Antiapoptotic Mechanism of Protein Phosphatase-1 in Lens

描述：蛋白质磷酸化和去磷酸化在调节眼透镜的各种细胞功能中起重要作用。虽然蛋白丝氨酸/苏氨酸激酶的功能已被充分研究，但对透镜系统中相应的磷酸酶的功能知之甚少。为此，我们在过去几年中研究了蛋白丝氨酸/苏氨酸磷酸酶-1（PP-1）和-2A（PP-2A）。我们的研究结果表明，在人类，大鼠和牛的晶状体，PR-1和PP-2A的表达与PP-1作为一个主要的丝氨酸/苏氨酸磷酸酶。在功能上，我们已经表明PP-1是维持人、兔和大鼠透镜上皮细胞存活所必需的。从机制上讲，PP-1似乎通过p53和Rb的去磷酸化来抑制细胞凋亡，p53和Rb两者在透镜发育中起关键作用。它们的功能失活导致小眼球或白内障。此外，p53是一个重要的转录因子。通过调节不同的靶基因，它控制细胞周期和凋亡。通过冈田酸或calyculin A抑制PP-1活性导致p53和Rb的过度磷酸化，随后激活凋亡程序，其中p53起关键作用，如以下结果所示。首先，通过冈田酸和calyculin A抑制PP-1诱导p53的强烈磷酸化，随后细胞凋亡。第二，用RNAi沉默p53表达大大减弱了冈田酸或calyculin A诱导的细胞凋亡。最后，PP-1的抑制导致Rb过度磷酸化，随后在冈田酸引发细胞凋亡之前上调p53及其靶基因bax。基于这些结果，我们推测PP-1促进透镜上皮细胞存活的主要功能机制是通过调节Rb磷酸化状态以上调p53，以及通过改变p53磷酸化状态以控制其转录活性。本研究的目的是阐明1）PP-1在p53中的去磷酸化位点; 2）PP-1去磷酸化对p53功能的影响; 3）Rb过度磷酸化诱导p53上调的机制。在实验上，人和兔透镜上皮细胞以及p53-/-小鼠透镜上皮细胞将用作测试系统。将使用各种分子和细胞生物学技术进行拟定研究。考虑到p53和Rb在透镜发育和病理学中的关键作用，我们的研究将揭示其功能途径和PP-1功能机制的新信息，这将有助于透镜发育和病理学的基本方面。

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.