Cell Signaling Technology

Product Pathways - MAPK Signaling

Phospho-p90RSK (Thr359) (D1E9) Rabbit mAb #8753

Applications Reactivity Sensitivity MW (kDa) Isotype
W IP IHC-P IF-IC H Mk (C) (X) (Dg) (Hr) Endogenous 90 Rabbit IgG

Applications Key:  W=Western Blotting  IP=Immunoprecipitation  IHC-P=Immunohistochemistry (Paraffin)  IF-IC=Immunofluorescence (Immunocytochemistry)
Reactivity Key:  H=Human  Mk=Monkey  C=Chicken  X=Xenopus  Dg=Dog  Hr=Horse
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-p90RSK (Thr359) (D1E9) Rabbit mAb recognizes endogenous levels of p90RSK1 protein when phosphorylated at Thr359. This antibody also detects p90RSK2 phosphorylated at Thr365 and p90RSK3 phosphorylated at Thr356. Phosphorylation of p90RSK isoforms at a proximal serine residue (Ser363 of p90RSK1, Ser369 of p90RSK2, and Ser360 of p90RSK3) does not affect the ability of this antibody to detect the phospho-Thr residue.

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Thr356 of human p90RSK3 protein.

Western Blotting

Western Blotting

Western blot analysis of extracts from HeLa, COS-7, and A-431 cells, starved overnight and either untreated (-) or treated (+) with TPA #4174 (200 nM, 15 min) or Human Epidermal Growth Factor (hEGF) #8916 (100 ng/mL, 15 min), using Phospho-p90RSK (Thr359) (D1E9) Rabbit mAb.

IP

IP

Immunoprecipitation of phospho-p90RSK (Thr359) from extracts of HeLa cells, serum starved overnight and either untreated (-) or treated with TPA #4174 (200 nM, 15 min; +), using Normal Rabbit IgG #2729 (lanes 5 and 6) or Phospho-p90RSK (Thr359) (D1E9) Rabbit mAb (lanes 3 and 4). Lanes 1 and 2 are 10% input. Western blot analysis was performed using RSK1/RSK2/RSK3 Antibody #9347.

IHC-P (paraffin)

IHC-P (paraffin)

Immunohistochemical analysis of paraffin-embedded human ovarian carcinoma, control (left) or λ phosphatase-treated (right), using Phospho-p90RSK (Thr359) (D1E9) Rabbit mAb.


IHC-P (paraffin)

IHC-P (paraffin)

Immunohistochemical analysis of paraffin-embedded A-431 cell pellets, untreated (left) or treated with hEGF #8916 (right), using Phospho-p90RSK (Thr359) (D1E9) Rabbit mAb.

IHC-P (paraffin)

IHC-P (paraffin)

Immunohistochemical analysis of paraffin-embedded colon carcinoma using Phospho-p90RSK (Thr359) (D1E9) Rabbit mAb in the presence of control peptide (left) or antigen-specific peptide (right).

IF-IC

IF-IC

Confocal immunofluorescent analysis of A-431 cells, serum-starved (left), treated with hEGF #8916 (100 ng/mL, 15 min; center), or treated with hEGF and λ phosphatase (right), using Phospho-p90RSK (Thr359) (D1E9) Rabbit mAb (green). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).


Background

The 90 kDa ribosomal S6 kinases (RSK1-4) are a family of widely expressed Ser/Thr kinases characterized by two nonidentical, functional kinase domains (1) and a carboxy-terminal docking site for extracellular signal-regulated kinases (ERKs) (2). Several sites both within and outside of the RSK kinase domain, including Ser380, Thr359, Ser363, and Thr573, are important for kinase activation (3). RSK1-3 are activated via coordinated phosphorylation by MAPKs, autophosphorylation, and phosphoinositide-3-OH kinase (PI3K) in response to many growth factors, polypeptide hormones, and neurotransmitters (3).

Upon mitogenic stimulation, p44/42 Erk1/2 and Erk5 MAP kinases cooperatively phosphorylate p90RSK at Thr573 (p90RSK1 numbering) located within the C-terminal kinase domain and at Thr359/Ser363 in the linker region between the two kinase domains (3). Phosphorylation at Thr573 within the activation loop of the p90RSK C-terminal kinase domain promotes activation and directs phosphorylation at Ser380 within the hydrophobic stretch of the linker region (4,5). When phosphorylated, Ser380 acts as a docking site for the constitutively active Ser/Thr kinase PDK1, which in turn phosphorylates p90RSK at Ser221 within the N-terminal kinase domain activation loop, resulting in full enzymatic activation of p90RSK (6). Antibodies against these phosphorylation sites are useful for understanding the kinetics and regulation of p90RSK activation.For more information regarding the phospho-regulatory sites within each RSK isoform, including more information regarding the seminal studies demonstrating the complex phosphorylation cascades involved, please see the references herein and PhosphoSitePlus® (www.phosphosite.org).

  1. Fisher, T.L. and Blenis, J. (1996) Mol Cell Biol 16, 1212-9.
  2. Smith, J.A. et al. (1999) J Biol Chem 274, 2893-8.
  3. Dalby, K.N. et al. (1998) J Biol Chem 273, 1496-505.
  4. Roux, P.P. et al. (2003) Mol Cell Biol 23, 4796-804.
  5. Cargnello, M. and Roux, P.P. (2011) Microbiol Mol Biol Rev 75, 50-83.
  6. Romeo, Y. et al. (2012) Biochem J 441, 553-69.

Application References

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

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