title: Identification of protein phosphatase 2C and confirmation of other protein phosphatases in the ocular lenses.
abstract: 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.
authors.affiliation: Faculty of Pharmaceutical Sciences, Teikyo University, 1090-1, Suwarashi, Sagamiko Tsukui, Kanagawa, Japan. io-umeda@pharm.teikyo-u.ac.jp
authors.forename: I Ogihara
authors.initials: IO
authors.lastname: Umeda
authors.forename: H
authors.initials: H
authors.lastname: Nakata
authors.forename: H
authors.initials: H
authors.lastname: Nishigori
mesh_terms: D000818:Animals; D002642:Chick Embryo; D002850:Chromatography, Gel / Q000379:methods; D003459:Crystallins / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000378:metabolism; D007908:Lens, Crystalline / Q000201:enzymology*; D008970:Molecular Weight; D019319:Okadaic Acid / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010766:Phosphorylation; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism
chemical_list: D003459:Crystallins; D004791:Enzyme Inhibitors; D011506:Proteins; D019319:Okadaic Acid; D010749:Phosphoprotein Phosphatases; D017027:Protein Tyrosine Phosphatases