Chandrasekher Gudiseva, Sailaja Dasetty
Department of Ophthalmology and Neuroscience Center, Louisiana State University Health Sciences Center, New Orleans, LA 70112, USA.
Curr Eye Res. 2004 Feb;28(2):135-44. doi: 10.1076/ceyr.28.2.135.26232.
Protein tyrosine phosphorylation is an important event in the cell signal transduction process. Phosphatidylinositol-3 kinase (PI-3K) is an intracellular signal mediator and plays a key role in many cellular functions. In this study we have examined the changes in lens protein tyrosine phosphorylation and its impact on phosphatidylinositol 3-kinase (PI-3K) signaling during selenite cataract development.
Cataract was induced in 10 days old rat pups by a single sub-cutaneous injection of sodium selenite (30 microM/Kg body weight) and lenses were collected at different stages of cataract development. Immunoprecipitation and Western immunoblotting were employed to determine protein tyrosine phosphorylation, PI-3K activity and protein in lens cell extracts. Tyrosine kinase activity in lens membrane preparations was assayed in the presence of a synthetic substrate peptide and [32P]ATP.
Protein tyrosine phosphorylation in the lens was disrupted before the onset of cataract. A decrease in tyrosine phosphorylation of lens proteins was observed within 2-3 days of selenite injection (pre-cataract stage). The effect was much more prominent with the progression of cataract. The decrease in protein tyrosine phosphorylation correlated with the decrease in tyrosine kinase activity associated with the lens membrane fraction. Stimulation of normal rat lenses in organ culture with insulin and IGF-1 caused an increase in the phosphorylation of proteins, whose tyrosine phosphorylation status appeared to be diminished during cataract development. Insulin and IGF-1 also stimulated rat lens PI-3K activity. While there was no change in total PI-3K activity during the onset of cataract, the activity of PI-3K associated with tyrosine phosphorylated proteins decreased markedly in pre-cataract lenses. Further, the ability of IGF-1 to stimulate PI-3K activity was significantly reduced in lens epithelial cells treated with selenium.
These studies show that signaling events involving the protein tyrosine phosphorylation process and activation of PI-3K are altered during selenite cataract formation and implicate defects in signal transduction mechanisms as contributing factors in the development of cataract.
蛋白酪氨酸磷酸化是细胞信号转导过程中的一个重要事件。磷脂酰肌醇-3激酶（PI-3K）是一种细胞内信号介质，在许多细胞功能中起关键作用。在本研究中，我们检测了亚硒酸钠诱导白内障形成过程中晶状体蛋白酪氨酸磷酸化的变化及其对磷脂酰肌醇3激酶（PI-3K）信号传导的影响。
通过皮下注射亚硒酸钠（30微摩尔/千克体重）诱导10日龄大鼠幼崽发生白内障，并在白内障发展的不同阶段收集晶状体。采用免疫沉淀和western免疫印迹法测定晶状体细胞提取物中的蛋白酪氨酸磷酸化、PI-3K活性和蛋白含量。在合成底物肽和[32P]ATP存在的情况下，测定晶状体膜制剂中的酪氨酸激酶活性。
在白内障发生之前，晶状体中的蛋白酪氨酸磷酸化就已受到破坏。在注射亚硒酸钠后2-3天（白内障前期），观察到晶状体蛋白酪氨酸磷酸化减少。随着白内障的进展，这种效应更为明显。蛋白酪氨酸磷酸化的减少与晶状体膜部分相关的酪氨酸激酶活性的降低相关。在器官培养中用胰岛素和IGF-1刺激正常大鼠晶状体，导致蛋白磷酸化增加，而这些蛋白的酪氨酸磷酸化状态在白内障发展过程中似乎有所降低。胰岛素和IGF-1也刺激大鼠晶状体PI-3K活性。虽然在白内障发生初期总PI-3K活性没有变化，但在白内障前期晶状体中，与酪氨酸磷酸化蛋白相关的PI-3K活性明显降低。此外，在用硒处理的晶状体上皮细胞中，IGF-1刺激PI-3K活性的能力显著降低。
这些研究表明，在亚硒酸钠诱导白内障形成过程中，涉及蛋白酪氨酸磷酸化过程和PI-3K激活的信号事件发生了改变，提示信号转导机制缺陷是白内障发生的促成因素。
