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.