Cell Signaling Technology

Product Pathways - Cell Cycle / Checkpoint

Phospho-cdc25C (Thr48) (D2H3) Rabbit mAb #12028

Applications Reactivity Sensitivity MW (kDa) Isotype
W IP IF-IC H (Mk) Endogenous 75 Rabbit IgG

Applications Key:  W=Western Blotting  IP=Immunoprecipitation  IF-IC=Immunofluorescence (Immunocytochemistry)
Reactivity Key:  H=Human  Mk=Monkey
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-cdc25C (Thr48) (D2H3) Rabbit mAb recognizes endogenous levels of cdc25C protein only when phosphorylated at Thr48.

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Thr48 of human cdc25C protein.

Western Blotting

Western Blotting

Western blot analysis of extracts from HT-29 cells, asynchronous (-) or synchronized in mitosis (thymidine block, nocodazole release, mitotic shake-off; +), using Phospho-cdc25C (Thr48) (D2H3) Rabbit mAb (upper) or cdc25C (5H9) Rabbit mAb #4688 (lower).

IF-IC

IF-IC

Confocal immunofluorescent analysis of HT-29 cells, untreated (left) or λ phosphatase-treated (right), using Phospho-cdc25C (Thr48) (D2H3) Rabbit mAb (green). Actin filaments were labeled with DY-554 phalloidin (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

Background

Cdc25 is a protein phosphatase responsible for dephosphorylating and activating cdc2, a crucial step in regulating the entry of all eukaryotic cells into mitosis (1). cdc25C is constitutively phosphorylated at Ser216 throughout interphase by c-TAK1, while phosphorylation at this site is DNA damage-dependent at the G2/M checkpoint (2). When phosphorylated at Ser216, cdc25C binds to members of the 14-3-3 family of proteins, sequestering cdc25C in the cytoplasm and thereby preventing premature mitosis (3). The checkpoint kinases Chk1 and Chk2 phosphorylate cdc25C at Ser216 in response to DNA damage (4,5).

Full activation of cdc25C involves phosphorylation at more than 12 different sites by cdc2/cyclin B and Polo-like kinase, and the activity of Pin1, a peptidyl-prolyl isomerase (PPI) (6,7). Pin1 contains a WW domain that binds phospho-Ser/Thr-Pro sites and a catalytic PPI region that induces a cis/trans isomerization at phospho-Ser/Thr-Pro bonds (8). Thr48 and Thr67 of cdc25C interact directly with the WW domain of Pin1 when these sites are phosphorylated (9). Thr48 phosphorylation also mediates binding to CKS/p13SUC1 (10).

  1. Jessus, C. and Ozon, R. (1995) Prog. Cell Cycle Res. 1, 215-228.
  2. Peng, C.Y. et al. (1997) Science 277, 1501-1505.
  3. Kumagai, A. and Dunphy, W.G. (1999) Genes Dev. 13, 1067-1072.
  4. Blasina, A. et al. (1999) Curr. Biol. 9, 1-10.
  5. Furnari, B. et al. (1999) Mol. Biol. Cell 10, 833-845.
  6. Izumi, T. and Maller, J.L. (1993) Mol Biol Cell 4, 1337-50.
  7. Stukenberg, P.T. and Kirschner, M.W. (2001) Mol Cell 7, 1071-83.
  8. Yaffe, M.B. et al. (1997) Science 278, 1957-60.
  9. Lu, P.J. et al. (1999) Science 283, 1325-8.
  10. Landrieu, I. et al. (2001) J Biol Chem 276, 1434-8.

Application References

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

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