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

Vasopressin controls water excretion through regulation of aquaporin-2 (AQP2) trafficking in renal collecting duct cells. Using mass spectrometry, we previously demonstrated four phosphorylated serines (Ser256, Ser261, Ser264, and Ser269) in the carboxyl-terminal tail of rat AQP2. Here, we used phospho-specific antibodies and protein mass spectrometry to investigate the roles of vasopressin and cyclic AMP in the regulation of phosphorylation at Ser269 and addressed the role of this site in AQP2 trafficking. The V2 receptor-specific vasopressin analog dDAVP increased Ser(P)269-AQP2 abundance more than 10-fold, but at a rate much slower than the corresponding increase in Ser256 phosphorylation. Vasopressin-mediated changes in phosphorylation at both sites were mimicked by cAMP addition and inhibited by protein kinase A (PKA) antagonists. In vitro kinase assays, however, demonstrated that PKA phosphorylates Ser256, but not Ser269. Phosphorylation of AQP2 at Ser269 did not occur when Ser256 was replaced by an unphosphorylatable amino acid, as seen in both S256L-AQP2 mutant mice and in Madin-Darby canine kidney cells expressing an S256A mutant, suggesting that Ser269 phosphorylation depends upon prior phosphorylation at Ser256. Immunogold electron microscopy localized Ser(P)269-AQP2 solely in the apical plasma membrane of rat collecting duct cells, in contrast to the other three phospho-forms (found in both apical plasma membrane and intracellular vesicles). Madin-Darby canine kidney cells expressing an S269D “phosphomimic” AQP2 mutant showed constitutive localization at the plasma membrane. The data support a model in which vasopressin-mediated phosphorylation of AQP2 at Ser269:(a) depends on prior PKA-mediated phosphorylation of Ser256 and (b) enhances apical plasma membrane retention of AQP2. 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. Aquaporins are molecular water channels that mediate rapid water transport across lipid membranes in a variety of cell types (1Kozono D. Yasui M. King L.S. Agre P. J. Clin. Investig. 2002; 109: 1395-1399Crossref PubMed Scopus (216) Google Scholar). Aquaporin-2 (AQP2), 3The abbreviations used are:AQP2aquaporin-2PKAprotein kinase AAVParginine vasopressinIMCDinner medullary collecting ductLC-MS/MSliquid chromatography-tandem mass spectrometrydDAVPdeamino-Cys,1D-Arg8vasopressinMRMmultiple reaction monitoringcpt-cAMP8-(4-chlorophenylthio)-cyclic adenosine monophosphateMDCKMadin-Darby canine kidneyWTwild typePBSphosphate-buffered salineERKextracellular signal-regulated kinaseJNKc-Jun NH2-terminal kinase. 3The abbreviations used are:AQP2aquaporin-2PKAprotein kinase AAVParginine vasopressinIMCDinner medullary collecting ductLC-MS/MSliquid chromatography-tandem mass spectrometrydDAVPdeamino-Cys,1D-Arg8vasopressinMRMmultiple reaction monitoringcpt-cAMP8-(4-chlorophenylthio)-cyclic adenosine monophosphateMDCKMadin-Darby canine kidneyWTwild typePBSphosphate-buffered salineERKextracellular signal-regulated kinaseJNKc-Jun NH2-terminal kinase. the vasopressin-regulated water channel, is responsible for control of water excretion by the kidney (2Nielsen S. Frokiaer J. Marples D. Kwon T.H. Agre P. Knepper M.A. Physiol. Rev. 2002; 82: 205-244Crossref PubMed Scopus (1005) Google Scholar). AQP2-mediated osmotic water transport from the lumens of the renal collecting ducts returns filtered water to the bloodstream under the control of the peptide hormone arginine vasopressin (AVP). This control is exerted through binding of AVP to the V2 receptor (a G protein-coupled receptor) in the collecting duct cells. The V2 receptor, working via Gs-mediated elevation of intracellular cAMP, increases epithelial osmotic water permeability via regulated trafficking of AQP2-containing intracellular vesicles to and from the apical plasma membrane (3Nielsen S. Chou C.L. Marples D. Christensen E.I. Kishore B.K. Knepper M.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1013-1017Crossref PubMed Scopus (872) Google Scholar). This process is impaired in several common disorders of water balance (2Nielsen S. Frokiaer J. Marples D. Kwon T.H. Agre P. Knepper M.A. Physiol. Rev. 2002; 82: 205-244Crossref PubMed Scopus (1005) Google Scholar), e.g. in congestive heart failure, in lithium-induced nephrogenic diabetes insipidus associated with treatment of bipolar disorder, and in the syndrome of inappropriate antidiuresis seen in many cancer patients. aquaporin-2 protein kinase A arginine vasopressin inner medullary collecting duct liquid chromatography-tandem mass spectrometry deamino-Cys,1D-Arg8vasopressin multiple reaction monitoring 8-(4-chlorophenylthio)-cyclic adenosine monophosphate Madin-Darby canine kidney wild type phosphate-buffered saline extracellular signal-regulated kinase c-Jun NH2-terminal kinase. aquaporin-2 protein kinase A arginine vasopressin inner medullary collecting duct liquid chromatography-tandem mass spectrometry deamino-Cys,1D-Arg8vasopressin multiple reaction monitoring 8-(4-chlorophenylthio)-cyclic adenosine monophosphate Madin-Darby canine kidney wild type phosphate-buffered saline extracellular signal-regulated kinase c-Jun NH2-terminal kinase. Exo- and endocytosis of AQP2 are believed to be independently regulated, and the amount of AQP2 in the plasma membrane is dependent on a balance between the two processes (4Knepper M.A. Nielsen S. Am. J. Physiol. 1993; 265: F214-F224PubMed Google Scholar, 5Nielsen S. Knepper M.A. Am. J. Physiol. 1993; 265: F204-F213PubMed Google Scholar, 6Brown D. Am. J. Physiol. 2003; 284: F893-F901Crossref PubMed Scopus (218) Google Scholar). Membrane trafficking processes that control the quantity of AQP2 in the apical plasma membrane have been proposed to depend on changes in phosphorylation of AQP2 at Ser256 (7Katsura T. Gustafson C.E. Ausiello D.A. Brown D. Am. J. Physiol. 1997; 272: F817-F822Crossref PubMed Google Scholar, 8Fushimi K. Sasaki S. Marumo F. J. Biol. Chem. 1997; 272: 14800-14804Abstract Full Text Full Text PDF PubMed Scopus (401) Google Scholar, 9van Balkom B.W. Graat M.P. van Raak M. Hofman E. van der S.P. Deen P.M. Am. J. Physiol. 2004; 286: C372-C379Crossref PubMed Scopus (29) Google Scholar). Recently, we have demonstrated by phosphoproteomic analysis of native rat renal inner medullary collecting duct (IMCD) cells that Ser256 is part of a polyphosphorylated region containing four phosphorylated serines (Ser256, Ser261, Ser264, and Ser269) within the last 16 amino acids of the AQP2 COOH-terminal tail (10Hoffert J.D. Pisitkun T. Wang G. Knepper M.A. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). have that the abundance of the of AQP2 is increased in to AVP G. M. D. Yasui M. A. Nielsen S. Am. J. Physiol. PubMed Google Scholar). In we have that the phosphorylation of AQP2 is J.D. Nielsen J. Pisitkun T. Nielsen S. Knepper M.A. Am. J. Physiol. PubMed Scopus Google Scholar), phosphorylation is increased by AVP J.D. Nielsen S. Knepper M.A. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). The role of the Ser269 phosphorylation site in AQP2 trafficking not been In this we a phospho-specific to liquid chromatography-tandem mass spectrometry in cells, as as mice expressing mutant of AQP2 to investigate the role of AQP2 phosphorylation at Ser269 in regulated trafficking of AQP2. The that vasopressin increases phosphorylation and that this phosphorylated is localized in the apical plasma membrane of collecting duct cells. in cells support the that Ser269 phosphorylation as a plasma membrane retention The that of phosphorylation on protein kinase A (PKA) is and is to for a phosphorylation at Ser256 prior to Ser269 phosphorylation. antibodies G. M. D. Yasui M. A. Nielsen S. Am. J. Physiol. PubMed Google Scholar), J.D. Nielsen J. Pisitkun T. Nielsen S. Knepper M.A. Am. J. Physiol. PubMed Scopus Google Scholar), and J.D. Nielsen S. Knepper M.A. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google were previously Here, an to Ser(P)269-AQP2 was a peptide corresponding to the of rat AQP2 that as J.D. Nielsen J. Pisitkun T. Nielsen S. Knepper M.A. Am. J. Physiol. PubMed Scopus Google Scholar). was by and A the amino of AQP2 of AQP2. AQP2 been previously Nielsen S. Christensen E.I. Knepper M.A. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). A of AQP2 in this was a peptide corresponding to amino acids in the from the polyphosphorylated region of rat AQP2 as M.A. S. Physiol. PubMed Scopus Google Scholar). The was the peptide to A was used for the were and the were by as (10Hoffert J.D. Pisitkun T. Wang G. Knepper M.A. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). were on an to an mass in as (10Hoffert J.D. Pisitkun T. Wang G. Knepper M.A. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). was as J.D. Wang G. Pisitkun T. Knepper M.A. J. PubMed Scopus Google Scholar). rat were as and in the of of dDAVP analog of for and the cell were in and a and were as previously J.D. Nielsen J. Pisitkun T. Nielsen S. Knepper M.A. Am. J. Physiol. PubMed Scopus Google that was not The were to the and The a and by a The were on the by a for phosphorylated of the AQP2 COOH-terminal peptide were and to were to both the and peptide for and of the AQP2 COOH-terminal tail data of both and were to to the of COOH-terminal of AQP2. were for retention and were for between controls and were from of as C.L. A. Knepper M.A. Am. J. Physiol. 1995; Scholar). were for the in the of In was for a prior to addition of to the of PKA In osmotic were in addition of the cells were by at for and in by via and as Nielsen S. Christensen E.I. Knepper M.A. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar), with and on the of a AQP2 carboxyl-terminal peptide the peptide at Ser256 were at for with and of the of of of of of of of of protein kinase of the were from Phosphorylation of the peptide was by both and the PubMed Scopus Google was to the The was to for and was on for as Nielsen S. Christensen E.I. Knepper M.A. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). AQP2 peptide was the AQP2 COOH-terminal the phosphorylated of the AQP2 peptide were with phospho-specific AQP2 mice with an B.W. F. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google and control mice from the were with dDAVP of of a in the kidney was and in were for as J. Marples D. Knepper M.A. Nielsen S. Am. J. Physiol. Google Scholar). to rat and water the were with of of dDAVP in of and four as the were and the were through the with in The were in in liquid in and as previously J. Marples D. Knepper M.A. Nielsen S. Am. J. Physiol. Google Scholar). The cells were to on with in for at with with on and in liquid Immunogold electron microscopy was on with an that AQP2. A for aquaporin-2 was by from inner the was used to AQP2 at Ser256 and Ser269 by of the were by cell were the The cell containing site J.D. P.M. Am. J. Physiol. 2004; 286: PubMed Scopus Google was with aquaporin-2 cells were cell were to by to A of two cell expressing the protein were for this cell a of AQP2 an in the cells were at and in with of and on were to in of the The cells were in and for by for at the cells were in at for in for at in and in for The were with at in and for with The were in and on A was used for of the cells. were a and a of between of AQP2 amino acids of the AQP2 COOH-terminal tail acids are in the four phosphorylated amino acids (10Hoffert J.D. Pisitkun T. Wang G. Knepper M.A. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). A phospho-specific was antibodies G. M. D. Yasui M. A. Nielsen S. Am. J. Physiol. PubMed Google Scholar), J.D. Nielsen J. Pisitkun T. Nielsen S. Knepper M.A. Am. J. Physiol. PubMed Scopus Google Scholar), and J.D. Nielsen S. Knepper M.A. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). of rat in the and of V2 vasopressin receptor dDAVP demonstrated a increase in phosphorylation of AQP2 at Ser269 The of this increase is with changes at the other three phosphorylation sites in for of the we previously J.D. Nielsen J. Pisitkun T. Nielsen S. Knepper M.A. Am. J. Physiol. PubMed Scopus Google Scholar), dDAVP increases AQP2 phosphorylation at Ser256 phosphorylation at AVP increases in phosphorylation at with a of and the increase in phosphorylation at Ser269 with a of the vasopressin-mediated increase in AQP2 phosphorylation at both and Ser269 more than the increase in Ser256 phosphorylation. of AMP and PKA in AQP2 of AVP to V2 an increase in intracellular cyclic AMP (2Nielsen S. Frokiaer J. Marples D. Kwon T.H. Agre P. Knepper M.A. Physiol. Rev. 2002; 82: 205-244Crossref PubMed Scopus (1005) Google Scholar). rat cell were to the cyclic AMP analog the of phosphorylation with dDAVP was that phosphorylation changes in to vasopressin are from cyclic for of the that through an increase in intracellular cAMP phosphorylation at four serines in a phosphorylation at Ser256, Ser264, and Ser269 was by the PKA a peptide corresponding to the polyphosphorylated region of the AQP2 COOH-terminal tail was with PKA in the of in phosphorylation at Ser256 as demonstrated both by with the and by analysis phosphorylation at the other three sites was not in when a peptide at Ser256 was with phosphorylation was PKA is to be responsible for the phosphorylation at Ser256 but not to the other sites of AQP2 COOH-terminal with several other and did not in protein kinase a at Ser256 Phosphorylation of AQP2 at and Ser269 on Phosphorylation at of AQP2 phosphorylation at and Ser269 by the PKA be the phosphorylation were by other but a for prior PKA-mediated phosphorylation of AQP2 at Ser256. this we the phospho-specific on kidney from mice with a S256L-AQP2 B.W. F. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google with the was an of for Ser(P)269-AQP2 in S256L-AQP2 with dDAVP dDAVP in increased in contrast to the seen in mice and in rat the cells were with AQP2 with an S256A The in an of Ser269 phosphorylation In cells phosphorylation at both and Ser269. the data from the in mice and the S256A in cells are with the that Ser256 phosphorylation is for phosphorylation at both and Ser269. of of AQP2 Using antibodies have the that phosphorylated amino acids are in phosphorylated of AQP2 are and in to the vasopressin analog we used protein mass spectrometry have demonstrated that cells of AQP2 phosphorylated at Ser256 (10Hoffert J.D. Pisitkun T. Wang G. Knepper M.A. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar), phosphorylated at Ser256 and (10Hoffert J.D. Pisitkun T. Wang G. Knepper M.A. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar), and phosphorylated at Ser256, Ser261, and J.D. Wang G. Pisitkun T. Knepper M.A. J. PubMed Scopus Google Scholar). and AQP2 in the rat in a we of the that a at that four phosphorylation Using an two phosphorylated that were at the and on under the for an the abundance of both to be increased in to of rat we not AQP2 that were phosphorylated at Ser269 but not at Ser256. AQP2 we used reaction monitoring In contrast to the other than with and that a peptide be in T. J.D. Knepper M.A. Physiol. PubMed Scopus Google Scholar), analysis of the in a to and several AQP2 from rat for with dDAVP an The prior that dDAVP the abundance of AQP2 and increases the abundance of phosphorylated AQP2 (10Hoffert J.D. Pisitkun T. Wang G. Knepper M.A. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). The abundance of was within of dDAVP showed that the abundance of the phosphorylated of AQP2 was increased by dDAVP but that of the increase in this the In AQP2 containing were not in via of the of with the associated with phosphorylated of AQP2. a to this containing the last three phosphorylation sites Ser264, and Ser269) to the phosphorylated AQP2 peptide in demonstrated that the abundance of this peptide was increased in to dDAVP but that of the increase was the Phosphorylation of AQP2 at Ser256 to Phosphorylation of of phosphorylation of AQP2 at Ser256 is to phosphorylation was in in to in the of AVP demonstrated that PKA be by via a J.D. P. Proc. Natl. Acad. Sci. U. S. A. 2002; PubMed Scopus Google Scholar). cells at an of were to a of at and were for and as a of increase in a increase in with a role for but did not phosphorylation at Ser269. Ser256 phosphorylation increase phosphorylation at the AQP2 at Ser269 to the Membrane in electron microscopy of cells was on renal inner medullary from AQP2 and were both the cell in and at the apical plasma membrane. In Ser(P)269-AQP2 was at the apical plasma membrane of collecting duct cells in Immunogold for Ser(P)269-AQP2 was in in the of vasopressin Phosphorylation of AQP2 at Ser269 the that Ser269 phosphorylation a role in the of retention of AQP2 in the apical plasma membrane. this cells were with AQP2 in which Ser269 was to the of to and were with cells cells were by microscopy a that of AQP2. was the cell and to both the apical and plasma membranes the seen in native collecting cells, the of phosphorylated was at the plasma membrane and a was seen the addition of Immunogold at the electron the of the S269D of AQP2 in the plasma membrane cells, AQP2 was intracellular in the of but with a with wild type cells, a with the was to the plasma that the regulated AQP2 process is not dependent on Ser269 phosphorylation intracellular to have the in to The are with the role of Ser269 phosphorylation as part of the responsible for regulated trafficking of AQP2 via AQP2 retention in the plasma membrane via of but with role in vasopressin-regulated protein mass phosphoproteomic analysis of rat renal collecting duct cells that the vasopressin-regulated water AQP2 is phosphorylated at four within the 16 amino acids of the COOH-terminal tail (10Hoffert J.D. Pisitkun T. Wang G. Knepper M.A. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). Here, we have demonstrated a role for the last of four phosphorylation which is within two amino acids of the COOH-terminal of the protein that through cyclic AMP and enhances AQP2 phosphorylation at Ser269 and that this phosphorylation a role in vasopressin-mediated aquaporin-2 trafficking. This of PKA PKA to Ser269 in and the Ser269 not a for by that the PKA of Ser269 phosphorylation is to the of PKA to Ser256 K. Sasaki S. Marumo F. J. Biol. Chem. 1997; 272: 14800-14804Abstract Full Text Full Text PDF PubMed Scopus (401) Google Scholar, M. K. Marumo F. Sasaki S. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar), which to be a phosphorylation for phosphorylation at Ser269. In this we upon and in of the and of AQP2 regulation as as disorders of water of AQP2 Phosphorylation by the four phosphorylation sites in AQP2 in prior phosphoproteomic (10Hoffert J.D. Pisitkun T. Wang G. Knepper M.A. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar), the Ser256 site was previously G. M. D. Yasui M. A. Nielsen S. Am. J. Physiol. PubMed Google Scholar). Ser256 is part of a for phosphorylation by the kinase PKA K. S. Marumo F. Sasaki S. 1993; PubMed Scopus Google Scholar). The PKA in in vitro kinase with peptide that PKA this site as demonstrated by and mass phosphorylation of AQP2 not the water of AQP2 M. K. Marumo F. Sasaki S. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar, M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google but been demonstrated to an role in cyclic regulation of AQP2 trafficking to the plasma membrane (7Katsura T. Gustafson C.E. Ausiello D.A. Brown D. Am. J. Physiol. 1997; 272: F817-F822Crossref PubMed Google Scholar, 8Fushimi K. Sasaki S. Marumo F. J. Biol. Chem. 1997; 272: 14800-14804Abstract Full Text Full Text PDF PubMed Scopus (401) Google Scholar, van Deen P.M. J. Biol. PubMed Scopus Google Scholar). in rat collecting duct that cyclic AMP addition the by addition of vasopressin both Ser256 showed a rapid increase in phosphorylation in than in cells. demonstrated rapid increases in Ser256 phosphorylation in to vasopressin G. M. D. Yasui M. A. Nielsen S. Am. J. Physiol. PubMed Google Scholar, C.L. Christensen S. J.D. P. Nielsen S. Knepper M.A. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). The increase in Ser256 phosphorylation was by slower increases in phosphorylation at and Ser269 in to cyclic AMP vasopressin AMP addition the previously demonstrated of vasopressin to phosphorylation J.D. Nielsen J. Pisitkun T. Nielsen S. Knepper M.A. Am. J. Physiol. PubMed Scopus Google Scholar). the between the changes by vasopressin and by cyclic AMP the as a of the of vasopressin to AQP2 phosphorylation at four with the from a of that the of vasopressin to water permeability in the renal collecting duct are by cyclic AMP (2Nielsen S. Frokiaer J. Marples D. Kwon T.H. Agre P. Knepper M.A. Physiol. Rev. 2002; 82: 205-244Crossref PubMed Scopus (1005) Google Scholar). of Phosphorylation in of AQP2 localization the phospho-specific antibodies to site Ser(P)269-AQP2 as the phosphorylated that was solely in the apical plasma membrane of renal collecting duct cells. of the other phosphorylated were seen both in intracellular vesicles and on the plasma membrane. been in the plasma membranes of collecting duct cells J.D. Nielsen S. Knepper M.A. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). The that Ser(P)269-AQP2 is in the apical plasma membrane to the that this phosphorylation an role in the that retention of AQP2 in the apical plasma membrane in the of This the for on the role of Ser269 in AQP2 trafficking. in the regulation of AQP2 trafficking by vasopressin is believed to two that AQP2 and that AQP2 endocytosis (4Knepper M.A. Nielsen S. Am. J. Physiol. 1993; 265: F214-F224PubMed Google Scholar, 5Nielsen S. Knepper M.A. Am. J. Physiol. 1993; 265: F204-F213PubMed Google Scholar). The abundance of AQP2 in the apical plasma membrane a balance between the of and which are in the of vasopressin D. Am. J. Physiol. 2003; 284: F893-F901Crossref PubMed Scopus (218) Google Scholar, C.E. T. M. Brown D. Am. J. Physiol. Scholar, M. Brown D. Am. J. Physiol. 2002; PubMed Scopus Google Scholar, K. M. Brown D. Am. J. Physiol. 2004; 286: PubMed Scopus Google and when Ser256 of AQP2 is replaced by the amino acid, K. M. Brown D. Am. J. Physiol. 2004; 286: PubMed Scopus Google Scholar). Phosphorylation of AQP2 at Ser269 the localization of Ser(P)269-AQP2 plasma membrane be more a model of regulated the phosphorylation at the apical plasma membrane and AQP2 for with this when we AQP2 to Ser269 with an a a and the mutant AQP2 in cells, the AQP2 was localized in the plasma to increase intracellular cyclic AMP The plasma membrane localization of was by microscopy the mutant AQP2 the to to the cell in to suggesting that Ser269 phosphorylation is not for regulation of AQP2 to the of a role in AQP2 cyclic phosphorylation of Ser269 the endocytosis of the water permeability of the apical plasma The of apical retention of AQP2 on Ser269 phosphorylation is with three AQP2 trafficking. The proposed by Sasaki and S. T. K. T. M. K. T. M. Sasaki S. 2004; PubMed Scopus Google Scholar), that trafficking of AQP2 depends on of a COOH-terminal by as an protein by S. T. K. T. M. K. T. M. Sasaki S. 2004; PubMed Scopus Google other as protein and which have been in with the apical plasma membrane of the renal inner medullary collecting duct in Pisitkun T. Wang G. Knepper M.A. Full Text Full Text PDF PubMed Scopus Google Scholar). a binding be by phosphorylation of a of the Phosphorylation of a as a for and membrane trafficking been previously for the protein with in the region of to trafficking to the plasma membrane. Phosphorylation of at an of the COOH-terminal that to the protein binding to and with J. 2002; PubMed Google Scholar, J. J. E. J. S. E. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). The proposed by Deen and G. M. van der S.P. J. Deen P.M. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar), is on the that amino of AQP2 is and that the of AQP2 from the apical plasma membrane. The of to be by phosphorylation of the AQP2 that AQP2 endocytosis by a process M. Brown D. Am. J. Physiol. 2002; PubMed Scopus Google Scholar), a between AQP2 with of the which are that protein via Rev. 2003; PubMed Scopus Google Scholar). a been demonstrated for endocytosis of transport protein in renal collecting duct cells, the epithelial J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). A from the that the protein to AQP2 that are not phosphorylated at Ser256 T. J. M. Brown D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). proposed when to AQP2 endocytosis via and that Ser256 phosphorylation the associated AQP2 other protein are by phosphorylation at Ser269 are to be of AQP2 have been in with nephrogenic diabetes insipidus Deen P.M. Am. J. Physiol. PubMed Scopus Google Scholar), a by of water excretion that are to In contrast to in other of the AQP2 in the COOH-terminal tail the polyphosphorylated region from Ser256 to are in an and in to the than plasma membrane Deen P.M. Am. J. Physiol. PubMed Scopus Google Scholar). is that of the COOH-terminal tail of AQP2 in this of phosphorylation and of AQP2. a the osmotic water permeability of the collecting duct cells not increase in to vasopressin in the water seen The COOH-terminal Ser256 phosphorylation by by with a phosphorylation at and several that the polyphosphorylated region of the COOH-terminal tail of AQP2. Ser256 Phosphorylation as a for responsible for phosphorylation at three of the four sites in vitro phosphorylation that Ser256 is phosphorylated by of the sites Ser264, and Ser269) have with PKA sites were phosphorylated by PKA in This with the of the PKA in cells, which phosphorylation at both and Ser269. This is by that Ser256 phosphorylation is for and Ser269 phosphorylation PKA phosphorylation at of the COOH-terminal Ser261, in contrast to and a in phosphorylation in to vasopressin J.D. Nielsen J. Pisitkun T. Nielsen S. Knepper M.A. Am. J. Physiol. PubMed Scopus Google cyclic AMP This is in S256L-AQP2 mutant mice suggesting that be dependent on phosphorylation at Ser256 that the a that of by a on the in this PKA-mediated phosphorylation of Ser256 be as a phosphorylation that a control for The of a phosphorylation is not phosphorylation of by at a of three sites and a phosphorylation at by kinase M. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). A is for phosphorylation of other P. S. Rev. Biol. PubMed Scopus Google Scholar). The by which Ser256 phosphorylation phosphorylation to be the phosphorylation of by kinase the at part of the for phosphorylation of in a of phosphorylation in which phosphorylates and A is for AQP2 but of the to from Ser256 to for the for Ser256 phosphorylation for phosphorylation a in the of the COOH-terminal tail Ser256 phosphorylation to to a kinase binding to Ser256 of in regulation of of AQP2 to membrane plasma membrane lipid containing E. D. J. K. P. M. E. P. S. E. J. Am. PubMed Scopus Google Scholar). with