Abstract
Vascular endothelial growth factor (VEGF)/VEGF receptor 2 (VEGFR2) signaling pathways are tightly regulated multistep chain reactions that involve a wide range of molecular interactions and enzymatic activities. The first signal induced by VEGF binding to VEGFR2, is the activation of the receptor tyrosine kinase and autophosphorylation of intracellular tyrosine residues of the receptor. In endothelial cells, five tyrosine residues in the VEGFR2 intracellular domain are essential in signal transmission and in the respective regulation of cellular processes. Because of their number and their localization on the receptor, it is challenging to locate the proteins with which these tyrosine residues interact that result in further downstream signaling cascades. In this chapter, we describe a method to precipitate phosphotyrosine binding proteins using phosphotyrosine-containing synthetic peptides immobilized to magnetic beads. The identity of the precipitated proteins is determined by mass spectrometry and the findings validated by Western blot. Using this method, we identified and verified two proteins, growth factor receptor binding-2 (GRB2) and phosphoinositide 3′-kinase (PI3Kp85), binding to the tyrosine 1214 of VEGFR2. Thereby, we can predict the signaling pathways downstream of pY1214.
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Acknowledgments
The author acknowledges the equipment and expert advice supplied by Prof. Lena Claesson-Welsh, Uppsala University, Sweden and the SciLifeLab Clinical Proteomics Mass Spectrometry facility, Solna, Sweden. This method was part of a larger study [5] that was made possible through grants to Prof. Lena Claesson-Welsh from the Swedish Research Council (2015-02375), the Swedish Cancer Foundation (CAN2016/578), and the Knut and Alice Wallenberg Foundation (KAW 2015.0030). KAW also supported LCW with a Wallenberg Scholar grant (2015.0275).
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Testini, C. (2022). SH2-Domain Protein Isolation Using Synthetic Phosphorylated Peptides to Study VEGFR2 Signaling. In: Fiedler, L.R., Pellet-Many, C. (eds) VEGF Signaling. Methods in Molecular Biology, vol 2475. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2217-9_6
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DOI: https://doi.org/10.1007/978-1-0716-2217-9_6
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