Phagocytic challenge induces changes in phosphorylation of retinal pigment epithelium proteins.
Changes in protein phosphorylation induced by phagocytic challenge were identified in cultured rat retinal pigment epithelium (RPE) following exposure to isolated rat rod outer segments (ROS) or to polystyrene latex microspheres (PSL). RPE phosphoproteins were characterized based on molecular weight and isoelectric point and 32P incorporation into phosphoproteins was quantified by digitized image analysis of two-dimensional gel autoradiograms. Changes in the phosphorylation of RPE proteins were determined by comparing 32P gel data from phagocytically challenged cultures with control cultures. ROS-specific changes were defined as those occurring only in response to ROS while nonspecific changes were those associated with either ROS or PSL phagocytosis. A parallel study was conducted to identify those proteins which also show increased phosphorylation following protein kinase C (PKC) activation by phorbol-12-myristate-13-acetate. ROS-specific increases in the phosphorylation of 2 RPE proteins were found, 1 of which also showed an increase with PKC activation. Nonspecific increases included the phosphorylation of 11 RPE proteins, 10 of which were also phosphorylated with PKC activation. ROS-specific decreases were observed in 12 RPE phosphoproteins while 3 proteins showed nonspecific decreases in their phosphorylation. These findings demonstrate that phagocytic challenge of the RPE with either specific or nonspecific particles is linked to the activation of phosphatases and kinases and that activation of PKC may play a role in phagocytosis of both particle types. The identification of two distinct groups of changes in phosphorylation supports the hypothesis that different pathways exist for phagocytosis of ROS-specific and nonspecific particles by the RPE.
Heth
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Berman-Gund Laboratory for the Study of Retinal Degenerations, Harvard Medical School, Massachusetts Eye and Ear Infirmary, Boston.
Schmidt
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D000818:Animals; D001345:Autoradiography; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D015180:Electrophoresis, Gel, Two-Dimensional; D004789:Enzyme Activation / Q000187:drug effects; D005136:Eye Proteins / Q000378:metabolism*; D007840:Latex; D008863:Microspheres; D010587:Phagocytosis / Q000502:physiology*; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D010857:Pigment Epithelium of Eye / Q000502:physiology*; D011493:Protein Kinase C / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D012374:Rod Cell Outer Segment; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D005136:Eye Proteins; D007840:Latex; D010750:Phosphoproteins; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate