Cell Signaling Technology

Product Pathways - Tyrosine Kinase/ Adaptors

c-Kit Kinase #7754

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 c-Kit kinase (Thr544-Val976), supplied as a GST fusion protein.

Source / Purification

The GST-c-Kit fusion protein was produced using a baculovirus expression system with a construct expressing human c-Kit (Thr544-Val976) with an amino-terminal GST tag. The protein was purified by one-step affinity chromatography using glutathione-agarose.

Gel Staining

Gel Staining

Figure 1. The purity of the GST-c-Kit fusion protein was analyzed using SDS/PAGE followed by anti-c-Kit Western blot (A) or Silver stain (B).

Kinase Assay - Radiometric

Kinase Assay - Radiometric

Figure 2. c-Kit 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, 2 µg/50 µl and 200 ng/50 µl Recombinant c-Kit.

Kinase Assay - DELFIA

Kinase Assay - DELFIA

Figure 3. Dose dependence curve of c-Kit kinase activity: DELFIA data gencerated using Phospho-Tyrosine mAb (P-Tyr-100) #9411 to detect phosphorylation of substrate peptide (#1364) by c-Kit kinase. In a 50ul reaction, increasing amounts of c-Kit and 1.5uM 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-c-Kit fusion protein is 75 kDa. The purified kinase was quality controlled for purity using SDS-PAGE followed by Silver stain and Western blot [Fig.1]. c-Kit kinase activity was determined using a radiometric assay [Fig.2]. A kinase dose dependency assay was performed to measure c-Kit activity using HTScan™ c-Kit Kinase Assay Kit #7755 [Fig.3].

Background

c-Kit is a member of the subfamily of receptor tyrosine kinases that includes PDGF, CSF-1 and FLT3/flk-2 receptors (1,2). It plays a critical role in activation and growth in a number of cell types including hematopoietic stem cells, mast cells, melanocytes and germ cells (3). Upon binding with its stem cell factor (SCF) ligand, c-Kit undergoes dimerization/oligomerization and autophosphorylation. Activation of c-Kit results in the recruitment and tyrosine phosphorylation of downstream SH2-containing signaling components including PLCγ, the p85 subunit of PI3 kinase, SHP2 and CrkL (4). Molecular lesions that impair the kinase activity of c-Kit are associated with a variety of developmental disorders (5), while mutations that constitutively activate c-Kit can lead to pathogenesis of mastocytosis and gastrointestinal stromal tumors (6). Tyr719 is located in the kinase insert region of the catalytic domain. c-Kit phosphorylated at Tyr719 binds to the p85 subunit of PI3 kinase in vitro and in vivo (7).

  1. Martin, F.H. et al. (1990) Cell 63, 203-11.
  2. Yarden, Y. et al. (1987) EMBO J 6, 3341-51.
  3. Gommerman, J.L. et al. (1997) J Biol Chem 272, 30519-25.
  4. Sattler, M. et al. (1997) J Biol Chem 272, 10248-53.
  5. Nocka, K. et al. (1990) EMBO J 9, 1805-13.
  6. Hirota, S. et al. (1998) Science 279, 577-80.
  7. Blume-Jensen, P. et al. (2000) Nat Genet 24, 157-62.

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Companion Products

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.

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