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YANGMass SpectrometryShotgun proteomicsNot availablePhosphoproteomicsPrm-msAmpkSilacMpkccdhttp://www.ebi.ac.uk/pride/archive/projects/PXD015719Epithelial CellKidneyThe cells were labeled by growing in complete SILAC medium (Thermo Fisher) containing either heavy (13C6 15N4 arginine and 13C6 lysine) and light (12C6 14N4 arginine and 12C6 lysine) amino acids. The cells were thawed on ice and treated with urea buffer (8 M urea, 50 mM Tris·HCl, 75 mM NaCl, 1× Halt protease and phosphatase inhibitors), scraped and sonicated (duration: 2 min, pulse/pause: 2 sec/2 sec, output level = 1) to solubilize proteins. Following measurement of protein concentration (BCA reagent), equal amounts of heavy- and light-labeled protein extracts were mixed (total, 2.5 ± 0.4 mg). The mixed samples were reduced with 20 mM DTT for 1 h at 37 °C, and then alkylated with 40 mM iodoacetamide for 1 h at 25 °C in the dark. The samples were diluted with 20 mM triethylammonium bicarbonate buffer (pH 8.5) to 1 M urea, and then digested with trypsin/LysC (Promega, Madison, WI, USA) (1:20 wt/wt) overnight at 37 °C. The peptides were desalted using hydrophilic–lipophilic–balanced extraction cartridges (Oasis, 1 cc, 30 mg). The peptide sample was divided into three parts: total peptide analysis (4%), heretofore referred to as ‘Total’ and phosphopeptide enrichments (48% × 2) using Fe-NTA and TiO2 columns. The enrichment protocols were as described in the manufacturer’s instructions with minor modifications. The enriched peptides from Fe-NTA columns were desalted using graphite columns. All three samples (Total, TiO2, Fe-NTA) were fractionated in a 96-well plate with off-line high-pH reverse-phase chromatography (Agilent 1200 HPLC system, Santa Clara, CA, USA) using a XBridge BEH C18 column (Waters; 130Å, 5 µm, 2.1 mm X 30 mm). For each SILAC experiment, eluted samples were pooled to 24 (Total), 12 (TiO2) and 12 (Fe-NTA) fractions, vacuum-dried and stored at −80 °C. The dried peptides were reconstituted with 0.1% (v/v) formic acid (FA) in LC-MS–grade water (J.T. Baker, Fisher Scientific) before mass spectrometry analysis.PrideSILACphosphorylated residueiodoacetamide derivatized residue6x(13)C labeled residueRaw data were analyzed using Proteome Discoverer 1.4 (version 1.4.0.288) in conjunction with two search engines; Mascot and SequestHT. The protein database used for SequestHT was the mouse Swiss-Prot (downloaded on April 16, 2017). The Mascot search engine and database were maintained by NIH Mascot server (https://biospec.nih.gov). The search criteria for all HCD raw files were set as follows: 1) precursor mass tolerance = 10 ppm, 2) fragment mass tolerance = 0.02 Da, 3) enzyme specificity was set as trypsin with two missed cleavages allowed. Carbamidomethylation of cysteine (C+57.021 Da) was set as a fixed modification. Variable modifications were as follows: a) isotope labeling of lysine (K+6.020 Da) and arginine (R+10.008 Da), b) oxidation of methionine (M+15.995 Da), c) deamination on glutamine and asparagine (N, Q+0.984 Da), d) phosphorylation of serine, threonine and tyrosine (S, T, Y+79.966 Da) and e) acetylation of protein N-terminal. The FDR was calculated by the target-decoy algorithm. The probabilities of the phosphorylation site localizations were calculated based on the given MS2 data using the PhosphoRS 3.0 module within Proteome Discoverer. The following data reduction filters was used; peptide confidence: high, peptide rank = 1. The FDR was calculated by the target decoy PSM validator. The FDR was set <0.01.ProteomicsMark A. KnepperOrbitrap Fusion LumosPrincipal Investigator Epithelial Systems Biology Laboratory Systems Biology Center National Heart, Lung and Blood Institute 10 CENTER DR MSC-1603 Building 10, Room 6N307 Bethesda Maryland 20892-1603 U.S.A.PARTIALMus Musculus (mouse)chin-rang.yang@nih.gov32219907 Datta A, Yang CR, Limbutara K, Chou CL, Rinschen MM, Raghuram V, Knepper MA. PKA-independent vasopressin signaling in renal collecting duct. FASEB J. 2020 34(5):6129-6146 10.1096/fj.201902982rNational Institutes of Health, USAUnited StatesVasopressin regulates renal water excretion by binding to a G<sub>α</sub> s-coupled receptor (V2R) in collecting duct cells, resulting in increased water permeability through regulation of the aquaporin-2 (AQP2) water channel. This action is widely accepted to be associated with cAMP-mediated activation of protein kinase A (PKA). Here, we use phosphoproteomics in collecting duct cells in which PKA has been deleted (CRISPR-Cas9) to identify PKA-independent responses to vasopressin. The results show that V2R-mediated vasopressin signaling is predominantly, but not entirely, PKA-dependent. Upregulated sites in PKA-null cells include Ser256 of AQP2, which is critical to regulation of AQP2 trafficking. In addition, phosphorylation changes in the protein kinases Stk39 (SPAK) and Prkci (an atypical PKC) are consistent with PKA-independent regulation of these protein kinases. Target motif analysis of the phosphopeptides increased in PKA-null cells indicates that vasopressin activates one or more members of the AMPK/SNF1-subfamily of basophilic protein kinases. In vitro phosphorylation assays using recombinant, purified SNF1-subfamily kinases confirmed postulated target specificities. Of interest, measured IBMX-dependent cAMP levels were an order of magnitude higher in PKA-null than in PKA-intact cells, indicative of a PKA-dependent feedback mechanism. Overall, the findings support the conclusion that V2-receptor mediated signaling in collecting duct cells is in part PKA-independent.PKA-independent vasopressin signaling in renal collecting duct.Datta Arnab A, Yang Chin-Rang CR, Limbutara Kavee K, Chou Chung-Lin CL, Rinschen Markus M MM, Raghuram Viswanathan V, Knepper Mark A MAglycogen synthase kinase activity, 6353, ribosomal S6 protein kinase activity, calcium/phospholipid-dependent protein kinase activity, beta Hypophamine, biological signaling, Dmel_CG3263, protein kinase A activity, glycogen synthase A kinase activity, Antidiuretic Hormone, glycogen synthase kinase 3 activity, Pk?5, PKA-C3, PKA-C-3, Vasopressin (USP), PKA-C1, ureteric tree, CG18677, phosphorylase b kinase kinase activity, BcDNA:GM01761, collecting duct system., CG12452, Prp4 protein kinase activity, Hormone, collecting tubule, Pka-RI, ATP-protein transphosphorylase activity, cAMP-dependent protein kinase, Pitressin, ATP:protein phosphotransferase (non-specific) activity, l(2)cos[[1]], Inyectable de vasopresina, serine(threonine) protein kinase activity, argipressin, mitogen-activated protein kinase activity, Dpck, BG02142, RI, AMPK, ribosomal protein S6 kinase II activity, 18304, T-antigen kinase activity, galactosyltransferase-associated kinase activity, C, protein-serine kinase activity, Solute injectable de vasopressine, Pka-C, PKAR, PkaR, CG4379, STK32, Pk72A, Antidiuretic hormone, 3', Antidiuretic, 5' cAMP-dependent protein kinase complex, PKa-R2, PKa-R1, PKAc, CG15862, Raf kinase activity, junctional tube, protein kinase (phosphorylating) activity, arcuate renal tubule, PKA-R1, serine kinase activity, DmelCG15862, 5'-cyclophosphate-dependent protein kinase activity, kidney collecting duct, PKA-R2, Cos, DmelCG6117, 5' cAMP-dependent protein kinase activity, CdkA, protein-cysteine kinase activity, atypical protein kinase C activity, Raf-1, signaling process, Hpr kinase activity, Vasopressin, Dc0, mitogen-activated S6 kinase activity, STK22, protein-aspartyl kinase activity, casein kinase (phosphorylating) activity, DRI, PKA-RI, single organism signaling, p82 kinase activity, PKA Cl, tubulus renalis colligens, ADH, renal collecting tubule, Cos1, 5'-cyclophosphate-dependent protein kinase complex, l(2)s4402, DC0, betaIIPKC, cyclic AMP-dependent protein kinase complex, DC2, kidney collecting tubule, dPKA-RI, CG42341, CG3263, CdkR, protein serine kinase activity, pka-C1, protein phosphokinase activity, M phase-specific cdc2 kinase activity, Wee-kinase activity, cos1, twitchin kinase activity, cyclic AMP-dependent protein kinase activity, PKAi, pkA, PKA, Pka, PKAr, pka-R1, PKC, MAPK, AP50 kinase activity, 5'-cAMP-dependent protein kinase complex, Cos-1, dcO, DCO, Dco, RII[[DR]], collecting duct, intrinsic catalyst activity, Antidiuretic Hormones, pka-c1, serine protein kinase activity, VP, AVP, protein kinase p58 activity, Pka-RII, serine/threonine protein kinase activity, threonine-specific protein kinase activity, 5-23, tubulus renalis arcuatus, PKA C, phosphorylase B kinase kinase activity, pka, pka C1, pKA, dr[[1]], PKA C3, protein glutamyl kinase activity, dco, non-specific serine/threonine protein kinase activity, CG6117, Dmel_CG12452, pka-RII, 5'-cAMP-dependent protein kinase activity, dsk1, PKA-RII, 5'-cyclic monophosphate-responsive protein kinase activity, atypical PKC activity, signalling, DmelCG42341, PKA CI, hydroxyalkyl-protein kinase activity, Vasopressini injectio, WEE1Hu, DmelCG4379, ATP:protein phosphotransferase (cAMP-dependent) activity, signalling process, PKA RII, serine-specific protein kinase activity, dPKA, epsilon PKC, PkaC1, l(2)01272, PKAcA, HIPK2, cytidine 3', beta-Hypophamine, arginine vasopressin, RII, Epa, Wee 1-like kinase activity, Dcpk, adenosine 3', protein serine-threonine kinase activity, PKAC1, A-kinase activitysodium salt, Hac-1/Dark, ammonium formate, 13C-labeled, IL1BC, cadmium salt, Lysine Hydrochloride, determination, L-arginine, DL-Arginine Acetate, DmelCG6829, Aminosaeure, CASP-1, classic hairy cell leukaemia, zinc formate, Amino acid, CG7788, Monohydrate, crice, lead salt, Visible Light, CSMF, Basodexan, Hydrogenchlorid, protein, magnesium formate, Apaf-1, clefted, zinc salt, Bca, peptide, Polypeptides, protein polypeptide chains, Lysine Acetate, halite, ammonium tetraformate, hydrogen chloride, peptido, ClvPrd, cobalt(II) formate dihydrate, hnu, B1, SEC, Urea, HCL-C, 2, Min, AGM4, Apaf1, Arg, protein aggregate, Il1bc, present in fewer numbers in organism, ARK, foton, DmelCG42273, L Lysine, bca, (S)-2-amino-5-guanidinopentanoic acid, amino acids, hac-1, peptides, K, E927b, (S)-2-Amino-5-guanidinovaleric acid, sec, R, hypoplasia, epsilon-diaminocaproic acid, min, arc, Arginine, mAPC, Processed cyclic AMP-responsive element-binding protein 3-like protein 1, Monohydrate DL-Arginine Acetate, proteins, copper, ark, IL-1BC, T1, HPLC, AI047805, cloruro sodico, salt, Wasserstoffchlorid, NOR-1, decreased, lithium salt, LYS, column, ICE, Lysin, sample, D1, Lyw-57, dApaf1, TEAB, phosphatase, ammonium (2:1) salt, reagents, gamma, nickel salt, L-Arginin, rock salt, Radiation, Caspase-1 subunit p10, subdivided, CHN, ice, iCE, D-Apaf-1, drice, cobalt (+2) salt, Aminokarbonsaeure, Arginine Hydrochloride, anon-53Fa, Dmel_CG7826, arginine, l(2)SH0173, Light, classic hairy cell leukemia, alpha-amino carboxylic acids, ur, DmelCG7788, SLP65, carbamide, Ly-57, common salt, sodium (4:1:1) salt, LIGHT, Dm1, magnesium salt, drIce, drICE, lysine, HCL, Chlorwasserstoff, leukemic reticuloendotheliosis, reactif, nickel (+2) salt, CG6829, Dmel_CG7835, forked, dark/hac-1/dapaf-1, L-Arginine, Visible Radiations, Mnb, MNB, Amino Acid, Acid, SLP-65, ammonium salt, Visible Radiation, divided, HVEML, Amino acids, BASH, LY57, P45, HCl, formate, cobaltous formate, pooled, reagent, cromium (+3), alpha, hac1, AW124434, natrii chloridum, septate, beta Trypsin, organ system, dapaf-1S, H2NC(O)NH2, apaf-1, light quantum, dapaf-1L, Acetamide, Drice, copper salt, p45, ammonium (4:1) salt, Acids, Polypeptide, chlorane, DRICE, lead (+2) salt, 1728, time, Old astrocyte specifically-induced substance, nickel formate dihydrate, Dapaf-1/HAC-1, lead formate, chlorure d'hydrogene, DrIce, DrICE, aluminum salt, cesium salt, Aminocarbonsaeure, L Isomer, Gene, high weight, Carbamide, alpha-amino acid, protein-containing complex, body system, CG7826, L-(+)-arginine, Ly113, Buffer, formic acid, Dark/Hac-1/dApaf1, Karbamid, period, Hac1, potassium salt, Dark/Hac-1/dApaf-1, (2S)-2-amino-5-(carbamimidamido)pentanoic acid, polypeptide chain, reduced, Ly57, subnumerary, Oasis, 14C-labeled, Harnstoff, heavy, CG7835, Gene Products, CG42273, system, Lysine, light, Graphene, tiny, Carmol, cloruro de hidrogeno, Pulses, Pro-Mega, Hydrogen Oxide., T6K12_14, anatomical systems, reactivo, AI573420, Hydrogen chloride, thallium (+1) salt, Lichtquant, Selb, PCE-2, dapaf-1, beta-Trypsin, rubidium salt, chlorure de sodium, dapaf, Visible, dark, decreased number, calcium formate, methanoic acid, hairy cell leukemia, Caspase-1 subunit p20, DARK, Kochsalz, photon, L-Arg, peptidos, cromium (+3) salt, TR2, dArk, Dapaf-1, Chromatographies, 2-iodo-, sodium formate, PTHB1, BLNK-S, small, dark/dapaf-1/hac-1, DYRK1, NOR1, Nor1, nickel formate, caspase 3, protein complex, dApaf-1, Proteins, strontium formate, apaf1, alpha-amino acids, chloridohydrogen, potassium formate, CE-2, copper (+2) salt, L-Isomer, strontium salt, DL Arginine Acetate, CD258, buffer, Cell, Peptide, polypeptide, [HCl], APAF1, secret agent, Experiment, native protein, Natriumchlorid, natural protein, L-Isomer Arginine, carbonyldiamide, Hac-1, Acetate, C18, cupric formate, Protein, chemical analysis, Hydrochloride, table salt, L Arginine, Dyrk1, CES2A1, Dark, Vacuums, connected anatomical system, NaCl, aluminum formate, L-Lysine, Dark/Apaf-I, T6K12.14, Radiations, AU020952, lithium formate, underdeveloped, MINOR, uree, HVEM-L, urea, (2S)-2-amino-5-guanidinopentanoic acid, CC1, Photoradiation, Tripcellim, OASIS, Dark/Dapaf-1/HAC1, phosphoric monoester hydrolase activity, IL-1 beta-converting enzyme, Interleukin-1 beta-converting enzyme, Amino, dApaf-1/DARK/HAC-1, sample population, chromic formate, LTg, UREA, ME-IV, Interleukin-1 beta convertase, Protein Gene Products, cAMP-responsive element-binding protein 3-like protein 1, Gene Proteins, Trypure, Photoradiations, 3.4.22.36, calcium salt, Peptid, TEC, assay, 6-diaminohexanoic acid, Enisyl, Minor, hydrochloric acid, dAPAF-1, sodium chloridebeta-hydroxy-beta-methylglutaryl-CoA reductase kinase activity, FALL39, aquaporin, Ass-1, CG18677, phosphorylation, heart-and-soul protein, Prp4 protein kinase activity, cell associated, AA408978, I-peptide kinase activity, protein polypeptide chains, collecting tubule, ATP-protein transphosphorylase activity, cAMP-dependent protein kinase, PASK, Pitressin, responsivity, Prkcl, ATP:protein phosphotransferase (non-specific) activity, CAP-18, symptoms, 5' Monophosphate, fold, Prkci, 3, Protein Kinases, cyclic 3', Kinase, Analysis, phosphodiesterase inhibitor, myd, Renal-type arginine vasopressin receptor, Cyclic AMP Dependent Protein Kinases, Protein Kinase A, mKIAA4165, Adenosine Cyclic 3, Disodium Salt, Solute injectable de vasopressine, C, HAMPKb, cAMP Dependent Protein Kinase, Analyses, down regulation of cyclic nucleotide phosphodiesterase activity, DI1, plant peltate hair, ATP:acetyl-CoA carboxylase phosphotransferase activity, Pk72A, Antidiuretic, Mbp-1, 4933403G17Rik, 5' cAMP-dependent protein kinase complex, Cyclic Monophosphate, proteins, down-regulation of cyclic nucleotide phosphodiesterase activity, Cyclic 3', cAMP-Dependent, Cyclic AMP Dependent Protein Kinase, ampk2, 3-Isobutyl-1-methyxanthine, kidney collecting duct, DmelCG6117, 5' cAMP-dependent protein kinase activity, Ampka2, 6-dione, Hal protein, DIR, ADHR, PKCiota, cAMP-Dependent Protein Kinases, 7-dihydro-1H-purine-2, Adenosine Cyclic 3', HMG-CoA reductase kinase activity, 3-isobutyl-1-methyl-3, single organism signaling, p82 kinase activity, screening, Rnl5, ADH, R74955, renal collecting tubule, downregulation of cyclic nucleotide phosphodiesterase activity, betaIIPKC, cyclic AMP-dependent protein kinase complex, CG42341, pka-C1, Spectrum Analysis, 5'-phosphate, HSL, Wee-kinase activity, pkc-iota, IBMX, Cyclic AMP, (R)-Isomer, mKIAA0609, RII[[DR]], PKC lambda, Antidiuretic Hormones, pka-c1, GEF-H1, Hydrogen Oxide, fg, PKA C, EG:132E8.2, CHAMP, expanded, Spectrometry, non-specific serine/threonine protein kinase activity, signs, Adenosine Cyclic Monophosphate-Dependent Protein Kinases, 5'-cyclic-AMP phosphodiesterase activity, Dmel_CG12452, pka-RII, cAMP Dependent Protein Kinases, VPV2R, human, DmelCG3051, Adenosine, MDC1D, PRKAA, Sodium Salt, AW556857, DmelCG4379, enr, PKA RII, serine-specific protein kinase activity, PkaC1, PKAcA, HIPK2, cytidine 3', acetyl-CoA carboxylase kinase 2 activity, beta-Hypophamine, dAMPKalpha, RII, ACK3, Epa, ACK2, Monophosphate, Wee 1-like kinase activity, adenosine 3', PKAC1, big, CRAMP, beta Hypophamine, prkci protein, Cyclic AMP-Dependent Protein Kinase, glycogen synthase A kinase activity, Antidiuretic Hormone, region or site annotation, nPKC-iota protein, glycogen synthase kinase 3 activity, peltate hair, number, Vasopressin (USP), PKA-C3, MECT1, PKA-C1, protein-containing complex, phosphorylase b kinase kinase activity, BcDNA:GM01761, PKClambda, 1H-Purine-2, Hormone, 5'-Cyclic AMP, large, Pka-RI, l(2)cos[[1]], V2R, ribosomal protein S6 kinase II activity, ND1, Pkcl protein, protein kinase C iota, mKIAA0616, protein-serine kinase activity, AW227544, Mect1, positional, Pka-C, DIR3, MDDGB6, ligand, DmAMPK alpha, Monosodium Salt, STK32, NDI, cAMP Protein Kinase, 5'-Monophosphate, prkaa1, 5'-cyclophosphate-dependent protein kinase activity, Adenosine Cyclic Monophosphate Dependent Protein Kinases, Atypical protein kinase C-lambda, Cos, Cyclic-3', CdkA, protein-cysteine kinase activity, STK29, Hpr kinase activity, STK22, scale (sensu Metazoa), DRI, heart and soul protein, FBgn0023169, iota protein, signalling., PKA Cl, findings, polypeptide motif, Phosphorylations, CG3263, Atypical protein kinase C iota, 5' cyclic nucleotide phosphodiesterase inhibitor, CdkR, protein serine kinase activity, motif, Cell, 3-hydroxy-3-methylglutaryl coenzyme A reductase kinase activity, protein phosphokinase activity, acetyl coenzyme A carboxylase kinase (phosphorylating) activity, hydroxymethylglutaryl-CoA reductase (NADPH) kinase activity, AMP, acetyl-coenzyme A carboxylase kinase activity, cyclic AMP-dependent protein kinase activity, pkA, PKA, Pka, lambda protein kinase C, native protein, PKC, 5'-cyclic phosphate, chemical analysis, Gprk-4, scales, intrinsic catalyst activity, protein kinase p58 activity, Mass Spectrum Analyses, epitheliocyte, nPKC-iota, serine/threonine protein kinase activity, pka, pka C1, pKA, dr[[1]], PKA C3, increased number, 3-isobutyl-1-methylxanthine, DCHT, Monopotassium Salt, 5'-cAMP-dependent protein kinase activity, dsk1, mKIAA0651, hydroxymethylglutaryl-CoA reductase kinase activity, hydroxymethylglutaryl coenzyme A reductase kinase activity, negative regulation of 3', negative regulation of cAMP phosphodiesterase activity, 5'-cyclic monophosphate-responsive protein kinase activity, atypical PKC activity, signalling, DmelCG42341, reductase kinase activity, PKA CI, signalling process, binding_or_interaction_site, apkc, Isolation of Nuclei TAgged in specific Cell Types, l(2)01272, ADENOSINE-3', regulation, Cyclic, Antidiuretic hormone receptor, cAMP-Dependent Protein Kinase, quantitative, 5' cyclic nucleotide phosphodiesterase activity, protein serine-threonine kinase activity, glycogen synthase kinase activity, 6353, cAMP phosphodiesterase inhibitor, scale tissue, Dmel_CG3263, protein kinase A activity, Lfc, 5'-cyclic-AMP phosphodiesterase inhibitor, 3 Isobutyl 1 methylxanthine, determination, 5'-cyclic monophosphate, Mbp1, AMPKa2, ureteric tree, protein, GEFH1, AMPKa1, ATP:hydroxymethylglutaryl-CoA reductase (NADPH) phosphotransferase activity, CG12452, binding site, iota, AA408052, mitogen-activated protein kinase activity, Dpck, RI, protein aggregate, Adenosine Cyclic Monophosphate, C13orf8, Mass Spectrum Analysis, PRKCI protein, increased, CG3051, cAMP, CG4379, Antidiuretic hormone, AMPK2, ZNF828, 3', LHS, acetyl-CoA carboxylase bound kinase activity, CG15862, Raf kinase activity, CCR1, AI427505, ASS, arcuate renal tubule, serine kinase activity, atypical protein kinase C activity, dAMPKa, aPKClambda, Accelerations, signaling process, mitogen-activated S6 kinase activity, protein-aspartyl kinase activity, snf1a, protein kinase C-iota, 3-hydroxy-3-methylglutaryl-CoA reductase kinase activity, Adenosine Cyclic, gyltl1b-b, prkcl, mouse, kidney collecting tubule, CAP18, XLfc, results, CAMP, M phase-specific cdc2 kinase activity, Spectroscopy, PAS14, epithelial, pka-R1, MAPK, MDDGA6, 5'-cAMP-dependent protein kinase complex, Cos-1, Prkcl protein, 1-methyl-3-(2-methylpropyl)-7H-purine-2, VP, serine protein kinase activity, AVP, acetyl-CoA carboxylase kinase (AMP-activated) activity, region, 5-23, phosphorylase B kinase kinase activity, Prkci protein, gyltl1b, positional polypeptide feature, protein glutamyl kinase activity, P40, Isobutyltheophylline, mdc1d, CG6117, prkaa, acetyl-CoA carboxylase kinase-3 (AMP-activated) activity, 1-methyl-3-isobutylxanthine, Permeabilities, protein kinase C, 7-dihydro-1-methyl-3-(2-methylpropyl)-, hydroxyalkyl-protein kinase activity, WEE1Hu, ATP:protein phosphotransferase (cAMP-dependent) activity, enlarged, dPKA, epsilon PKC, ampka1, Adenosine 3', acetyl-CoA carboxylase kinase activity, Protein Kinase, Dcpk, cell bound, zebrafish, accessory, TORC1, ribosomal S6 protein kinase activity, calcium/phospholipid-dependent protein kinase activity, biological signaling, AMPKalpha, ampk, 1 Methyl 3 isobutylxanthine, Pk?5, PKA-C-3, Spectrum Analyses, presence, supernumerary, froggy, Gyltl1a, CAT1, RF005, polypeptide chain, GEF, Cyclic adenylic acid, Mass, AI413414, Inyectable de vasopresina, argipressin, serine(threonine) protein kinase activity, BG02142, AMPK, 18304, Mass Spectroscopy, acetyl-CoA carboxylase kinase (cAMP-independent) activity, T-antigen kinase activity, ampkalpha, acetyl-CoA carboxylase kinase-2 activity, galactosyltransferase-associated kinase activity, reactivity, Has protein, TORC-1, HAF3, PKAR, PkaR, negative regulation of cyclic nucleotide phosphodiesterase activity, STK5, PKa-R2, 2310021H13Rik, PKa-R1, PKAc, Ampk, ATP-protein phosphotransferase, LFP40, junctional tube, SPAK, BPFD#36, protein kinase (phosphorylating) activity, PKA-R1, DmelCG15862, PKA-R2, Raf-1, great, Vasopressin, Gprk4, Dc0, site, 5 Monophosphate, casein kinase (phosphorylating) activity, PKA-RI, tubulus renalis colligens, Monoammonium Salt, PKCI, Cos1, 5'-cyclophosphate-dependent protein kinase complex, l(2)s4402, protein complex, DC0, PKC iota, DC2, negative regulation of cGMP phosphodiesterase activity, dPKA-RI, inhibition of cyclic nucleotide phosphodiesterase activity, cos1, count in organism, twitchin kinase activity, PKAi, AVPR V2, Phosphopeptide, 1-methyl-3-(2-methylpropyl)-3, Adenosine Cyclic-3', PKAr, natural protein, AP50 kinase activity, Protein, collecting duct system, dcO, DCO, Dco, collecting duct, Pka-RII, WAMTP1, threonine-specific protein kinase activity, tubulus renalis arcuatus, Mass Spectrum, DXS1179E, LL37, scale, FALL-39, hydroxymethylglutaryl-CoA reductase (NADPH2) kinase activity, Kinases, INSDC_feature:misc_binding, Lbcl1, dco, 5'-(hydrogen phosphate), GLC2, AOMS4, 3-Isobutyl-1-methylxanthine, hydroxymethylglutaryl coenzyme A reductase kinase (phosphorylating) activity, 5'-CYCLIC-MONOPHOSPHATE, PKA-RII, Pkcl, present in greater numbers in organism, Vasopressini injectio, Pkci, DEL, arginine vasopressin, assay, response, A-kinase activity, presence or absence in organismFALL39, beta-hydroxy-beta-methylglutaryl-CoA reductase kinase activity, aquaporin, PKC-53B, cPKCgamma, CG18677, phosphorylation, Prp4 protein kinase activity, I-peptide kinase activity, collecting tubule, Generic Action, InaC, INAC, DmelCG12298, ATP-protein transphosphorylase activity, cAMP-dependent protein kinase, PASK, Pitressin, Prkcl, CAP-18, ATP:protein phosphotransferase (non-specific) activity, symptoms, SCRAMBLED, Prkci, 3, Protein Kinases, IFITMD1, Kinase, Prkcc, Renal-type arginine vasopressin receptor, Cyclic AMP Dependent Protein Kinases, Protein Kinase A, mKIAA4165, Solute injectable de vasopressine, C, nPKCtheta, HAMPKb, cAMP Dependent Protein Kinase, RATPKCI, DI1, ATP:acetyl-CoA carboxylase phosphotransferase activity, Pk72A, Antidiuretic, PKCbeta, 5' cAMP-dependent protein kinase complex, BFIC2, cAMP-Dependent, Cyclic AMP Dependent Protein Kinase, ampk2, 3-Isobutyl-1-methyxanthine, kidney collecting duct, DmelCG6117, 5' cAMP-dependent protein kinase activity, Ampka2, 6-dione, DIR, PKC-53E, ADHR, PKCiota, cAMP-Dependent Protein Kinases, 7-dihydro-1H-purine-2, HMG-CoA reductase kinase activity, 3-isobutyl-1-methyl-3, single organism signaling, p82 kinase activity, screening, Rnl5, ADH, renal collecting tubule, STRUBBELIG, betaIIPKC, cyclic AMP-dependent protein kinase complex, CG42341, pka-C1, 5'-phosphate, Wee-kinase activity, pkc-iota, IBMX, Cyclic AMP, RII[[DR]], Antidiuretic Hormones, pka-c1, activation, Hydrogen Oxide, diacylglycerol-activated phospholipid-dependent protein kinase C activity, PKA C, EG:132E8.2, CHAMP, PKC-98F, non-specific serine/threonine protein kinase activity, signs, Adenosine Cyclic Monophosphate-Dependent Protein Kinases, Dmel_CG12452, pka-RII, cAMP Dependent Protein Kinases, VPV2R, DmelCG3051, PRKAA, AW556857, DmelCG4379, PKA RII, serine-specific protein kinase activity, PkaC1, PKAcA, HIPK2, cytidine 3', acetyl-CoA carboxylase kinase 2 activity, beta-Hypophamine, dAMPKalpha, RII, ACK3, Epa, ACK2, Feedbacks, Wee 1-like kinase activity, adenosine 3', PKAC1, CRAMP, atypia, beta Hypophamine, 98F, Cyclic AMP-Dependent Protein Kinase, glycogen synthase A kinase activity, Antidiuretic Hormone, glycogen synthase kinase 3 activity, PKC activity, Vasopressin (USP), PKA-C3, PKA-C1, PKCalpha, phosphorylase b kinase kinase activity, BcDNA:GM01761, PKClambda, 1H-Purine-2, Hormone, 5'-Cyclic AMP, PKC 98F, Pka-RI, l(2)cos[[1]], CG6622, atypical, V2R, ribosomal protein S6 kinase II activity, ND1, PKC 53B, PKC 53E, protein-serine kinase activity, AW227544, Pka-C, DIR3, ligand, mei-1794, DmAMPK alpha, STK32, NDI, cAMP Protein Kinase, prkaa1, 5'-cyclophosphate-dependent protein kinase activity, Adenosine Cyclic Monophosphate Dependent Protein Kinases, Cos, CdkA, protein-cysteine kinase activity, DmelCG6518, STK29, Hpr kinase activity, dPKC98F, STK22, STK24, DRI, FBgn0023169, PKA Cl, dPKC53E(br), DmelCG6622, findings, polypeptide motif, Phosphorylations, CG3263, CdkR, defective, protein serine kinase activity, motif, Cell, 3-hydroxy-3-methylglutaryl coenzyme A reductase kinase activity, protein phosphokinase activity, acetyl coenzyme A carboxylase kinase (phosphorylating) activity, hydroxymethylglutaryl-CoA reductase (NADPH) kinase activity, dPKC53E(ey), acetyl-coenzyme A carboxylase kinase activity, cyclic AMP-dependent protein kinase activity, pkA, PKA, Pka, PKC, 5'-cyclic phosphate, chemical analysis, Gprk-4, intrinsic catalyst activity, protein kinase p58 activity, nPKC-iota, serine/threonine protein kinase activity, pka, pka C1, pKA, dr[[1]], PKA C3, increased number, 3-isobutyl-1-methylxanthine, Dub, DCHT, eye PKC, 5'-cAMP-dependent protein kinase activity, dsk1, CG6518, hydroxymethylglutaryl-CoA reductase kinase activity, hydroxymethylglutaryl coenzyme A reductase kinase activity, 5'-cyclic monophosphate-responsive protein kinase activity, atypical PKC activity, signalling, DmelCG42341, reductase kinase activity, PKA CI, signalling process, apkc, Isolation of Nuclei TAgged in specific Cell Types, l(2)01272, ADENOSINE-3', regulation, Antidiuretic hormone receptor, cAMP-Dependent Protein Kinase, protein serine-threonine kinase activity, glycogen synthase kinase activity, 6353, Dmel_CG3263, protein kinase A activity, 3 Isobutyl 1 methylxanthine, determination, 5'-cyclic monophosphate, AMPKa2, ureteric tree, AMPKa1, ATP:hydroxymethylglutaryl-CoA reductase (NADPH) phosphotransferase activity, nPKCepsilon, CG12452, SUB, mitogen-activated protein kinase activity, Dpck, cPKCbeta, RI, KIF20A, C13orf8, cPKCalpha, nPKCdelta, increased, BFIS2, CG3051, nPKC, cPKC, cAMP, CG4379, Antidiuretic hormone, ZNF828, AMPK2, 3', acetyl-CoA carboxylase bound kinase activity, CG15862, Dpkc2, Dpkc3, Raf kinase activity, Dpkc1, AI427505, CCR1, arcuate renal tubule, serine kinase activity, atypical protein kinase C activity, dAMPKa, aPKClambda, signaling process, mitogen-activated S6 kinase activity, protein-aspartyl kinase activity, snf1a, 5'-cAMP-dependent protein kinase activity., associated, DSPB3, ATP:protein phosphotransferase (diacylglycerol-dependent) activity, PKC53E, 3-hydroxy-3-methylglutaryl-CoA reductase kinase activity, aberrant, prkcl, dPKC53E, kidney collecting tubule, CAP18, results, PKC98E, CAMP, Pkc1p, M phase-specific cdc2 kinase activity, PAS14, pka-R1, MAPK, 5'-cAMP-dependent protein kinase complex, Cos-1, 1-methyl-3-(2-methylpropyl)-7H-purine-2, VP, serine protein kinase activity, AVP, acetyl-CoA carboxylase kinase (AMP-activated) activity, 5-23, Action, phosphorylase B kinase kinase activity, DPKC, PKCdelta, protein glutamyl kinase activity, Isobutyltheophylline, CG6117, prkaa, acetyl-CoA carboxylase kinase-3 (AMP-activated) activity, 1-methyl-3-isobutylxanthine, ICCA, Prkc, Permeabilities, 7-dihydro-1-methyl-3-(2-methylpropyl)-, hydroxyalkyl-protein kinase activity, WEE1Hu, ATP:protein phosphotransferase (cAMP-dependent) activity, dPKC, dPKA, epsilon PKC, ampka1, Adenosine 3', acetyl-CoA carboxylase kinase activity, eye-PKC, Protein Kinase, Dcpk, accessory, PKCgamma, AT1G11140, ribosomal S6 protein kinase activity, calcium/phospholipid-dependent protein kinase activity, biological signaling, AMPKalpha, ampk, ePKC, 1 Methyl 3 isobutylxanthine, Pk?5, PKA-C-3, supernumerary, CG12298, CAT1, RF005, Cyclic adenylic acid, diacylglycerol-activated phospholipid-dependent PKC activity, Inyectable de vasopresina, pkc-2, argipressin, serine(threonine) protein kinase activity, BG02142, AMPK, 18304, acetyl-CoA carboxylase kinase (cAMP-independent) activity, T-antigen kinase activity, ampkalpha, acetyl-CoA carboxylase kinase-2 activity, galactosyltransferase-associated kinase activity, HAF3, PKAR, PkaR, STK5, PKa-R2, 2310021H13Rik, PKa-R1, PKCzeta, PKAc, Ampk, DYT10, ATP-protein phosphotransferase, junctional tube, SPAK, protein kinase (phosphorylating) activity, PKA-R1, DmelCG15862, PKA-R2, protein kinase Cepsilon activity, Raf-1, nPKCeta, Nc98F, Vasopressin, Gprk4, Dc0, SRF9, casein kinase (phosphorylating) activity, PKA-RI, CG1954, tubulus renalis colligens, PKCI, Cos1, 5'-cyclophosphate-dependent protein kinase complex, l(2)s4402, PKC d98F, pkc53e, DC0, DC2, dPKA-RI, STRUBBELIG-RECEPTOR FAMILY 9, cos1, twitchin kinase activity, PKAi, AVPR V2, Phosphopeptide, 1-methyl-3-(2-methylpropyl)-3, PKAr, PKCepsilon, Pkc1, PKC1, AP50 kinase activity, Protein, Pkc3, collecting duct system, Pkc2, dcO, DCO, Dco, EKD1, collecting duct, Pka-RII, threonine-specific protein kinase activity, tubulus renalis arcuatus, DXS1179E, LL37, FALL-39, DmelCG1954, hydroxymethylglutaryl-CoA reductase (NADPH2) kinase activity, Kinases, dco, 5'-(hydrogen phosphate), GLC2, 3-Isobutyl-1-methylxanthine, hydroxymethylglutaryl coenzyme A reductase kinase (phosphorylating) activity, 5'-CYCLIC-MONOPHOSPHATE, PKA-RII, Pkcl, present in greater numbers in organism, Vasopressini injectio, Pkci, PKCi, arginine vasopressin, assay, T19D16.8, SCM, A-kinase activitySH2-Bb, Bru, IPP2A2, para Tyrosine, Raw, Lysine Hydrochloride, RANK, L-arginine, DL-Arginine Acetate, Irip, Monohydrate, TRANCER, L-Threonine, protein, TALL-2, NAALAdase, phosphorylation, L-(+)-glutamine, binding site, 2-Amino-3-hydroxypropionic acid, 5730420M11Rik, peptide, Polypeptides, NAALADase I, protein polypeptide chains, L-Isomer Methionine, FOLH, Lysine Acetate, L-glutamine, Drug Tolerance, Methionine, L-Glutaminsaeure-5-amid, ClvPrd, peptido, 2, L-2-aminoglutaramic acid, C530001K22Rik, 3.4.17.21, Arg, protein aggregate, WMS, Isotopically-Coded Affinity Tagging, Threonin, L Lysine, C79691, mRANK, Svc, DL-Asparagine, SH2-B, somitogenic mesoderm, SET, (S)-2-amino-5-guanidinopentanoic acid, asparagina, i2pp2a., peptides, asparagine, Membrane glutamate carboxypeptidase, TAF-I, K, M, N, phosphatase 2A inhibitor I2PP2A, L-Asparagine, 2-amino-3-(4-hydroxyphenyl)propanoic acid, 3-(p-Hydroxyphenyl)alanine, TRDL-1, (S)-2-Amino-5-guanidinovaleric acid, D-Glutamine, Q, E430039A18Rik, R, epsilon-diaminocaproic acid, Arginine, proteins, Monohydrate DL-Arginine Acetate, NAALAD1, Y, ASN, Asn, Folylpoly-gamma-glutamate carboxypeptidase, Lccp, DmelCG4299, set, IGAAD, LYS, 2-amino-3-hydroxypropanoic acid, DmelCG10574, Rank, ppm, Lysin, Tyr, house mouse, Half Cystine, OPTB7, Tagging, GPHYSD2, Racemethionine, drug tolerance, Stable Isotope Labeling, SGS, L-Arginin, phapii, Zinc Cysteinate, unsegmented mesenchyme, immune system tolerance, T5E21.12, SSP29, acetylation, mouse, Arginine Hydrochloride, 2-Amino-4-(methylthio)butyric acid, 2-Amino-3-(p-hydroxyphenyl)propionic acid, StF-IT-1, arginine, Search, para-Tyrosine, SH2-B PH domain-containing signaling mediator 1, ACMICD, Tyrosine, HCD, 3-Hydroxyalanine, PSM, Psm, Algorithm, lysine, Immunologic Tolerance, Methionin, methionine, Hmet, presumptive somite mesoderm, Prostate-specific membrane antigen, tyrosine, region, L Glutamine, T5E21_12, parent ion, L-Arginine, Pteroylpoly-gamma-glutamate carboxypeptidase, Serine, HLA-DR-associated protein II, positional polypeptide feature, DI-2, MS/MS, mice, I-2Dm, FGCP, 2310026N09Rik, L-isomer, MASCOT, L-Cysteine, CG4299, MASS, alpha, inhibitor of granzyme A-activated DNase, 2-amino-3-carbamoylpropanoic acid, (S)-2, beta Trypsin, L isomer, I-2PP1, L-glutamic acid gamma-amide, Pro-rich, TAF-IBETA, Self Tolerance, precursor ion, Tolerance, PSMA, L Serine, Mouse, TAF-Ibeta, L-Tyrosine, Polypeptide, L-Serine, i2pp2a, Liquimeth, PDB2, GLUTAMINE, SH2 domain-containing protein 1B, Cysteine Hydrochloride, 2-amino-4-(methylsulfanyl)butanoic acid, AW549739, region or site annotation, 5-diamino-5-oxopentanoic acid, unsegmented paraxial mesoderm, L Isomer, FBN, Levoglutamide, Hasp, precursor, (2S)-2-amino-4-carbamoylbutanoic acid, protein-containing complex, Isotope-Coded Affinity, ZTNF2, somitomeric mesoderm, GCPII, PHAPII, L-(+)-arginine, polypeptide chain, template-activating factor I, (2S)-2-amino-5-(carbamimidamido)pentanoic acid, ECTOL1, deamination, L-Methionine, N-acetylated-alpha-linked acidic dipeptidase I, mKIAA1299, Del(8)44H, Lysine, Half-Cystine, Isotopically-Coded Affinity, Pedameth, Search Engines, SH2B, positional, ODFR, OSTS, A proliferation-inducing ligand, TNFRSF11A, Ly109, L Cysteine, (2S)-2, MS2, receptor activator of NF-KB, OFE, ipp2a2, TNF-related death ligand 1, beta-Trypsin, Acetylations, 2pp2a, mGCP, L Threonine, CG10574, Probabilities, CD156, OCTD, Asparagin, mKIAA0989, 2PP2A, 2-amino-4-(methylthio)butanoic acid, 10^[-6], Stable, L Tyrosine, taf-ibeta, Isotope-Coded Affinity Tagging, dSET, dSet, L-Arg, FEO, site, L-Glutamine, peptidos, Tyrosin, metionina, serine, data, DL-Methionine, protein complex, igaad, Phosphorylations, L-2-Aminoglutaramic acid, LOH18CR1, L-Isomer, TALL2, DL Arginine Acetate, Labeling, CD256, Peptide, Engine, Trdl1, GCP2, Glutamate carboxypeptidase II, polypeptide, Isotope, Serin, TRANCE-R, Cell growth-inhibiting gene 27 protein, TNF- and APOL-related leukocyte expressed ligand 2, native protein, Immune Tolerance, natural protein, Mus, I-2PP2A, L-Isomer Arginine, tirosina, Stable Isotope, Acetate, Protein, April, Dm I-2, I2PP2A, Hydrochloride, Glutamic acid 5-amide, MFS1, L Arginine, L-Glutamin, segmental plate, Sh2bpsm1, L-Lysine, Isotope Coded Affinity Tagging, tandem MS, Data Base, WMS2, Met, CD265, PH and SH2 domain-containing signaling mediator, Col4a-1, Tall2, Glutamic acid amide, INSDC_feature:misc_binding, (2S)-2-amino-5-guanidinopentanoic acid, Tripcellim, Deaminations, Isotope Labeling, CD156a, Affinity Tagging, 4-diamino-4-oxobutanoic acid, osteoclast differentiation factor receptor, Isotopically-Coded, Folate hydrolase 1, APRIL, Trypure, dSET/TAF-Ibeta, 2610030F17Rik, D Glutamine, binding_or_interaction_site, PHAPI2, SSKS, AI425885, alpha-amino-gamma-methylmercaptobutyric acid, Isotope-Coded, Peptid, threonine, variable, AA407739, 6-diaminohexanoic acid, Enisyl, Immunological Toleranceglycogen synthase kinase activity, 6353, ribosomal S6 protein kinase activity, calcium/phospholipid-dependent protein kinase activity, beta Hypophamine, biological signaling, Dmel_CG3263, protein kinase A activity, glycogen synthase A kinase activity, Antidiuretic Hormone, glycogen synthase kinase 3 activity, Pk?5, PKA-C3, PKA-C-3, Vasopressin (USP), PKA-C1, ureteric tree, CG18677, phosphorylase b kinase kinase activity, BcDNA:GM01761, collecting duct system., CG12452, Prp4 protein kinase activity, Hormone, collecting tubule, Pka-RI, ATP-protein transphosphorylase activity, cAMP-dependent protein kinase, Pitressin, ATP:protein phosphotransferase (non-specific) activity, l(2)cos[[1]], Inyectable de vasopresina, serine(threonine) protein kinase activity, argipressin, mitogen-activated protein kinase activity, Dpck, BG02142, RI, AMPK, ribosomal protein S6 kinase II activity, 18304, T-antigen kinase activity, galactosyltransferase-associated kinase activity, C, protein-serine kinase activity, Solute injectable de vasopressine, Pka-C, PKAR, PkaR, CG4379, STK32, Pk72A, Antidiuretic hormone, 3', Antidiuretic, 5' cAMP-dependent protein kinase complex, PKa-R2, PKa-R1, PKAc, CG15862, Raf kinase activity, junctional tube, protein kinase (phosphorylating) activity, arcuate renal tubule, PKA-R1, serine kinase activity, DmelCG15862, 5'-cyclophosphate-dependent protein kinase activity, kidney collecting duct, PKA-R2, Cos, DmelCG6117, 5' cAMP-dependent protein kinase activity, CdkA, protein-cysteine kinase activity, atypical protein kinase C activity, Raf-1, signaling process, Hpr kinase activity, Vasopressin, Dc0, mitogen-activated S6 kinase activity, STK22, protein-aspartyl kinase activity, casein kinase (phosphorylating) activity, DRI, PKA-RI, single organism signaling, p82 kinase activity, PKA Cl, tubulus renalis colligens, ADH, renal collecting tubule, Cos1, 5'-cyclophosphate-dependent protein kinase complex, l(2)s4402, DC0, betaIIPKC, cyclic AMP-dependent protein kinase complex, DC2, kidney collecting tubule, dPKA-RI, CG42341, CG3263, CdkR, protein serine kinase activity, pka-C1, protein phosphokinase activity, M phase-specific cdc2 kinase activity, Wee-kinase activity, cos1, twitchin kinase activity, cyclic AMP-dependent protein kinase activity, PKAi, pkA, PKA, Pka, PKAr, pka-R1, PKC, MAPK, AP50 kinase activity, 5'-cAMP-dependent protein kinase complex, Cos-1, dcO, DCO, Dco, RII[[DR]], collecting duct, intrinsic catalyst activity, Antidiuretic Hormones, pka-c1, serine protein kinase activity, VP, AVP, protein kinase p58 activity, Pka-RII, serine/threonine protein kinase activity, threonine-specific protein kinase activity, 5-23, tubulus renalis arcuatus, PKA C, phosphorylase B kinase kinase activity, pka, pka C1, pKA, dr[[1]], PKA C3, protein glutamyl kinase activity, dco, non-specific serine/threonine protein kinase activity, CG6117, Dmel_CG12452, pka-RII, 5'-cAMP-dependent protein kinase activity, dsk1, PKA-RII, 5'-cyclic monophosphate-responsive protein kinase activity, atypical PKC activity, signalling, DmelCG42341, PKA CI, hydroxyalkyl-protein kinase activity, Vasopressini injectio, WEE1Hu, DmelCG4379, ATP:protein phosphotransferase (cAMP-dependent) activity, signalling process, PKA RII, serine-specific protein kinase activity, dPKA, epsilon PKC, PkaC1, l(2)01272, PKAcA, HIPK2, cytidine 3', beta-Hypophamine, arginine vasopressin, RII, Epa, Wee 1-like kinase activity, Dcpk, adenosine 3', protein serine-threonine kinase activity, PKAC1, A-kinase activityfalsePKA-Independent Vasopressin Signaling in Renal Collecting DuctVasopressin regulates renal water excretion by binding to the Gs-coupled vasopressin receptor (V2R) in collecting duct cells, resulting in cyclic AMP-dependent increases in epithelial water permeability through regulation of the aquaporin-2 (AQP2) water channel. Our prior studies showed that CRISPR-mediated deletion of protein kinase A (PKA) in cultured mpkCCD cells largely eliminates these regulatory events. These PKA-null cells provide a means of identifying PKA-independent signaling downstream from the V2 receptor. We carried out large-scale quantitative protein mass spectrometry (SILAC) to identify PKA-independent phosphorylation changes in response to V2R-selective vasopressin analog, dDAVP. The results show that V2R-mediated vasopressin signaling is predominantly, but not entirely, PKA-dependent. Target motif analysis of the phosphopeptides increased in response to dDAVP in PKA-null cells indicates that the vasopressin activates of one or more members of the AMPK/SNF1 subfamily of basophilic protein kinases. Among the upregulated phosphorylation sites were three known targets of SNF1-subfamily kinases, namely Lipe (S559), Crtc1 (S151) and Arhgef2 (S151). One of the phosphorylation sites that increased in occupancy in PKA-null cells was Ser256 of AQP2, a site critical for vasopressin-mediated trafficking of AQP2 to the cell surface. Beyond this, PKA-independent active site phosphorylation changes were also seen for protein kinases Stk39 (SPAK) and Prkci (Protein kinase C iota). Cyclic AMP levels were ~10-fold higher in PKA-null than in PKA-intact cells in the presence of phosphodiesterase inhibitor IBMX, consistent with a marked acceleration of cAMP production in PKA-null cells. The findings are indicative of substantial PKA-independent signaling downstream from the Gs-coupled V2 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