Cell Signaling Technology

Product Pathways - MAPK Signaling

Phospho-c-Raf (Ser289/296/301) Antibody #9431

Applications Reactivity MW (kDa) Source
W H M 74 Rabbit

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

Specificity / Sensitivity

Phospho-c-Raf (Ser289/296/301) Antibody detects endogenous levels of c-Raf protein only when phosphorylated at either Ser289, Ser296 or Ser301.

Source / Purification

Polyclonal antibodies are produced by immunizing rabbits with a synthetic phospho-peptide (KLH-coupled) corresponding to residues surrounding Ser289/296/301 of human c-Raf. Antibodies are purified by protein A and peptide affinity chromatography.

Western Blotting

Western Blotting

Western blot analysis of extracts from untreated or TPA-treated 293 and NIH/3T3 cells, using Phospho-c-Raf (Ser289/296/301) Antibody.

Background

A-Raf, B-Raf and c-Raf (Raf-1) are the main effectors recruited by GTP-bound Ras to activate the MEK-MAP kinase pathway (1). Activation of c-Raf is the best understood and involves phosphorylation at multiple activating sites including Ser338, Tyr341, Thr491, Ser494, Ser497 and Ser499 (2). p21-activated protein kinase (PAK) has been shown to phosphorylate c-Raf at Ser338 and the Src family phosphorylates Tyr341 to induce c-Raf activity (3,4). Ser338 of c-Raf corresponds to similar sites in A-Raf (Ser299) and B-Raf (Ser445), although this site is constitutively phosphorylated in B-Raf (5). Inhibitory 14-3-3 binding sites on c-Raf (Ser259 and Ser621) can be phosphorylated by Akt and AMPK, respectively (6,7). While A-Raf, B-Raf and c-Raf are similar in sequence and function, differential regulation has been observed (8). Of particular interest, B-Raf contains three consensus Akt phosphorylation sites (Ser364, Ser428 and Thr439) and lacks a site equivalent to Tyr341 of c-Raf (8,9). The B-Raf mutation V600E results in elevated kinase activity and is commonly found in malignant melanoma (10). Six residues of c-Raf (Ser29, Ser43, Ser289, Ser296, Ser301 and Ser642) become hyperphosphorylated in a manner consistent with c-Raf inactivation. The hyperphosphorylation of these six sites is dependent on downstream MEK signaling and renders c-Raf unresponsive to subsequent activation events (11).

  1. Avruch, J. et al. (1994) Trends Biochem. Sci. 19, 279-283.
  2. Chong, H. et al. (2001) EMBO J. 20, 3716-3727.
  3. King, A.J. et al. (1998) Nature 396, 180-183.
  4. Fabian, J.R. et al. (1993) Mol. Cell Biol. 13, 7170-7179.
  5. Mason, C.S. et al. (1999) EMBO J. 18, 2137-2148.
  6. Zimmermann, S. and Moelling, K. (1999) Science 286, 1741-1744.
  7. Sprenkle, A.B. et al. (1997) FEBS Lett. 403, 254-258.
  8. Marais, R. et al. (1997) J. Biol. Chem. 272, 4378-4383.
  9. Guan, K.L. et al. (2000) J. Biol. Chem. 275, 27354-27359.
  10. Davies, H. et al. (2002) Nature 417, 949-954.
  11. Dougherty, M.K. et al. (2005) Mol. Cell 17, 215-224.

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