Protein phosphatase-2A is a target of epigallocatechin-3-gallate and modulates p53-Bak apoptotic pathway.
(-)-Epigallocatechin-3-gallate (EGCG) is a well-known chemoprevention factor. Recent studies have revealed that EGCG triggers cancer cells undergoing apoptosis through p53-dependent pathway. How EGCG activates p53-dependent apoptosis is not fully understood. In the present study using JB6 cell as a model system, we have shown that EGCG can negatively regulate protein serine/threonine phosphatase-2A (PP-2A) to positively regulate p53-dependent apoptosis. First, EGCG at physiologic levels down-regulates PP-2A at the protein and enzyme activity levels. Second, EGCG induces apoptosis of JB6 cells, which is associated with hyperphosphorylation of p53 and up-regulation of the proapoptotic gene, Bak. DNA sequence analysis, gel mobility shifting, chromatin immunoprecipitation, and reporter gene activity assays revealed that p53 directly controls Bak in JB6 cells. Knockdown of p53 and Bak expression with RNAi substantially inhibits EGCG-induced apoptosis. Third, PP-2A directly interacts with p53 and dephosphorylates p53 at Ser-15 in vitro and in vivo. Fourth, overexpression of the catalytic subunit for PP-2A down-regulates p53 phosphorylation at Ser15, attenuates expression of the downstream proapoptotic gene, Bak, and antagonizes EGCG-induced apoptosis. Inhibition of PP-2A activity enhances p53 phosphorylation at Ser-15 and up-regulates Bak expression to promote EGCG-induced apoptosis. Finally, in the p53(-/-) H1299 and p53(+/+) H1080 cells, EGCG down-regulates PP-2A similarly but induces differential apoptosis. In summary, our results show that (a) PP-2A directly dephosphorylates p53 at Ser-15; (b) P53 directly controls Bak expression; and (c) EGCG negatively regulates PP-2A. Together, our results show that EGCG-mediated negative regulation of PP-2A is an important molecular event for the activation of p53-dependent apoptosis during its chemoprevention.
Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, Nebraska 68198-5870, USA.
Jichao
J
Qin
He-Ge
HG
Chen
Qin
Q
Yan
Mi
M
Deng
Jinping
J
Liu
Stephan
S
Doerge
Weiya
W
Ma
Zigang
Z
Dong
Wan-Cheng David
DW
Li
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D001483:Base Sequence; D015153:Blotting, Western; D002392:Catechin / Q000031:analogs & derivatives* / Q000494:pharmacology; D002460:Cell Line; D047369:Chromatin Immunoprecipitation; D017931:DNA Primers; D005434:Flow Cytometry; D007150:Immunohistochemistry; D051379:Mice; D010766:Phosphorylation; D054648:Protein Phosphatase 2 / Q000187:drug effects* / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*; D050998:bcl-2 Homologous Antagonist-Killer Protein / Q000378:metabolism*
D017931:DNA Primers; D016159:Tumor Suppressor Protein p53; D050998:bcl-2 Homologous Antagonist-Killer Protein; D002392:Catechin; C045651:epigallocatechin gallate; D054648:Protein Phosphatase 2