title: Use of LC-MS/MS and Bayes' theorem to identify protein kinases that phosphorylate aquaporin-2 at Ser256.
abstract: 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.
authors.affiliation: Epithelial Systems Biology Laboratory, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland.
authors.forename: Davis
authors.initials: D
authors.lastname: Bradford
authors.affiliation: Epithelial Systems Biology Laboratory, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland.
authors.forename: Viswanathan
authors.initials: V
authors.lastname: Raghuram
authors.affiliation: Epithelial Systems Biology Laboratory, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland.
authors.forename: Justin L L
authors.initials: JL
authors.lastname: Wilson
authors.affiliation: Epithelial Systems Biology Laboratory, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland.
authors.forename: Chung-Lin
authors.initials: CL
authors.lastname: Chou
authors.affiliation: Epithelial Systems Biology Laboratory, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland.
authors.forename: Jason D
authors.initials: JD
authors.lastname: Hoffert
authors.affiliation: Epithelial Systems Biology Laboratory, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland knepperm@nhlbi.nih.gov.
authors.forename: Mark A
authors.initials: MA
authors.lastname: Knepper
authors.affiliation: Epithelial Systems Biology Laboratory, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland.
authors.forename: Trairak
authors.initials: T
authors.lastname: Pisitkun
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D051399:Aquaporin 2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001499:Bayes Theorem; D002853:Chromatography, Liquid / Q000379:methods*; D019295:Computational Biology; D003627:Data Interpretation, Statistical; D015971:Gene Expression Regulation, Enzymologic; D007685:Kidney Tubules, Collecting / Q000201:enzymology; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011494:Protein Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D012694:Serine / Q000378:metabolism; D053719:Tandem Mass Spectrometry / Q000379:methods*; D059467:Transcriptome; D014667:Vasopressins / Q000378:metabolism
chemical_list: C496025:Aqp2 protein, rat; D051399:Aquaporin 2; D047428:Protein Kinase Inhibitors; D014667:Vasopressins; D012694:Serine; D011494:Protein Kinases
keywords: Bayes' theorem; aquaporin-2; inner medullary collecting duct; urea channel UT-A; vasopressin