Cell Signaling Technology

Product Pathways - Kinases

c-Kit D816V Kinase #7924

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Description

Purified recombinant human c-Kit D816V (Thr544-Val976) 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 c-Kit D816V (Thr544-Val976) (GenBank Accession No. NM_000222) 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 D816V fusion protein was analyzed using SDS-PAGE followed by Coomassie stain.

Kinase Assay - Radiometric

Kinase Assay - Radiometric

Figure 2. c-Kit D816V kinase activity was measured in a radiometric assay using the following reaction conditions: 60 mM HEPES-NaOH, pH 7.5, 5 mM MgCl2, 5 mM MnCl2, 3 µM Na-orthovanadate, 1.2 mM DTT, 100 μM ATP, 100 μM KDR (Tyr996) Biotinylated Peptide #1364 and variable amount of recombinant c-Kit D816V. The reaction mixture was incubated at room temperature for 15 minutes.

Kinase Assay - DELFIA

Kinase Assay - DELFIA

Figure 3. Dose dependence curve of c-Kit D816V kinase activity: DELFIA® data generated using Phospho-Tyrosine Mouse mAb (P-Tyr-100) #9411 to detect phosphorylation of substrate peptide (#1364) by c-Kit D816V kinase. In a 50 µl reaction, increasing amounts of c-Kit D816V and 1.5 µM substrate peptide were used per reaction at room temperature for 30 minutes. (DELFIA® is a registered trademark of PerkinElmer, Inc.)


Kinase Assay - DELFIA

Kinase Assay - DELFIA

Figure. 4. Sensitivities of wild type and mutant c-Kit D816V kinase to Staurosporine (A) and Gleevec (B) were compared: DELFIA® data generated using Phospho-Tyrosine mAb (P-Tyr-100) #9411 to detect phosphorylation of c-Kit substrate peptide (#1364) by the c-Kit kinases. In a 50 µl reaction, 100 ng of c-Kit D816V kinase, 1.5 µM substrate peptide, 5 μM ATP and increasing concentration of indicated inhibitors were used per reaction well 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 D816V fusion protein is 75 kDa. The purified kinase was quality controlled for purity using SDS-PAGE followed by Coomassie stain [Fig.1]. c-Kit D816V kinase activity was determined using a radiometric assay [Fig.2]. A kinase dose dependency assay was performed to measure c-Kit D816V activity using HTScan® c-Kit Kinase Assay Kit #7755 [Fig.3]. Sensitivity of c-Kit D816V to Gleevec was determined using the c-Kit substrate peptide #1364 and detection antibody #9411 provided in HTScan® c-Kit Kinase Assay Kit #7755 [Fig.4].

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).

c-Kit D816V, a constitutively active Kit mutant, has been found in adult-type human mastocytosis and is resistant to Gleevec (Imatinib), which has been successfully used to treat c-Kit kinase associated chronic myeloid leukemia (CML) (8,9).

  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.
  8. Foster, R. et al. (2004) J. Mol. Graph Model 23, 139-152.
  9. Ma, Y. et al. (2002) Blood 99, 1741-1744.

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This product is intended for research purposes only. The product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.

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