migration is an important in physiological of VEGF, endothelial cells and to in the angiogenic The of Shb to regulate this process that of Shb signaling have a We that VEGF the VEGFR-2 is Src with the and the SH2 domain of Shb binds to phosphorylated tyrosine 1175 in the C-terminal tail of the VEGFR-2. Src which the activation of PI 3-kinase and phosphorylation of FAK at tyrosine in the kinase This regulate focal fiber formation the and of the response in endothelial cells.