Protein phosphorylation in retinal pigment epithelium of Long-Evans and Royal College of Surgeons rats.
Cultures of retinal pigment epithelium (RPE) from normal Long-Evans (LE) and dystrophic Royal College of Surgeons (RCS) rats were incubated with 32P-orthophosphate and then phagocytically challenged with isolated rod outer segments (ROS) or polystyrene latex spheres (PSL). The 32P incorporation into individual proteins was quantified by image analysis of two-dimensional gel autoradiograms, and changes in phosphorylation were identified by comparison with unchallenged control cultures. Phosphorylation changes that were similar in response to either ROS or PSL were classified as nonspecific and omitted from further analysis; those associated solely with ROS exposure were classified as ROS specific and compared between the two strains. None of the 30 ROS-specific changes in protein phosphorylation identified in normal LE RPE were the same as in RCS RPE. However, unique ROS-specific changes in phosphorylation were observed in 13 RCS RPE proteins. Three RCS proteins showed ROS-specific decreases; ten showed ROS-specific increases. Six of these ten RCS proteins with increased phosphorylation showed ROS-specific decreases in LE RPE. No other correspondence in ROS-specific changes was found among the remaining LE or RCS RPE proteins, but several RCS proteins were phosphorylated at abnormal levels under control conditions. Even though ROS-induced changes in phosphorylation were aberrant in RCS RPE, their presence indicated that a ROS-specific transmembrane signal was generated after interaction with ROS. The abnormal increases and decreases observed in ROS-specific phosphorylation in the RCS suggested that the defect in ROS phagocytosis was associated with the misregulation or malfunction of both protein kinases and phosphatases.
Heth
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Berman-Gund Laboratory for the Study of Retinal Degenerations, Harvard Medical School, Massachusetts Eye and Ear Infirmary, Boston 02114.
Schmidt
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D000818:Animals; D004195:Disease Models, Animal; D015180:Electrophoresis, Gel, Two-Dimensional; D005136:Eye Proteins / Q000378:metabolism*; D008715:Methionine / Q000378:metabolism; D008863:Microspheres; D010587:Phagocytosis / Q000502:physiology; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D010857:Pigment Epithelium of Eye / Q000378:metabolism*; D051381:Rats; D011922:Rats, Mutant Strains; D012162:Retinal Degeneration / Q000378:metabolism*; D012374:Rod Cell Outer Segment / Q000378:metabolism; D013462:Sulfur Radioisotopes
D005136:Eye Proteins; D010761:Phosphorus Radioisotopes; D013462:Sulfur Radioisotopes; D008715:Methionine