Global discovery of high-NaCl-induced changes of protein phosphorylation

Abstract

High extracellular NaCl, such as in the renal medulla, can perturb and even kill cells, but cells mount protective responses that enable them to survive and function. Many high-NaCl-induced perturbations and protective responses are known, but the signaling pathways involved are less clear. Change in protein phosphorylation is a common mode of cell signaling, but there was no unbiased survey of protein phosphorylation in response to high NaCl. We used stable isotopic labeling of amino acids in cell culture coupled to mass spectrometry to identify changes in protein phosphorylation in human embryonic kidney (HEK 293) cells exposed to high NaCl. We reproducibly identify >8,000 unique phosphopeptides in 4 biological replicate samples with a 1% false discovery rate. High NaCl significantly changed phosphorylation of 253 proteins. Western analysis and targeted ion selection mass spectrometry confirm a representative sample of the phosphorylation events. We analyze the affected proteins by functional category to infer how altered protein phosphorylation might signal cellular responses to high NaCl, including alteration of cell cycle, cyto/nucleoskeletal organization, DNA double-strand breaks, transcription, proteostasis, metabolism of mRNA, and cell death.

PTGER2-β-Catenin Axis Links High Salt Environments to Autoimmunity by Balancing IFNγ and IL-10 in FoxP3+Regulatory T cells

“…6c, d). These data, along with the evidence from non-immune cells 48 , supports our hypothesis that the β-catenin/SGK1/Foxo1 axis is activated by high salt stimulation.…”

“…7a, b). Although a salinity stress sensor has not been fully described in mammalian cells, a number of pathways contributing to the salt stress response have been identified 48, 49 . Within these pathways, we focused on AKT kinase because it is well known to regulate β-catenin signaling via direct phosphorylation of β-catenin 50 or indirectly through GSK3β, which is a negative regulator of β-catenin 51 .…”

Parallel evolution and adaptation to environmental factors in a marine flatfish: Implications for fisheries and aquaculture management of the turbot (Scophthalmus maximus)

“…As mentioned above, two of these markers (7033_88 and 5397_68) are linked to several growth‐related loci (Robledo et al., 2016; Rodríguez‐Ramilo et al., 2014; Sánchez‐Molano et al., 2011), and also to some candidate genes associated with osmotic stress response (Norman et al., 2014; Ortells et al., 2012). Two other outlier loci reside close to genes related to osmoregulation: 1587_12 on LG13 near RAC1 and PRKCA (Di Ciano et al., 2002; Zhuang, Hirai, & Ohno, 2000), and 2921_40 on LG16 near NAP1L1 (Wang et al., 2014). …”

Database of osmoregulated proteins in mammalian cells

“…The other was from a study of changes in phosphorylation of proteins in HEK293 cells in response to hypertonicity (Wang et al. ). To curate the data, we initially organized the details about each study in an electronic spreadsheet ( Excel, Microsoft).…”

“…The first was from a study of changes in protein abundance in the nucleus of HEK293 cells in response to hypertonicity (Li et al 2012). The other was from a study of changes in phosphorylation of proteins in HEK293 cells in response to hypertonicity (Wang et al 2014). To curate the data, we initially organized the details about each study in an electronic spreadsheet (Excel, Microsoft).…”