Identification of vascular endothelial growth factor receptor-1 tyrosine phosphorylation sites and binding of SH2 domain-containing molecules.
Receptor tyrosine phosphorylation is crucial for signal transduction by creating high affinity binding sites for Src homology 2 domain-containing molecules. By expressing the intracellular domain of Flt-1/vascular endothelial growth factor receptor-1 in the baculosystem, we identified two major tyrosine phosphorylation sites at Tyr-1213 and Tyr-1242 and two minor tyrosine phosphorylation sites at Tyr-1327 and Tyr-1333 in this receptor. This pattern of phosphorylation of Flt-1 was also detected in vascular endothelial growth factor-stimulated cells expressing intact Flt-1. In vitro protein binding studies using synthetic peptides and immunoblotting showed that phospholipase C-gamma binds to both Y(p)1213 and Y(p)1333, whereas Grb2 and SH2-containing tyrosine protein phosphatase (SHP-2) bind to Y(p)1213, and Nck and Crk bind to Y(p)1333 in a phosphotyrosine-dependent manner. In addition, unidentified proteins with molecular masses around 74 and 27 kDa bound to Y(p)1213 and another of 75 kDa bound to Y(p)1333 in a phosphotyrosine-dependent manner. SHP-2, phospholipase C-gamma, and Grb2 could also be shown to bind to the intact Flt-1 intracellular domain. These results indicate that a spectrum of already known as well as novel phosphotyrosine-binding molecules are involved in signal transduction by Flt-1.
Ito
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Department of Medical Biochemistry and Microbiology, Uppsala University, Biomedical Center, Box 575, S-751 23 Uppsala, Sweden.
Wernstedt
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C
Engström
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U
Claesson-Welsh
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L
D016475:3T3 Cells; D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001665:Binding Sites; D004730:Endothelium, Vascular; D051380:GRB2 Adaptor Protein; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D051379:Mice; D008958:Models, Molecular; D010446:Peptide Fragments / Q000378:metabolism; D010449:Peptide Mapping; D010748:Phosphopeptides / Q000032:analysis; D010766:Phosphorylation; D011485:Protein Binding; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism; D011506:Proteins / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D054593:SH2 Domain-Containing Protein Tyrosine Phosphatases; D015398:Signal Transduction; D013552:Swine; D010738:Type C Phospholipases / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism; D040281:Vascular Endothelial Growth Factor Receptor-1; D018909:src Homology Domains
D048868:Adaptor Proteins, Signal Transducing; D051380:GRB2 Adaptor Protein; C495948:GRB2 protein, human; C495949:Grb2 protein, mouse; D047908:Intracellular Signaling Peptides and Proteins; D010446:Peptide Fragments; D010748:Phosphopeptides; D011506:Proteins; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D014443:Tyrosine; D020794:Receptor Protein-Tyrosine Kinases; D040281:Vascular Endothelial Growth Factor Receptor-1; C516724:PTPN11 protein, human; C509249:PTPN6 protein, human; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6; D017027:Protein Tyrosine Phosphatases; C516725:Ptpn11 protein, mouse; C509248:Ptpn6 protein, mouse; D054593:SH2 Domain-Containing Protein Tyrosine Phosphatases; D010738:Type C Phospholipases