title: Light-induced phosphorylation of crystallins in the retinal pigment epithelium.
abstract: Protein phosphorylations have essential regulatory roles in visual signaling. Previously, we found that phosphorylation of several proteins in the retina and retinal pigment epithelium (RPE) is involved in anti-apoptotic signaling under oxidative stress conditions, including light exposure. In this study, we used a phosphoprotein enrichment strategy to evaluate the light-induced phosphoproteome of primary bovine RPE cells. Phosphoprotein-enriched extracts from bovine RPE cells exposed to light or dark conditions for 1h were separated by 2D SDS-PAGE. Serine and tyrosine phosphorylations were visualized by 2D phospho Western blotting and specific phosphorylation sites were analyzed by tandem mass spectrometry. Light induced a marked increase in tyrosine phosphorylation of beta crystallin A3 and A4. The most abundant light-induced up-regulated phosphoproteins were crystallins of 15-25 kDa, including beta crystallin S and zeta crystallin. Phosphorylation of beta crystallin suggests an anti-apoptotic chaperone function of crystallins in the RPE. Other chaperones, cytoskeletal proteins, and proteins involved in energy balance were expressed at higher levels in the dark. A detailed analysis of RPE phosphoproteins provides a molecular basis for understanding of light-induced signal transduction and anti-apoptosis mechanisms. Our data indicates that phosphorylation of crystallins likely represents an important mechanism for RPE shielding from physiological and pathophysiological light-induced oxidative injury.
authors.affiliation: Department of Ophthalmology, University of South Carolina, Columbia, SC 29208, USA.
authors.forename: Hyunju
authors.initials: H
authors.lastname: Lee
authors.forename: Hyewon
authors.initials: H
authors.lastname: Chung
authors.forename: Sung Haeng
authors.initials: SH
authors.lastname: Lee
authors.forename: Wan Jin
authors.initials: WJ
authors.lastname: Jahng
mesh_terms: D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D002417:Cattle; D003459:Crystallins / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D008027:Light; D010766:Phosphorylation / Q000528:radiation effects; D010768:Phosphoserine / Q000378:metabolism; D019000:Phosphotyrosine / Q000378:metabolism; D020543:Proteome / Q000378:metabolism; D015203:Reproducibility of Results; D055213:Retinal Pigment Epithelium / Q000166:cytology / Q000378:metabolism* / Q000528:radiation effects*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D003459:Crystallins; D020543:Proteome; D010768:Phosphoserine; D019000:Phosphotyrosine; D000469:Alkaline Phosphatase