Articles in PresS. Am J Physiol Renal Physiol (April 5, 2017). doi:10.1152/ajprenal.00004.

2017

1 Protein phosphatase 2C (PP2C) is responsible for VP-induced

2 dephosphorylation of AQP2 serine 261

4 Pui W. Cheung*, Lars Ueberdiek*, Jack Day, Richard Bouley and Dennis Brown

6 Center for Systems Biology, Program in Membrane Biology and Division of Nephrology,

7 Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, USA,

8
9
10
11 Address for Correspondence:
12 Dr. Dennis Brown
13 Massachusetts General Hospital
14 Program in Membrane Biology and Division of Nephrology
15 Simches Research Center
16 185 Cambridge St,
17 Boston, MA 02114
18 Phone: (617-726-5665)
19 E-mail: Brown.Dennis@mgh.harvard.edu
20
21
22 Conflict of interest statement
23 The authors have declared that no conflict of interest exists.
24
25 P.W.C and L.U. contributed equally to this work
26

Copyright © 2017 by the American Physiological Society.

27 Abstract

28 AQP2 trafficking is regulated by phosphorylation and dephosphorylation of serine

29 residues in the AQP2 c-terminus. Vasopressin (VP) binding to its receptor (V2R) leads to a

30 cascade of events that result in the phosphorylation of serine 256 (S256), S264 and S269,

31 but dephosphorylation of S261. To identify which phosphatase is responsible for VP-

32 induced S261 dephosphorylation, we pretreated cells with different phosphatase inhibitors

33 before VP stimulation. Only sanguinarine, a specific protein phosphatase 2C (PP2C)

34 inhibitor, abolished VP-induced S261 dephosphorylation, but not inhibitors of PP1, PP2A

35 (okadaic acid) or PP2B (cyclosporine). However, both sanguinarine and VP significantly

36 increased phosphorylation of ERK, a kinase that can phosphorylate S261; inhibition of ERK

37 by PD98059 partially decreased baseline S261 phosphorylation. These data support a role

38 of ERK in S261 phosphorylation, but suggest that upon VP treatment, increased

39 phosphatase activity overcomes the increase in ERK activity, resulting in overall

40 dephosphorylation of S261. We also found that sanguinarine abolished VP-induced S261

41 dephosphorylation in cells expressing mutated AQP2 S256A, suggesting that the

42 phosphorylation state of S261 is independent of S256. Sanguinarine alone did not induce

43 AQP2 membrane trafficking, nor did it inhibit VP-induced AQP2 membrane accumulation

44 in cells and kidney tissues, suggesting that S261 does not play an observable role in acute

45 AQP2 membrane accumulation. In conclusion, PP2C activity is required for S261 AQP2

46 dephosphorylation upon VP stimulation, and this occurs independent of S256

47 phosphorylation. Understanding the pathways involved in modulating PP2C will help

48 obtain a deeper understanding of the role of S261 in cellular events involving AQP2.

49
50 Introduction

51 The ability to concentrate urine is essential for adaptation to terrestrial life. In

52 response to vasopressin (VP) stimulation, aquaporin-2 (AQP2) water channels accumulate

53 on the apical membrane of kidney principal cells and increase water reabsorption from the

54 collecting duct (7, 12, 29). AQP2 trafficking is regulated by phosphorylation and

55 dephosphorylation of its c-terminus on four different serine residues: serine 256 (S256),

56 serine 261 (S261), serine 264 (S264) and serine 269 (S269) (5, 18). Under physiological

57 conditions, binding of VP to the vasopressin receptor type 2 (V2R) increases cAMP levels

58 (21), which leads to activation of protein kinase A (PKA) (19) and subsequently to

59 phosphorylation of the key S256 residue (12, 16, 40). S264 and S269 are also

60 phosphorylated in response to VP stimulation or hypertonicity (5, 8, 16, 18). On the

61 contrary, S261 is dephosphorylated in response to VP stimulation (5, 17, 30, 32, 40). It is

62 well known that protein phosphorylation and signaling is a balance between the actions of

63 kinases and phosphatases. However, while there is a wealth of knowledge and studies on

64 the kinases that can phosphorylate some of these essential serine residues on AQP2 (5, 33),

65 the other side of the balance - the phosphatases - is less well explored.

66 There are 3 major groups in the Ser/Thr phosphatase family: phosphoprotein

67 phosphatases (PPPs), metal-dependent protein phosphatases (PPMs), and the aspartate

68 based phosphatases targeting RNA polymerase II. Representative members of the PPP

69 family include protein phosphatase 1 (PP1) (PPP1CA gene), PP2A (PPP2CA gene), PP2B

70 (PPP3CB gene, commonly known as calcineurin), PP4 (PPP4C gene), PP5 (PPP5C gene),

71 PP6 (PPP6C gene) and PP7 (2). The PPM family includes protein phosphatases dependent
72 on manganese/magnesium ions, such as PP2C (PPM1A gene), while FCPs and SCPs

73 represent the aspartate-based phosphatases (6, 34).

74 In this study we focused on the effect of phosphatase inhibition on both AQP2

75 phosphorylation and trafficking using in vitro and in situ models. As the only serine residue

76 that is dephosphorylated when VP binds to V2R, we aimed to identify the phosphatase that

77 is responsible for VP induced dephosphorylation of S261. Using a specific antibody against

78 phosphorylated S261-AQP2, we showed that VP induced S261 dephosphorylation is

79 dependent on protein phosphatase 2C activity, and this dephosphorylation of S261 occurs

80 independently of S256.

81
 82 Materials and Methods
83
84 Reagents

85 All cell culture media were purchased from Invitrogen (Grant Island, NY) while the

86 fetal bovine serum (FBS) was purchased from Atlanta Biological (Laurenceville, GA). All

87 chemical reagents were purchased from Sigma-Aldrich (St. Louis, MO). Other providers are

88 specified in the text.

89

90 Cell culture

91 LLC-PK1 cells stably expressing c-myc tagged AQP2 (LLC-AQP2) or an AQP2 S256A

92 mutant (LLC-AQP2 S256A) were grown in Dulbecco’s Modified Eagle Medium (DMEM), FBS

93 10% and additional L-glutamine (15). Cells were passaged twice a week and DAPI staining

94 was used weekly to test for mycoplasma contamination.

95

96 Cell treatment and immunocytochemistry

97 Cells were plated onto glass coverslips (Ted Pella, Redding, Ca) 3 days prior to the

98 experiment at a density of 70,000 cells/ml. Cells were starved for at least 1h in DMEM

99 without FBS before treatment. Cells were treated with okadaic acid (OA, 1 μM)

100 (Calbiochem, Billerica, MA), cyclosporine A (CsA, 70 nM) (Selleckchem, Houston, TX) or

101 sanguinarine (S, 15 μM) (Tocris, Minneapolis, MN) diluted in dimethyl sulfoxide (DMSO) for

102 40 minutes. Cells were treated with PD98059 (PD, 30 μM) (Tocris) for 30 minutes. After

103 pre-treatment with various phosphatase inhibitors and PD98059, lysine-vasopressin (10

104 nM) was added. Treatment of cells with VP alone for 10 minutes served as a positive

105 control, and treatment of cells with DMSO served as a negative control.
106 After treatment, cells were fixed in 4% paraformaldehyde containing 5% sucrose,

107 followed by 3 washes with PBS. Cells were permeabilized in 0.1% Triton X-100 for 4

108 minutes. After 3 washes with PBS (5 minutes each), non-specific binding was blocked by

109 1% bovine serum albumin (BSA) in PBS. Cells were incubated with mouse anti c-myc IgG

110 for 1 hour at room temperature to detect c-myc tagged-AQP2 in LLC-AQP2 cells. After

111 washing 3 x 5 min in PBS, the secondary antibody, donkey anti mouse Alexa-488 (15

112 μg/ml) (Jackson ImmunoResearch, West Grove, PA), was applied also for 1h at room

113 temperature. LLC-AQP2 cells were co-stained with rhodamine-conjugated wheat germ

114 agglutinin (WGA 2 μg/ml; Lectin Kit, Vector Labs, Burlingame, Ca) for 10 min as a plasma

115 membrane marker for subsequent quantification of specific AQP2 membrane labeling.

116 Finally, cells were mounted in Vectashield (Vector Labs) with DAPI diluted 2:1 in 0.1 M

117 Tris-HCl, pH 8.0. Images of LLC-AQP2 cells were acquired with a Nikon 90i microscope.

118

119 Kidney tissue preparation and immunohistochemistry

120 The kidney tissue slices were prepared as previously described (3, 4). All animal

121 experiments were approved by the Massachusetts General Hospital Institutional

122 Committee on Research Animal Care in accordance with the guide published by the

123 National Institutes of Health for the Care and Use of Laboratory Animals. In brief, adult

124 Sprague Dawley rats were anesthetized with 2% isofluorane inhalation. The left ventricle

125 was punctured and kidneys were perfused with PBS (37°C) until clear PBS was leaking out

126 of an incision in the inferior vena cava (about 1 minute). Kidneys were removed and slices

127 of approximately 0.5 mm were cut with a Stadie-Riggs microtome. Before treatment, all

128 kidney slices were equilibrated in CO2 saturated Hanks’ balanced salt solution (HBSS: NaCl
129 110 mM, KCl 5 mM, MgSO4 1.2 mM, CaCl2 1.8 mM, NaOAc 4 mM, C6H7NaO7 1 mM, D-glucose

130 6 mM, L-alanine 6mM, NaH2PO4 1 mM, Na2HPO4 3 mM and NaHCO3 25 mM) for 15 minutes.

131 Drugs were added directly into the HBSS to obtain their final concentration. When the

132 experiment ended, slices were fixed in 4% paraformaldehyde-lysine-periodate fixative

133 (PLP) overnight. The slices were then washed with PBS 5 times and the tissues were

134 cryoprotected with PBS containing 30% sucrose overnight before tissues were embedded

135 in O.C.T. compound 4583 (Tissue-Tek; Miles Inc., Elkhart, IN). Cryosections (5 μm) were cut

136 using a Leica CM3050S microtome (Leica Microsystems, Deerfield, Il). Sections were

137 placed onto positive charged slides (Denville Scientific Inc., Metuchen, NJ) and stored at -

138 20°C until use. Before staining, sections were rehydrated in PBS for 20 minutes and then

139 permeabilized with 0.1% Triton X-100 for 10 minutes. After washing 3 times, non-specific

140 binding was blocked with 1% BSA in PBS for 15 minutes, and the tissues were incubated

141 with goat anti-AQP2 (C17) (0.2 μg/ml) (Santa Cruz, Dallas, TX) overnight at 4°C. Sections

142 were then incubated with secondary antibodies, donkey anti-goat conjugated to Alexa-488

143 or Cy3 (15 μg/ml or 1.9 μg/ml, respectively) for 1 hour at room temperature. After three

144 washes in PBS, sections were mounted in Vectashield/DAPI diluted 2:1 in 0.1 M Tris-HCl,

145 pH 8.0. Pictures were acquired using a Nikon 90i epifluorescence microscope or a Nikon

146 A1R confocal laser-scanning microscope.

147

148 Western blotting analysis

149 LLC-AQP2 cells were grown on 35mm tissue culture dishes (Corning, Corning, NY)

150 for 3 days. Before treatment, cells were starved in DMEM without serum for 1 hour. After

151 treatment cells were washed with cold PBS and cells were lysed in RIPA buffer (Boston
152 Bioproducts, Ashland, MA) supplemented with a protease inhibitor cocktail (Complete

153 mini, Roche Diagnostics, Mannheim, Germany) and phosphatase inhibitors NaF (1 mM), 5

154 mM EDTA and sodium orthovanadate (1 mM). Cell lysates were mixed for 30 minutes at

155 4°C followed by a centrifugation for 15 minutes at 17,000 g. Protein concentration was

156 determined using the BCA kit according to the manufacturer’s manual (Pierce, Rockford, Il).

157 Samples were mixed with NuPage SDS sample buffer and NuPage sample reducing buffer

158 and the samples were then incubated at 70oC for 10 minutes. Samples were centrifuged for

159 15 minutes at 17,000 g, then 20 μg of protein was loaded in the each well of NuPage 4-12%

160 Bis-Tris gels (Invitrogen). Then, protein was transferred onto a Sequi-blot PVDF membrane

161 (Bio-rad, Hercules, CA). Membranes were blocked with either PBS-Tween-20 0.1% (PBS-T)

162 containing 5% non-fat milk or 1% BSA for 1 hour at room temperature. Primary antibodies

163 were incubated overnight at 4°C. Primary antibodies were anti-AQP2 (C-17), anti-phospho-

164 AQP2 (pSer261) (Symansism, Temecula, CA), anti-phospho-AQP2 (pSer256) (Abcam,

165 Cambridge, MA), anti-phospho-p44/42 MAPK (Erk 1/2) (Thr202/Tyr204) and anti-p44/42

166 MAPK (Erk 1/2) (Thr202/Tyr204) (Cell Signaling Technology, Danvers, MA). After

167 incubation, the membranes were washed 5 times with PBS-T 5 minutes each before

168 secondary anti-rabbit IgG antibodies conjugated to horseradish peroxidase (Jackson

169 ImmunoResearch) were applied (0.16 μg/ml). Membranes were incubated in

170 chemiluminescence Western Lightning ECL (Amersham, Arlington Heights, MA) and were

171 exposed to Hyblot ES film (Denville Scientific, Metuchen, NJ). Afterwards, membranes were

172 stripped with western blot stripping buffer (Thermo Scientific, Rockford, IL) for at least 15

173 minutes. Band intensities in the different experimental conditions were quantified using
174 Image J (NIH, Bethesda, MD). Band intensity of the phospho-serine antibodies was

175 corrected according to the band intensity of total AQP2 on the same membrane.

176

177 Quantification of AQP2 at the cell membrane

178 Acquired images were imported into Volocity software (PerkinElmer, Waltham,

179 MA). The quantification of AQP2 membrane accumulation was performed under blinded

180 conditions in which the region of interest (ROI) for quantification was determined using

181 WGA membrane staining while the Alexa-488 channel (containing the AQP2 signal) was

182 shut down. First, the DAPI staining was used to mark the nucleus. Then the plasma

183 membrane was outlined by the WGA staining. Quantification was performed on cells that

184 were not adjacent to each other. Using this approach we then quantified the fluorescence of

185 Alexa 488 (AQP2) in the three ROIs, e.g. the cell membrane, the cytoplasm and the nucleus,

186 respectively. The mean fluorescence in the cell membrane was corrected using the

187 fluorescence in the nucleus as a measure of nonspecific background labeling. Each

188 treatment condition was performed in triplicate in each experiment, and for image

189 quantifications, 30 cells from at least 10 different images were used, and these analyses

190 were repeated on three independent experiments.

191

192 PKA activity assay

193 The effect of cyclosporine A (CsA) and sanguinarine (S) on PKA activity was tested

194 by using a PKA kinase activity ELISA kit. The assay was performed according to the

195 manufacturer’s protocol (Enzo Life Sciences Inc. Farmingdale, NY). Briefly, after treatment

196 cells were lysed in buffer prepared according to the manufacturer’s protocol. Protein
197 content was determined using the BCA method and samples were diluted to 2 μg crude

198 protein per reaction sample. The reaction was initiated by adding ATP onto the plate. ELISA

199 results were measured at 450 nm using a DTX880 multimode plate reader (Beckman

200 Coulter, Fullerton, CA). Each condition was performed in duplicate and results were

201 averaged from 3 independent experiments.

202

203 Data analysis

204 Images of immunostained cells and tissues were analyzed using Volocity software

205 (PerkinElmer, Waltham, USA). Western blotting results were quantified with Image J (NIH,

206 Bethesda MD). Data are expressed as means ± SEM. Statistical analyses were performed as

207 appropriate using the one-way ANOVA Tukey test using PRISM software. Differences were

208 considered to be significant at P values <0.05. All experiments were done at least in

209 triplicate.

210
211 Results

212 As described previously, AQP2 accumulated on the basolateral plasma membrane

213 after VP treatment of LLC-AQP2 cells (20, 44). In contrast to a previous study using AQP2-

214 transfected renal CD8 cells from rabbit cortical collecting ducts (39), we found that

215 incubation with okadaic acid (OA) did not result in AQP2 membrane accumulation in our

216 LLC-AQP2 cells. Instead, AQP2 accumulated in the perinuclear region (Figure 1A).

217 Quantification showed that AQP2 accumulated on the plasma membrane only when cells

218 were exposed to VP. Pretreatment with OA did not affect the extent of the VP induced AQP2

219 plasma membrane accumulation (Figure 1B). These results were confirmed in Sprague-

220 Dawley rat kidney slices. In our in situ model, OA treated tissues had a cytoplasmic AQP2

221 staining, similar to control tissues (Figure 1C). In contrast, VP treatment led to a clear

222 apical AQP2 staining in the cortical and medullary regions of the collecting duct (Figure

223 1C).

224 Western blotting was used to analyze the phosphorylation state of S261 and S256

225 (Figure 2A, B and C). Specific phospho-antibodies revealed that OA treatment alone did not

226 significantly affect the phosphorylation of S261, and OA did not prevent VP-induced

227 dephosphorylation of S261 (Figure 2A). While the phosphorylation of S256 was slightly

228 increased, this difference was not significant. PKA activity analysis suggested that OA did

229 not have any effect on PKA activity by itself, and OA did not affect the activation of PKA by

230 VP (Figure 2D). In order to determine which phosphatase is responsible for the VP induced

231 dephosphorylation of S261 we searched for OA insensitive serine/threonine protein

232 phosphatases. We found that two phosphatases were reportedly OA insensitive, PP2B
233 (PPP3CB) in the PPP family and PP2C (PPM1A) in the PPM family. We used cyclosporine A

234 (CsA) (10, 11) for PP2B inhibition and sanguinarine (S) for PP2C inhibition (1).

235 Western blotting with specific phospho-antibodies against S261 showed that similar

236 to OA, CsA alone did not affect phosphorylation of S261, and it did not inhibit S261

237 dephosphorylation induced by VP (Figure 3A and B). Similar to OA, CsA alone did not

238 increase PKA activity, nor did it inhibit the activation of PKA induced by VP. There was no

239 difference in PKA activity between cells treated with VP alone and cells pretreated with CsA

240 before VP stimulation (Figure 3C). In LLC-AQP2 cells, CsA treated cells showed cytoplasmic

241 AQP2 staining similar to control, and AQP2 accumulated on the plasma membrane in cells

242 treated with VP, and both CsA and VP (Figure 4A). The quantification of the membrane

243 fluorescence confirmed that CsA alone did not induce membrane accumulation of AQP2

244 and did not inhibit VP induced AQP2 membrane accumulation (Figure 4B).

245 Based on our analysis of the inhibition profiles of known phosphatases thus far, we

246 hypothesized that the OA and CsA-insensitive phosphatase PP2C might be responsible for

247 VP induced dephosphorylation of S261. Previous data have shown that PP2C is expressed

248 in collecting duct principal cells (38) and using RT-qPCR we found that LLC-AQP2 cells also

249 highly express PP2C (PPM1A) (data not shown). Therefore, we treated LLC-AQP2 cells with

250 the specific PP2C inhibitor sanguinarine (25μM) for 30 minutes (1). We found that

251 sanguinarine pretreatment before VP stimulation completely inhibited the VP induced

252 dephosphorylation of S261 (Figure 5A and 5B). Sanguinarine alone did not increase

253 phosphorylation of S261 compared to control cells (Figure 5A), and sanguinarine alone did

254 not affect the phosphorylation of S256 compared to control cells (Figure 5A and C). While
255 sanguinarine did not affect the level of S256 phosphorylation under basal conditions or

256 upon VP stimulation (Figure 5C), we were interested to find whether the VP/PKA signaling

257 pathway was intact or whether sanguinarine activated another pathway that

258 phosphorylates S256. We found that sanguinarine alone did not increase PKA activity and

259 pre-treatment with sanguinarine did not block the activation of PKA induced by VP (Figure

260 5D), indicating that the cellular VP signaling machinery remains intact under sanguinarine

261 treatment. We conclude that inhibition of PP2C by sanguinarine independently blocked

262 dephosphorylation of S261 while S256 phosphorylation continued to be induced by VP.

263 This was confirmed by immunocytochemistry. Cells treated with sanguinarine alone had a

264 cytoplasmic AQP2 staining that was comparable to control conditions (Figure 6A). AQP2

265 accumulated on the plasma membrane in VP treated cells as expected, and sanguinarine

266 pre-treatment did not abolish VP induced membrane accumulation (Figure 6B), again

267 indicating that VP-signaling remains intact in cells when treated with sanguinarine. In

268 addition, sanguinarine also blocked the dephosphorylation of S261 in LLC-AQP2 S256A

269 mutant cells, suggesting that the dephosphorylation of S261 is S256 independent (Figure 7

270 A and B)

271 Bioinformatic analysis (5) and phosphoproteomic studies (33) both suggested that

272 S261 could be phosphorylated by ERK; therefore, we asked whether sanguinarine has any

273 effect on ERK phosphorylation. We found that sanguinarine alone significantly increases

274 ERK phosphorylation (Figure 8A), suggesting that PP2C could play a role in regulating the

275 phosphorylation state of ERK. We also found that VP treatment significantly increases ERK

276 phosphorylation (Fig. 8A). To investigate whether ERK is indeed involved in S261

277 phosphorylation, we used a specific MAPK/ERK inhibitor PD98059 to evaluate whether it

278 affects S261 phosphorylation. PD98059 suppressed ERK phosphorylation, and decreased

279 S261 phosphorylation under baseline conditions compared to control. However, the

280 decrease in phospho-S261 did not reach the very low level seen after VP treatment of cells,

281 even though PD98059 completely abolished VP-induced ERK phosphorylation indicating

282 that the inhibitor was fully active under these conditions (Figure 8A and 8B).

283 Using kidney tissue slice preparations, we confirmed the results seen in LLC-AQP2

284 cells. Pretreatment with the phosphatase inhibitors CsA and sanguinarine did not inhibit

285 VP-induced AQP2 accumulation in the apical membrane of cortical and medullary

286 collecting duct principal cells (Figure 9).

287
288 Discussion

289 Our study shows that dephosphorylation of the S261 c-terminal residue of AQP2 is

290 achieved via the activity of protein phosphatase PP2C, which appears to act both by

291 directly dephosphorylating S261, and by decreasing phosphorylation of ERK. We also show

292 that the dephosphorylation of S261 AQP2 induced by VP does not require phosphorylation

293 of S256 AQP2, and the phosphorylation state of S261 does not have a detectable impact on

294 acute AQP2 membrane accumulation.

295 While the mechanistic pathway linking VP to PP2C is unclear, in rat parotid acinar

296 cells, it is thought that PP2C is activated by elevated cAMP levels (43). Therefore, a similar

297 mechanism may also exist in the VP signaling pathway; as intracellular cAMP increases

298 under VP stimulation, PP2C would be activated to dephosphorylate S261. PP2C has been

299 described in other signaling pathways to indirectly terminate some effectors such as BMP

300 or TNF-αby acting on their respective key kinases such as SMAD or IKKb (22, 35). PP2C

301 has also been shown to deactivate p38, JNK and MAP kinases (9, 36), which were

302 previously implicated in the phosphorylation of S261 (28). Our study now shows that the

303 PP2C inhibitor sanguinarine significantly increases the phosphorylation of ERK, and

304 presumably activates this kinase, which can also phosphorylate S261 (5, 33). Furthermore,

305 PD98059, a specific MAPK/ERK inhibitor, causes a partial decrease in S261

306 phosphorylation under basal (no VP) conditions, again supporting a role of ERK in S261

307 phosphorylation. Our finding that sanguinarine increases ERK phosphorylation

308 significantly under basal conditions also suggests that PP2C is at least partially active in the

309 absence of VP stimulation. However, although inhibiting ERK by PD98059 decreases S261

310 phosphorylation, it does not cause the more extensive dephosphorylation seen after VP
311 treatment. This suggests that ERK is not the only kinase involved in S261 phosphorylation,

312 as also indicated above. Increasing the complexity of this system is our finding that VP

313 greatly increases phosphorylation of ERK (as much as PP2C inhibition by sanguinarine),

314 but still induces a drastic S261 dephosphorylation. This suggests that in the presence of VP,

315 activation of PP2C can overcome increased ERK activity (and possibly the action of other

316 kinases) to favor the ultimate dephosphorylation of S261. Taken together, our data suggest

317 that PP2C is involved in S261 dephosphorylation in two ways. It can directly

318 dephosphorylate S261, and it can act indirectly by decreasing ERK phosphorylation, which

319 may contribute to the observed decrease S261 phosphorylation.

320 PP2C does not seem to play a direct role in the canonical V2R signaling pathway, as

321 sanguinarine does not inhibit the VP induced cAMP increase, PKA activation or S256

322 phosphorylation. Furthermore, sanguinarine alone does not induce AQP2 plasma

323 membrane accumulation, nor does it affect VP induced AQP2 membrane trafficking. This

324 confirms previous findings (24) that the phosphorylation state of S261 does not affect

325 AQP2 trafficking, at least acutely. In addition, we showed that dephosphorylation of S261 is

326 not dependent on S256 phosphorylation, and this may again imply that S261, unlike other

327 serine residues, eg. S264 and S269, has regulatory role(s) in AQP2 function other than in

328 membrane trafficking (5, 25, 26). In this respect, PP2C is a magnesium or manganese

329 dependent protein phosphatase, and members of this family have been implicated in stress

330 signaling, protein ubiquitination, death and survival signaling (23). Indeed, Deen et al.

331 showed that S261 phosphorylation regulates degradation of AQP2 by stabilizing K279

332 ubiquitination (37), and S261 seems to play a role in the long-term regulation of AQP2 (25).
333 We also investigated in parallel the effect of phosphatase inhibitors on the

334 phosphorylation of S256. Some studies reported that phosphatases such as PP1A, PP2A

335 and PP2B affect AQP2 trafficking in cell cultures. For example, Valenti et al showed that OA,

336 an inhibitor of PP1 and PP2A, causes AQP2 membrane accumulation in cultured rabbit

337 collecting duct cells (39), and Gooch et al suggested that PP2B signaling plays a role in

338 AQP2 localization and phosphorylation in IMCD cells (13). We were unable to reproduce

339 their results on AQP2 trafficking in our LLC-AQP2 cells, and were unable to detect a

340 significant increase in S256 phosphorylation upon OA or cyclosporine treatment (data not

341 shown). Instead, we found that OA treatment resulted in a marked perinuclear

342 accumulation of AQP2 in LLC-AQP2 cells. The cellular compartment involved in this OA-

343 induced process has not yet been identified. One possible explanation for this discrepancy

344 may be that the distribution of phosphatases and phosphorylation patterns of AQP2 could

345 vary in different segments of the collecting duct and other cell types (14, 33), thus

346 producing inconsistent results across different cell lines (14, 33, 41, 42). We validated our

347 results obtained from LLC-AQP2 cells by examining bona fide principal cells in in situ rat

348 kidney slices that have allowed us to uncover alternative signaling pathway in previous

349 studies (3, 4). We were again unable to observe accumulation of AQP2 at the plasma

350 membrane or an inhibition of VP induced AQP2 membrane accumulation in the presence of

351 any of the phosphatase inhibitors used in this study, thus confirming our results obtained

352 from cells. More recently, Ren et al. showed that calyculin A, another inhibitor of PP1 and

353 PP2A increased AQP2 membrane accumulation in rats, and increased phosphorylation of

354 S256. However, as the authors discussed in their study, due to systemic toxicity, calyculin A

355 was infused through iliac artery followed by clamping of the renal arteries and veins for 30
356 minutes, and it was difficult to rule out the effect of tissue ischemia on AQP2 localization.

357 The same group also showed that tacrolimus, another PP2B or calcineurin inhibitor

358 increased AQP2 membrane accumulation 45 minutes after systemic administration in rats

359 (31), which was not observed in our cell and tissue models. The reason for such

360 discrepancy is unclear, but systemic administration of tacrolimus can acutely cause afferent

361 arteriolar vasoconstriction (27), mimicking ischemia similar to the method used for

362 calyculin A administration. Our tissue slice model, however, eliminates this possible

363 confounder. In addition, similar to our data, Ren et al showed that PP2B inhibition by

364 tacrolimus did not induce a significant increase in S256 phosphorylation, but unlike our

365 study, tacrolimus caused an increase S261 phosphorylation. This inconsistency is not well

366 understood, but may provide us some hints on the physiological role of S261 AQP2, as CsA

367 and tacrolimus, although both inhibiting calcineurin and NFAT pathways have different

368 clinical efficacies and side effect profiles.

369 In conclusion, PP2C activity results in S261 AQP2 dephosphorylation upon VP

370 stimulation, and this effect occurs independent of S256 phosphorylation. Understanding

371 the potential pathways involved in modulating PP2C could help us obtain a deeper

372 understanding of the physiological role of S261 in cellular events involving AQP2.

373

374 Acknowledgements

375
376 This work was supported by NIH grant DK096586 (D. Brown). Lars Ueberdiek was

377 supported by the Kolff Student Fellowship Abroad Grant of the Dutch Kidney Foundation.

378 P. W. Cheung was supported by T32 grant 5T32DK007540-29. Jack Day was supported by
379 an undergraduate summer research award from the American Physiological Society. R.

380 Bouley was supported by a MGH/ECOR interim support fund. The Nikon A1R confocal in

381 the PMB Microscopy Core was purchased using an NIH Shared Instrumentation Grant S10

382 RR031563-01 (DB). Additional support for the Program in Membrane Biology Microscopy

383 Core came from the Boston Area Diabetes and Endocrinology Research Center (DK057521)

384 and the MGH Center for the Study of Inflammatory Bowel Disease (DK043351).

385 Disclosure
386
387 None
388
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526

527
528 Legends to Figures

529 Figure 1: OA treatment produces a perinuclear patch of AQP2 but does not cause

530 plasma membrane accumulation.

531 (A) LLC-AQP2 cells were treated for 40 minutes with OA (1 μM). After OA pre-treatment,

532 some cells were incubated with VP (10 nM) for 10 minutes and others were treated with

533 DMSO as negative controls. Cells stimulated with VP alone were used as a positive control.

534 Images are representative of at least three independent experiments. (B) Quantification of

535 images showed that only cells treated with VP alone or OA plus VP showed significantly

536 increased AQP2 plasma membrane accumulation. OA alone did not affect AQP2

537 accumulation on the plasma membrane but resulted in a marked perinuclear accumulation

538 of AQP2. Data were analyzed using the one-way ANOVA Tukey test (mean±SEM, n=3,

539 **P<0.01, ****P<0.0001). (C) AQP2 did not accumulate on the apical plasma membrane of

540 cortical or medullary collecting duct principal cells after OA treatment of tissue slices in

541 vitro. Kidney slices from adult Sprague-Dawley rats were incubated with OA (1 μM) for 40

542 minutes or with VP (100 nM) for 15 minutes. In both the cortex and outer medulla, AQP2

543 accumulated on the apical plasma membrane after VP treatment. In contrast, both control

544 (CT) slices and OA treated slices showed mostly cytoplasmic AQP2 staining. Scale bar = 25

545 μm. These images are representative of three independent experiments.

546

547 Figure 2: OA does not affect the phosphorylation of AQP2 at S256 and S261, and does

548 not inhibit VP induced S261 dephosphorylation.

549 (A) Specific anti-phospho-AQP2 antibodies were used on western blots with cell lysates

550 from LLC-AQP2 cells treated with either VP (10 nM), OA (1 μM) or OA plus VP. (B, C)
551 Intensities of phospho-serine bands were normalized to the band intensity of total AQP2.

552 OA slightly increased S256 phosphorylation but this was not significant. OA did not prevent

553 VP induced S261 dephosphorylation. Data were analyzed using the one-way ANOVA Tukey

554 test (mean±SEM, n=4, *P<0.05, **P<0.01, ***P<0.001). (D) PKA activity was not altered by

555 OA. PKA activity was assessed using a PKA ELISA kit and was increased only in cells treated

556 with VP, but not after OA pre-treatment. Results are the average of 3 independent

557 experiments performed in triplicate. (mean±SEM, n=3, ****P<0.0001).

558

559 Figure 3: CsA does not prevent VP induced dephosphorylation of S261. (A) Specific

560 antibodies against pS261 were used on western blots with cell lysates of LLC-AQP2 cells

561 treated with CsA (70 nM), VP (10 nM) or CsA plus VP. (B) Intensities of phospho-serine

562 bands were normalized to the band intensity of total AQP2. CsA did not prevent

563 dephosphorylation of S261 after VP treatment. Data were analyzed using the one-way

564 ANOVA Tukey test (mean±SEM, n=5, **P<0.01, ***P<0.001). (C) PKA activity was not

565 altered by CsA. (mean±SEM, n=3, **P<0.01, ***P<0.001, ****P<0.0001).

566

567 Figure 4: CsA does not affect AQP2 membrane accumulation. (A) LLC-AQP2 cells were

568 treated with CsA (70nM) or DMSO for 40 minutes. After 30 minutes pretreatment, cells

569 were either incubated with buffer alone or VP (10 nM). Cells treated with VP alone were

570 used as a positive control. Confocal images were taken and are representative of at least

571 three independent experiments. (B) Quantification of the images showed that only cells

572 treated with VP alone or CsA plus VP showed significantly increased AQP2 plasma

573 membrane accumulation. CsA pretreatment before VP stimulation did not prevent AQP2
574 plasma membrane accumulation. Data were analyzed using the one-way ANOVA Tukey test

575 (mean±SEM, n=2, *P<0.05). Scale bar = 25 μm.

576

577 Figure 5: Sanguinarine inhibits VP induced dephosphorylation of S261.

578 (A) Specific anti-phospho-AQP2 antibodies were used on western blots to determine the

579 phosphorylation state of S261 and S256. Cell lysates from LLC-AQP2 cells treated with VP

580 (10nM), sanguinarine (S) (25 μM) or SA+VP were used (B, C). Intensity of phospho-serine

581 bands was normalized to the band intensity of total AQP2. 30 minutes pretreatment with S

582 prevented VP induced S261 dephosphorylation. Data were analyzed using one-way ANOVA

583 Tukey test (mean±SEM, n=4, *P<0.05, **P<0.01). (D) PKA activity was not altered by

584 sanguinarine. Only VP alone or sanguinarine plus VP increased PKA activity (mean±SEM,

585 n=3, **P<0.01, ***P<0.001).

586

587 Figure 6: Sanguinarine did not alter AQP2 membrane accumulation.

588 (A) LLC-AQP2 cells were treated with sanguinarine (S) (25 μM) for 40 minutes. After 30

589 minutes pretreatment, cells were either treated with DMSO or VP (10 nM). Cells treated

590 with VP alone were used as a positive control. Images were taken using a confocal

591 microscope and are representative of at least three independent experiments. (B)

592 Quantification of images showed that only cells treated with VP alone or S plus VP showed

593 significantly increased AQP2 plasma membrane accumulation. Data were analyzed using

594 the one-way ANOVA Tukey test (mean±SEM, n=3, *P<0.05). Scale bar = 25 μm.

595
596 Figure 7: Sanguinarine inhibits the VP induced dephosphorylation of AQP2 at S261 in

597 LLC-AQP2 S256A cells.

598 (A) Specific anti-phospho-AQP2 antibodies were used on western blots with cell lysates

599 from LLC-AQP2 S256A cells treated with VP (10nM), sanguinarine (25 μM) or sanguinarine

600 plus VP. (B) Intensities of phospho-serine bands were normalized to the band intensity of

601 total AQP2. Sanguinarine prevents VP induced S261 dephosphorylation in these cells.

602 Results are the average of 4 independent experiments performed in triplicate. Data were

603 analyzed using the one-way ANOVA Tukey test (mean±SEM, n=4, *P<0.05)

604

605 Figure 8: Sanguinarine and VP increase ERK phosphorylation, and inhibiting ERK

606 decreases S261 phosphorylation.

607 (A) Treatment of LLC-AQP2 cells with sanguinarine (25 μM) or VP significantly increases

608 ERK phosphorylation.). VP induced ERK phosphorylation was significantly suppressed by

609 the MAPK/ERK inhibitor PD98059 (PD) (mean±SEM, n=3). (B) Phosphorylation of

610 S261AQP2 is decreased significantly with PD, but not to the same low level as VP-induced

611 dephosphorylation of S261. Adding VP to cells treated with PD98059 for 30 minutes did

612 not cause further dephosphorylation of S261 compared to VP alone (mean±SEM, n=5).

613 Results are all performed in duplicate, and data were analyzed using one-way ANOVA

614 Tukey test (*P<0.05. **P <0.01, ***P<0.001, ****P<0.0001).

615

616 Figure 9: Pre-treatment with Sanguinarine or CsA does not prevent VP induced AQP2

617 plasma membrane accumulation in principal cells of the collecting duct.

618 Kidney slices from adult Sprague-Dawley rats were incubated with sanguinarine (25 μM)

619 or cyclosporine A (140 nM) for 40 minutes. 15 minutes before the experiment ended, VP

620 (100 nM) was added to the kidney slices. Upper panel: In the cortex AQP2 accumulates on

621 the apical plasma membrane after VP treatment. This effect was not affected by pre-

622 treatment with CsA or sanguinarine. CT slices showed a cytoplasmic AQP2 staining. Similar

623 results were seen in the inner medulla (lower panel). Pictures are representative of three

624 independent experiments and scale bar = 25 μm.

625