title: Phosphoproteomic profiling reveals vasopressin-regulated phosphorylation sites in collecting duct.
abstract: Protein phosphorylation is an important component of vasopressin signaling in the renal collecting duct, but the database of known phosphoproteins is incomplete. We used tandem mass spectrometry to identify vasopressin-regulated phosphorylation events in isolated rat inner medullary collecting duct (IMCD) suspensions. Using multiple search algorithms to identify the phosphopeptides from spectral data, we expanded the size of the existing collecting duct phosphoproteome database from 367 to 1187 entries. Label-free quantification in vasopressin- and vehicle-treated samples detected a significant change in the phosphorylation of 29 of 530 quantified phosphopeptides. The targets include important structural, regulatory, and transporter proteins. The vasopressin-regulated sites included two known sites (Ser-486 and Ser-499) present in the urea channel UT-A1 and one previously unknown site (Ser-84) on vasopressin-sensitive urea channels UT-A1 and UT-A3. In vitro assays using synthetic peptides showed that purified protein kinase A (PKA) could phosphorylate all three sites, and immunoblotting confirmed the PKA dependence of Ser-84 and Ser-486 phosphorylation. These results expand the known list of collecting duct phosphoproteins and highlight the utility of targeted phosphoproteomic approaches.
authors.affiliation: National Institutes of Health, 10 Center Drive, Building 10, Room 6N260, Bethesda, MD 20892-1603, USA.
authors.forename: Amar D
authors.initials: AD
authors.lastname: Bansal
authors.forename: Jason D
authors.initials: JD
authors.lastname: Hoffert
authors.forename: Trairak
authors.initials: T
authors.lastname: Pisitkun
authors.forename: Shelly
authors.initials: S
authors.lastname: Hwang
authors.forename: Chung-Lin
authors.initials: CL
authors.lastname: Chou
authors.forename: Emily S
authors.initials: ES
authors.lastname: Boja
authors.forename: Guanghui
authors.initials: G
authors.lastname: Wang
authors.forename: Mark A
authors.initials: MA
authors.lastname: Knepper
mesh_terms: D000465:Algorithms; D000818:Animals; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D003894:Deamino Arginine Vasopressin / Q000494:pharmacology; D007685:Kidney Tubules, Collecting / Q000187:drug effects / Q000378:metabolism*; D023421:Models, Animal; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D040901:Proteomics; D051381:Rats; D011910:Rats, Brattleboro; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction; D014667:Vasopressins / Q000378:metabolism*; D051176:beta Catenin / Q000378:metabolism
chemical_list: D010750:Phosphoproteins; D051176:beta Catenin; D014667:Vasopressins; D017868:Cyclic AMP-Dependent Protein Kinases; D003894:Deamino Arginine Vasopressin