Figure 4. PDGFR alpha kinase activity was measured in a radioisotopic filtration 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(AEKY) 2.5 ug/50 ul, Recombinant PDGFR alpha: 20 Units/50 ul.
Figure 2. Dose dependence curve of GST-PDGFR-alpha kinase activity: DELFIA® data generated using Phospho-Tyrosine mAb P-Tyr-100 #9411 to detect phosphorylation of substrate peptide (#1305) by PDGFR-alpha kinase. In a 50 ul reaction, increasing amounts of PDGFR-alpha and 1.5 uM substrate peptide were used per reaction well at 25ºC for 30 minutes. Background reading is 1624. (DELFIA® is a registered trademark of PerkinElmer, Inc.)
Figure 5. Staurosporine inhibition of PDGFR-alpha kinase activity: DELFIA® data generated using Phospho-Tyrosine mAb P-Tyr-100 #9411 to detect phosphorylation of PDGFR-alpha substrate peptide (#1305) by GST-PDGFR-alpha kinase. In a 50 ul reaction, 10 Units PDGFRalpha kinase, 1.5 uM substrate peptide, 20 uM ATP and increasing amounts of staurosporine were used per reaction well at 25ºC room temperature for 30 minutes. (DELFIA® is a registered trademark of PerkinElmer, Inc.)
Figure 3. Peptide concentration dependence of PDGFR-alpha kinase activity: DELFIA® data generated using Phospho-Tyrosine mAb P-Tyr-100 #9411 to detect phosphorylation of substrate peptide (#1305) by PDGFR-alpha kinase. In a 50 ul reaction, 10 Units of PDGFR-alpha and increasing concentrations of substrate peptide were used per reaction well at 25ºC for 30 minutes. Background reading is 597. (DELFIA® is a registered trademark of PerkinElmer, Inc.)
Figure 1. Time course of PDGFR-alpha kinase activity: DELFIA® data generated using Phospho-Tyrosine mAbP-Tyr-100 #9411 to detect phosphorylation of PDGFR-alpha substrate peptide (#1305) by PDGFR-alpha kinase. In a 50 ul reaction, 10 Units PDGFR alpha and 1.5 uM substrate peptide were used per reaction well. Background reading is 2421. (DELFIA® is a registered trademark of PerkinElmer, Inc.)
Quality Control: Biotin-FLT3 (Tyr589) peptide was selected as PDGFR-alpha kinase substrate from screening a panel of 150 pairs of tyrosine containing peptides as potential substrate candidates. Phospho-Tyrosine mAb #9411 was used for detection. The quality of the biotinylated peptides was evaluated by reverse-phase HPLC and by mass spectrometry.
Purified PDGFR-alpha kinase was quality controlled for purity by SDS-PAGE followed by silver stain and Western blot.
Assay conditions (time course [Fig.1], kinase dose-dependence [Fig.2], substrate dose-dependence [Fig.3] and staurosporine sensitivity [Fig.4]) for PDGFR-alpha kinase activity were verified using the PDGFR-alpha substrate peptide provided in this kit.
PDGFR-alpha kinase Vmax and Km values were measured by radiometric filtration assay using polyAEKY as a substrate [Fig.5].
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).
Description: The kit provides a means of performing enzymatic assays with active human PDGFR-alpha kinase. It includes active PDGFR-alpha kinase (supplied as a GST fusion protein), a biotinylated substrate peptide and a phospho-tyrosine monoclonal antibody for detection of the phosphorylated form of the substrate peptide.
Unit Definition: 10 Units is defined as the amount of PDGFR-alpha kinase required to maximally phosphorylate 75 pmol of FLT3 (Tyr589) biotinylated substrate peptide (#1305) in 30 minutes at 25ºC in a total reaction volume of 50 ul quantified by DELFIA® to achieve signal/background=25 or greater.
Peptide Core Sequence: NEY*FY*V
Molecular Weights: Peptide substrate, Biotin-FLT3 (Tyr589): 1,945 Daltons, GST-PDGFR-alpha Kinase domain: 87,851 Daltons
Storage: Antibodies are supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 ug/ml BSA and 50% glycerol. Do not aliquot the antibodies. Peptides are supplied at 6 uM in 0.001% DMSO carbonate buffer solution (3 mM Na2CO3, 7mM NaHCO3, PH9.6). Enzymes are supplied in 50 mM Tris-HCL (pH 8.0), 100 mM NaCl, 5 mM DTT, 15 mM reduced glutathione and 20% glycerol. Store at -80ºC.
Keep enzymes on ice during use.
Avoid repeated freeze-thaw cycles.
Amount of kinase: GST-PDGFR-alpha10 Units/50 ul.
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