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J. Biol. Chem., Vol. 279, Issue 21, 22267-22275, May 21, 2004

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

Kristina Holmqvist, Michael. J. Cross¶, Charlotte Rolny, Robert Hägerkvist, Nader Rahimi||, Taro Matsumoto**, Lena Claesson-Welsh, and Michael Welsh

From the Department of Medical Cell Biology, Uppsala University, Uppsala 75123, Sweden and the Department of Genetics and Pathology, Uppsala University, Uppsala 75185, Sweden, and ^||Department of Ophthalmology, School of Medicine, Boston University, Boston, Massachusetts 02118, and the ^**Division of Cell Regeneration and Transplantation, Nihon University School of Medicine, Tokyo 173-8610, Japan

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.

Received for publication, November 20, 2003 , and in revised form, March 10, 2004.

^* This work was supported by grants from the Juvenile Diabetes Foundation International, the JDRF-SRC-SDA joint program on stem cell research, the Swedish Medical Research Council Grant 31X-10822, the Swedish Diabetes Association, the Novo-Nordisk Foundation, the Family Ernfors Fund, and the Association for International Cancer Research (to M. J. C. and L. C.-W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ Present address: NWCRF Institute, University of Wales, Bangor, LL57 2UW, UK.

To whom correspondence should be addressed: Dept. of Medical Cell Biology, Box 571, Husargatan 3, 75123, Uppsala, Sweden. Tel.: 46-18-4714447; Fax: 46-18-4714059; E-mail: michael.welsh{at}medcellbiol.uu.se.

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