Jason D. Hoffert

Large-Scale Proteomics and Phosphoproteomics of Urinary Exosomes

Normal human urine contains large numbers of exosomes, which are 40- to 100-nm vesicles that originate as the internal vesicles in multivesicular bodies from every renal epithelial cell type facing the urinary space. Here, we used LC-MS/MS to profile the proteome of human urinary exosomes. Overall, the analysis identified 1132 proteins unambiguously, including 177 that are represented on the Online Mendelian Inheritance in Man database of disease-related genes, suggesting that exosome analysis is a potential approach to discover urinary biomarkers. We extended the proteomic analysis to phosphoproteomic profiling using neutral loss scanning, and this yielded multiple novel phosphorylation sites, including serine-811 in the thiazide-sensitive Na-Cl co-transporter, NCC. To demonstrate the potential use of exosome analysis to identify a genetic renal disease, we carried out immunoblotting of exosomes from urine samples of patients with a clinical diagnosis of Bartter syndrome type I, showing an absence of the sodium-potassium-chloride co-transporter 2, NKCC2. The proteomic data are publicly accessible at http://dir.nhlbi.nih.gov/papers/lkem/exosome/.

Vasopressin-stimulated Increase in Phosphorylation at Ser269 Potentiates Plasma Membrane Retention of Aquaporin-2

Vasopressin controls water excretion through regulation of aquaporin-2 (AQP2) trafficking in renal collecting duct cells. Using mass spectrometry, we previously demonstrated four phosphorylated serines (Ser256, Ser261, Ser264, and Ser269) in the carboxyl-terminal tail of rat AQP2. Here, we used phospho-specific antibodies and protein mass spectrometry to investigate the roles of vasopressin and cyclic AMP in the regulation of phosphorylation at Ser269 and addressed the role of this site in AQP2 trafficking. The V2 receptor-specific vasopressin analog dDAVP increased Ser(P)269-AQP2 abundance more than 10-fold, but at a rate much slower than the corresponding increase in Ser256 phosphorylation. Vasopressin-mediated changes in phosphorylation at both sites were mimicked by cAMP addition and inhibited by protein kinase A (PKA) antagonists. In vitro kinase assays, however, demonstrated that PKA phosphorylates Ser256, but not Ser269. Phosphorylation of AQP2 at Ser269 did not occur when Ser256 was replaced by an unphosphorylatable amino acid, as seen in both S256L-AQP2 mutant mice and in Madin-Darby canine kidney cells expressing an S256A mutant, suggesting that Ser269 phosphorylation depends upon prior phosphorylation at Ser256. Immunogold electron microscopy localized Ser(P)269-AQP2 solely in the apical plasma membrane of rat collecting duct cells, in contrast to the other three phospho-forms (found in both apical plasma membrane and intracellular vesicles). Madin-Darby canine kidney cells expressing an S269D “phosphomimic” AQP2 mutant showed constitutive localization at the plasma membrane. The data support a model in which vasopressin-mediated phosphorylation of AQP2 at Ser269:(a) depends on prior PKA-mediated phosphorylation of Ser256 and (b) enhances apical plasma membrane retention of AQP2.

Quantitative phosphoproteomic analysis reveals vasopressin V2-receptor–dependent signaling pathways in renal collecting duct cells

Vasopressin’s action in renal cells to regulate water transport depends on protein phosphorylation. Here we used mass spectrometry–based quantitative phosphoproteomics to identify signaling pathways involved in the short-term V2-receptor–mediated response in cultured collecting duct cells (mpkCCD) from mouse. Using Stable Isotope Labeling by Amino acids in Cell culture (SILAC) with two treatment groups (0.1 nM dDAVP or vehicle for 30 min), we carried out quantification of 2884 phosphopeptides. The majority (82%) of quantified phosphopeptides did not change in abundance in response to dDAVP. Analysis of the 273 phosphopeptides increased by dDAVP showed a predominance of so-called “basophilic” motifs consistent with activation of kinases of the AGC family. Increases in phosphorylation of several known protein kinase A targets were found. In addition, increased phosphorylation of targets of the calmodulin-dependent kinase family was seen, including autophosphorylation of calmodulin-dependent kinase 2 at T286. Analysis of the 254 phosphopeptides decreased in abundance by dDAVP showed a predominance of so-called “proline-directed” motifs, consistent with down-regulation of mitogen-activated or cyclin-dependent kinases. dDAVP decreased phosphorylation of both JNK1/2 (T183/Y185) and ERK1/2 (T183/Y185; T203/Y205), consistent with a decrease in activation of these proline-directed kinases in response to dDAVP. Both ERK and JNK were able to phosphorylate residue S261of aquaporin-2 in vitro, a site showing a decrease in phosphorylation in response to dDAVP in vivo. The data support roles for multiple vasopressin V2-receptor–dependent signaling pathways in the vasopressin signaling network of collecting duct cells, involving several kinases not generally accepted to regulate collecting duct function.

Polyphosphoinositides inhibit the interaction of vinculin with actin filaments

Binding of vinculin to adhesion plaque proteins is restricted by an intramolecular association of vinculin’s head and tail regions. Results of previous work suggest that polyphosphoinositides disrupt this interaction and thereby promote binding of vinculin to both talin and actin. However, data presented here show that phosphatidylinositol 4,5-bisphosphate (PI4,5P2) inhibits the interaction of purified tail domain with F-actin. Upon re-examining the effect of PI4,5P2 on the actin and talin-binding activities of intact vinculin, we find that when the experimental design controls for the effect of magnesium on aggregation of PI4,5P2 micelles, polyphosphoinositides promote interactions with the talin-binding domain, but block interactions of the actin-binding domain. In contrast, if vinculin is trapped in an open confirmation by a peptide specific for the talin-binding domain of vinculin, actin binding is allowed. These results demonstrate that activation of the actin-binding activity of vinculin requires steps other than or in addition to the binding of PI4,5P2.

Acute regulation of aquaporin-2 phosphorylation at Ser-264 by vasopressin

By phosphoproteome analysis, we identified a phosphorylation site, serine 264 (pS264), in the COOH terminus of the vasopressin-regulated water channel, aquaporin-2 (AQP2). In this study, we examined the regulation of AQP2 phosphorylated at serine 264 (pS264–AQP2) by vasopressin, using a phospho-specific antibody (anti-pS264). Immunohistochemical analysis showed pS264–AQP2 labeling of inner medullary collecting duct (IMCD) from control mice, whereas AQP2 knockout mice showed a complete absence of labeling. In rat and mouse, pS264–AQP2 was present throughout the collecting duct system, from the connecting tubule to the terminal IMCD. Immunogold electron microscopy, combined with double-labeling confocal immunofluorescence microscopy with organelle-specific markers, determined that the majority of pS264 resides in compartments associated with the plasma membrane and early endocytic pathways. In Brattleboro rats treated with [deamino-Cys-1, d-Arg-8]vasopressin (dDAVP), the abundance of pS264–AQP2 increased 4-fold over controls. Additionally, dDAVP treatment resulted in a time-dependent change in the distribution of pS264 from predominantly intracellular vesicles, to both the basolateral and apical plasma membranes. Sixty minutes after dDAVP exposure, a proportion of pS264–AQP2 was observed in clathrin-coated vesicles, early endosomal compartments, and recycling compartments, but not lysosomes. Overall, our results are consistent with a dynamic effect of AVP on the phosphorylation and subcellular distribution of AQP2.

Proteomic analysis of lithium-induced nephrogenic diabetes insipidus: mechanisms for aquaporin 2 down-regulation and cellular proliferation.

Lithium is a commonly prescribed mood-stabilizing drug. However, chronic treatment with lithium induces numerous kidney-related side effects, such as dramatically reduced aquaporin 2 (AQP2) abundance, altered renal function, and structural changes. As a model system, inner medullary collecting ducts (IMCD) isolated from rats treated with lithium for either 1 or 2 weeks were subjected to differential 2D gel electrophoresis combined with mass spectrometry and bioinformatics analysis to identify (i) signaling pathways affected by lithium and (ii) unique candidate proteins for AQP2 regulation. After 1 or 2 weeks of lithium treatment, we identified 6 and 74 proteins with altered abundance compared with controls, respectively. We randomly selected 17 proteins with altered abundance caused by lithium treatment for validation by immunoblotting. Bioinformatics analysis of the data indicated that proteins involved in cell death, apoptosis, cell proliferation, and morphology are highly affected by lithium. We demonstrate that members of several signaling pathways are activated by lithium treatment, including the PKB/Akt-kinase and the mitogen-activated protein kinases (MAPK), such as extracellular regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK), and p38. Lithium treatment increased the intracellular accumulation of β-catenin in association with increased levels of phosphorylated glycogen synthase kinase type 3β (GSK3β). This study provides a comprehensive analysis of the proteins affected by lithium treatment in the IMCD and, as such, provides clues to potential lithium targets in the brain.

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 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.

PhosphoScore: An Open-Source Phosphorylation Site Assignment Tool for MSn Data

Correct phosphorylation site assignment is a critical aspect of phosphoproteomic analysis. Large-scale phosphopeptide data sets that are generated through liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS) analysis often contain hundreds or thousands of phosphorylation sites that require validation. To this end, we have created PhosphoScore, an open-source assignment program that is compatible with phosphopeptide data from multiple MS levels (MS(n)). The algorithm takes into account both the match quality and normalized intensity of observed spectral peaks compared to a theoretical spectrum. PhosphoScore produced >95% correct MS(2) assignments from known synthetic data, > 98% agreement with an established MS(2) assignment algorithm (Ascore), and >92% agreement with visual inspection of MS(3) and MS(4) spectra.

Dynamics of the G protein-coupled vasopressin V2 receptor signaling network revealed by quantitative phosphoproteomics

G protein-coupled receptors (GPCRs) regulate diverse physiological processes, and many human diseases are due to defects in GPCR signaling. To identify the dynamic response of a signaling network downstream from a prototypical Gs-coupled GPCR, the vasopressin V2 receptor, we have carried out multireplicate, quantitative phosphoproteomics with iTRAQ labeling at four time points following vasopressin exposure at a physiological concentration in cells isolated from rat kidney. A total of 12,167 phosphopeptides were identified from 2,783 proteins, with 273 changing significantly in abundance with vasopressin. Two-dimensional clustering of phosphopeptide time courses and Gene Ontology terms revealed that ligand binding to the V2 receptor affects more than simply the canonical cyclic adenosine monophosphate-protein kinase A and arrestin pathways under physiological conditions. The regulated proteins included key components of actin cytoskeleton remodeling, cell-cell adhesion, mitogen-activated protein kinase signaling, Wnt/β-catenin signaling, and apoptosis pathways. These data suggest that vasopressin can regulate an array of cellular functions well beyond its classical role in regulating water and solute transport. These results greatly expand the current view of GPCR signaling in a physiological context and shed new light on potential roles for this signaling network in disorders such as polycystic kidney disease. Finally, we provide an online resource of physiologically regulated phosphorylation sites with dynamic quantitative data (http://helixweb.nih.gov/ESBL/Database/TiPD/index.html).

CALMODULIN IS REQUIRED FOR VASOPRESSIN-STIMULATED INCREASE IN CYCLIC AMP PRODUCTION IN INNER MEDULLARY COLLECTING DUCT

Calmodulin plays a critical role in regulation of renal collecting duct water permeability by vasopressin. However, specific targets for calmodulin action have not been thoroughly addressed. In the present study, we investigated whether Ca2+/calmodulin regulates adenylyl cyclase activity in the renal inner medullary collecting duct. Rat inner medullary collecting duct suspensions were incubated in the presence or absence of 0.1 nm vasopressin and the calmodulin inhibitors, monodansylcadaverine, W-7, and trifluoperazine, followed by measurement of cAMP. Vasopressin-stimulated cAMP elevation was significantly attenuated in the presence of calmodulin inhibitors. Analysis of transglutaminase 2 knock-out mice confirmed that these compounds were not acting through inhibition of transglutaminase 2 activity. Calmodulin inhibitors also blocked both cholera toxin- and forskolin-stimulated cAMP accumulation. In isolated perfused tubules, W-7 reversibly blocked vasopressin-stimulated urea permeability, a process that requires a rise in intracellular cAMP but does not appear to involve protein trafficking to the apical plasma membrane. These results suggest that calmodulin is required for vasopressin-stimulated adenylyl cyclase activity in the intact inner medullary collecting duct. Reverse transcription-PCR, immunoblotting, and immunohistochemistry revealed the presence of the calmodulin-sensitive adenylyl cyclase type 3 in the rat collecting duct, an isoform previously not known to be expressed in the collecting duct. Long-term treatment of Brattleboro rats with a vasopressin analog markedly decreased adenylyl cyclase type 3 protein abundance, providing an explanation for long-term down-regulation of vasopressin response in the collecting duct. These studies demonstrate the importance of calmodulin in the regulation of collecting duct adenylyl cyclase activity and transport function.