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Z. Faghiri, N.G. Bazan; Proteasome-Mediated PAF-Induced Down-Regulation of PKC in ARPE-19 Cells . Invest. Ophthalmol. Vis. Sci. 2003;44(13):389.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Protein kinases C (PKC) are key signaling isozymes implicated in physiologic and pathophysiologic processes. PKC α, ß, and δ are expressed in RPE. The messenger platelet-activating factor (PAF) has been implicated in RPE cell functions. Here we tested the hypothesis that PAF promotes translocation and down-regulation of PKCα in the RPE cell line ARPE-19. Methods: We studied expression and translocation patterns of PKCα-GFP fusion protein by fluorescence microscopy before and after cell stimulation by either PMA or PAF. PKCα-GFP accumulation in Golgi was examined by BODIPY TR ceramide staining, pretreatment of cells with a Golgi inhibitor, and immunostaining of cells with antibodies to p230 and GM130. Translocation of PKCα was confirmed by Western blot analysis of cytosolic and membrane fractions of ARPE-19. Down-regulation of PKCα and the role of proteasomes in its degradation were assayed by Western blot of whole-cell lysates of ARPE-19 stimulated with PAF with or without proteasome inhibitor lactacystin or MG-132. Results: PKCα was constitutively expressed in ARPE-19 cells with a rather diffuse cytoplasmic distribution. Upon stimulation with either PMA or PAF, PKCα was translocated to the plasma membrane and later accumulated within the Golgi as shown by BODIPY TR ceramide staining and immunostaining with antibodies to p230 and GM130. PAF antagonists BN50739, CV3988, or BN52021 prevented translocation of PKCα to Golgi. PLC inhibitor U-7312021 abolished PAF-induced PKCα-GFP translocation to the Golgi, indicating a G-protein-coupled receptor. Golgi inhibitor Brefeldin A prevented accumulation of PKCα in Golgi without affecting its membrane relocalization. PKCα activation is required for PAF-induced translocation to the Golgi, since pretreatment with PKC inhibitor calphostin prevented this translocation. Western blots of nontransfected ARPE-19 cells also showed translocation of constitutive PKCα from cytoplasm to membrane and down-regulation of its expression after stimulation by either PMA or PAF. Proteasome inhibitors prevented the depletion of PKCα in response to PAF or PMA. Conclusion: PAF induces activation and translocation of PKCα to the cytoplasmic membrane and Golgi through a G-protein-coupled receptor-specific mechanism involving the PLC-DAG pathway for PKCα activation. This activation and translocation leads to proteasome-dependent degradation of PKCα, suggesting the involvement of the ubiquitination pathway in degradation of PKCα in ARPE-19. These signaling events may be critical during RPE oxidative stress, when PAF production is enhanced. Supported by NIH EY05121
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