Cell Signaling Technology

Product Pathways - Tyrosine Kinase / Adaptors

Phospho-EGF Receptor (Thr669) (D2F1) Rabbit mAb #8808

Applications Reactivity Sensitivity MW (kDa) Isotype
W IP H (M) (R) Endogenous 175 Rabbit IgG

Applications Key:  W=Western Blotting  IP=Immunoprecipitation
Reactivity Key:  H=Human  M=Mouse  R=Rat
Species cross-reactivity is determined by western blot. Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Protocols

Specificity / Sensitivity

Phospho-EGF Receptor (Thr669) (D2F1) Rabbit mAb detects endogenous levels of EGFR protein only when phosphorylated at Thr669. While the literature refers to this residue as Thr669, it is Thr693 of human EGFR (UniProt sequence P00533) and corresponds to Thr695 of mouse EGFR or Thr694 of rat EGFR.

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Thr693 of human EGFR protein.

Western Blotting

Western Blotting

Western blot analysis of extracts from A-431 cells serum starved overnight, and left untreated (-) or treated (+) with EGF (100 ng/ml, 15 min), using Phospho-EGF Receptor (Thr669) (D2F1) Rabbit mAb (upper) and EGF Receptor (D38B1) XP® Rabbit mAb #4267 (lower).

Background

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).

Thr669 (equivalent to Thr693 of human EGFR) is phosphorylated by p38 MAP kinase following EGF stimulation (11). Phosphorylation of EGFR at Thr669 may be involved in regulation of ligand induced receptor internalization through interaction with specific downstream EGFR tyrosine kinase substrates (11).

  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. (1994) 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.
  11. Winograd-Katz, S.E. and Levitzki, A. (2006) Oncogene 25, 7381-90.

Application References

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For Research Use Only. Not For Use In Diagnostic Procedures.

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