The p53 gene is a tumor suppressor gene that is activated whenever a cell is subjected to cell damage. The p53 gene determines whether a cell should survive a damage or should undergo apoptosis. These decisions are regulated by posttranslational modifications. Phosphorylation of the serine 46 residue is one such modification that is relevant to this study. However, it is unclear why a Ser46 phosphorylated p53 favors apoptosis over survival (Feng et al. 2006, p. 2812; Taira et al. 2014, p. 717).
Studies have shown that the elusive molecular mechanism of Ser46 phosphorylation of p53 induce apoptosis in the tumor cells, which makes the molecular mechanism a potential target for cancer therapy. Elucidation of the molecular mechanism could lead to the development of new cancer drugs that could artificially induce apoptosis in the tumor cells without damaging the healthy cells (Carvajal and Manfredi 2013, 414; Taira et al. 2014, p. 717).
A critical connection between miRNA, p53, tumor suppression and DDX5 has been established by a few recent researches (Suzuki et al. 2009, p. 529; Newman and Hammond 2010, p. 1086). Based on the current study, a novel target, palmdelphin (PALMD), is being explored as a possible cancer therapy target (Dashzeveg et al. 2014, p. 1).
2. Summary of the findings
The first step in proving the hypothesis was to establish the fact that Ser46 phosphorylated p53 binds to AREG. To prove this, the researchers used p53-deficient H1299 lung adenocarcinoma cells and SaOS-2 osteosarcoma cells transfected with three vector-constructs, namely, empty Flag vector, Flag-p53 wild type (WT) and mutant S46A p53. The mutant construct contained Ala46 in place of Ser46. Microarray analysis of the WT revealed that of the 54, 000 probes, only one probe was common to both the cell lines that showed more 1. 5 fold increase. The probe coded for AREG, and the corresponding mRNA and protein levels confirmed the same. This experiment established that Ser46 phosphorylated p53 indeed binds to AREG and causes up-regulation of AREG expression. To pinpoint the region where the Ser46 phosphorylated p53 might bind to AREG promoter, the DNA sequence of p53 was matched to the promoter region. The scientists obtained two possible sites within a span of 500 base pairs. To confirm the p53 consensus element’s binding to the AREG promoter region in vivo, chromatins were gathered from the three types of transfected cells for immunoprecipitation (Taira et al. 2014, p. 718).
Finally, the authors had to establish that AREG regulates the miRNA processing, which leads to apoptosis. To prove this, the miRNA precursors miR15a, miR34 and miR 143 were studied. Expression levels of the transfected cells with relevant constructs proved that AREG regulates miRNA expression and its biogenesis (Taira et al. 2014, p. 721).
3. Critique and analysis
The paper talks about the role of Ser46 phosphorylated p53 in inducing apoptosis by up-regulating AREG expression, which in turn regulates the biogenesis of apoptosis-inducing miRNA. The other members of the EGF family are known proliferators of tumorigenesis; however, AREG has dual functions as a cell growth inhibitor as well as a cell growth proliferator. AREG binds to the EGF receptor present on the plasma membrane to promote growth but also promotes apoptosis by regulating miRNA. However, to regulate miRNA expression for apoptosis, AREG needs to be localized in the nuclear membrane. Researches dating back to a couple of decades have proven that AREG has binding sites in the nuclear membrane, where it regulates the pri-miRNA processing (Taira et al. 2014, p. 717).
The paper is very well researched and has been designed to address each phase of the hypothesis systematically. The authors have broken down the hypothesis into smaller, manageable and critical parts that could be proven using specific experiments. The authors, however, did not mention that the phosphorylation of p53 at Ser46 requires the enzyme ataxia-telangiectasia mutated (ATM) kinase during the early-phase response to DNA damage. ATM induces phosphorylation in the Ser46 residue before it activates the enzyme DYRK2 to continue the phosphorylation (Kodama et al. 2010, p. 1632). They also did not mention the base pair sequence of the putative p53 consensus elements. The paper could have been elaborated on the sequences used for real time RT PCR and semi-quantitative PCR. A statistical error analysis of the data could have been done to provide authenticity to the entire work.
The experiments help in understanding that phosphorylated p53 binds to AREG promoter region, the phosphorylation occurs during DNA damage, the binding of p53 to AREG is biochemically relevant, and that AREG regulates miRNA biogenesis. Despite correlating the Ser46 phosphorylation of p53 to a miRNA-mediated cell death, the experiments do not reveal why a phosphorylated p53 tagged cell undergoes apoptosis. Though it is not clear why or how the Ser46 phosphorylation p53 induces apoptosis, it is evident from research that Ser46 phosphorylation increases the affinity of the p53 to bind to proapoptotic genes such as p53AIP1. The affinity oriented binding also up-regulates the expression of the proapoptotic gene (Taira et al. 2014, p. 722). A study by a group of scientists identified a protein called Apak (ATM and p53-associated KZNF protein) that competed with p53 to bind to p53AIP1 gene, thereby protecting the healthy, undamaged DNA. However, during cell damage, Apak protein unhinges itself from the DNA to let p53 take over the repair or apoptotic pathway (Yuan et al. 2012, p. 363).
In order to develop new cancer drugs and identify potential targets for cancer therapy, it is essential to understand why phosphorylated p53 changes affinity to proapoptotic genes and to identify the intracellular localization mechanism of AREG.
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Carvajal, L. A., & Manfredi, J. J. (2013). Another fork in the road—life or death decisions by the tumour suppressor p53. EMBO reports. Vol. 14, no. 5, pp. 414-421.
Dashzeveg, N., Taira, N., Lu, Z. G., Kimura, J., & Yoshida, K. (2013). Palmdelphin, a novel target of p53 with Ser46 phosphorylation, controls cell death in response to DNA damage. Cell death & disease. Vol. 5, pp. e1221-e1221.
Feng, L., Hollstein, M., & Xu, Y. (2006). Ser46 phosphorylation regulates p53-dependent apoptosis and replicative senescence. Cell Cycle. Vol. 5, no. 23, pp. 2812-2819.
Kodama, M., Otsubo, C., Hirota, T., Yokota, J., Enari, M., & Taya, Y. (2010). Requirement of ATM for rapid p53 phosphorylation at Ser46 without Ser/Thr-Gln sequences. Molecular and cellular biology. Vol. 30, no. 7, pp. 1620-1633.
Newman, M. A., & Hammond, S. M. (2010). Emerging paradigms of regulated microRNA processing. Genes & development Vol. 24, no. 11, pp. 1086-1092.
Suzuki, H. I., Yamagata, K., Sugimoto, K., Iwamoto, T., Kato, S., & Miyazono, K. (2009). Modulation of microRNA processing by p53. Nature. Vol. 460, no. 7254, pp. 529-533.
Taira, N., Mimoto, R., Kurata, M., Yamaguchi, T., Kitagawa, M., Miki, Y., & Yoshida, K. (2012). DYRK2 priming phosphorylation of c-Jun and c-Myc modulates cell cycle progression in human cancer cells. The Journal of clinical investigation. Vo. 122, no. 3, pp. 859-872.
Taira, N., Yamaguchi, T., Kimura, J., Lu, Z. G., Fukuda, S., Higashiyama, S., & Yoshida, K. (2014). Induction of amphiregulin by p53 promotes apoptosis via control of microRNA biogenesis in response to DNA damage. Proceedings of the National Academy of Sciences. Vol. 111, no. 2, pp. 717-722.
Yuan, L., Tian, C., Wang, H., Song, S., Li, D., Xing, G., & Zhang, L. (2012). Apak competes with p53 for direct binding to intron 1 of p53AIP1 to regulate apoptosis. EMBO reports. Vol. 13, no. 4, pp. 363-370.
Zhang, X., Wan, G., Berger, F. G., He, X., & Lu, X. (2011). The ATM kinase induces microRNA biogenesis in the DNA damage response. Molecular cell. Vol. 41, no. 4, pp. 371-383.