title: The adaptor protein shb binds to tyrosine 1175 in vascular endothelial growth factor (VEGF) receptor-2 and regulates VEGF-dependent cellular migration.
abstract: 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.
authors.affiliation: Department of Medical Cell Biology, Uppsala University, Uppsala 75123, Sweden.
authors.forename: Kristina
authors.initials: K
authors.lastname: Holmqvist
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Cross
authors.forename: Charlotte
authors.initials: C
authors.lastname: Rolny
authors.forename: Robert
authors.initials: R
authors.lastname: Hägerkvist
authors.forename: Nader
authors.initials: N
authors.lastname: Rahimi
authors.forename: Taro
authors.initials: T
authors.lastname: Matsumoto
authors.forename: Lena
authors.initials: L
authors.lastname: Claesson-Welsh
authors.forename: Michael
authors.initials: M
authors.lastname: Welsh
mesh_terms: D000199:Actins / Q000378:metabolism; D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D001011:Aorta / Q000166:cytology; D002465:Cell Movement; D003598:Cytoskeletal Proteins / Q000378:metabolism; D004730:Endothelium, Vascular / Q000166:cytology; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D005786:Gene Expression Regulation; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D008856:Microscopy, Fluorescence; D009154:Mutation; D051419:Paxillin; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000737:chemistry; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism / Q000502:physiology*; D034741:RNA, Small Interfering / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013552:Swine; D019098:Telomerase / Q000378:metabolism; D013997:Time Factors; D014443:Tyrosine / Q000737:chemistry* / Q000378:metabolism; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000378:metabolism*
chemical_list: D000199:Actins; D048868:Adaptor Proteins, Signal Transducing; D003598:Cytoskeletal Proteins; C496061:PXN protein, human; D051419:Paxillin; D010750:Phosphoproteins; D011518:Proto-Oncogene Proteins; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins; C490081:SHB protein, human; D042461:Vascular Endothelial Growth Factor A; D019000:Phosphotyrosine; D014443:Tyrosine; D005982:Glutathione Transferase; D019869:Phosphatidylinositol 3-Kinases; D011505:Protein-Tyrosine Kinases; D040301:Vascular Endothelial Growth Factor Receptor-2; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496050:PTK2 protein, human; D019098:Telomerase