H.-G. Chen

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.

Protein Phosphatase-1 Modulates the Function of Pax-6, a Transcription Factor Controlling Brain and Eye Development

Pax-6 is an evolutionarily conserved transcription factor and acts high up in the regulatory hierarchy controlling eye and brain development in humans, mice, zebrafish, and Drosophila. Previous studies have shown that Pax-6 is a phosphoprotein, and its phosphorylation by ERK, p38, and homeodomain-interacting protein kinase 2 greatly enhances its transactivation activity. However, the protein phosphatases responsible for the dephosphorylation of Pax-6 remain unknown. Here, we present both in vitro and in vivo evidence to show that protein serine/threonine phosphatase-1 is a major phosphatase that directly dephosphorylates Pax-6. First, purified protein phosphatase-1 directly dephosphorylates Pax-6 in vitro. Second, immunoprecipitation-linked Western blot revealed that both protein phosphatase-1α and protein phosphatase-1β interact with Pax-6. Third, overexpression of protein phosphatase-1 in human lens epithelial cells leads to dephosphorylation of Pax-6. Finally, inhibition of protein phosphatase-1 activity by calyculin A or knockdown of protein phosphatase-1α and protein phosphatase-1β by RNA interference leads to enhanced phosphorylation of Pax-6. Moreover, our results also demonstrate that dephosphorylation of Pax-6 by protein phosphatase-1 significantly modulates its function in regulating expression of both exogenous and endogenous genes. These results demonstrate that protein phosphatase 1 acts as a major phosphatase to dephosphorylate Pax-6 and modulate its function.

Transcriptional Regulation of PP2A-Aα Is Mediated by Multiple Factors Including AP-2α, CREB, ETS-1, and SP-1

Protein phosphatases-2A (PP-2A) is a major serine/threonine phosphatase and accounts for more than 50% serine/threonine phosphatase activity in eukaryotes. The holoenzyme of PP-2A consists of the scaffold A subunit, the catalytic C subunit and the regulatory B subunit. The scaffold subunits, PP2A-Aα/β, provide a platform for both C and B subunits to bind, thus playing a crucial role in providing specific PP-2A activity. Mutation of the two genes encoding PP2A-Aα/β leads to carcinogenesis and likely other human diseases. Regulation of these genes by various factors, both extracellular and intracellular, remains largely unknown. In the present study, we have conducted functional dissection of the promoter of the mouse PP2A-Aα gene. Our results demonstrate that the proximal promoter of the mouse PP2A-Aα gene contains numerous cis-elements for the binding of CREB, ETS-1, AP-2α, SP-1 besides the putative TFIIB binding site (BRE) and the downstream promoter element (DPE). Gel mobility shifting assays revealed that CREB, ETS-1, AP-2α, and SP-1 all bind to PP2A-Aα gene promoter. In vitro mutagenesis and reporter gene activity assays reveal that while SP-1 displays negative regulation, CREB, ETS-1 and AP-2Aα all positively regulate the promoter of the PP2A-Aα gene. ChIP assays further confirm that all the above transcription factors participate the regulation of PP2A-Aα gene promoter. Together, our results reveal that multiple transcription factors regulate the PP2A-Aα gene.

Contrast expression patterns of JNK1 during sex reversal of the rice-field eel

The c-Jun N-terminal kinases (JNKs) are members of the mitogen-activated protein kinase family. Their functions in regulating animal development have been well studied in both invertebrates and vertebrates. However, it remains to be determined whether they play a role in sex determination. Here we present first evidence to show that expression of JNK1 displays distinct patterns during sex reversal of rice-field eel. Molecular cloning reveals that JNK1 is well conserved among rice-field eel and other vertebrates. Both quantitative real-time polymerase chain reaction and Western blot analysis demonstrate that JNK1 is highly expressed in the ovary of the female individual and reduced to a substantial degree at the later stage of the intersex. However, when the intersex individual develops into the stage of male, expression of the JNK1 in the testis of the male individual is distinctly downregulated. Associated with the contrast JNK1 expression pattern in female and male gonads, several stem cell marker genes including Nanog, Oct-3/4, and Sox-2 were also differentially expressed in female and male germinal stem cells. Together, these results suggest it is possible that JNK1 plays an important role in sexual reversal of the rice-field eel.

The PP2A-Aβ gene is regulated by multiple transcriptional factors including Ets-1, SP1/SP3, and RXRα/β

Protein phosphatase-2A (PP-2A) is a major serine/threonine phosphatase abundantly expressed in eukaryotes. PP-2A is a heterotrimer that contains a 65 kD scaffold A subunit, a 36 kD catalytic C subunit, and a regulatory B subunit of variable isoforms ranging from 54-130 kDs. The scaffold subunits, PP2A-Aα/β, act as platforms for both the C and B subunits to bind, and thus are key structural components for PP-2A activity. Mutations in both genes encoding PP2A-Aα and PP2A-Aβ lead to carcinogenesis and likely other human diseases. Our previous work showed that the gene coding for PP2A-Aα is positively regulated by multiple transcription factors including Ets-1, CREB, and AP-2α but negatively regulated by SP-1/SP-3. In the present study, we have functionally dissected the promoter of the mouse PP2A-Aβ gene. Our results demonstrate that three major cis-elements, including the binding sites for Ets-1, SP1/SP3, and RXRα/β, are present in the proximal promoter of the mouse PP2A-Aβ gene. Gel mobility shifting assays reveal that Ets-1, SP1/SP3, and RXRα/β all bind to PP2A-Aβ gene promoter. In vitro mutagenesis and reporter gene activity assays demonstrate that while Ets-1 displays negative regulation, SP1/SP3 and RXRα/β positively regulate the promoter of the PP2A-Aβ gene. Co-expression of the cDNAs encoding Ets-1, SP1/SP3, or RXRα/β and the luciferase reporter gene driven by PP2A-Aβ promoter further confirm their control over the PP2A-Aβ promoter. Finally, ChIP assays demonstrate that Ets-1, SP1/SP3, and RXRα/β can all bind to the PP2A-Aβ gene promoter. Together, our results reveal that multiple transcription factors regulate the PP2A-Aβ gene. Moreover, our results provide important information explaining why PP2A-Aα and PP2A-Aβ display distinct expression levels.