Quantitative phosphoproteomic analysis reveals cAMP/vasopressin-dependent signaling pathways in native renal thick ascending limb cells

Abstract

Quantitative mass spectrometry was used to identify hormonedependent 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 β-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.protein phosphatase | glucose transporters | mass spectrometry | ion transporters | protein kinase T he thick ascending limb (TAL) of Henle's loop is a nephron segment that plays a critical role in the control of mammalian water excretion. Active NaCl transport by the medullary TAL (mTAL) drives the countercurrent multiplication process that concentrates the urine (1). Hormones that increase the concentration of the intracellular second messenger, cAMP, have been shown to enhance the rate of NaCl transport in mTAL cells (2). These hormones include parathyroid hormone (PTH), calcitonin, glucagon, and vasopressin (2). Among these, only vasopressin plays a selective role in regulation of water balance. The molecular targets for cAMP-mediated regulation in the mTAL include the apical Na + : K + :2Cl − cotransporter NKCC2 (gene symbol: Slc12a1) and the apical Na + :H + exchanger NHE3 (Slc9a3) (3).The signaling network that accounts for cAMP-dependent regulation of these transporters is largely unk...

G Protein–Coupled Receptor Endocytosis Confers Uniformity in Responses to Chemically Distinct Ligands

“…Three lines of evidence strongly suggest that the 54 phosphorylated sites we discovered are indeed induced in response to b2-AR/cAMP signaling. First, 9 of the 54 phosphosites we identified are previously known cAMP-stimulated sites (Berwick et al, 2002;Gunaratne et al, 2010;Lundby et al, 2013;Yip et al, 2014). Second, we found enrichment of the amino acid motif R/K-X-pS (p , 1.0 Â 10 2 8 by Fisher's exact test; Supplemental Fig.…”

Quantitative Proteomics Identifies Vasopressin-Responsive Nuclear Proteins in Collecting Duct Cells

“…18 Prior studies showed that b-catenin phosphorylation at Ser-552 is regulated by vasopressin. [19][20][21] However, none of these studies addressed changes in subcellular localization. Here, immunoblotting confirmed that b-catenin significantly increased in nuclear abundance in response to dDAVP ( Figure 7B) and further showed parallel changes in b-catenin phosphorylated at Ser-552 ( Figure 7C).…”

Dynamics of the G Protein-coupled Vasopressin V2 Receptor Signaling Network Revealed by Quantitative Phosphoproteomics

“…Akt and PKA, which are both activated by vasopressin (30), phosphorylate ␤-catenin at Ser-552, which induces ␤-catenin accumulation in the nucleus and increases its ability to regulate gene transcription (63)(64)(65). Prior studies have demonstrated that phosphorylation of ␤-catenin at Ser-552 increases with vasopressin (9) (8,11). We confirmed this increase in phosphorylation by LC-MS/MS and demonstrated through quantitative immunoblotting that this increase occurs within 0.5 min of vasopressin exposure (Fig.…”

“…2 10.1074/mcp.M111.014613-5 demonstrated both in a mouse cortical collecting duct cell line (8) and in rat kidney thick ascending limb (11) in the presence of dDAVP. These general motifs are apparent in many of the peptide sequences presented in Table I.…”