Cell Signaling Technology

Product Pathways - MAPK Signaling

Phospho-p44/42 MAPK (Erk1) (Tyr204)/(Erk2) (Tyr187) (D1H6G) Mouse mAb #5726

Applications Reactivity Sensitivity MW (kDa) Isotype
W IF-IC F H M R Mk (C) (Dm) (X) (Z) (B) (Ce) Endogenous 42, 44 Mouse IgG2a

Applications Key:  W=Western Blotting  IF-IC=Immunofluorescence (Immunocytochemistry)  F=Flow Cytometry
Reactivity Key:  H=Human  M=Mouse  R=Rat  Mk=Monkey  C=Chicken  Dm=D. melanogaster  X=Xenopus  Z=Zebrafish  B=Bovine  Ce=C. elegans
Species cross-reactivity is determined by western blot. Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Protocols

* Product-specific protocol.

Specificity / Sensitivity

Phospho-p44/42 MAPK (Erk1) (Tyr204)/(Erk2) (Tyr187) (D1H6G) Mouse mAb recognizes endogenous levels of p44/42 MAPK/Erk protein when phosphorylated at Tyr204 of p44 MAPK/Erk1 (Tyr187 of p42 MAPK/Erk2). This antibody detects dual-phosphorylated p44 MAPK/Erk1 (Thr202/Tyr204)/p42 MAPK/Erk2 (Thr185/Tyr187), but does not detect threonine mono-phosphorylated p44/42 MAPK/Erk. This antibody does not cross-react with any other MAP kinases.

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Tyr187 of human Erk2 protein.

Western Blotting

Western Blotting

Western blot analysis of extracts from various cell lines, serum-starved overnight and untreated (-) or treated with U0126 #9903, hEGF #8916, or TPA #4174 as indicated, using Phospho-p44/42 MAPK (Erk1) (Tyr204)/(Erk2) (Tyr187) (D1H6G) Mouse mAb (upper) or p44/42 MAPK (Erk1/2) (L34F12) Mouse mAb #4696 (lower).

Flow Cytometry

Flow Cytometry

Flow cytometric analysis of Jurkat cells, treated with U0126 #9903 (10 μM, 2 hr) (blue) or TPA #4174 (200 nM, 15 min) (green), using Phospho-p44/42 MAPK (Erk1) (Tyr204)/(Erk2) (Tyr187) (D1H6G) Mouse mAb. Anti-mouse IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4408 was used as a secondary antibody.

IF-IC

IF-IC

Confocal immunofluorescent analysis of HeLa cells, treated with PDBu (100 nM, 15 min; left) or U0126 #9903 (10 μM, 2 hr; right), using Phospho-p44/42 MAPK (Erk1) (Tyr204)/(Erk2) (Tyr187) (D1H6G) Mouse mAb (green) and β-Actin (13E5) Rabbit mAb #4970 (red).


Background

Mitogen-activated protein kinases (MAPKs) are a widely conserved family of serine/threonine protein kinases involved in many cellular programs, such as cell proliferation, differentiation, motility, and death. The p44/42 MAPK (Erk1/2) signaling pathway can be activated in response to a diverse range of extracellular stimuli including mitogens, growth factors, and cytokines (1-3), and research investigators consider it an important target in the diagnosis and treatment of cancer (4). Upon stimulation, a sequential three-part protein kinase cascade is initiated, consisting of a MAP kinase kinase kinase (MAPKKK or MAP3K), a MAP kinase kinase (MAPKK or MAP2K), and a MAP kinase (MAPK). Multiple p44/42 MAP3Ks have been identified, including members of the Raf family, as well as Mos and Tpl2/COT. MEK1 and MEK2 are the primary MAPKKs in this pathway (5,6). MEK1 and MEK2 activate p44 and p42 through phosphorylation of activation loop residues Thr202/Tyr204 and Thr185/Tyr187, respectively. Several downstream targets of p44/42 have been identified, including p90RSK (7) and the transcription factor Elk-1 (8,9). p44/42 are negatively regulated by a family of dual-specificity (Thr/Tyr) MAPK phosphatases, known as DUSPs or MKPs (10), along with MEK inhibitors, such as U0126 and PD98059.

The "activation loop" of MAPK family members contains two phosphorylation sites, typically a threonine and a tyrosine separated by a single amino acid, designated the T-x-Y motif. Phosphorylation on both residues has been shown to be required for full activation of kinase activity, but it has been appreciated for some time that mono-phosphorylation of the T-x-Y motif occurs, resulting in partial activation of catalytic acitvity and priming for subsequent, dual-phosphorylation (11,12). The crystal structures of non-, mono-, and dual-phospho MAPK/Erk demonstrate that each phospho-isomer assumes an independent conformation (13). In addition, mono-phosphorylation of MAPK/Erk2 at Tyr187 reveals that phosphorylation at this site serves to configure the ATP binding site, while phosphorylation of both Tyr and Thr residues is required to completely stabilize the substrate binding site (14). Furthermore, T-x-Y mutational analysis of members of the Erk and p38 MAP kinases appears to suggest that mono-phosphorylation of the T-x-Y motif confers differential activity and substrate preference (15,16). Taken together, these data suggest an important and underappreciated role for Thr- and Tyr- mono-phosphorylation of the T-x-Y motif among MAP kinases.

  1. Roux, P.P. and Blenis, J. (2004) Microbiol Mol Biol Rev 68, 320-44.
  2. Baccarini, M. (2005) FEBS Lett 579, 3271-7.
  3. Meloche, S. and Pouysségur, J. (2007) Oncogene 26, 3227-39.
  4. Roberts, P.J. and Der, C.J. (2007) Oncogene 26, 3291-310.
  5. Rubinfeld, H. and Seger, R. (2005) Mol Biotechnol 31, 151-74.
  6. Murphy, L.O. and Blenis, J. (2006) Trends Biochem Sci 31, 268-75.
  7. Dalby, K.N. et al. (1998) J Biol Chem 273, 1496-505.
  8. Marais, R. et al. (1993) Cell 73, 381-93.
  9. Kortenjann, M. et al. (1994) Mol Cell Biol 14, 4815-24.
  10. Owens, D.M. and Keyse, S.M. (2007) Oncogene 26, 3203-13.
  11. Seger, R. et al. (1991) Proc Natl Acad Sci U S A 88, 6142-6.
  12. Robbins, D.J. et al. (1993) J Biol Chem 268, 5097-106.
  13. Kinoshita, T. et al. (2008) Biochem Biophys Res Commun 377, 1123-7.
  14. Prowse, C.N. et al. (2001) J Biol Chem 276, 40817-23.
  15. Zhou, B. and Zhang, Z.Y. (2002) J Biol Chem 277, 13889-99.
  16. Zhang, Y.Y. et al. (2008) J Biol Chem 283, 26591-601.

Application References

Have you published research involving the use of our products? If so we'd love to hear about it. Please let us know!

Companion Products

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

Products