Phosphoproteomic response to epidermal growth factor in native rat inner medullary collecting duct

Abstract

Epidermal growth factor (EGF) has important effects in the renal collecting duct to regulate salt and water transport. To identify elements of EGF-mediated signaling in the rat renal inner medullary collecting duct (IMCD), we carried out phosphoproteomic analysis. Biochemically isolated rat IMCD suspensions were treated with 1 µM of EGF or vehicle for 30 min. We performed comprehensive quantitative phosphoproteomics using tandem mass tag (TMT)-labeling of tryptic peptides followed by protein mass spectrometry. We present a data resource reporting all detected phosphorylation sites and their changes in response to EGF. For a total of 29,881 unique phosphorylation sites, 135 sites were increased and 119 sites were decreased based on stringent statistical analysis. The data are provided to users at https://esbl.nhlbi.nih.gov/Databases/EGF-phospho/. The analysis demonstrated that EGF signals through canonical EGF pathways in the renal IMCD. Analysis of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways in which EGF-regulated phosphoproteins are over-represented in native rat IMCD cells confirmed mapping to RAF-MEK-extracellular signal-regulated kinase (ERK) signaling but also pointed to a role for EGF in the regulation of protein translation. A large number of phosphoproteins regulated by EGF contained PDZ domains that are key elements of epithelial polarity determination. We also provide a collecting duct EGF-network map as a user-accessible web resource at https://esbl.nhlbi.nih.gov/Databases/EGF-network/. Overall, the phosphoproteomic data presented provide a useful resource for experimental design and modeling of signaling in the renal collecting duct.NEW & NOTEWORTHY EGF negatively regulates transepithelial water and salt transport across the kidney collecting duct. This study identified phosphoproteins affected by EGF stimulation in normal rat collecting ducts, providing insights into global cell signaling mechanisms. Bioinformatic analyses highlighted enhanced canonical ERK signaling alongside a diminished activity in the PI3K-Akt pathway, which is crucial for cell proliferation and survival. This EGF response differs somewhat from prior studies where both pathways were prominently activated.

Keywords: growth factor; kidney; mass spectrometry; phosphorylation; protein kinase.