The Shc-related adaptor protein, Sck, forms a complex with the vascular-endothelial-growth-factor receptor KDR in transfected cells.
Despite much progress in recent years, the precise signalling events triggered by the vascular-endothelial-growth-factor (VEGF) receptors, fms-like tyrosine kinase (Flt1) and kinase insert domain-containing receptor (KDR), are incompletely defined. Results obtained when Flt1 and KDR are individually expressed in fibroblasts or porcine aortic endothelial cells have not been entirely consistent with those observed in other endothelial cells expressing both receptors endogenously. It has also been difficult to demonstrate VEGF-induced phosphorylation of Flt1, which has led to speculation that KDR may be the more important receptor for the mitogenic action of VEGF on endothelial cells. In an attempt to identify physiologically important effectors which bind to KDR, we have screened a yeast two-hybrid mouse embryo library with the cytoplasmic domain of KDR. Here we describe the identification of the adaptor protein, Shc-like protein (Sck), as a binding partner for KDR. We demonstrate that this interaction requires phosphorylation of KDR, and identify the binding site for the Src-homology 2 (SH2) domain as tyrosine-1175 of KDR. We have also shown that the SH2 domain of Sck, but not that of Src-homology collagen protein (Shc), can precipitate phosphorylated KDR from VEGF-stimulated porcine aortic endothelial cells expressing KDR, and that an N-terminally truncated Sck protein can associate with KDR, in a phosphorylation-dependent fashion, when co-expressed in human embryonic kidney 293 cells. Furthermore, we demonstrate that in the two-hybrid assay, both Shc and Sck SH2 domains can associate with the related receptor Flt1.
Warner
A J
AJ
Department of Biochemistry, University of Leicester, Leicester LE1 7RH, UK.
Lopez-Dee
J
J
Knight
E L
EL
Feramisco
J R
JR
Prigent
S A
SA
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D001665:Binding Sites; D002460:Cell Line; D016228:Endothelial Growth Factors / Q000494:pharmacology; D004789:Enzyme Activation; D006801:Humans; D008222:Lymphokines / Q000494:pharmacology; D009154:Mutation / Q000235:genetics; D010766:Phosphorylation / Q000187:drug effects; D011233:Precipitin Tests; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism; D020794:Receptor Protein-Tyrosine Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017978:Receptors, Growth Factor / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D040262:Receptors, Vascular Endothelial Growth Factor; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D055765:Shc Signaling Adaptor Proteins; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D000071426:Src Homology 2 Domain-Containing, Transforming Protein 2; D014162:Transfection; D020798:Two-Hybrid System Techniques; D014443:Tyrosine / Q000378:metabolism; D042461:Vascular Endothelial Growth Factor A; D040281:Vascular Endothelial Growth Factor Receptor-1; D042442:Vascular Endothelial Growth Factors; D018909:src Homology Domains
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D016228:Endothelial Growth Factors; D008222:Lymphokines; D011506:Proteins; D011518:Proto-Oncogene Proteins; D017978:Receptors, Growth Factor; D011993:Recombinant Fusion Proteins; C528024:SHC1 protein, human; C099021:SHC2 protein, human; D055765:Shc Signaling Adaptor Proteins; C528034:Shc1 protein, mouse; C486295:Shc2 protein, mouse; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D000071426:Src Homology 2 Domain-Containing, Transforming Protein 2; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D014443:Tyrosine; D020794:Receptor Protein-Tyrosine Kinases; D040262:Receptors, Vascular Endothelial Growth Factor; D040281:Vascular Endothelial Growth Factor Receptor-1