Large-scale phosphoproteomic analysis of membrane proteins in renal proximal and distal tubule

Marina Feric, Boyang Zhao, Jason D. Hoffert, Trairak Pisitkun, Mark A. Knepper

Research output: Contribution to journalArticlepeer-review

35 Scopus citations

Abstract

Recent advances in mass spectrometry (MS) have provided means for large-scale phosphoproteomic profiling of specific tissues. Here, we report results from large-scale tandem MS [liquid chromatography (LC)-MS/MS]- based phosphoproteomic profiling of biochemically isolated membranes from the renal cortex, with focus on transporters and regulatory proteins. Data sets were filtered (by target-decoy analysis) to limit false-positive identifications to 2%. A total of 7,125 unique nonphosphorylated and 743 unique phosphorylated peptides were identified. Among the phosphopeptides identified were sites on transporter proteins, i.e., solute carrier (Slc, n 63), ATP-binding cassette (Abc, n 4), and aquaporin (Aqp, n 3) family proteins. Database searches reveal that a majority of the phosphorylation sites identified in transporter proteins were previously unreported. Most of the Slc family proteins are apical or basolateral transporters expressed in proximal tubule cells, including proteins known to mediate transport of glucose, amino acids, organic ions, and inorganic ions. In addition, we identified potentially important phosphorylation sites for transport proteins from distal nephron segments, including the bumetanide-sensitive Na-K-2Cl cotransporter (Slc12a1 or NKCC2) at Ser 87, Thr 101, and Ser 126 and the thiazide-sensitive Na-Cl cotransporter (Slc12a3 or NCC) at Ser 71 and Ser 124. A subset of phosphorylation sites in regulatory proteins coincided with known functional motifs, suggesting specific regulatory roles. An online database from this study (http://dir.nhlbi.nih.gov/papers/lkem/rcmpd/) provides a resource for future studies of transporter regulation.

Original languageEnglish (US)
Pages (from-to)C755-C770
JournalAmerican Journal of Physiology - Cell Physiology
Volume300
Issue number4
DOIs
StatePublished - Apr 2011

All Science Journal Classification (ASJC) codes

  • Physiology
  • Cell Biology

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