Antiapoptotic Mechanism of Protein Phosphatase-1 in Lens

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DESCRIPTION: Protein phosphorylation and dephosphorylation play an essential role in regulating various cellular functions in the ocular lens. While the functions of protein serine/threonine kinases have been well-studied, much less is known about the functions of the corresponding phosphatases in lens system. For this reason, we have studied the protein serine/threonine phosphatases-1 (PP-1) and - 2A (PP-2A) in the past several years. Our results have shown that in human, rat and bovine lenses, both PR-1 and PP-2A are expressed with PP-1 as a major serine/threonine phosphatase. Functionally, we have shown that PP-1 is necessary to maintain survival of human, rabbit and rat lens epithelial cells. Mechanistically, PP-1 appears to suppress apoptosis through dephosphorylation of p53 and Rb, both of which play key roles in lens development. Inactivation of their function leads to microphthalmia or cataractogenesis. In addition, p53 is an important transcription factor. By regulating different target genes, it controls both cell cycle and apoptosis. Inhibition of PP-1 activity by okadaic acid or calyculin A causes hyperphosphorylation of p53 and Rb, followed by activation of the apoptotic program in which p53 plays a critical role as demonstrated from the following results. First, inhibition of PP-1 by okadaic acid and calyculin A induces strong phosphorylation of p53 followed by apoptosis. Second, silence of p53 expression with RNAi greatly attenuates apoptosis induced by okadaic acid or calyculin A. Finally; inhibition of PP-1 causes Rb hyperphosphorylation followed by upregulation of p53 and its target gene, bax before initiation of apoptosis by okadaic acid. Based on these results, we hypothesize that the major functional mechanism for PP-1 to promote survival of lens epithelial cells occurs through modulating Rb phosphorylation status to upregulate 53 and also via altering p53 phosphorylation status to control its transcriptional activity. The present proposal aims at delineating 1) the PP-1 dephosphorylation sites in p53; 2 ) the effect of PP-1 dephosphorylation on p53 function and 3) the mechanism by which Rb hyperphosphorylation induces p53 upregulation. Experimentally, human and rabbit lens epithelial ceils, and p53-/-mouse lens epithelial cells will be used as testing systems. Various molecular and cellular biology techniques will be used to conduct the proposed studies. Considering the critical roles of p53 and Rb in lens development and pathology, our proposed studies will reveal novel information on their functional pathways and also on the functional mechanisms of PP-1, which will contribute to fundamental aspects of lens development and pathology.