Product Pathways - Tyrosine Kinase/ Adaptors
EphB3 Kinase #7715
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Description
Purified recombinant human EphB3 kinase (Gln585-Val1998), 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 EphB3 (Gln585-Val998) (GenBank accession No. NM_004443) 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-EphB3 fusion protein was analyzed using SDS/PAGE followed by anti-EphB3 Western blot (A) or Silver stain (B).
Kinase Assay - Radiometric
Figure 2. EphB3 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, 0.5 µg/50 µl and 25 ng/50 ul Recombinant EphB3.
Kinase Assay - DELFIA
Figure 3. Dose dependence curve of EphB3 kinase activity: DELFIA® data generated using Phospho-Tyrosine mAb (P-Tyr-100) #9411 to detect phosphorylation of substrate peptide (#1330) by EphB3 kinase. In a 50 µl reaction, increasing amounts of EphB3 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-EphB3 fusion protein is 80 kDa. The purified kinase was quality controlled for purity using SDS-PAGE followed by Silver stain and Western blot [Fig.1]. EphB3 kinase activity was determined using a radiometric assay [Fig.2]. A kinase dose dependency assay was performed to measure EphB3 activity using HTScan™ EphB3 Kinase Assay Kit #7716 [Fig.3].
Background
The Eph receptors are the largest known family of receptor tyrosine kinases (RTKs). They can be divided into two groups based on sequence similarity and on their preference for a subset of ligands: EphA receptors bind to a glycosylphosphatidylinositol-anchored ephrin A ligand, and EphB receptors bind to ephrin B proteins that have a transmembrane and cytoplasmic domain (1,2). Eph receptors and ligands may be involved in many diseases including cancer (3). Both ephrin A and ephrin B ligands have dual functions. As RTK ligands, the ephrins stimulate the kinase activity of the Eph receptors and activate signaling pathways in receptor-expressing cells. The ephrin extracellular domain is sufficient for this function as long as it is clustered (4). The second function of ephrins has been described as ?reverse signaling,? whereby the cytoplasmic domain becomes tyrosine phosphorylated, allowing interactions with other proteins that may activate signaling pathways in the ligand-expressing cells (5). Various stimuli can induce tyrosine phosphorylation of ephrin B, including binding to EphB receptors, activation of Src kinase and stimulation by PDGF and FGF (6). Tyrosines 324/327 have been identified as major phosphorylation sites of ephrin B1 in vivo (7).
- Wilkinson, D.G. (2000) Int. Rev. Cytol. 196, 177-244.
- Klein, R. (2001) Curr. Opin. Cell Biol. 13, 196-203.
- Dodelet, V.C. and Pasquale, E.B. (2000) Oncogene 19, 5614-5619.
- Holder, N. and Klein, R. (1999) Development 126, 2033-2044.
- Bruckner, K. et al. (1997) Science 275, 1640-1643.
- Palmer, A. et al. (2002) Mol. Cell 9, 1-20.
- Kalo, M.S. et al. (2001) J. Biol. Chem. 276, 38940-38948.
Application References
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