|Western blot, immunofluorescence, and immunohistochemistry|
Host Species and Isotype:
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 (Ref 1). p53 is phosphorylated at multiple sites in vivo and by several different protein kinases in vitro (Ref 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 (Ref 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 (Ref 4,7). Chk2 and Chk1 can phosphorylate p53 at Ser20, enhancing its tetramerization, stability, and activity (Ref 8,9). p53 is phosphorylated at Ser392 in vivo (Ref 10,11) and by CAK in vitro (Ref 11). Phosphorylation of p53 at Ser392 is increased in human tumors (Ref 12) and has been reported to influence the growth suppressor function, DNA binding and transcriptional activation of p53 (Ref 10,13,14). p53 is phosphorylated at Ser6 and Ser9 by CK1-delta and CK1-epsilon both in vitro and in vivo (Ref 3,15). Phosphorylation of p53 at Ser46 regulates the ability of p53 to induce apoptosis (Ref 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 (Ref 17). Following DNA damage, human p53 becomes acetylated at Lys382 (Lys379 in mouse) in vivo to enhance p53-DNA binding (Ref 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 (Ref 19). DNA damage and stress-inducing agents can also result in SAPK/JNK-mediated phosphorylation of p53 at Thr81, and a Thr81Ala mutant p53 does not permit SAPK/JNK-dependent p53 transcriptional activation and apoptosis (Ref 17).
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