Purchase this article with an account.
Z Faghiri, PK Mukherjee, NG Bazan; Subcellular Distribution of Protein Kinase C Alpha in Human Retinal Pigment Epithelial Cells using PKC-Alpha-GFP Fusion Protein . Invest. Ophthalmol. Vis. Sci. 2002;43(13):2426.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Purpose:The protein kinase C (PKC) family includes eight subspecies in the retina, six of which (α, ß1, ß2, δ, ϵ, E) are expressed in retinal pigment epithelial cells (RPE). PKC isoforms display different patterns of subcellular localization. Subcellular localization of PKC isoforms is believed to play an important role in their functions. PKCα translocates to the nucleus in response to physiological stimuli as well as to exogenous ligands such as phorbol esters. To investigate PKCα's subcellular distribution in RPE cells we used a fusion protein consisting of PKCα and the green fluorescent protein (GFP). Methods:In this study we transfected (DOTAP method) ARPE-19 cells with PKCα-GFP fusion protein. Four hours later, transfected cells were fed with normal medium and incubated 10-12 hours before being serum-starved. The cells were serum-starved 8 hours and challenged with either TPA (50 nM) or TNF-α (10ng/ml). Results:The results showed that TPA and TNF-α both induced PKCα-GFP expression in RPE cells. The initial expression was detected in the cytoplasmic region. The GFP fluorescence moved to the perinuclear region within 1-2 hours when induced with physiologic concentration of TPA (50 nM). On the other hand it took 5-10 minutes with 200-250 nM TPA. Stimulation for at least 30-45 minutes with TNFα is required for an increased expression of PKCα in the cytoplasm without obvious translocation to the nucleus. Conclusion:The localization profile of PKCα in ARPE-19 cells as revealed with the GFP fusion protein in this study indicates another degree of heterogeneity in PKC response to ligands. Since in RPE cells PKCα could be resident or translocated to the nucleus, involvement of this enzyme in signaling that modulates nuclear protein phosphorylation, which in turn regulates gene expression, is an important step in RPE survival. Supported by NIH EY05121.
This PDF is available to Subscribers Only