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Protein Phosphatase-2A Is a Target of Epigallocatechin-3-Gallate and Modulates p53-Bak Apoptotic Pathway

Departments of ¹ Biochemistry and Molecular Biology and ² Ophthalmology and Visual Sciences, University of Nebraska Medical Center, Omaha, Nebraska; ³ key Laboratory of Protein Chemistry and Developmental Biology of National Education Ministry of China, College of Life Sciences, Hunan Normal University, Changsha, Hunan, China; and ⁴ Hormel Institute, University of Minnesota, Austin, Minnesota

Requests for reprints: David Wan-Cheng Li, Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, 985870 Nebraska Medical Center, Omaha, NE 68198-5870. Phone: 402-559-5073; Fax: 402-559-6650; E-mail: dwli1688{at}hotmail.com.

Key Words: (–)-Epigallocatechin-3-gallate (EGCG) • chemoprevention • JB6 Cl41 cell • protein serine/threonine phosphatase-2A • p53 • Bak • phosphorylation/dephosphorylation • apoptosis

(-)-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. [Cancer Res 2008;68(11):4150–62]

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				Correction: EGCG Negatively Regulates PP-2A to Promote Apoptosis Cancer Res., August 15, 2008; 68(16): 6859 - 6859. [Full Text] [PDF]