Ubiquitin-dependent degradation of p53 protein despite phosphorylation at its N terminus by acetaminophen.
We previously reported that acetaminophen (APAP, 4-hydroxyacetanilide) caused apoptosis of C6 glioma cells. Therefore, we hypothesized that the level of p53, which usually stimulates apoptosis, might be increased after APAP exposure. However, APAP exposure for 24 h markedly decreased the p53 content and its downstream target p21 in a concentration-dependent manner. Reduction of p53 was not accompanied by a decrease in p53 mRNA in C6 glioma cells, suggesting that p53 was mainly affected at the protein level. Unexpectedly, APAP stimulated phosphorylation of p53 at Ser15, Ser20, and Ser37, which usually elevates p53 content. However, phosphorylation of these residues did not prevent APAP-induced decrease in p53. The p53 reduction was independent from the level of phospho-Akt, which is known to promote p53 degradation. Immunoblot analysis of the immunoprecipitated p53 revealed that increased amounts of murine double minute 2 (mdm2) and ubiquitin were bound to p53 during its degradation. Lactacystin and N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal (MG132), inhibitors of proteasomal proteolysis, prevented the decrease, supporting the proteasomal degradation of p53 upon APAP exposure. Pretreatment with chlormethiazole, an inhibitor of ethanol-inducible CYP2E1, significantly lowered the CYP2E1 enzyme activity and the rate of APAP-induced cell death while it prevented the reduction of p53 and p21 in C6 glioma cells. A nontoxic analog of APAP, 3-hydroxyacetanilde, did not reduce p53 and p21 contents in C6 glioma cells and LLC-PK1 porcine kidney cells. Taken together, our results show that APAP or its reactive metabolite(s) can directly reduce the p53 content through mdm2-mediated ubiquitin conjugation, despite phosphorylation of p53 at its N terminus.
Lee
Yun-Sik
YS
Laboratory of Membrane Biochemistry and Biophysics, National Institute on Alcohol Abuse and Alcoholism, Bethesda, MD 20892-9410, USA.
Wan
Jie
J
Kim
Bong-Jo
BJ
Bae
Myung-Ae
MA
Song
Byoung J
BJ
D000082:Acetaminophen / Q000494:pharmacology*; D018712:Analgesics, Non-Narcotic / Q000494:pharmacology*; D000818:Animals; D016923:Cell Death / Q000187:drug effects; D045744:Cell Line, Tumor; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000378:metabolism; D019392:Cytochrome P-450 CYP2E1 / Q000378:metabolism; D015536:Down-Regulation; D018374:LLC-PK1 Cells; D010766:Phosphorylation; D051381:Rats; D013552:Swine; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*; D025801:Ubiquitin / Q000502:physiology*
D018712:Analgesics, Non-Narcotic; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016159:Tumor Suppressor Protein p53; D025801:Ubiquitin; D000082:Acetaminophen; D019392:Cytochrome P-450 CYP2E1