Phosphoproteomic profiling reveals vasopressin-regulated phosphorylation sites in collecting duct.
ABSTRACT: 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.
Project description:Protein phosphorylation plays a key role in vasopressin signaling in the renal-collecting duct. Large-scale identification and quantification of phosphorylation events triggered by vasopressin is desirable to gain a comprehensive systems-level understanding of this process. We carried out phosphoproteomic analysis of rat inner medullary collecting duct cells by using a combination of phosphopeptide enrichment by immobilized metal affinity chromatography and phosphorylation site identification by liquid chromatography-mass spectrometry(n) neutral loss scanning. A total of 714 phosphorylation sites on 223 unique phosphoproteins were identified from inner medullary collecting duct samples treated short-term with either calyculin A or vasopressin. A number of proteins involved in cytoskeletal reorganization, vesicle trafficking, and transcriptional regulation were identified. Previously unidentified phosphorylation sites were found for membrane proteins essential to collecting duct physiology, including eight sites among aquaporin-2 (AQP2), aquaporin-4, and urea transporter isoforms A1 and A3. Through label-free quantification of phosphopeptides, we identified a number of proteins that significantly changed phosphorylation state in response to short-term vasopressin treatment: AQP2, Bclaf1, LRRC47, Rgl3, and SAFB2. In the presence of vasopressin, AQP2 monophosphorylated at S256 and diphosphorylated AQP2 (pS256/261) increased in abundance, whereas AQP2 monophosphorylated at S261 decreased, raising the possibility that both sites are involved in vasopressin-dependent AQP2 trafficking. This study reveals the practicality of liquid chromatography-mass spectrometry(n) neutral loss scanning for large-scale identification and quantification of protein phosphorylation in the analysis of cell signaling in a native mammalian system.
Project description:Vasopressin controls water balance largely through PKA-dependent effects to regulate the collecting duct water channel aquaporin-2 (AQP2). Although considerable information has accrued regarding the regulation of water and solute transport in collecting duct cells, information is sparse regarding the signaling connections between PKA and transport responses. Here, we exploited recent advancements in protein mass spectrometry to perform a comprehensive, multiple-replicate analysis of changes in the phosphoproteome of native rat inner medullary collecting duct cells in response to the vasopressin V2 receptor-selective agonist 1-desamino-8D-arginine vasopressin. Of the 10,738 phosphopeptides quantified, only 156 phosphopeptides were significantly increased in abundance, and only 63 phosphopeptides were decreased, indicative of a highly selective response to vasopressin. The list of upregulated phosphosites showed several general characteristics: 1) a preponderance of sites with basic (positively charged) amino acids arginine (R) and lysine (K) in position -2 and -3 relative to the phosphorylated amino acid, consistent with phosphorylation by PKA and/or other basophilic kinases; 2) a greater-than-random likelihood of sites previously demonstrated to be phosphorylated by PKA; 3) a preponderance of sites in membrane proteins, consistent with regulation by membrane association; and 4) a greater-than-random likelihood of sites in proteins with class I COOH-terminal PDZ ligand motifs. The list of downregulated phosphosites showed a preponderance of those with proline in position +1 relative to the phosphorylated amino acid, consistent with either downregulation of proline-directed kinases (e.g., MAPKs or cyclin-dependent kinases) or upregulation of one or more protein phosphatases that selectively dephosphorylate such sites (e.g., protein phosphatase 2A). The phosphoproteomic data were used to create a web resource for the investigation of G protein-coupled receptor signaling and regulation of AQP2-mediated water transport.
Project description:Vasopressin regulates osmotic water transport in the renal collecting duct by protein kinase A (PKA)-mediated control of the water channel aquaporin-2 (AQP2). Collecting duct principal cells express two seemingly redundant PKA catalytic subunits, PKA catalytic ? (PKA-C?) and PKA catalytic ? (PKA-C?). To identify the roles of these two protein kinases, we carried out deep phosphoproteomic analysis in cultured mpkCCD cells in which either PKA-C? or PKA-C? was deleted using CRISPR-Cas9-based genome editing. Controls were cells carried through the genome editing procedure but without deletion of PKA. TMT mass tagging was used for protein mass spectrometric quantification. Of the 4,635 phosphopeptides that were quantified, 67 phosphopeptides were significantly altered in abundance with PKA-C? deletion, whereas 21 phosphopeptides were significantly altered in abundance with PKA-C? deletion. However, only four sites were changed in both. The target proteins identified in PKA-C?-null cells were largely associated with cell membranes and membrane vesicles, whereas target proteins in PKA-C?-null cells were largely associated with the actin cytoskeleton and cell junctions. In contrast, in vitro incubation of mpkCCD proteins with recombinant PKA-C? and PKA-C? resulted in virtually identical phosphorylation changes. In addition, analysis of total protein abundances in in vivo samples showed that PKA-C? deletion resulted in a near disappearance of AQP2 protein, whereas PKA-C? deletion did not decrease AQP2 abundance. We conclude that PKA-C? and PKA-C? serve substantially different regulatory functions in renal collecting duct cells and that differences in phosphorylation targets may be due to differences in protein interactions, e.g., mediated by A-kinase anchor proteins, C-kinase anchoring proteins, or PDZ binding.
Project description:In the renal collecting duct, binding of AVP to the V2 receptor triggers signaling changes that regulate osmotic water transport. Short-term regulation of water transport is dependent on vasopressin-induced phosphorylation of aquaporin-2 (AQP2) at Ser256. The protein kinase that phosphorylates this site is not known. We use Bayes' theorem to rank all 521 rat protein kinases with regard to the likelihood of a role in Ser256 phosphorylation on the basis of prior data and new experimental data. First, prior probabilities were estimated from previous transcriptomic and proteomic profiling data, kinase substrate specificity data, and evidence for kinase regulation by vasopressin. This ranking was updated using new experimental data describing the effects of several small-molecule kinase inhibitors with known inhibitory spectra (H-89, KN-62, KN-93, and GSK-650394) on AQP2 phosphorylation at Ser256 in inner medullary collecting duct suspensions. The top-ranked kinase was Ca2+/calmodulin-dependent protein kinase II (CAMK2), followed by protein kinase A (PKA) and protein kinase B (AKT). Liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based in vitro phosphorylation studies compared the ability of three highly ranked kinases to phosphorylate AQP2 and other inner medullary collecting duct proteins, PKA, CAMK2, and serum/glucocorticoid-regulated kinase (SGK). All three proved capable of phosphorylating AQP2 at Ser256, although CAMK2 and PKA were more potent than SGK. The in vitro phosphorylation experiments also identified candidate protein kinases for several additional phosphoproteins with likely roles in collecting duct regulation, including Nedd4-2, Map4k4, and 3-phosphoinositide-dependent protein kinase 1. We conclude that Bayes' theorem is an effective means of integrating data from multiple data sets in physiology.
Project description: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.
Project description: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.
Project description:Prior studies have implicated myosin light chain kinase (MLCK) in the regulation of aquaporin-2 (AQP2) in the renal collecting duct. To discover signaling targets of MLCK, we used CRISPR-Cas9 to delete the MLCK gene (<i>Mylk</i>) to obtain MLCK-null mpkCCD cells and carried out comprehensive phosphoproteomics using stable isotope labeling with amino acids in cell culture for quantification. Immunocytochemistry and electron microscopy demonstrated a defect in the processing of AQP2-containing early endosomes to late endosomes. The phosphoproteomics experiments revealed that, of the 1,743 phosphopeptides quantified over multiple replicates, 107 were changed in abundance by MLCK deletion (29 decreased and 78 increased). One of the decreased phosphopeptides corresponded to the canonical target site in myosin regulatory light chain. Network analysis indicated that targeted phosphoproteins clustered into distinct structural/functional groups: actomyosin, signaling, nuclear envelope, gene transcription, mRNA processing, energy metabolism, intermediate filaments, adherens junctions, and tight junctions. There was significant overlap between the derived MLCK signaling network and a previously determined PKA signaling network. The presence of multiple proteins in the actomyosin category prompted experiments showing that MLCK deletion inhibits the normal effect of vasopressin to depolymerize F-actin, providing a potential explanation for the AQP2 trafficking defect. Changes in phosphorylation of multiple proteins in the nuclear envelope prompted measurement of nuclear size, showing a significant increase in average nuclear volume. We conclude that MLCK is part of a multicomponent signaling pathway in both the cytoplasm and nucleus that includes much more than simple regulation of conventional nonmuscle myosins through myosin regulatory light chain phosphorylation.
Project description:Large-scale phosphoproteomic analysis employing liquid chromatography-tandem mass spectrometry (LC-MS/MS) often requires a significant amount of manual manipulation of phosphopeptide datasets in the post-acquisition phase. To assist in this process, we have created software, PhosphoPIC (PhosphoPeptide Identification and Compilation), which can perform a variety of useful functions including automated selection and compilation of phosphopeptide identifications from multiple MS levels, estimation of dataset false discovery rate, and application of appropriate cross-correlation (XCorr) filters. In addition, the output files generated by this program are compatible with downstream phosphorylation site assignment using the Ascore algorithm, as well as phosphopeptide quantification via QUOIL. In this report, we utilized this software to analyze phosphoproteins from short-term vasopressin-treated rat kidney inner medullary collecting duct (IMCD). A total of 925 phosphopeptides representing 173 unique proteins were identified from membrane-enriched fractions of IMCD with a false discovery rate of 1.5%. Of these proteins, 106 were found only in the membrane-enriched fraction of IMCD cells and not in whole IMCD cell lysates. These identifications included a number of well-studied ion and solute transporters including ClC-1, LAT4, MCT2, NBC3, and NHE1, all of which contained novel phosphorylation sites. Using a label-free quantification approach, we identified phosphoproteins that changed in abundance with vasopressin exposure including aquaporin-2 (AQP2), Hnrpa3, IP3 receptor 3, and pur-beta.
Project description:Satavaptan (SR121463) is a vasopressin V2 receptor antagonist that has been shown to improve hyponatremia in patients with cirrhosis, congestive heart failure, and syndrome of inappropriate antidiuresis. While known to inhibit adenylyl cyclase-mediated accumulation of intracellular cyclic AMP and potentially recruit ?-arrestin in kidney cell lines, very little is known regarding the signaling pathways that are affected by this drug. To this end, we carried out a global quantitative phosphoproteomic analysis of native rat inner medullary collecting duct cells pretreated with satavaptan or vehicle control followed by the V2 receptor agonist desmopressin (dDAVP) for 0.5, 2, 5, or 15 min. A total of 2,449 unique phosphopeptides from 1,160 proteins were identified. Phosphopeptides significantly changed by satavaptan included many of the same kinases [protein kinase A, phosphoinositide 3-kinase, mitogen-activated protein kinase kinase kinase 7 (TAK1), and calcium/calmodulin-dependent kinase kinase 2] and channels (aquaporin-2 and urea transporter UT-A1) regulated by vasopressin. Time course clustering and kinase motif analysis suggest that satavaptan blocks dDAVP-mediated activation of basophilic kinases, while also blocking dDAVP-mediated inhibition of proline-directed kinases. Satavaptan affects a variety of dDAVP-mediated processes including regulation of cell-cell junctions, actin cytoskeleton dynamics, and signaling through Rho GTPases. These results demonstrate that, overall, satavaptan acts as a selective V2 receptor antagonist and affects many of the same signaling pathways regulated by vasopressin. This study represents the first "systems-wide" analysis of a "vaptan"-class drug and provides a wealth of new data regarding the effects of satavaptan on vasopressin-mediated phosphorylation events.
Project description:Activation of V2 receptors (V2R) during antidiuresis increases the permeability of the inner medullary collecting duct to urea and water. Extracellular osmolality is elevated as the concentrating capacity of the kidney increases. Osmolality is known to contribute to the regulation of collecting duct water (aquaporin-2; AQP2) and urea transporter (UT-A1, UT-A3) regulation. AQP1KO mice are a concentrating mechanism knockout, a defect attributed to the loss of high interstitial osmolality. A V2R-specific agonist, deamino-8-D-arginine vasopressin (dDAVP), was infused into wild-type and AQP1KO mice for 7 days. UT-A1 mRNA and protein abundance were significantly increased in the medullas of wild-type and AQP1KO mice following dDAVP infusion. The mRNA and protein abundance of UT-A3, the basolateral urea transporter, was significantly increased by dDAVP in both wild-type and AQP1KO mice. Semiquantitative immunoblots revealed that dDAVP infusion induced a significant increase in the medullary expression of the endoplasmic reticulum (ER) chaperone GRP78. Immunofluorescence studies demonstrated that GRP78 expression colocalized with AQP2 in principal cells of the papillary tip of the renal medulla. Using immunohistochemistry and immunogold electron microscopy, we demonstrate that vasopressin induced a marked apical targeting of GRP78 in medullary principal cells. Urea-sensitive genes, GADD153 and ATF4 (components of the ER stress pathway), were significantly increased in AQP1KO mice by dDAVP infusion. These findings strongly support an important role of vasopressin in the activation of an ER stress response in renal collecting duct cells, in addition to its role in activating an increase in UT-A1 and UT-A3 abundance.