Prideapplication/xmlftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/03/PXD014985/Mylk2_IMAC_SILACK6R10_Mascot_Sprot.msfftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/03/PXD014985/Mylk2_Total_SILACK6R10_Mascot_SprotMouse.msfftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/03/PXD014985/Mylk2_IMAC_SILACK6R10_SquestHT_Sprot.msfftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/03/PXD014985/Mylk3_TiO2_SILACK6R10_SequestHT_Sprot.msfftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/03/PXD014985/Mylk2_TiO2_SILACK6R10_SquestHT_Sprot.msfftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/03/PXD014985/Mylk3_IMAC_SILACK6R10_SequestHT_Sprot.msfftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/03/PXD014985/Mylk2_TiO2_SILACK6R10_Mascot_Sprot.msfftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/03/PXD014985/Mylk1_TiO2_SILACK6R10_SequestHT_Sprot.msfftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/03/PXD014985/Mylk3_Total_SILACK6R10_SequestHT_Sprot.msfftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/03/PXD014985/Mylk2_Total_SILACK6R10_SequestHT_SprotMouse.msfftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/03/PXD014985/Mylk1_Total_SILACK6R10_SequestHT_Sprot.msfftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/03/PXD014985/Mylk1_Total_SILACK6R10_Mascot_Sprot.msfftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/03/PXD014985/Mylk1_IMAC_SILACK6R10_Mascot_Sprot.msfftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/03/PXD014985/Mylk3_IMAC_SILACK6R10_Mascot_Sprot.msfftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/03/PXD014985/Mylk3_TiO2_SILACK6R10_Mascot_Sprot.msfftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/03/PXD014985/Mylk1_TiO2_SILACK6R10_Mascot_Sprot.msfftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/03/PXD014985/Mylk3_Total_SILACK6R10_Mascot_Sprot.msfftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/03/PXD014985/Mylk1_IMAC_SILACK6R10_SequestHT_Sprot.msfftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/03/PXD014985/Mylk3_TiO2_05.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/03/PXD014985/Mylk2_Total_08.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/03/PXD014985/Mylk2_Fe_04.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/03/PXD014985/Mylk2_TiO2_05.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/03/PXD014985/Mylk1_TiO2_11.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/03/PXD014985/Mylk3_Fe_01.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/03/PXD014985/Mylk1_TiO2_05.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/03/PXD014985/Mylk1_Fe_05.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/03/PXD014985/Mylk1_Total_09.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/03/PXD014985/Mylk1_TiO2_07.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/03/PXD014985/Mylk2_Fe_02.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/03/PXD014985/Mylk3_Total_09.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/03/PXD014985/Mylk2_Total_12.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/03/PXD014985/Mylk3_Fe_03.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/03/PXD014985/Mylk3_TiO2_03.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/03/PXD014985/Mylk3_Total_11.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/03/PXD014985/Mylk1_Total_02.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/03/PXD014985/Mylk3_Total_02.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/03/PXD014985/Mylk2_TiO2_03.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/03/PXD014985/Mylk1_Total_11.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/03/PXD014985/Mylk1_TiO2_03.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/03/PXD014985/Mylk2_Fe_06.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/03/PXD014985/Mylk3_Fe_12.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/03/PXD014985/Mylk1_Fe_09.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/03/PXD014985/Mylk2_TiO2_11.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/03/PXD014985/Mylk1_Fe_07.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/03/PXD014985/Mylk3_TiO2_11.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/03/PXD014985/Mylk2_Total_06.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/03/PXD014985/Mylk3_TiO2_01.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/03/PXD014985/Mylk2_Fe_12.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/03/PXD014985/Mylk1_Total_04.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/03/PXD014985/Mylk2_Total_04.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/03/PXD014985/Mylk3_Fe_05.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/03/PXD014985/Mylk3_Fe_10.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/03/PXD014985/Mylk1_Fe_02.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/03/PXD014985/Mylk3_Total_05.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/03/PXD014985/Mylk3_Total_03.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/03/PXD014985/Mylk2_Fe_07.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/03/PXD014985/Mylk1_TiO2_01.rawftp://ftp.pride.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l_07.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/03/PXD014985/Mylk1_TiO2_09.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/03/PXD014985/Mylk3_Fe_08.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/03/PXD014985/Mylk2_TiO2_09.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/03/PXD014985/Mylk3_Total_06.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/03/PXD014985/Mylk1_Fe_11.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/03/PXD014985/Mylk1_Fe_03.rawprimaryOK200knepperm@nhlbi.nih.govCHIN-RANG YANGMass SpectrometryShotgun proteomicsNot availableEndosomal traffickingNuclear membraneSted microscopyMass spectrometryAqp2Vasopressinhttp://www.ebi.ac.uk/pride/archive/projects/PXD014985Epithelial CellAll SILAC reagents were purchased from Invitrogen. All three pairs of Mylk + and Mylk – cells were grown in culture medium containing the amino acids arginine and lysine labeled with stable isotopes, “light”: 12C6 14N4 arginine, 12C6 lysine and “Heavy”: 13C6 15N4 arginine, 13C6 lysine, respectively for 17 days (five passages). Cells were lysed and sonicated with 8 M urea buffer [8M urea, 50 mM Tris-HCl, 75 mM NaCl, protease and phosphatase inhibitors (Cat. No. 78440, Thremo Fisher Scientific)]. Then, samples were reduced with 20 mM dithiothreitol (Cat. No. 20291, Thremo Fisher Scientific) for 1 hour at 25 oC and then alkylated with iodoacetamide (Cat. No. 90034, Thermo Fisher Scientific) (final concentration 40 mM) for 1 hour at 25 oC in dark. The samples were further diluted to final concentration less than 1M urea with 20 mM triethylammonium bicarbonate (TEAB) buffer (pH8.5) (Cat. No.T7408, Sigma), and digested in solution with Trypsin/LysC (Cat. No. V5072, Promega) (1:20 by weight) for overnight at 37oC. The peptides were desalted using hydrophilic-lipophilic-balanced (HLB) extraction cartridges (Cat. No.WAT094225. Oasis, Milford, MA). The samples were vacuum-dried and subjected to high pH reversed phase fractionation (12 fractions per sample).Peptides were fractionated across an XBridge BEH C18 reversed-phase column (4.6 mm x 250 mm; Waters). Fractions were collected every minute and pooled into 12 samples which were dried for further analysis. Each fraction was divided into three parts for total peptide analysis (2%) and two phospho-peptide enrichment analyses (49% X 2) using Fe-NTA (Cat. No. 88300, Thermo Fisher Scientific) column and TiO2 columns (Cat. No. 88301, Thermo Fisher Scientific) per manufacture’s protocols, then vacuum-dried and stored at -80 oC. Before mass spectrometry analysis, the samples were dissolved in 0.1% formic acid (Cat. No. 28905, Thermo Fisher Scientific) in LC-MS grade water (Cat. No.9831, J.T. Baker chemicals, PA.). Total peptides and phosphopeptides were analyzed using an EKSIGENT nanoLC system connected to an Orbitrap Elite mass spectrometer (Thermo Fisher Scientific).PrideSILACphosphorylated residueiodoacetamide derivatized residuemonohydroxylated residuedeamidated residueMS spectra was analyzed using Proteome Discoverer 1.4 (Thermo Fisher Scientific). Two peptide-spectra match software, Mascot (47) and SEQUEST (48) were performed. Swiss-Prot mouse protein database (Jul 10, 2016) was used in search. The target-decoy algorithm (49) was used to calculate the peptide false discovery rate (FDR). The PhosphoRS (50) was used to calculate individual probability values for each putatively phosphorylated site based on the given MS/MS data, Phosphorylation sites with probability > 80% were labelled with the corresponding phosphorylation site identified; the phospho-peptides with probability < 80% were labelled as ambiguous. The search results from Mascot and SequestHT were integrated using Proteome Discoverer with FDR <0.01 and peptide rank = 1. Relative quantification of unique total and phospho-peptides was performed using the “Precursor Ions Quantifier” module within Myosin Light Chain Kinase in Renal Collecting Duct 23 Proteome Discoverer, which calculates light channel (Mylk-) over heavy channel (Mylk+) intensity (area under curve) ratios of total and phospho-peptides from the corresponding MS1 peaks. The missing channel values were replaced with the minimum intensity detected of that channel.ProteomicsMark A KnepperLTQ Orbitrap ElitePARTIALEpithelial Systems Biology Laboratory Systems Biology Center Division of Intramural Research NHLBI, NIH USAMus Musculus (mouse)31904282 Isobe K, Raghuram V, Krishnan L, Chou CL, Yang CR, Knepper MA. CRISPR-Cas9/phosphoproteomics identifies multiple noncanonical targets of myosin light chain kinase. Am J Physiol Renal Physiol. 2020 318(3):F600-F616 10.1152/ajprenal.00431.2019chin-rang.yang@nih.govNational Institutes of Health, USAUnited StatesPrior 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.CRISPR-Cas9/phosphoproteomics identifies multiple noncanonical targets of myosin light chain kinase.Isobe Kiyoshi K, Raghuram Viswanathan V, Krishnan Laya L, Chou Chung-Lin CL, Yang Chin-Rang CR, Knepper Mark A MAsodium salt, Hac-1/Dark, Rps17, ammonium formate, 13C-labeled, cadmium salt, Lysine Hydrochloride, determination, L-arginine, DL-Arginine Acetate, DmelCG6829, Aminosaeure, classic hairy cell leukaemia, RPS17, zinc formate, Amino acid, Monohydrate, Cs-1, lead salt, Basodexan, Hydrogenchlorid, magnesium formate, Apaf-1, clefted, zinc salt, M(3)67, peptide, Polypeptides, Lysine Acetate, M(3L)i, halite, ammonium tetraformate, hydrogen chloride, peptido, ClvPrd, domestic cat, cobalt(II) formate dihydrate, B1, Urea, HCL-C, 1, 2, Apaf1, Arg, 2210418N07, present in fewer numbers in organism, ARK, bacterial catalase-peroxidase activity, DmelCG42273, L Lysine, (R*, (S)-2-amino-5-guanidinopentanoic acid, amino acids, hac-1, peptides, K, CT21282, M, E927b, l(3)67BDo, (S)-2-Amino-5-guanidinovaleric acid, R, hypoplasia, epsilon-diaminocaproic acid, arc, min, Arginine, Processed cyclic AMP-responsive element-binding protein 3-like protein 1, Monohydrate DL-Arginine Acetate, copper, ark, T1, cloruro sodico, MLCK, salt, Wasserstoffchlorid, haem catalase activity, decreased, lithium salt, LYS, h, column, Sputolysin, Lysin, sample, D1, dApaf1, TEAB, D9, phosphatase, M(3)RpS17, ammonium (2:1) salt, nickel salt, L-Arginin, AI315345, catalase-peroxidase activity, equilase activity, rock salt, subdivided, D-Apaf-1, cobalt (+2) salt, Aminokarbonsaeure, MLCK108, Arginine Hydrochloride, anon-53Fa, M(3)67C, Dmel_CG7826, arginine, l(2)SH0173, classic hairy cell leukemia, 2.3.1.7, AAT7, alpha-amino carboxylic acids, ur, AI195249, A330009E03Rik, carbamide, common salt, sodium (4:1:1) salt, catalase reaction, Cleland, weight, Dm1, magnesium salt, MLCK1, M(3)i(55), lysine, HCL, Chlorwasserstoff, leukemic reticuloendotheliosis, nickel (+2) salt, GAT, CG6829, Dmel_CG7835, Hydrogen Oxide, forked, dark/hac-1/dapaf-1, L-Arginine, ACGNAT, Mnb, MNB, rpS17, Amino Acid, Acid, ammonium salt, divided, Amino acids, HCl, Rp S17, hydrogen-peroxide:hydrogen-peroxide oxidoreductase activity, formate, cobaltous formate, pooled, cromium (+3), 3-Butanediol, alpha, hac1, natrii chloridum, Felis silvestris catus, septate, beta Trypsin, organ system, dapaf-1S, H2NC(O)NH2, apaf-1, dapaf-1L, MLCK210, Acetamide, copper salt, MSTP083, ammonium (4:1) salt, Acids, Polypeptide, chlorane, DROCATHPO, lead (+2) salt, 1728, bs36h11.y1, U00145, MYLK1, Old astrocyte specifically-induced substance, Cleland Reagent, Cas1, Carnitine acetylase, nickel formate dihydrate, Dapaf-1/HAC-1, lead formate, chlorure d'hydrogene, Reagent, aluminum salt, cesium salt, Aminocarbonsaeure, L Isomer, 4-dimercapto-, Carbamide, alpha-amino acid, R*)-, CG7826, L-(+)-arginine, Buffer, formic acid, Dark/Hac-1/dApaf1, Karbamid, CrAT, culture filtrate, Hac1, M(3)S33, potassium salt, Dark/Hac-1/dApaf-1, manganese catalase activity, M(3)i, (2S)-2-amino-5-(carbamimidamido)pentanoic acid, reduced, subnumerary, Oasis, 14C-labeled, Harnstoff, CG7835, CG42273, system, Lysine, caperase activity, tiny, Carmol, cloruro de hidrogeno, Pro-Mega, anatomical systems, Hydrogen chloride, thallium (+1) salt, dapaf-1, beta-Trypsin, rubidium salt, chlorure de sodium, CatA, CATA, Minute, dapaf, dark, decreased number, l(3)dtOA4, calcium formate, methanoic acid, Solution, DmelCG6871, hairy cell leukemia, DmelCG3922, Catl, DARK, Kochsalz, M(3)i[55], L-Arg, cats, peptidos, cromium (+3) salt, dArk, Dapaf-1, 2-iodo-, sodium formate, PTHB1, small, dark/dapaf-1/hac-1, DYRK1, DMCATHPO, culture medium, heme catalase activity, nickel formate, dApaf-1, Clelands Reagent, strontium formate, apaf1, alpha-amino acids, chloridohydrogen, potassium formate, copper (+2) salt, backward, L-Isomer, strontium salt, DL Arginine Acetate, buffer, Carnitine acetyltransferase, Cell, Peptide, polypeptide, Isotope, [HCl], APAF1, Phosphopeptide, Natriumchlorid, L-Isomer Arginine, carbonyldiamide, Hac-1, Acetate, C18, body system., Felis domesticus, cupric formate, chemical analysis, Hydrochloride, table salt, BcDNA:RE44119, L Arginine, Dyrk1, Dark, Vacuums, CAT, connected anatomical system, NaCl, aluminum formate, L-Lysine, Dark/Apaf-I, HLB, smMLCK, optidase activity, CS1, Cleland's, Cas-1, lithium formate, KRP, underdeveloped, uree, culture supernatant, urea, (2S)-2-amino-5-guanidinopentanoic acid, M(3)q, Tripcellim, OASIS, Dark/Dapaf-1/HAC1, phosphoric monoester hydrolase activity, CG6871, cat, Amino, dApaf-1/DARK/HAC-1, anon-EST:Posey48, sample population, chromic formate, UREA, CG3922, ME-IV, Cleland's Reagent, cAMP-responsive element-binding protein 3-like protein 1, Trypure, Cat01, concentration, M(3L)i[55], calcium salt, Peptid, assay, 6-diaminohexanoic acid, Enisyl, hydrochloric acid, dAPAF-1, reversed, sodium chlorideWater, LVM, deglutamylated form, 2.7.11.18, Metabolic Concepts, BOUND WATER, Visible Light, nonselective vesicle endocytosis, oxidane, CG18677, phosphorylation, WATER, Prp4 protein kinase activity, HOH, Myo, endocytic import into cell, protein polypeptide chains, collecting tubule, ATP-protein transphosphorylase activity, cAMP-dependent protein kinase, Pitressin, hnu, RLC2, ATP:protein phosphotransferase (non-specific) activity, Endosome., MLRC, Kinase, MYL2, Metabolism Concept, Fs(3)Hor, dro MLC-2, calcium/calmodulin-dependent myosin light chain kinase activity, integumentum commune, average, C, Solute injectable de vasopressine, anon-WO02059370.56, catabolism, Pk72A, Myosin ATPase, Antidiuretic, 5' cAMP-dependent protein kinase complex, metabolic process resulting in cell growth, proteins, beta Actin, NTef2, Van der Knaap syndrome, myosin-light-chain kinase activity, Biotinylations, kidney collecting duct, ch, decreased, DmelCG6117, 5' cAMP-dependent protein kinase activity, N-Actin, ATP:myosin-light-chain O-phosphotransferase activity, Telokin, G-Actin, ATP Phosphotransferases, transcription from bacterial-type RNA polymerase promoter, SIMPLE, megalencephalic leukoencephalopathy with subcortical cysts 1, DmMLC2, Myo II, single organism signaling, p82 kinase activity, ADH, Radiation, renal collecting tubule, external covering of organism, DNA-dependent transcription, Cytoplasms, NMHC-II-C, metabolism resulting in cell growth, alpha Isoactin, MLCK108, betaIIPKC, cyclic AMP-dependent protein kinase complex, Actin Activated ATPase, CG42341, sgh, Light, Normalcy, pka-C1, signal transduction by trans-phosphorylation, Fs(3)Sz11, Wee-kinase activity, H2O, LIGHT, PIG7, megalencephalic leukodystrophy, actin polymerization, NMHC II-C, secretion, RII[[DR]], Antidiuretic Hormones, pka-c1, myosin light-chain kinase (phosphorylating) activity, PKA C, HVEML, Myosin Adenosine Triphosphatase, non-specific serine/threonine protein kinase activity, RMLC, gamma-Actin, INSDC_feature:gene, Dmel_CG12452, pka-RII, porin, light quantum, DmelCG4379, signaling pathway, MLCK210, PKA RII, serine-specific protein kinase activity, PkaC1, PKAcA, HIPK2, cytidine 3', CG2684, beta-Hypophamine, RII, MyoII, Epa, Ifm(3)99Eb, Wee 1-like kinase activity, adenosine 3', PKAC1, MYLK1, transcription, beta Hypophamine, advanced, STK18, Vacuolating megalencephalic leukoencephalopathy with subcortical cysts, glycogen synthase A kinase activity, region or site annotation, Antidiuretic Hormone, Processes, glycogen synthase kinase 3 activity, megalencephaly-cystic leukodystrophy, cg6647, PKA-C3, Vasopressin (USP), PKA-C1, Metabolic Processes, protein-containing complex, phosphorylase b kinase kinase activity, BcDNA:GM01761, DmelCG2184, agua, Ly113, Hormone, surface, myosin light-chain kinase activity, Pka-RI, l(2)cos[[1]], Gene Products, CG3595, ribosomal protein S6 kinase II activity, alpha-Actin, Normalities, protein-serine kinase activity, DmelCG6647, positional, network topology analysis, Pka-C, DmVDAC, RLC, Myo-II, Lichtquant, STK32, Metabolic Concept, 5'-cyclophosphate-dependent protein kinase activity, Cos, ATPase, clustered, myosin, CdkA, protein-cysteine kinase activity, DmMRLC_C, Hpr kinase activity, photon, STK22, TR2, DRI, mlc-2, DmelCG3595, PKA Cl, cellular transcription, degradation, plasma membrane invagination, CG2184, Proteins, Phosphorylations, sqh/RLC, CG3263, CdkR, protein serine kinase activity, LC2, CD258, murein sacculus, Cell, Endosome, protein phosphokinase activity, Concept, cyclic AMP-dependent protein kinase activity, pkA, PKA, Pka, native protein, PKC, Sqh, spg, MLCkase activity, peptidoglycan, F-actin, Squh, Squ, Receptosomes, signalling cascade, intrinsic catalyst activity, metabolism, protein kinase p58 activity, Metabolic Phenomenon, smMLCK, serine/threonine protein kinase activity, multicellular organism metabolic process, MYOSIN, G Actin, pka, pka C1, pKA, dr[[1]], PKA C3, myosin light chain protein kinase activity, HVEM-L, increased number, caMLCK, DNA-templated, squ, muscle, Actomyosin ATPase, 5'-cAMP-dependent protein kinase activity, dsk1, Lds, 5'-cyclic monophosphate-responsive protein kinase activity, atypical PKC activity, signalling, DmelCG42341, plan specification, actin polymerizing activity, PKA CI, Gene Proteins, Actomyosin, Photoradiations, signalling process, binding_or_interaction_site, l(2)01272, Protoplasms, F Actin, CG6647, regulation, MLC 2, [OH2], MRLC, protein serine-threonine kinase activity, Anabolism, biochemical pathways, glycogen synthase kinase activity, 6353, Porin, Metabolic Process, Dmel_CG3263, protein kinase A activity, MLC1, MLC2, ssq, PORIN, beta-Actin, ureteric tree, protein, binding site, CG12452, Protoplasm, Actin Activated, gamma Actin, DmPorin1, Concepts, envelope, mitogen-activated protein kinase activity, Dpck, RI, protein aggregate, Phenomenon, present in fewer numbers in organism, foton, mlc2, signal transduction by protein phosphorylation, Mlck, increased, DmelCG2684, N Actin, smooth muscle, megalencephaly-cystic leukodystrophy syndrome, CG4379, Antidiuretic hormone, 3', CG15862, myosin light-chain kinase (phosphorylating), Raf kinase activity, MLCK, arcuate renal tubule, DNA-dependent, Isoactin, serine kinase activity, Adenosinetriphosphatase, VDAC-1, atypical protein kinase C activity, signaling process, NMM, dihydridooxygen, mitogen-activated S6 kinase activity, DVDAC, biotransformation, protein-aspartyl kinase activity, MHC16, dihydrogen oxide, KMLC, Catabolism, ATP, gamma, MLC, Process, aqua, kidney collecting tubule, signal transduction by conformational transition, late, organism surface, myoII, AAT7, M phase-specific cdc2 kinase activity, Electron Microscopy, vesicle endocytosis, signaling cascade, pka-R1, MLCK1, MLCK2, MAPK, 5'-cAMP-dependent protein kinase complex, Cos-1, precocious, overlap, smooth-muscle-myosin-light-chain kinase activity, VL, serine protein kinase activity, VP, AVP, region, D830007F02Rik, DFNA4, Visible Radiations, 5-23, Transphosphorylases, phosphorylase B kinase kinase activity, Visible Radiation, outer membrane exporter porin, positional polypeptide feature, protein glutamyl kinase activity, hydrogen hydroxide, CG6117, Actin, skMLCK, acqua, early, muscle element, hydroxyalkyl-protein kinase activity, WEE1Hu, Health, ATP:protein phosphotransferase (cAMP-dependent) activity, integumentary system, Horka, bacterial transcription, dPKA, epsilon PKC, MSTP083, Fs(3)Horka, Klmc, Wasser, Dcpk, dermal system, accessory, ribosomal S6 protein kinase activity, biological signaling, calcium/phospholipid-dependent protein kinase activity, DFNA4A, Myosin Adenosinetriphosphatase, Pk?5, Gene, PKA-C-3, Endocytoses, supernumerary, method, MYH17, Myosin light chain kinase, myosin light-chain kinase, myosin 1, Actin-Activated ATPase, reduced, polypeptide chain, subnumerary, Metabolism, method used in an experiment, Inyectable de vasopresina, serine(threonine) protein kinase activity, argipressin, l(3)99Eb, light, DmF2, BG02142, AMPK, Metabolism Phenomena, Phosphotransferase, 18304, T-antigen kinase activity, lod, galactosyltransferase-associated kinase activity, MLC-2, PKAR, PkaR, Phalloidin, Receptosome, F-Actin, clumped, PKa-R2, labeling, PKa-R1, Transphosphorylase, eau, Vdac, Visible, PKAc, decreased number, myosin kinase activity, junctional tube, protein kinase (phosphorylating) activity, PKA-R1, DmelCG15862, signalling pathway, PKA-R2, Raf-1, musculus, l(2)k05123, Vasopressin, Dc0, site, signal transduction by cis-phosphorylation, VDAC, casein kinase (phosphorylating) activity, PKA-RI, alpha Actin, TP53I7, tubulus renalis colligens, Actomyosin Adenosinetriphosphatase, Cos1, 5'-cyclophosphate-dependent protein kinase complex, l(2)s4402, protein complex, DC0, DC2, dPKA-RI, Kinase-related protein, Myosin, POR-1, Phosphotransferases, Metabolic Phenomena, Dmlc2, Metabolism Concepts, cos1, twitchin kinase activity, PKAi, Phosphopeptide, PKAr, natural protein, water, AP50 kinase activity, Protein, collecting duct system, Phenomena, dcO, DCO, Dco, collecting duct, Pka-RII, Radiations, Dmlc, threonine-specific protein kinase activity, tubulus renalis arcuatus, PNMHH, Adenosine Triphosphatase, KRP, distinct, biodegradation, Metabolic, megalencephalic leukoencephalopathy with subcortical cysts type 1, Normality, Kinases, body surface, INSDC_feature:misc_binding, Photoradiation, dco, PKA-RII, ATP:myosin-light-chain O-phosphotransferase, LTg, alpha-Isoactin, Protein Gene Products, present in greater numbers in organism, Vasopressini injectio, single-organism metabolic process, DEL, arginine vasopressin, A-kinase activity, Actin-ActivatedTransphosphorylases, Kinase, ATP Phosphotransferases, Transphosphorylase, Phosphotransferase, Kinases, Phosphotransferases, ATP.MYLK1, PDB2, ion, RANK, AW549739, False, region or site annotation, TRANCER, high weight, Under Curves, Visible Light, protein, ureteric tree, Computer, protein-containing complex, phosphorylation, binding site, Ly113, peptide, Polypeptides, protein polypeptide chains, collecting tubule, ClvPrd, peptido, Curve, polypeptide chain, Ms1, Area Under, hnu, heavy, Software Engineering, Kinase, light, protein aggregate, Phosphotransferase, foton, Computer Program, Application, C79691, mRANK, Open Source Softwares, proportion, positional, ODFR, F, OSTS, peptides, MS1, TNFRSF11A, Ly109, proportionality to, Software Application, iones, Lichtquant, receptor activator of NF-KB, Open, OFE, Open Source Software, Computer Programs and Programming, proteins, Transphosphorylase, Visible, T6G21.3, ions, Probabilities, Computer Software Application, Lccp, junctional tube, mKIAA0989, arcuate renal tubule, MLCK, kidney collecting duct, Tools, Rank, photon, FEO, site, peptidos, TR2, Area Under Curves, house mouse, OPTB7, ATP Phosphotransferases, ATP, gamma, Under Curve, Curves, Applications Software, tubulus renalis colligens, Open Source, data, Stars, Radiation, Computer Software, renal collecting tubule, protein complex, T5E21.12, AUC, mouse, MLCK108, Phosphorylations, kidney collecting tubule, Light, LOH18CR1, CD258, male sterility 1., Peptide, Source Softwares, results, Phosphotransferases, AAT7, Software Tools, Tool, Programs, polypeptide, Program, Computer Applications, Software Tool, TRANCE-R, LIGHT, native protein, natural protein, Ionen, Mus, MLCK1, Algorithm, Computer Applications Software, Protein, collecting duct system, Computer Applications Softwares, Area, collecting duct, Softwares, Software, region, Data Base, T5E21_12, CD265, Visible Radiations, Radiations, smMLCK, tubulus renalis arcuatus, Transphosphorylases, Visible Radiation, Software Applications, positional polypeptide feature, HVEML, KRP, MALE STERILITY 1 PROTEIN, Kinases, HVEM-L, Source Software, mice, INSDC_feature:misc_binding, Engineering, proportionality, Photoradiation, Striated muscle activator of Rho-dependent signaling, rate, MASCOT, osteoclast differentiation factor receptor, LTg, Computer Programs, Applications, light quantum, Applications Softwares, Photoradiations, binding_or_interaction_site, Ion, MLCK210, MSTP083, quotient, Peptid, Mouse, Polypeptide, STARS, Computer Software ApplicationsTransitional Epithelial Cell, Transitional, Adenomatous Epithelial Cell, Transphosphorylases, Squamous Cells, Adenomatous Epithelial Cells, biological signaling, Squamous Cell, Epithelial Cell, Kinases, Glandular Epithelial Cell., Glandular Epithelial Cells, Adenomatous, Adenomatous Epithelial, Glandular Epithelial, Transphosphorylase, Squamous, Glandular, Transitional Epithelial, Phosphotransferases, Cell, signalling, Kidneys, Squamous Epithelial Cells, signalling process, Cuboidal Glandular Epithelial Cells, Epithelial Cells, Transitional Epithelial Cells, signaling process, Cells, Kinase, Columnar Glandular Epithelial Cells, ATP Phosphotransferases, Squamous Epithelial Cell, Phosphotransferase, Squamous Epithelial, ATP, single organism signaling, reniculate kidney, Epithelialdeglutamylated form, Metabolisms, 2.7.11.18, Visible Light, CG18677, phosphorylation, Prp4 protein kinase activity, protein polypeptide chains, collecting tubule, ATP-protein transphosphorylase activity, cAMP-dependent protein kinase, Pitressin, hnu, Tight, ATP:protein phosphotransferase (non-specific) activity, Expenditure, Fs(3)Hor, calcium/calmodulin-dependent myosin light chain kinase activity, average, C, Solute injectable de vasopressine, regulation of aquaporin permeability, Pk72A, Myosin ATPase, Antidiuretic, 5' cAMP-dependent protein kinase complex, proteins, beta Actin, NTef2, myosin-light-chain kinase activity, actomyosin structure, kidney collecting duct, ch, decreased, DmelCG6117, 5' cAMP-dependent protein kinase activity, N-Actin, ATP:myosin-light-chain O-phosphotransferase activity, Adherens Junction, Telokin, G-Actin, SIMPLE, single organism signaling, p82 kinase activity, ADH, Radiation, renal collecting tubule, Cytoplasms, NMHC-II-C, alpha Isoactin, MLCK108, betaIIPKC, cyclic AMP-dependent protein kinase complex, Actin Activated ATPase, CG42341, Light, Normalcy, pka-C1, signal transduction by trans-phosphorylation, Fs(3)Sz11, Wee-kinase activity, LIGHT, PIG7, NMHC II-C, RII[[DR]], Antidiuretic Hormones, pka-c1, myosin light-chain kinase (phosphorylating) activity, PKA C, HVEML, Occluden, regulation of aquaporin, Myosin Adenosine Triphosphatase, non-specific serine/threonine protein kinase activity, gamma-Actin, INSDC_feature:gene, Tonofilaments, Dmel_CG12452, pka-RII, mRNA maturation, Nuclear Membrane, light quantum, DmelCG4379, signaling pathway, MLCK210, PKA RII, serine-specific protein kinase activity, PkaC1, PKAcA, HIPK2, CG2684, cytidine 3', beta-Hypophamine, RII, Epa, Wee 1-like kinase activity, adenosine 3', PKAC1, MYLK1, Zonula Occluden, advanced, actomyosin complex, beta Hypophamine, Nuclear Membranes, STK18, glycogen synthase A kinase activity, region or site annotation, Antidiuretic Hormone, glycogen synthase kinase 3 activity, number, PKA-C3, Vasopressin (USP), Kern, PKA-C1, protein-containing complex, phosphorylase b kinase kinase activity, BcDNA:GM01761, Occluding Junctions, Ly113, Hormone, Filament, myosin light-chain kinase activity, Pka-RI, l(2)cos[[1]], Gene Products, nucleo atomico, ribosomal protein S6 kinase II activity, Tight Junction, alpha-Actin, Normalities, protein-serine kinase activity, positional, network topology analysis, Pka-C, Lichtquant, STK32, Energy, Expenditures, 5'-cyclophosphate-dependent protein kinase activity, Cos, ATPase, clustered, myosin, CdkA, protein-cysteine kinase activity, Hpr kinase activity, photon, STK22, TR2, DRI, PKA Cl, Proteins, Phosphorylations, CG3263, CdkR, Occludens, protein serine kinase activity, CD258, Cell, Endosome, protein phosphokinase activity, cyclic AMP-dependent protein kinase activity, Zonula Adherens, pkA, PKA, Pka, native protein, PKC, MLCkase activity, F-actin, Zonula, Receptosomes, signalling cascade, intrinsic catalyst activity, protein kinase p58 activity, Energy Expenditure, smMLCK, serine/threonine protein kinase activity, MYOSIN, G Actin, pka, pka C1, pKA, dr[[1]], PKA C3, myosin light chain protein kinase activity, HVEM-L, increased number, caMLCK, Energy Expenditures, nucleus of neuraxis, Actomyosin ATPase, 5'-cAMP-dependent protein kinase activity, Lds, dsk1, neuraxis nucleus, 5'-cyclic monophosphate-responsive protein kinase activity, atypical PKC activity, signalling, DmelCG42341, PKA CI, Gene Proteins, Actomyosin, Photoradiations, signalling process, binding_or_interaction_site, l(2)01272, Protoplasms, F Actin, regulation, quantitative, protein serine-threonine kinase activity, glycogen synthase kinase activity, 6353, Dmel_CG3263, protein kinase A activity, Bioenergetics, beta-Actin, ureteric tree, protein, Nuclear Envelopes, Neurofilaments, Atomkern, binding site, CG12452, Protoplasm, Actin Activated, gamma Actin, horsetail nucleus, mitogen-activated protein kinase activity, Dpck, RI, protein aggregate, present in fewer numbers in organism, foton, signal transduction by protein phosphorylation, Mlck, increased, neuronal nucleus, nucleus atomi, DmelCG2684, N Actin, smooth muscle, Envelope, CG4379, Antidiuretic hormone, 3', CG15862, Phosphoprotein, myosin light-chain kinase (phosphorylating), Raf kinase activity, MLCK, arcuate renal tubule, Isoactin, serine kinase activity, Adenosinetriphosphatase, atypical protein kinase C activity, signaling process, Actomyosins, mitogen-activated S6 kinase activity, protein-aspartyl kinase activity, MHC16, KMLC, gamma, Occluding, kidney collecting tubule, signal transduction by conformational transition, late, AAT7, M phase-specific cdc2 kinase activity, Electron Microscopy, signaling cascade, pka-R1, MLCK1, MLCK2, size of nucleus, MAPK, 5'-cAMP-dependent protein kinase complex, Cos-1, precocious, cell nucleus, overlap, smooth-muscle-myosin-light-chain kinase activity, serine protein kinase activity, VP, Occluding Junction, AVP, region, D830007F02Rik, DFNA4, Visible Radiations, 5-23, phosphorylase B kinase kinase activity, Visible Radiation, positional polypeptide feature, protein glutamyl kinase activity, CG6117, Filaments, Actin, Tonofilament, skMLCK, Junction, early, Adherens, hydroxyalkyl-protein kinase activity, Energy Metabolisms, WEE1Hu, Health, ATP:protein phosphotransferase (cAMP-dependent) activity, Horka, dPKA, epsilon PKC, MSTP083, Fs(3)Horka, Klmc, Dcpk, accessory, ribosomal S6 protein kinase activity, biological signaling, calcium/phospholipid-dependent protein kinase activity, noyau atomique, DFNA4A, Myosin Adenosinetriphosphatase, Pk?5, Gene, PKA-C-3, supernumerary, presence, MYH17, Myosin light chain kinase, myosin light-chain kinase, myosin 1, Actin-Activated ATPase, Zonula Occludens, reduced, polypeptide chain, subnumerary, Metabolism, Inyectable de vasopresina, serine(threonine) protein kinase activity, argipressin, DmF2, light, BG02142, AMPK, 18304, T-antigen kinase activity, lod, nuclei, galactosyltransferase-associated kinase activity, PKAR, PkaR, Receptosome, nucleus of CNS, noyau, nucleo, Envelopes, F-Actin, clumped, PKa-R2, PKa-R1, Visible, PKAc, decreased number, myosin kinase activity, Junctions, junctional tube, protein kinase (phosphorylating) activity, PKA-R1, DmelCG15862, signalling pathway, PKA-R2, nucleus, Raf-1, Vasopressin, Dc0, site, signal transduction by cis-phosphorylation, casein kinase (phosphorylating) activity, PKA-RI, alpha Actin, TP53I7, tubulus renalis colligens, Actomyosin Adenosinetriphosphatase, Cos1, 5'-cyclophosphate-dependent protein kinase complex, l(2)s4402, protein complex, DC0, DC2, dPKA-RI, Intermediate, Kinase-related protein, Myosin, cos1, count in organism, twitchin kinase activity, PKAi, Phosphopeptide, PKAr, natural protein, nervous system nucleus, AP50 kinase activity, Protein, collecting duct system, dcO, DCO, Dco, collecting duct, Nuclear, Pka-RII, Radiations, threonine-specific protein kinase activity, tubulus renalis arcuatus, Membranes, PNMHH, Adenosine Triphosphatase, KRP, distinct, Normality, INSDC_feature:misc_binding, Photoradiation, dco, Membrane, PKA-RII, ATP:myosin-light-chain O-phosphotransferase, LTg, Bioenergetic, alpha-Isoactin, Protein Gene Products, present in greater numbers in organism, Vasopressini injectio, DEL, Neurofilament, arginine vasopressin, phosphorylation., Intermediate Filament, A-kinase activity, Actin-Activated, presence or absence in organismfalseCRISPR-Cas9/phosphoproteomics identifies Myosin Light Chain Kinase dependent signaling in kidney epithelial cellsPrior studies have implicated myosin light chain kinase (MLCK) in the regulation of aqua-porin-2 (AQP2) in the renal collecting duct. To discover signaling targets of MLCK, we deleted the Mylk- gene to obtain MLCK-null mpkCCD cells and carried out comprehensive phosphopro-teomics using the SILAC method for quantification. Only 107 phosphopeptides were changed in abundance by MLCK deletion out of 1743 quantified over multiple replicates (29 decreased and 78 increased). One of the decreased phosphopeptides corresponded to the canonical target site in myosin regulatory light chain (MLC). Network analysis indicated that targeted phosphopro-teins clustered into distinct structural/functional groups: “acto-myosin,” “signaling,” “nuclear envelope,” “gene transcription,” “mRNA processing,” “energy metabolism,” “intermediate fila-ments,” “adherens junctions” and “tight junctions.” There was significant overlap between the derived MLCK signaling network and a previously determined PKA signaling network. Phalloidin labeling revealed that MLCK deletion inhibited the normal effect of vasopressin to depolymer-ize F-actin. Immunocytochemistry and electron microscopy demonstrated a defect in pro-cessing of early endosomes to late endosomes. In contrast, surface biotinylation studies showed no effect of MLCK deletion on AQP2 endocytosis. We conclude that MLCK is part of a multi-component signaling pathway that includes much more than simple regulation of conven-tional non-muscle myosins through MLC phosphorylation. Furthermore, vasopressin signaling in collecting duct cells involves both MLCK-dependent signaling and PKA-dependent signaling, which display significant overlap. Mapping MLCK targets revealed potential roles in both nu-cleus and cytoplasm. The latter includes proteins that regulate both actin polymerization and AQP2-endosomal processing from early to late endosomes.2020-03-042019-08-12PXD014985445446415249917559311738804333092866432359839062887051194599327160246196246197160329358113635894532758385118794757230713512251177505855571468353649167387215891030629159291583641515849388580332715910022638886915135588870435930945146479692877133216413328038103430436545833398580818894621432059118259063295717493469381247272563227321928672813954513294914081691804541619345374571390363453011431140206411981087135547714245073264421043555127443214967146945597293751461575464784224220896413923174471270208963139011431933829355082307411148119466948969352937251264690727580240NCBITaxon_5476550721659936658121659529412821331283296543128212803395232181843183292043531522811257554787575930797253623114367331159556444214321381849223948824261914548140479109697622615515311179573347205968798662330980058884558626528173630943179698936160488566416242567825317513320496303555432046565050632633NCBITaxon:1313456586068860769938942468879554535120148877959216129105884410026420328909694569410167631076228531643518543121574577760445246794144242324113NCBITaxon:177392979310239101178709NCBITaxon:40559749907991365261122734982171701873674510116569156931328426103609833413367951089448663303NCBITaxon:49329925883910005898732994044586115357114716122491134305311563120175544219329935872631787236669872464261513458872742157547624223141461151041607913055468911180066125522856213616299767982510216998231174673897641167610807723264241155744689415367767446859600393765209611822635735784582730688086363266689NCBITaxon:269704910312NCBITaxon:1389838192011241368108931997596123077319747946522832917619662776689605960615729513722117110125673712334351052311009040544931799863878310429863578840193733496155087711982159739757487822971972154078211908206189985076480256318418985623951655190264946454635286746360NCBITaxon:110719646195051375939288357461287460113450664912538458814594319722152638NCBITaxon:615797787237953440812632269003803945264114595361235178616722724323074691123869978540979541NCBITaxon:2954424327762791292497036125180276067476795961532848123136254264282573131504752697961029938865990346941713443386263475154155403632943648697966287732396440426289930629548657558462823823522862871736231399463601068842043601043994796858355847954440426071017316780781440772NCBITaxon:619184023171993128118361003610502318557919080218787837499070384766416922596345910659212257862607046183260705334542260707NCBITaxon:780NCBITaxon:15024515168853187029211586910371647209285747078124966842528619212638541094343197383379192875NCBITaxon:1035928291268063956029598704483006418022427522664373105552423544322430814336146900815025754129839642711913645169963281041216979272623272624300852123899364368010977913562233474718213142697049583313618580308268829760527796381858334240906490911477871274414153681639588858130767494022432640817212816140559204254687613837081274423434271285099011912744201274426519534911370249240543734131641660817562615282352567506028532189517731193501846451450511492412153239564818822970915800257125614801549031384736031423166385962859631015101450520493227591428693732664179332648373153NCBITaxon:96152972210976771121114493760833341911079276283332100658123756126662555592924847914869177492003600946143534073120325835247225944751750375451649908515151438992NCBITaxon:1155237451715256374731904282