L29	VEGF signalling.
L.Claesson-Welsh
Uppsala University, Dept. of Genetics and Pathology, Vascular Biology Unit, Uppsala, SE.

Blocking of vascular endothelial growth factor (VEGF) by neutralizing antibodies prolongs survival in a number of human tumor diseases, notably colorectal and kidney cancer. The main transducer of VEGF signals in endothelial cells is VEGF receptor-2 (VEGFR-2). This receptor is expressed also on non-endothelial cells. Thus, long-term treatment with neutralizing VEGF antibodies may affect other compartments than the vascular bed. Moreover, normal vessels in healthy tissue may be affected, dependent on the dose and duration of the treatment. Thus, it may be necessary to pinpoint treatment to relevant signal transduction pathways downstream of VEGFR-2 of importance for the angiogenic response, such as cytoskeletal rearrangements required for formation of a vessel lumen. We have therefore mapped tyrosine phosphorylation sites in the non-catalytic part of the intracellular domain of VEGFR-2. Three major phosphorylation sites were identified by phosphopeptide mapping, Yp951 in the kinase insert, and Yp1175 and Yp1214 in the C-terminal tail. The usage of these phosphorylation sites in VEGFR-2 was examined in intact vessels by use of phosphosite-specific antibodies. All endothelial cells expressing VEGFR-2 were phosphorylated on Y1175 and Y1214. In contrast, phosphorylation of tyrosine residue 951 was selective, and anti-phospho951 antibodies identified subsets of endothelial cells that were devoid of perivascular smooth muscle cell coating. Introduction of phosphorylated 951 peptide into endothelial cells specifically blocked VEGF-A-induced actin stress fibers and endothelial cell migration, but not DNA synthesis. Yp951 bound and mediated tyrosine phosphorylation of the adaptor molecule T-cell specific adaptor (TSAd), which in turn coupled to Src. TSAd is an adaptor molecule equipped with a central Src Homology 2 domain and a C-terminal proline-rich domain. Transfection with TSAd-specific siRNA also blocked VEGF-A-induced stress fibers and endothelial cell migration. Fibrosarcoma growth rate and vascular density was reduced in TSAd knock out mice supporting a role for TSAd in pathological angiogenesis. In conclusion, VEGFR-2 phosphorylation site mapping identified three major phosphorylation sites in the non-catalytic part of the receptor, of which Yp951 appears to be utilized in endothelial cells engaged in active angiogenesis and critical for VEGF-induced regulation of the actin cytoskeleton and endothelial cell migration. References: Cross MJ, Dixelius J, Matsumoto T, Claesson-Welsh L. (2003) VEGF-receptor signal transduction.Trends Biochem Sci. 28, 488-494 Matsumoto T, Bohman S, Dixelius J, Berge T, Dimberg A, Magnusson P, Wang L, Wikner C, Qi JH, Wernstedt C, Wu J, Bruheim S, Mugishima H, Mukhopadhyay D, Spurkland A, Claesson-Welsh L. VEGF receptor-2 Y951 signaling and a role for the adapter molecule TSAd in tumor angiogenesis EMBO J, June 16 2005

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