Product Pathways - Kinases
VEGF Receptor 2 Kinase #7407
Cell Signaling Technology offers a full line of protein kinases, substrates, antibody detection reagents and HTScan® kits. Browse our "Reagents for High-Throughput Screening" product listing or contact us at drugdiscovery@cellsignal.com.
Description
Purified recombinant human VEGFR2 (Val789-Val1356) kinase, supplied as a GST fusion protein.
Source / Purification
The GST-Kinase fusion protein was produced using a baculovirus expression system with a construct expressing human VEGFR2 (Val789-Val1356) (GenBank Accession No. NM_002253) with an amino-terminal GST tag. The protein was purified by one-step affinity chromatography using glutathione-agarose.
Gel Staining
Figure 1. The purity of the GST-VEGFR2 fusion protein was analyzed using SDS/PAGE followed by Coomassie stain.
Kinase Assay - Radiometric
Figure 2. VEGFR2 kinase activity was measured in a radiometric assay using the following reaction conditions: 4 mM MOPS, pH 7.2, 2.5 mM β-glycerophosphate, 1 mM EGTA, 0.4 mM EDTA, 4 mM MgCl2, 0.05 mM DTT, 50 μM ATP, Substrate: MBP 200 ng/μL, and recombinant VEGFR2: variable.
Quality Control
The theoretical molecular weight of the GST-VEGFR-2 fusion protein is 110 kDa. The purified kinase was quality controlled for purity using SDS-PAGE followed by Coomassie stain [Fig.1]. VEGFR-2 kinase activity was determined using a radiometric assay [Fig.2].
Background
Vascular endothelial growth factor receptor 2 (VEGFR2, KDR, Flk-1) is a major receptor for VEGF-induced signaling in endothelial cells. Upon ligand binding, VEGFR2 undergoes autophosphorylation and becomes activated (1). Major autophosphorylation sites of VEGFR2 are located in the kinase insert domain (Tyr951/996) and in the tyrosine kinase catalytic domain (Tyr1054/1059) (2). Activation of the receptor leads to rapid recruitment of adaptor proteins, including Shc, GRB2, PI-3 kinase, Nck and the protein tyrosine phosphatases SHP-1 and SHP-2 (3). The phosphorylation of Tyr1212 provides a docking site for Grb2 binding and phospho-Tyr1175 binds with the p85 subunit of PI-3 kinase and PLCγ, as well as Shb (1,4,5). Signaling from VEGFR2 is necessary for the execution of VEGF-stimulated proliferation, chemotaxis and sprouting, as well as survival of cultured endothelial cells in vitro and angiogenesis in vivo (6-8).
- Meyer, M. et al. (1999) EMBO J. 18, 363-374.
- Dougher-Vermazen, M. et al. (1994) Biochem. Biophys. Res. Commun. 205, 728-738.
- Kroll, J. and Waltenberger, J. (1997) J. Biol. Chem. 272, 32521-32527.
- Takahashi, T. et al. (2001) EMBO J. 20, 2768-2778.
- Holmqvist, K. et al. (2004) J. Biol. Chem. 279, 22267-22275.
- Karkkainen, M.J. and Petrova, T. (2000) Oncogene 19, 5598-5605.
- Rahimi, N. et al. (2000) J. Biol. Chem. 275, 16986-16992.
- Claesson-Welsh, L. (2003) Biochem. Soc. Trans. 31, 20-24.
Application References
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Companion Products
- 9805 HTScan® Tyrosine Kinase Buffer (4X)
- 9804 ATP (10 mM)
- 7450 Tyrosine Kinase Substrate Screening Kit
- 7788 HTScan® VEGF Receptor 2 Kinase Assay Kit
- 1310 Gastrin Precursor (Tyr87) Biotinylated Peptide
This product is for in vitro research use only and is not intended for use in humans or animals. This product is not intended for use as therapeutic or in diagnostic procedures.
