晶状体中蛋白磷酸酶2C的鉴定及其他蛋白磷酸酶的确认。

Umeda I Ogihara, Nakata H, Nishigori H

Faculty of Pharmaceutical Sciences, Teikyo University, 1090-1, Suwarashi, Sagamiko Tsukui, Kanagawa, Japan.

Exp Eye Res. 2004 Dec;79(6):385-92.

The reversible phosphorylation of proteins plays essential roles in regulating various cellular events, and is regulated by the opposing actions of protein kinases and protein phosphatases. Protein kinases in the lens system have been well studied, but very little is known about lens protein phosphatases. Protein phosphatases can be divided several families, such as protein phosphatase types 1, 2A, 2B and 2C (PP1, PP2A, PP2B and PP2C) and protein tyrosine phosphatases (PTP). In this study we evaluated what kinds of protein phosphatases are present in the lens by using various specific substrates and inhibitors. Samples were prepared from lenses of 17-day-old chick embryos, and fractionated by high-resolution gel permeation column chromatography, then the fractions were assayed for phosphatase activities. The results with 32P-labeled glycogen phosphorylase A, okadaic acid and inhibitor-1, which are a specific substrate and inhibitors of PP1 and/or PP2A, showed that PP1 activities were present in the 500-, 115- and 45-kDa fractions of the lens protein. The 115-kDa fraction also contained PP2A activity. By using a phosphothreonine-containing peptide as a substrate, three peaks of phosphatase activities were found at around 115, 55 and 35 kDa. Based on their response to various phosphatase inhibitors and their metal dependency, the fractions of 115 and 35 kDa were concluded to contain PP2A, while the 55-kDa fraction contained PP2C. Immunoblot using specific antibodies against PP1, PP2A and PP2C confirmed that each fraction above contained corresponding protein phosphatases as proteins. When a phosphotyrosine-containing peptide substrate was examined at pH 7.4, we observed a major peak at 500 kDa, which was presumed to contain receptor-like PTP(s). On the other hand, at pH 5.5, we observed a peak of 18 kDa, which was confirmed to contain a low-molecular-weight PTP. These protein phosphatases have recently been suggested to be involved in stress response and apoptosis. Their physiological roles in the lens are of much interest.

蛋白质的可逆磷酸化在调节各种细胞活动中起着至关重要的作用，并且受蛋白激酶和蛋白磷酸酶的相反作用调控。晶状体系统中的蛋白激酶已得到充分研究，但对于晶状体蛋白磷酸酶却知之甚少。蛋白磷酸酶可分为几个家族，如蛋白磷酸酶1型、2A型、2B型和2C型（PP1、PP2A、PP2B和PP2C）以及蛋白酪氨酸磷酸酶（PTP）。在本研究中，我们通过使用各种特异性底物和抑制剂来评估晶状体中存在哪些类型的蛋白磷酸酶。从17日龄鸡胚的晶状体中制备样品，并通过高分辨率凝胶渗透柱色谱进行分级分离，然后对各馏分进行磷酸酶活性测定。使用32P标记的糖原磷酸化酶A、冈田酸和抑制剂-1（它们分别是PP1和/或PP2A的特异性底物和抑制剂）所得到的结果表明，PP1活性存在于晶状体蛋白的500 kDa、115 kDa和45 kDa馏分中。115 kDa馏分中也含有PP2A活性。通过使用含磷酸苏氨酸的肽作为底物，在约115 kDa、55 kDa和35 kDa处发现了三个磷酸酶活性峰。根据它们对各种磷酸酶抑制剂的反应及其对金属的依赖性，得出115 kDa和35 kDa馏分含有PP2A，而55 kDa馏分含有PP2C的结论。使用针对PP1、PP2A和PP2C的特异性抗体进行免疫印迹证实，上述每个馏分都含有相应的蛋白磷酸酶作为蛋白质。当在pH 7.4条件下检测含磷酸酪氨酸的肽底物时，我们在500 kDa处观察到一个主峰，推测其含有受体样PTP。另一方面，在pH 5.5时，我们观察到一个18 kDa的峰，经证实其含有低分子量PTP。最近有人提出这些蛋白磷酸酶参与应激反应和细胞凋亡。它们在晶状体中的生理作用备受关注。