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Epidermal Growth Factor‐Induced Phosphorylation Changes in Rat Inner Medullary Collecting Duct
Author(s) -
Chou ChungLin,
Knepper Mark A
Publication year - 2019
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2019.33.1_supplement.750.4
Subject(s) - phosphorylation , epidermal growth factor , mapk/erk pathway , phosphopeptide , microbiology and biotechnology , chemistry , protein kinase b , protein kinase a , pi3k/akt/mtor pathway , protein phosphorylation , kinase , signal transduction , biology , biochemistry , receptor
Epidermal growth factor (EGF) is a potent mitogenic agent promoting cell proliferation, differentiation and survival. The kidney thick ascending limb of Henle's loop and distal convoluted tubule were identified as major sites of EGF synthesis. Downstream from these sites, EGF has been shown to modulate the osmotic water permeability of the collecting duct. The present study sought to determine the EGF‐elicited phosphorylation changes in inner medullary collecting duct (IMCD). Rat IMCD suspensions were treated with 1 μM EGF or vehicle for 30 min. Samples from three replicates were analyzed by TMT isobaric labeling quantitative protein mass spectrometry after phosphopeptide enrichment. A total of 12260 phosphopeptides with unique phosphorylation sites were quantified in EGF‐treated IMCD, with 78 peptides increased and 100 peptides decreased in phosphorylation based on dual statistical criteria. The enriched motif of upregulated phosphopeptides is consistent with activation of proline‐directed kinases, with a unique motif of [S/T]‐P‐K. DAVID analysis of 178 peptides (in 142 proteins) that underwent significant phosphorylation change shows ErbB as the prominent KEGG signaling pathway, confirming that EGF activates ERK mitogen‐activated protein kinase (MAPK), Mapk1 at T183/Y185 and Mapk3 at T203/Y205, via the canonical Ras‐Raf‐MEK pathway. There are five proteins (Atf2, eIF4ebp1, Raf1, Slc9a1, SP1) with phosphorylation sites that are known ERK targets. Among cytosolic docking proteins that can direct the intracellular effects of EGF, several proteins that underwent phosphorylation changes are associated with the PI3k‐Akt‐Mtor pathway, which regulates translation initiation complex eIF4F formation and the translation elongation processes (Ago2, eIF2ak3, eIF2ak4, eIF4ebp1, Eef2, Eef2k, Fbxo6, Pdcd4). In addition, several proteins associated with the Plcγ calcium signaling pathway were significantly altered in phosphorylation. These include Plcg1, Plcb4, Itpr2, Camk2g, Ppp3cb (calcineurin B) and Nos1. EGF had no significant effect on known vasopressin‐regulated phosphorylation sites of the aquaporin‐2 water channel (AQP2) (S256, S261, S264, S269) or the vasopressin‐regulated urea channel (UT‐A1) (S84, S486, S499). However, several modulators of cytoskeleton rearrangement (Cfl1, Fnbp1, Macf1, Parva, Plec, Sipa1l1, Specc1l, Trip10) and trafficking proteins (Dnajc5, Epn3, Mvb12a, Stx6, Ston2, Sytl5, Trak2) underwent phosphorylation changes. EGF also induced phosphorylation changes in several RNA polymerase II‐associated nuclear proteins (Cdk12, Gtf2f1, Leo1, Iws1, Rbm5, Rpap3, SP1). This data can be useful in further investigation of the EGF's action in renal collecting duct cells. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .