Phospho-p53 (Ser46) Antibody

2521:

Flow , Immunofluorescence , Immunoprecipitation , Western Blotting

Phospho-p53 (Ser46) Antibody detects endogenous levels of p53 only when phosphorylated at serine 46. The antibody does not cross-react with p53 phosphorylated at other sites.

Polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser46 of human p53. Antibodies are purified by protein A and peptide affinity chromatography.

Western Blotting

Western blot analysis of extracts from MCF-7 cells treated with etoposide for the indicated times, using Phospho-p53 (Ser46) Antibody.

IP

Immunoprecipitation of extracts from MCF-7 cells treated with etoposide under nondenaturing conditions, using Phospho-p53 (Ser46) Antibody, followed by Western blot analysis using a monoclonal p53 antibody.

Flow Cytometry

Flow cytometric analysis of HT-29 cells, untreated (blue) or UV-treated (green), using Phospho-p53 (Ser46) Antibody.

IF-IC

Confocal immunofluorescent analysis of MCF-7 cells, untreated (left) or etoposide-treated (right), using Phospho-p53 (Ser46) Antibody (green). Actin filaments have been labeled with DY-554 phalloidin (red).

The p53 tumor suppressor protein plays a major role in cellular response to DNA damage and other genomic aberrations. Activation of p53 can lead to either cell cycle arrest and DNA repair or apoptosis (1). p53 is phosphorylated at multiple sites in vivo and by several different protein kinases in vitro (2,3). DNA damage induces phosphorylation of p53 at Ser15 and Ser20 and leads to a reduced interaction between p53 and its negative regulator, the oncoprotein MDM2 (4). MDM2 inhibits p53 accumulation by targeting it for ubiquitination and proteasomal degradation (5,6). p53 can be phosphorylated by ATM, ATR, and DNA-PK at Ser15 and Ser37. Phosphorylation impairs the ability of MDM2 to bind p53, promoting both the accumulation and activation of p53 in response to DNA damage (4,7). Chk2 and Chk1 can phosphorylate p53 at Ser20, enhancing its tetramerization, stability, and activity (8,9). p53 is phosphorylated at Ser392 in vivo (10,11) and by CAK in vitro (11). Phosphorylation of p53 at Ser392 is increased in human tumors (12) and has been reported to influence the growth suppressor function, DNA binding, and transcriptional activation of p53 (10,13,14). p53 is phosphorylated at Ser6 and Ser9 by CK1δ and CK1ε both in vitro and in vivo (13,15). Phosphorylation of p53 at Ser46 regulates the ability of p53 to induce apoptosis (16). Acetylation of p53 is mediated by p300 and CBP acetyltransferases. Inhibition of deacetylation suppressing MDM2 from recruiting HDAC1 complex by p19 (ARF) stabilizes p53. Acetylation appears to play a positive role in the accumulation of p53 protein in stress response (17). Following DNA damage, human p53 becomes acetylated at Lys382 (Lys379 in mouse) in vivo to enhance p53-DNA binding (18). Deacetylation of p53 occurs through interaction with the SIRT1 protein, a deacetylase that may be involved in cellular aging and the DNA damage response (19).

Homeodomain-interacting protein kinase 2 (HIPK2) Phosphorylates Ser46 off p53 in vitro and in vivo (17,18), and p38 can phosphorylate this site in vitro (19).

1. Levine, A.J. (1997) Cell 88, 323-331.
2. Meek, D.W. (1994) Semin. Cancer Biol. 5, 203-210.
3. Milczarek, G.J. et al. (1997) Life Sci. 60, 1-11.
4. Shieh, S.Y. et al. (1997) Cell 91, 325-334.
5. Chehab, N.H. et al. (1999) Proc. Natl. Acad. Sci. USA 96, 13777-13782.
6. Honda, R. et al. (1997) FEBS Lett. 420, 25-27.
7. Tibbetts, R.S. et al. (1999) Genes Dev. 13, 152-157.
8. Shieh, S.Y. et al. (1999) EMBO J. 18, 1815-1823.
9. Hirao, A. et al. (2000) Science 287, 1824-1827.
10. Hao, M. et al. (1996) J. Biol. Chem. 271, 29380-29385.
11. Lu, H. et al. (1997) Mol. Cell. Biol. 17, 5923-5934.
12. Ullrich, S.J. et al. (1993) Proc. Natl. Acad. Sci. USA 90, 5954-5958.
13. Kohn, K.W. (1999) Mol. Biol. Cell 10, 2703-2734.
14. Lohrum, M. and Scheidtmann, K.H. (1996) Oncogene 13, 2527-2539.
15. Knippschild, U. et al. (1997) Oncogene 15, 1727-1736.
16. Oda, K. et al. (2000) Cell 102, 849-862.
17. Ito, A. et al. (2001) EMBO J. 20, 1331-1340.
18. Sakaguchi, K. et al. (1998) Genes Dev. 12, 2831-2841.
19. Solomon, J.M. et al. (2006) Mol. Cell. Biol. 26, 28-38.
20. D'Orazi, G. et al. (2002) Nat. Cell Biol. 4, 11-19.
21. Hofmann, T. G. et al. (2002) Nat. Cell Biol. 4, 1-10.
22. Bulavin, D. V. et al. (1999) EMBO J. 18, 6845-6854.

• Wang, C. and Chen, J. (2003) Phosphorylation and hsp90 binding mediate heat shock stabilization of p53. J. Biol. Chem. 278, 2066-2071. Applications: Western Blotting

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