2008 JASN IMPACT FACTOR 7.505	HOME   AUTHOR INFO   EDITORIAL BOARD   SUBSCRIBE   FEEDBACK   ALERTS   HELP
advanced	CURRENT ISSUE	ARCHIVES	JASN Express	ONLINE SUBMISSION

This Article Full Text Full Text (PDF) Supplemental Data All Versions of this Article: ASN.2009070728v1 21/2/303    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Google Scholar Articles by Bansal, A. D. Articles by Knepper, M. A. PubMed PubMed Citation Articles by Bansal, A. D. Articles by Knepper, M. A.

Phosphoproteomic Profiling Reveals Vasopressin-Regulated Phosphorylation Sites in Collecting Duct

Amar D. Bansal^*, Jason D. Hoffert^*, Trairak Pisitkun^*, Shelly Hwang^*, Chung-Lin Chou^*, Emily S. Boja, Guanghui Wang and Mark A. Knepper^*

*Epithelial Systems Biology Laboratory, and
Proteomics Core Facility, Division of Intramural Research, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland

Correspondence: Dr. Mark A. Knepper, National Institutes of Health, 10 Center Drive, Building 10, Room 6N260, Bethesda, MD 20892-1603. Phone: 301-496-3064; Fax: 301-402-1443; E-mail: knep{at}helix.nih.gov

Received for publication July 15, 2009. Accepted for publication November 2, 2009.

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.

Copyright © 2009 by the American Society of Nephrology. Online ISSN: 1533-3450 Print ISSN: 1046-6673