Product Pathways - Tyrosine Kinase/ Adaptors
Csk Kinase #7801
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 Csk kinase (Met1-Leu450), supplied as a GST fusion protein.
Source / Purification
Source/Purification: The GST-Kinase fusion protein was produced using a baculovirus expression system with a construct expressing full length human Csk Kinase (Met1-Leu 450) (GenBank Accession No.NM_004383) with an amino-terminal GST tag. The protein was then purified by one-step affinity chromatography using glutathione-agarose.
Gel Staining
Figure 1. The purity of the GST-Csk fusion protein was analyzed using SDS/PAGE followed by anti-Csk Western blot (A) or Coomassie stain (B).
Kinase Assay - Radiometric
Figure 2. Csk 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 uM Na-orthovanadate, 1.2 mM DTT, ATP (variable), 2.5 ug/50 ul PEG20,000, Substrate: Poly(EY) 1 ug/50 ul, Recombinant CSK: 40 bng /50 µl.
Kinase Assay - DELFIA
Figure 3. Dose dependence curve of Csk kinase activity: DELFIA® data generated using Phospho-Tyrosine Monoclonal Antibody (P-Tyr-100) #9411 to detect phosphorylation of substrate peptide (#1370) by Csk kinase. In a 50 ul reaction, increasing amounts of Csk and 1.5 uM 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-Csk kinase fusion protien is 80 kDa. The purified kinase was quality controlled for purity using SDS-PAGE followed by Coomassie stain and Western blot [Fig.1]. Csk kinase activity was determined using a radiometric assay [Fig.2]. A kinase dose dependency assay was performed to measure Csk activity using HTScan® Csk Kinase Assay Kit #7802 [Fig.3].
Background
Carboxy-terminal Src kinase (Csk) is a ubiquitously expressed nonreceptor tyrosine kinase that negatively regulates the Src family kinases (SFKs) by phosphorylation of the SFK carboxy-terminal tyrosine (1,2). Phosphorylated carboxy-terminal tyrosine binds to the SH2 domain of SFK intramolecularly and leads to folding and inactivation of the SFK (3). This Csk-catalyzed SFK tyrosine phosphorylation is highly specific and exclusive. The SFK carboxy-terminal tyrosine is the only known physiological substrate of Csk (4).Csk consists of an SH2, an SH3, and a kinase domain. There is evidence that the SH2 and SH3 domains are essential for the regulation of SFK, and Csk can be recruited to the membrane where SFKs are in an active state. This process is mediated by a Csk-binding protein (Cbp, also called PAG), which binds tightly to the SH2 domain of Csk (5). Activation of SFK by extracellular stimuli leads to the tyrosine phosphorylation of Cbp, generating docking sites for Csk. The recruitment of Csk forms a feedback mechanism for termination of SFK function (6).
- Nada, S. et al. (1991) Nature 351, 69-72.
- Nada, S. et al. (1993) Cell 73, 1125-1135.
- Lee, S. et al. (2003) Proc. Natl. Acad. Sci. USA 100, 14707-14712.
- Imamoto, A. and Soriano, P. (1993) Cell 73, 1117-1124.
- Kawabuchi, M. et al. (2000) Nature 404, 999-1003.
- Matsuoka, H. et al. (2004) J. Biol. Chem. 279, 5975-5983.
Application References
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Companion Products
- 7450 Tyrosine Kinase Substrate Screening Kit
- 9953 Staurosporine
- 9805 HTScan® Tyrosine Kinase Buffer (4X)
- 9804 ATP (10 mM)
- 9411 Phospho-Tyrosine Mouse mAb (P-Tyr-100)
- 7802 HTScan® Csk Kinase Assay Kit
- 1370 Pyk2 (Tyr881) 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.
