Abstract
Purpose: :
Protein phosphorylation and dephosphorylation have an essential role in the regulatory events in visual signaling. We previously found that phosphorylation of several proteins in the retinal pigment epithelial cells (RPE) is involved in anti-apoptotic signaling in oxidative stress including light exposure. We hypothesize that phosphorylation of key proteins may protect the RPE against light-induced damage.
Methods: :
Proteins from excised bovine eyes exposed to light for 1 hr or kept in the dark were separated by 2D SDS-PAGE. RPE proteins in the light vs. dark were visualized by silver staining. Serine- or tyrosine-specific phosphorylations were visualized by 2D Western blotting using anti-phosphoserine and anti-phosphotyrosine antibodies, respectively. To overcome the difficulties in detecting less abundant, low stoichiometric phosphoproteins in the RPE, an enrichment procedure was used.
Results: :
Phosphorylations at tyrosine residue of beta crystalline A3 and A4 in the light, were dramatically increased as shown by phosphortyrosine-specific Western blotting. Enzymes related to phosphorylation or energy metabolism, including Ser/Thr protein kinase 4 and ATP, were up-regulated in the dark. After enrichment of phosphoproteins, major phosphoproteins of MW 15-25 kDa were identified as belonging to the crystallin family including beta crystalline S and zeta crystalline. Other chaperones, cytoskeletal proteins or proteins related to energy metabolism are up-regulated in the dark from 1.5- to 6-fold compared to proteins in the light. Tandem mass spectrometry revealed specific phosphorylation sites of crystallin alphaA and alphaB.
Conclusions: :
We found that specific phosphorylations of the crystallin family were up-regulated in the RPE under light exposure. Phosphorylation of beta crystalline may suggest its role as a potential anti-apoptotic chaperone function in the RPE. The detailed results of phosphorylated proteins in the RPE provide a basis for understanding the molecular mechanisms of signal transduction and anti-apoptosis in the light. Our data support the likelihood that crystallin phosphorylations represent an important RPE shield from physiologic and pathophysiologic light-induced oxidative injury.
Keywords: crystallins • phosphorylation • retinal pigment epithelium