The specific function of each crystallin, particularly -crystallin in the RPE, is still largely unknown, although levels of B-crystallin are increased after heat shock and oxidative stress [28], and B-crystallin immunoreactivity has been found in both rod outer segments and the RPE after light exposure [25]. and A4. The most abundant light-induced up-regulated phosphoproteins were crystallins of 1525-kDa, including beta crystallin S and zeta crystallin. Phosphorylation of beta crystallin suggests an anti-apoptotic chaperone function 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 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. Keywords: phosphoproteome, retinal pigment epithelium, crystalline, 2D electrophoresis, mass spectrometry == Introduction == Phosphorylation of specific amino acids is an important modulator of protein function that regulates protein subcellular localization, proteinprotein interactions, conformational changes, and signal transduction [1]. The change in charge that accompanies phosphorylation SB-505124 HCl creates a switch mechanism, allowing reversible phosphorylation to modify intracellular signaling in response to specific microenvironmental conditions and thereby act as a basic survival tool. Phosphorylation is mediated by the action of protein kinases, which add phosphate to serine, threonine and tyrosine residues at a relative ratio of 1000 to 100 to 1 [2]. This differential modification reflects the distinct pKa values of these amino acids, and the distinct properties of their respective specific Ser/Thr kinases and Tyr kinases. Equally important in the dynamic regulation of protein phosphorylation is the activity of specific protein phosphatases. Phosphorylation may also interact with other post-translational modifications such as ubiquitination through positive or negative SB-505124 HCl feedback mechanisms, a crosstalk that adds an additional dimension to the combinatorial possibilities of this switch mechanism [3]. The retinal pigment epithelium (RPE) is a cellular monolayer that provides crucial support to the retina, and transports oxygen, nutrients, and vitamin A. RPE is essential to the visual cycle responsible for maintaining sight, which is in turn mediated Oaz1 by the continuous regeneration of 11-cis-retinaldehyde as a rhodopsin chromophore. Loss of RPE cells is thought to be an early event in retinal degeneration [6]. Many critical reactions involved in RPE responses to environmental changes, such as oxidative stress, light exposure and changes in nutrients, especially those involving a rapid response, are mediated by phosphorylation events rather than by changes at the transcriptional level [7, 8]. For example , we have demonstrated that oxidative stress, such as bright light exposure, increases the phosphorylation of Janus kinase 2 (Jak2), which is the downstream regulator of erythropoietin previously [9]. Typically, phosphoproteins have been studied using chemical and affinity-based methods [4, 5]. However , the rapid and dynamic nature of the underlying changes and the low abundance of phosphoproteins reflecting their substoichiometry present challenges to the quantitative study of the phosphoproteome. Thus, the isolation of the phosphoproteome or phosphopeptidome represents a potentially advanced step in this analysis. In this study, we introduced a system-wide, unbiased, and high-throughput approach to investigate global phosphoproteomes of light- and dark-exposed RPE using a phosphoproteome enrichment method. This detailed analysis of the phosphoproteome provides a basis for understanding the molecular mechanisms of phosphorylation-dependent signaling in the RPE. == Materials and Methods == == Preparation of RPE proteins == Fresh bovine eyes were obtained from a local abattoir (Brown Packing Company, Gaffney, SC) immediately after excision from the animal. The post-mortem stability, procedures for preparing RPE cells and general proteomic techniques have been described in detail previously [1115]. Briefly, bovine eyes were opened 5 mm posterior to the limbus, and the vitreous and retina were removed. After washing with a phosphate-buffered saline (PBS), eye-cups were incubated in 0. 25% trypsin in Dulbeccos minimum essential medium (DMEM; Gibco, Grand Island, NY) for 60 minutes at 37C. RPE cells were collected under a dissecting microscope using a Pasteur pipette. After adding the culture medium (DMEM/F12) containing 10% fetal bovine serum (FBS), cells were centrifuged and resuspended in a culture medium, and plated into 6-well plates (Nunc). Second passage cells were used for experiments. Cells were incubated under dark or light (700 lux) conditions in a humidified chamber at room temperature SB-505124 HCl SB-505124 HCl for 1 hour. RPE cells were incubated in lysis buffer consisting of 20 mM Tris-Cl (pH 7. 4), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2 . 5 mM sodium pyrophosphate, 1 mM Na3VO4, 1 mM NaF,.