Quantitative phosphoproteomic analysis reveals cAMP/vasopressin-dependent signaling pathways in native renal thick ascending limb cells.
Quantitative mass spectrometry was used to identify hormone-dependent signaling pathways in renal medullary thick ascending limb (mTAL) cells via phosphoproteomic analysis. Active transport of NaCl across the mTAL epithelium is accelerated by hormones that increase cAMP levels (vasopressin, glucagon, parathyroid hormone, and calcitonin). mTAL suspensions from rat kidneys were exposed (15 min) to a mixture of these four hormones. Tryptic phosphopeptides (immobilized metal affinity chromatography-enriched) were identified and quantified by mass spectrometry (LTQ-Orbitrap) using label-free methodology. We quantified a total of 654 phosphopeptides, of which 414 were quantified in three experimental pairs (hormone vs. vehicle). Of these phosphopeptides, 82% were statistically unchanged in abundance in response to the hormone mixture. In contrast, 48 phosphopeptides were significantly increased, whereas 28 were significantly decreased. The population of up-regulated phosphopeptides was highly enriched in basophilic kinase substrate motifs (AGC or calmodulin-sensitive kinase families), whereas the down-regulated sites were dominated by "proline-directed" motifs (cyclin-dependent or MAP kinase families). Bioinformatic classification uncovered overrepresentation of transmembrane transporters, protein phosphatase regulators, and cytoskeletal binding proteins among the regulated proteins. Immunoblotting with phospho-specific antibodies confirmed cAMP/vasopressin-dependent phosphorylation at Thr96, Ser126, and Ser874 of the Na(+):K(+):2Cl(-) cotransporter NKCC2, at Ser552 of the Na(+):H(+) exchanger NHE3, and at Ser552 of beta-catenin. Vasopressin also increased phosphorylation of NKCC2 at both Ser126 (more than fivefold) and Ser874 (more than threefold) in rats in vivo. Both sites were phosphorylated by purified protein kinase A during in vitro assays. These results support the view that, although protein kinase A plays a central role in mTAL signaling, additional kinases, including those that target proline-directed motifs, may be involved.
Gunaratne
Ruwan
R
Epithelial Systems Biology Laboratory, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Braucht
Drew W W
DW
Rinschen
Markus M
MM
Chou
Chung-Lin
CL
Hoffert
Jason D
JD
Pisitkun
Trairak
T
Knepper
Mark A
MA
D000818:Animals; D001665:Binding Sites; D002116:Calcitonin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism; D005934:Glucagon / Q000494:pharmacology; D015151:Immunoblotting; D007679:Kidney Medulla / Q000166:cytology / Q000378:metabolism*; D008297:Male; D013058:Mass Spectrometry / Q000379:methods; D010281:Parathyroid Hormone / Q000494:pharmacology; D010750:Phosphoproteins / Q000032:analysis*; D010766:Phosphorylation / Q000187:drug effects; D011494:Protein Kinases / Q000378:metabolism; D020543:Proteome / Q000032:analysis*; D040901:Proteomics / Q000379:methods*; D051381:Rats; D011910:Rats, Brattleboro; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers / Q000378:metabolism; D028021:Sodium-Potassium-Chloride Symporters / Q000378:metabolism; D064466:Solute Carrier Family 12, Member 1; D014667:Vasopressins / Q000494:pharmacology
D010281:Parathyroid Hormone; D010750:Phosphoproteins; D020543:Proteome; C578716:Slc12a1 protein, rat; C000613387:Slc9a3 protein, rat; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers; D028021:Sodium-Potassium-Chloride Symporters; D064466:Solute Carrier Family 12, Member 1; D014667:Vasopressins; D002116:Calcitonin; D005934:Glucagon; D000242:Cyclic AMP; D011494:Protein Kinases; D017868:Cyclic AMP-Dependent Protein Kinases