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PhosphoSitePlus® Resource

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Specificity / Sensitivity

Quality Control: PTP1B peptide was selected by using Tyrosine Kinase Substrates Screening Kit #7450 to screen for EGF-R kinase substrates. Phospho-Tyrosine Monoclonal Antibody #9411 was used for detection (fig.2). The quality of the biotinylated peptides was evaluated by reverse-phase HPLC and by mass spectrometry.

Purified EGF-R kinase was quality controlled for purity using silver stain SDS-PAGE and Western blot. EGF-R kinase Vmax and Km values were measured to determine specific enzymatic activity (fig.5).

Assay conditions (time course [fig.1], kinase dose-dependence [fig.3], substrate dose-dependence [fig.4] and inhibitor sensitivity [fig.6]) for EGFR kinase activity were verified using the EGF-R substrate peptide provided in this kit.

The epidermal growth factor (EGF) receptor is a transmembrane tyrosine kinase that belongs to the HER/ErbB protein family. Ligand binding results in receptor dimerization, autophosphorylation, activation of downstream signaling, internalization, and lysosomal degradation (1,2). Phosphorylation of EGF receptor (EGFR) at Tyr845 in the kinase domain is implicated in stabilizing the activation loop, maintaining the active state enzyme, and providing a binding surface for substrate proteins (3,4). c-Src is involved in phosphorylation of EGFR at Tyr845 (5). The SH2 domain of PLCγ binds at phospho-Tyr992, resulting in activation of PLCγ-mediated downstream signaling (6). Phosphorylation of EGFR at Tyr1045 creates a major docking site for the adaptor protein c-Cbl, leading to receptor ubiquitination and degradation following EGFR activation (7,8). The GRB2 adaptor protein binds activated EGFR at phospho-Tyr1068 (9). A pair of phosphorylated EGFR residues (Tyr1148 and Tyr1173) provide a docking site for the Shc scaffold protein, with both sites involved in MAP kinase signaling activation (2). Phosphorylation of EGFR at specific serine and threonine residues attenuates EGFR kinase activity. EGFR carboxy-terminal residues Ser1046 and Ser1047 are phosphorylated by CaM kinase II; mutation of either of these serines results in upregulated EGFR tyrosine autophosphorylation (10).

Description: The kit provides a means of performing enzymatic assays with active human EGF-R kinase. It includes active EGF-R 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 EGF-R kinase required to maximally phosphorylate 75 pmol of PTP1B (Tyr66) #C03-1727 biotinylated substrate peptide in 30 minutes at 25ºC in a total reaction volume of 50 µl quantified by DELFIA®.

Peptide Core Sequence: NDY*IN

Molecular Weights: Peptide Substrate, Biotin-PTP1B (Tyr66): 2141 Daltons, GST-EGF-R Kinase domain: 91,163 Daltons

Storage: Antibodies are supplied in in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA and 50% glycerol. Do not aliquot the antibodies. Peptides are supplied at 6 µM in 0.001% DMSO. Enzymes are supplied in 50 mM Tris-HCL (pH 8.0), 100 mM NaCl, 5 mM DTT, 15 mM reduced glutathion and 20% glycerol. Store at -80ºC.

Keep enzymes on ice during use.

Avoid repeated freeze-thaw cycles.

1.  Hackel, P.O. et al. (1999) Curr Opin Cell Biol 11, 184-9.

2.  Zwick, E. et al. (1999) Trends Pharmacol Sci 20, 408-12.

3.  Cooper, J.A. and Howell, B. (1993) Cell 73, 1051-4.

4.  Hubbard, S.R. et al. Nature 372, 746-54.

5.  Biscardi, J.S. et al. (1999) J Biol Chem 274, 8335-43.

6.  Emlet, D.R. et al. (1997) J Biol Chem 272, 4079-86.

7.  Levkowitz, G. et al. (1999) Mol Cell 4, 1029-40.

8.  Ettenberg, S.A. et al. (1999) Oncogene 18, 1855-66.

9.  Rojas, M. et al. (1996) J Biol Chem 271, 27456-61.

10.  Feinmesser, R.L. et al. (1999) J Biol Chem 274, 16168-73.

Entrez-Gene Id 1956
Swiss-Prot Acc. P00533

Data Sheets & Documentation

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.

HTScan® EGFR Kinase Assay Kit