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7915
HTScan® PDGF Receptor α D842V Kinase Assay Kit
Cellular Assay Kits
Assay Kit

HTScan® PDGF Receptor α D842V Kinase Assay Kit #7915

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HTScan® PDGF Receptor α D842V Kinase Assay Kit: Image 1

Figure 3. Dose dependence curve of PDGFRalpha D842V kinase activity: DELFIA® data generated using Phospho-Tyrosine Mouse mAb (P-Tyr-100) #9411 to detect phosphorylation of substrate peptide (#1305) by PDGFRalpha D842V kinase. In a 50 µl reaction, increasing amounts of PDGFRalpha D842V and 1.5 µM substrate peptide were used per reaction at room temperature for 30 minutes. (DELFIA® is a registered trademark of PerkinElmer, Inc.)

HTScan® PDGF Receptor α D842V Kinase Assay Kit: Image 2

Figure 5. Sensitivities of wild type and mutant PDGFRalpha D842V kinases to Staurosporine (A) and Gleevec (B) were compared: DELFIA® data generated using Phospho-Tyrosine mAb (P-Tyr-100) #9411 to detect phosphorylation of PDGFRalpha substrate peptide (#1305) by the PDGFRalpha kinases. In a 50 µl reaction, 100 ng of kinase, 1.5 µM substrate peptide, 20 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.)

HTScan® PDGF Receptor α D842V Kinase Assay Kit: Image 3

Figure 4. Peptide concentration dependence of PDGFRalpha D842V kinase activity: DELFIA® data generated using Phospho-Tyrosine Mouse mAb (P-Tyr-100) #9411 to detect phosphorylation of substrate peptide (#1305) by PDGFRalpha D842V kinase. In a 50 µl reaction, 100 ng of PDGFRalpha D842V and increasing concentrations of substrate peptide were used per reaction at room temperature for 30 minutes. (DELFIA® is a registered trademark of PerkinElmer, Inc.)

HTScan® PDGF Receptor α D842V Kinase Assay Kit: Image 4

Figure 2. Time course of PDGFRalpha D842V kinase activity: DELFIA® data generated using Phospho-Tyrosine Mouse mAb (P-Tyr-100) #9411 to detect phosphorylation of PDGFRalpha D842V substrate peptide (#1305) by PDGFRalpha D842V kinase. In a 50 µl reaction, 100 ng PDGFRalpha D842V and 1.5 µM substrate peptide were used per reaction. (DELFIA® is a registered trademark of PerkinElmer, Inc.)

HTScan® PDGF Receptor α D842V Kinase Assay Kit: Image 5

Figure 1. PDGFRalpha D842V 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 FLT3 (Tyr589) Biotinylated Peptide (#1305) and variable amount of Recombinant PDGFRalpha D842V. Reaction mixture incubated at room temperature for 10 minutes.

To Purchase # 7915

Product Description

The kit provides a means of performing kinase activity assays with recombinant human PDGFRalpha D842V kinase. It includes active PDGFRalpha D842V kinase (supplied as a GST fusion protein), a biotinylated peptide substrate and a phospho-tyrosine monoclonal antibody for detection of the phosphorylated form of the substrate peptide.

Molecular Formula

Peptide substrate, Biotin-FLT3 (Tyr589): 1945 Daltons. GST-PDGFRalpha D842V Kinase: 76 kDa.

Background

Platelet derived growth factor (PDGF) family proteins exist as several disulphide-bonded, dimeric isoforms (PDGF AA, PDGF AB, PDGF BB, PDGF CC, and PDGF DD) that bind in a specific pattern to two closely related receptor tyrosine kinases, PDGF receptor α (PDGFRα) and PDGF receptor β (PDGFRβ). PDGFRα and PDGFRβ share 75% to 85% sequence homology between their two intracellular kinase domains, while the kinase insert and carboxy-terminal tail regions display a lower level (27% to 28%) of homology (1). PDGFRα homodimers bind all PDGF isoforms except those containing PDGF D. PDGFRβ homodimers bind PDGF BB and DD isoforms, as well as the PDGF AB heterodimer. The heteromeric PDGF receptor α/β binds PDGF B, C, and D homodimers, as well as the PDGF AB heterodimer (2). PDGFRα and PDGFRβ can each form heterodimers with EGFR, which is also activated by PDGF (3). Various cells differ in the total number of receptors present and in the receptor subunit composition, which may account for responsive differences among cell types to PDGF binding (4). Ligand binding induces receptor dimerization and autophosphorylation, followed by binding and activation of cytoplasmic SH2 domain-containing signal transduction molecules, such as GRB2, Src, GAP, PI3 kinase, PLCγ, and NCK. A number of different signaling pathways are initiated by activated PDGF receptors and lead to control of cell growth, actin reorganization, migration, and differentiation (5). Tyr751 in the kinase-insert region of PDGFRβ is the docking site for PI3 kinase (6). Phosphorylated pentapeptides derived from Tyr751 of PDGFRβ (pTyr751-Val-Pro-Met-Leu) inhibit the association of the carboxy-terminal SH2 domain of the p85 subunit of PI3 kinase with PDGFRβ (7). Tyr740 is also required for PDGFRβ-mediated PI3 kinase activation (8).

A subset of gastrointestinal stromal tumors (GIST) are associated with PDGFRalpha activating mutations (8,9). The most common mutation is D842V which located in the kinase activation loop domain and results in contitutively active PDGFRalpha (8,9). This mutation also renders the kinase resistant to Gleevec (Imatinib) inhibition (9,10) and predicts poor clinical response of the patients to Gleevec (10).

  1. Deuel, T.F. et al. (1988) Biofactors 1, 213-217.
  2. Bergsten, E. et al. (2001) Nat. Cell Biol. 3, 512-516.
  3. Betsholtz, C. et al. (2001) Bioessays 23, 494-507.
  4. Coughlin, S.R. et al. (1988) Prog. Clin. Biol. Res. 266, 39-45.
  5. Ostman, A. and Heldin, C.H. (2001) Adv. Cancer Res. 80, 1-38.
  6. Panayotou, G. et al. (1992) EMBO J. 11, 4261-4272.
  7. Ramalingam, K. et al. (1995) Bioorg. Med. Chem. 3, 1263-1272.
  8. Kashishian, A. et al. (1992) EMBO J. 11, 1373-1382.
  9. Heinrich, M.C. et al. (2003) Science 299, 708-10.
  10. Corless, C.L. et al. (2005) J. Clin. Oncol. 23, 5357-64.
  11. Heinrich, M.C. et al. (2003) J. Clin. Oncol. 21, 4342-9.

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For Research Use Only. Not For Use In Diagnostic Procedures.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
DELFIA is a registered trademark of PerkinElmer, Inc.