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D. W. Li, H.-G. Chen, M. Deng, J. Liu, Q. Yan, D. Yuan, H. Feng, Y. Liu; Protein Phosphatases-1 and -2A Dephosphorylate p53 at Multiple Sites and Regulate Different Downstream Genes to Promote Survival of Human Lens Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2008;49(13):1900.
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Protein phosphorylation and dephosphorylation are the fundamental mechanisms to regulate various cellular activities such as gene expression, cell proliferation, differentiation, apoptosis, organgenesis, and tissue homeostasis. The protein serine/threonine phosphatases-1 and -2A are the major cellular phosphatases and contribute to more than 90% serine/threonine phosphatase activity in eukaryotes. In the mature vertebrate (human, bovine, rat, mouse and fish) lens, PP-1 is a more abundant phosphatase than PP-2A and playing important roles in promoting survival of lens epithelial cells (Li et al., 2001. IOVS. 42:2603-2609). Inhibition of PP-1 and PP-2A activity leads to apoptosis of lens epithelial cells (Li et al., 2001. Exp. Cell Res.266:279-291). Mechanistically, through dephosphorylation, PP-1 and PP-2A are actively modulating the functions of p53 (Li et al., 2006. Oncogene. 25:3006-3022), an extremely important regulator of lens differentiation and pathology. Knockout of p53 or inactivation of p53 by expression of viral gene E6 leads to cataractogenesis. In the present study, we demonstrate that PP-1 and/or PP-2A dephosphorylate p53 directly at multiple residues, which lead to modulation of the expression pattern of numerous proapoptotic and anti-apoptotic members of the Bcl-2 gene family and also other genes.
Dephosphorylation of p53 by protein phosphatase-1 and -2A was explored with both in vitro and in vivo dephosphorylation assays, co-immunoprecipitation, RNAi, immunocytochemistry and tet-on induction. The regulation of p53 downstream genes was explored by gel mobility shifting assay, RT-PCR, Western blot analysis, immunocytochemistry, and luciferase reporter gene assays. The apoptosis was examined using cell flow cytometry and Hoechst staining.
PP-1 and/or PP-2A can directly dephosphorylate p53 at multiple residues including Ser-15, Ser-20, Ser-37, Ser-46, etc. Dephosphorylation of p53 at these sites distinctly modulates its ability for DNA binding and thus changes its control over a panel of downstream genes including p21, PCNA, Bcl-2, Bax, Bak, etc involved in regulation of cell proliferation, differentiation, senescence and apoptosis.
One of the major mechanisms for PP-1 to promote survival of lens epithelial cells and thus to maintain transparency of the ocular lens is to negatively regulate the function of p53, which plays a key role in controlling both lens development and cataractogenesis.
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