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

Abstract

Abstract(-)-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 À/À 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 EGCGmediated 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]

Protein phosphatase-1 regulates Akt1 signal transduction pathway to control gene expression, cell survival and differentiation

“…5 PP-2A is also found to modulate the phosphorylation status of p53 at these sites to suppress stress-induced apoptosis. 6,7 In this study, we present the first evidence to show that PP-1 has a critical role during the activation process of Akt1 signaling pathway.…”

“…After dephosphorylation, the free 32 P released from substrate proteins by PP-1 or PP-2A was measured in a scintillation counter. As shown in Figure 2c, in vitro enzyme concentration-dependent assays showed that PP-1 was more efficient than PP-2A in dephosphorylating the labeled substrate with the same amount of enzyme (PP-1 and PP-2A activities were calibrated as described previously 5,6 ). Similarly, time-dependent assays with 1 Unit of PP-1 or PP-2A showed that the free 32 P released by PP-1 was much more prominent than PP-2A (Figure 2d).…”

“…When the growth factors are withdrawn, inactivation of the Akt pathway occurs at two or pCI-AKT1-T450D were treated with a single dose of 100 nM staurosporine for 6 h and then harvested for cell flow cytometry analysis as described previously (also see Supplementary Figure 4). 6,63 It must be noted that AKT1-T450D-transfected cells showed the highest level of survival. In contrast, the AKT1-T450A-transfected cells displayed a level of apoptosis similar to vector-transfected cells, suggesting that Akt1-T450A lost most of the survival-promotion functions of AKT1.…”

“…The mutant Akt1 expression constructs, pCI-neo-AKT-T450A and pCI-AKT-T450D were generated using PCR-linked in vitro mutagenesis as described previously. 5,6,61 Both wild-type and mutant constructs were cloned into pCI-neo vector at the EcoRI (5 0 ) and SalI (3 0 ) sites. The human PP-1a, PP-1b, PP-1g, PP-2Aa and PP-2Ab catalytic subunits were purchased from ATCC.…”

“…In this regard, we have recently shown that both PP-1 and PP-2A have important roles in modulating apoptosis. 5,6 PP-1 promotes survival through its negative regulation of p53, which is a master regulator of apoptosis. It directly dephosphorylates p53 at Ser-15 and Ser-37 to attenuate its transcriptional and apoptotic activities.…”