Product Pathways - Tyrosine Kinase/ Adaptors
FGF Receptor 3 Kinase #7733
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 FGFR-3 kinase (Glu322-Thr806), 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 FGFR-3 (Glu322-Thr806) (GenBank Accession No. NM_000142) 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-FGFR-3 fusion protein was analyzed using SDS/PAGE followed by anti-FGFR-3 Western blot (A) or Silver stain (B).
Kinase Assay - Radiometric
Figure 2. FGFR-3 kinase activity was measured in a radiometric assay using the following reaction conditions: 60 mM HEPES-NaOH, pH 7.5, 3 mM MgCl2, 3 mM MnCl2, 3 µM Na-orthovanadate, 1.2 mM DTT, ATP (variable), 2.5 µg/50 µl PEG20.000, Substrate: PolyEY, 5 µg/50 µl and 100 ng/50 µl Recombinant FGFR-3.
Kinase Assay - DELFIA
Figure 3. Dose dependence curve of FGFR-3 kinase activity: DELFIA® data generated using Phospho-Tyrosine mAb (P-Tyr-100) #9411 to detect phosphorylation of substrate peptide (#1305) by FGFR-3 kinase. In a 50 µl reaction, increasing amounts of FGFR-3 and 1.5 µM substrate peptide were used per reaction at room temperature for 30 minutes. (DELFIA® is a registered trademark of PerkinElmer, Inc.)
Quality Control
The theoretical molecular weight of the GST-FGFR-3 fusion protein is 82 kDa. The purified kinase was quality controlled for purity using SDS-PAGE followed by Silver stain and Western blot [Fig.1]. FGFR-3 kinase activity was determined using a radiometric assay [Fig.2]. A kinase dose dependency assay was performed to measure FGFR-3 activity using HTScan™ FGF Receptor 3 Kinase Assay Kit #7734 [Fig.3].
Background
Fibroblast growth factors (FGFs) produce mitogenic and angiogenic effects in target cells by signaling through cell surface receptor tyrosine kinases. There are four members of the FGF receptor family: FGFR-1 (flg), FGFR-2 (bek, KGFR), FGFR-3 and FGFR-4. Each receptor contains an extracellular ligand binding domain, a transmembrane domain and a cytoplasmic kinase domain (1). Following ligand binding and dimerization, the receptors are phosphorylated at specific tyrosine residues (2). Seven tyrosine residues in the cytoplasmic tail of FGFR-1 can be phosphorylated: Tyr463, Tyr583, Tyr585, Tyr653, Tyr654, Tyr730 and Tyr766. Tyrosines 653 and 654 are important for catalytic activity of activated FGFR and are essential for signaling (3). The other phosphorylated tyrosine residues may provide docking sites for downstream signaling components such as Crk and PLCγ (4,5).
- Powers, C.J. et al. (2000) Endocr Relat Cancer 7, 165-97.
- Reilly, J.F. et al. (2000) J Biol Chem 275, 7771-8.
- Mohammadi, M. et al. (1996) Mol Cell Biol 16, 977-89.
- Mohammadi, M. et al. (1991) Mol Cell Biol 11, 5068-78.
- Larsson, H. et al. (1999) J Biol Chem 274, 25726-34.
Application References
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Companion Products
- 7734 HTScan® FGF Receptor 3 Kinase Assay Kit
- 1305 FLT3 (Tyr589) Biotinylated Peptide
- 9411 Phospho-Tyrosine Mouse mAb (P-Tyr-100)
- 9805 HTScan® Tyrosine Kinase Buffer (4X)
- 9804 ATP (10 mM)
- 9953 Staurosporine
- 7450 Tyrosine Kinase Substrate Screening Kit
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.
