Phosphatidylinositol 3-kinase in bovine lens and its stimulation by insulin and IGF-1.
PURPOSE: To identify and characterize phosphatidylinositol 3-kinase ( PI-3K) in the lens and to study its involvement as a signal mediator in lens epithelial cells exposed to insulin and insulin-like growth factor (IGF)-1, which are known to induce lens epithelial cell proliferation and differentiation into fiber cells. METHODS: Concentric fiber cell layers from single bovine lens were prepared by dissolution in buffer. PI-3K activity in capsule-epithelium and fiber cell layers was determined after immunoprecipitation with antibodies against p85, the regulatory subunit of PI-3K. High-performance liquid chromatography on an ion exchange column (Partisil-SAX; Whatman, Maidstone, United Kingdom) was used to identify PI-3K reaction products. Cultured bovine lens epithelial cells were stimulated with insulin or IGF-1, and PI-3K activity was determined after immunoprecipitation with antibody against phosphotyrosine. Association of p85 with other proteins after stimulation was determined in anti-p85 immunoprecipitates by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western immunoblot analysis using anti-phosphotyrosine antibody. RESULTS: PI-3K activity was found in both lens epithelial cells and fiber cells. The highest specific activity was found in the capsule-epithelium, but there was considerable activity in other fiber cell layers. Insulin and IGF-1 stimulated the PI-3K activity in epithelial cells in culture by more than 100%, and activation of the enzyme resulted in tyrosine phosphorylation of the p85 subunit. After stimulation, the p85 subunit of PI-3K was associated with 100- and 180-kDa tyrosine phosphorylated proteins. CONCLUSIONS: The activation of PI-3K and its association with specific tyrosine- phosphorylated proteins may be important in insulin and IGF-1 signal transduction in lens epithelial cells. The presence of significant PI-3K activity throughout the lens further suggests that this signal transduction enzyme is sustained in fiber cells.[1]References
- Phosphatidylinositol 3-kinase in bovine lens and its stimulation by insulin and IGF-1. Chandrasekher, G., Bazan, H.E. Invest. Ophthalmol. Vis. Sci. (2000) [Pubmed]
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