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.

Fig. 1. Analysis of light-associated changes in protein tyrosine/serine phosphorylation by 2D gel electrophoresis

Primary bovine RPE cells were incubated under light or dark conditions for 1 hour at room temperature. The retinal pigment epithelium proteins were isolated and analyzed by 2D electrophoresis and western blotting using anti-pSer and anti-pTyr antibodies. (A) Light-treated RPE cells. Proteins were separated by 2D-electrophoresis and visualized by silver staining. Phosphorylated proteins were visualized by pSer (B) or pTyr (C) western blot. (D) Dark-induced proteins were visualized by silver staining, pSer (E), and pTyr (F) western blot, respectively.

Fig. 2. Analysis of the phospho-enriched RPE proteome

Total RPE proteins were enriched by affinity purification using a Ga³⁺ column. The phosphoproteome of light-induced (A) and dark-incubated control (B) RPE cells are shown. Proteins analyzed by mass spectrometry are shown in numbers. Identified proteins are listed in Table 2 and 3.

Fig. 2. Analysis of the phospho-enriched RPE proteome

Total RPE proteins were enriched by affinity purification using a Ga³⁺ column. The phosphoproteome of light-induced (A) and dark-incubated control (B) RPE cells are shown. Proteins analyzed by mass spectrometry are shown in numbers. Identified proteins are listed in Table 2 and 3.

Fig. 3. MALDI-TOF-TOF mass analysis of phosphoprotein-enriched RPE proteome

Phosphoproteins were analyzed by tandem mass spectrometry to identify phosphorylated amino acid residues. Mass spectra analyzed by MALDI-TOF-TOF shows amino acid sequence and phosphorylated serine residues. The peptide sequence and parental protein names are shown and phosphate groups on serine within the parent peptide sequence are denoted by pS. X axis represents mass to charge ratio (m/z) and Y axis shows relative ion intensity.

Fig. 4. Confirmation of phosphoproteins in the RPE by alkaline phosphatase treatment

The phosphoproteome of light-treated (A, B) and dark-incubated (C, D) RPE cells were split into two aliquots and incubated with alkaline phosphatase (+ AP) or without alkaline phosphatase (− AP). The samples were separated by 2D gel electrophoresis and proteins were visualized by Coomassie blue staining. In alkaline phosphatase-treated samples, phosphorylated protein spots were down-regulated or disappeared entirely. However, major phosphorylated spots were still remaining implying crystalline family might be resistant to non-specific phosphatase reaction.

Fig. 5