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Two Peeling Methods for the Isolation of Photoreceptor Cell Compartments in the Mouse Retina for Protein Analysis
Published on: December 7, 2021
Selective relocalization and proteasomal downregulation of PKCalpha induced by platelet-activating factor in retinal
Zahra Faghiri1, Nicolas G Bazan
1LSU Neuroscience Center of Excellence and Department of Ophthalmology, Louisiana State University Health Sciences Center School of Medicine, New Orleans, Louisiana 70112, USA.
Purpose:
Protein kinases C (PKCs) are key cell-signaling mediators in retinal physiology and pathophysiology. The cellular localization of PKC isoforms is important in defining their activity and specificity; the present study investigated the modulatory potential of the proinflammatory mediator platelet-activating factor (PAF) on the subcellular distribution of PKCalpha, beta, and delta isotypes.
Methods:
This study used real-time visualization of green fluorescent protein fused to PKCalpha, beta, or delta in the human retinal pigment epithelial (RPE) cell line ARPE-19.
Results:
In PAF-stimulated ARPE-19 cells, PKCalpha translocated to the plasma membrane and then colocalized with Golgi markers p230 and GM130; PKCbeta translocated to the plasma membrane but not to the Golgi; and PKCdelta translocated to the Golgi. Pretreatment with PKC inhibitor calphostin C abolished the PAF-induced translocation of PKCalpha to the plasma membrane or to the Golgi, but the Golgi inhibitor Brefeldin A only prevented the accumulation of PKCalpha in Golgi, without affecting its membrane relocalization. PAF promoted depletion of PKCalpha and delta isoforms but not that of PKCbeta. Proteasome inhibitors lactacystin and MG-132 prevented the PAF-induced depletion of PKCalpha, but the inhibitor of lysosomal proteolysis E-64d was ineffective in rescuing PKCalpha.
Conclusions:
These results suggest that the PAF-induced downregulation of PKCalpha occurs principally through the proteasomal pathway. This remarkable PAF-mediated diversity in PKC translocation and downregulation highlights the significance of isotype-specific PKC activation in signaling pathways in ARPE-19 cells. These signaling events may be critical during RPE responses to oxidative stress, inflammation, and retinal degenerations, when PAF production is enhanced.
