©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
cAMP-dependent Phosphorylation Stimulates Water Permeability of Aquaporin-collecting Duct Water Channel Protein Expressed in Xenopus Oocytes(*)

Michio Kuwahara , Kiyohide Fushimi , Yoshio Terada , Liqun Bai , Fumiaki Marumo , Sei Sasaki (§)

From the (1) Second Department of Internal Medicine, School of Medicine, Tokyo Medical and Dental University, Tokyo 113, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Among water channel proteins (aquaporins), aquaporin-collecting duct (AQP-CD) is the vasopressin-regulated water channel. Vasopressin causes cAMP production in the renal collecting duct cells, and this is believed to lead to exocytic insertion of water channel into the apical membrane (shuttle hypothesis). AQP-CD contains a consensus sequence for cAMP-dependent protein kinase, residues at positions 253-256 ( Arg- Arg-Gln- Ser). To determine the role of this site, Ser-256 was substituted for Ala, Leu, Thr, Asp, or Glu by site-directed mutagenesis. In Xenopus oocytes injected with wild-type or mutated AQP-CD cRNAs, osmotic water permeability (Pf) was 4.8-7.7 times higher than Pf of water-injected oocytes. Incubation with cAMP plus forskolin or direct cAMP injection into the oocytes increased Pf of wild-type, but not mutated, AQP-CD-expressing oocytes, whereas the amounts of AQP-CD expression were similar in wild and mutated types as identified by Western blot analysis. In vitro phosphorylation studies of AQP-CD proteins expressed in oocyte showed that cAMP-dependent protein kinase phosphorylated wild-type, but not mutated, AQP-CD proteins. Phosphoamino acid analysis revealed that this phosphorylation occurred at the serine residue. Moreover, phosphorylation of AQP-CD protein in intact rat kidney medulla tissues was stimulated by incubation with cAMP. Our data suggest that cAMP stimulates water permeability of AQP-CD by phosphorylation. This process may contribute to the vasopressin-regulated water permeability of collecting duct in addition to the apical insertion of AQP-CD by exocytosis.


INTRODUCTION

A water channel of collecting duct, aquaporin-collecting duct (AQP-CD),() was cloned from the rat kidney using a reverse transcribed polymerase chain reaction-based strategy (1) . The cDNA for AQP-CD encodes 271-amino-acid protein ( M 28,928) with 59 and 42% sequence identity with rat eye lens major intrinsic protein (MIP) and human AQP-channel-forming integral membrane protein (AQP-CHIP), respectively. Because AQP-CD is immunohistochemically confined to the apical region of the collecting duct cells, it was suggested to be a vasopressin-sensitive water channel (1) . Recent studies demonstrated that AQP-CD is present exclusively in principal cells and inner medullary collecting duct cells (2, 3) . AQP-CD is diffusely distributed in the cytoplasm in euhydrated condition, whereas apical staining of AQP-CD is intensified in dehydrated condition (2, 3, 4) . Moreover, the immunoblot of the collecting duct cell membranes demonstrated an increased expression of AOP-CD protein in dehydrated rat (2, 4) .

Phosphorylation is an important regulatory process responsible for the modulation of channel proteins (see Ref. 5 for review). The same holds true for MIP family members as well. For example, cAMP-dependent protein kinase phosphorylates MIP possibly at Ser-243 (6, 7) . Phosphorylation increases the voltage-dependent closure of MIP channel (6) . When Ser-262 in nodulin-26, a MIP family in the peribacteroid membrane of symbiotic root nodules, is phosphorylated by a Ca-dependent calmodulin-independent protein kinase, malate uptake is stimulated (8, 9) . In the kidney, the presence of three AQPs, AQP-CHIP, AQP-CD, and AQP 3 has been observed (1, 10, 11) . Of these AQPs, a potential phosphorylation site for cAMP-dependent protein kinase is present in AQP-CD (1, 12) , whereas other AQPs lack such a site. Because the role of this site is not established at present, we examined whether phosphorylation by cAMP-dependent protein kinase is involved in the regulation of AQP-CD.


EXPERIMENTAL PROCEDURES

Site-directed Mutagenesis and in Vitro RNA Synthesis

An in vitro mutagenesis system (Promega) was used for site-directed mutagenesis. Ser-256 in the amino acid sequence of AQP-CD cDNA was altered to Ala (S256A), Leu (S256L), Thr (S256T), Asp (S256D), or Glu (S256E) with mutation primers, GCGGCGGCAGGCGGTGGAGCTCC, GCGGCGGCAGTTGGTGGAGCTCC, GCGGCGGCAGACGGTGGAGCTCC, GCGGCGGCAGGACGTGGAGCTCC, and GCGGCGGCAGGAGGTGGAGCTCC, respectively. Mutations were confirmed by a fluorescence DNA sequencer (Applied Biosystems 373A). Capped RNA transcripts were synthesized in vitro with T3 RNA polymerase using NotI-digested AQP-CD expression vector DNA (3) .

Measurement of Osmotic Water Permeability of Oocyte

Oocytes at stages V-VI were obtained from Xenopuslaevis(1) . Each oocyte was injected with either 40 nl of water or 4 ng of wild-type or mutated AQP-CD cRNAs. After 48 h of incubation, the osmotic water permeability (Pf) of oocyte was measured at 20 °C from the time course of osmotic cell swelling as described (1, 3) . Because the time course of cell swelling was principally linear during the initial 15 s, Pf of oocytes was calculated from this 15-s response. In some experiments, oocytes were incubated in Barth's buffer containing 0.5 mM 8-(4-chlorophenylthio)-cAMP plus 50 µM forskolin, or oocytes were directly injected with 20 nl of 1 mM cAMP before the assay.

Western Blot Analysis

Oocyte membranes were isolated as described (13) . The membrane fraction of rat inner medulla was prepared as described (14) . Western blot was performed as described (14) using an affinity-purified antibody against 15 COOH-terminal amino acids of AQP-CD (3) . In our previous studies, Western blot with this anti-AQP-CD antibody specifically identified a band at 29 kDa, which was an expected molecular size of AQP-CD protein (3, 4) . Moreover, immunohistochemical study of the collecting duct with the antibody revealed that the staining was confined to the apical membrane of the principal cells and the inner medullary collecting duct cells (3) , both of which are known to be vasopressin-responsive cells (2, 20) . These results suggested that the antibody used in this study was specific for AQP-CD protein.

In Vitro Phosphorylation

Phosphorylated membrane proteins of oocyte and kidney medulla were analyzed by SDS-PAGE and autoradiography essentially as described (21) . Protein G-agarose (Protein G PLUS-Agarose, Oncogene Science) and affinity-purified anti-AQP-CD antibody (1:200 dilution) were used for immunoprecipitation. Immune complexes were resuspended in a 50-µl volume of phosphorylation buffer and incubated with 0.2 µg of catalytic subunit of cAMP-dependent protein kinase (Sigma; specific activity, 39 nmol of phosphate/min/mg of protein) in the presence of 0.5 mM ATP containing 10 µCi of [-P]ATP (6000 Ci/mmol, DuPont NEN). In one protocol, the incubation was performed in the presence of 10 µM H89 (Seikagaku Corp.), an inhibitor of cAMP-dependent protein kinase (15, 16) . Autoradiograms were quantified by a dual-wavelength densitometer (Shimadzu CS-9000).

Quantitative Evaluation of AQP-CD Protein Expressed on the Oocyte Membrane

To quantify AQP-CD protein on the oocyte membrane, an anti-AQP-CD antibody against the external domain of AQP-CD was made using synthetic peptide corresponding to amino acid residues at positions 113-127 as described (3) . Immunohistochemical study in the rat kidney and Western blot analysis of AQP-CD protein expressed in oocytes with this antibody showed essentially similar results as obtained by an anti-AQP-CD antibody against the COOH-terminal domain of AQP-CD (3) . Oocytes (10-20 in each group) were incubated in Barth's buffer with or without 0.5 mM 8-(4-chlorophenylthio)-cAMP plus 50 µM forskolin at 20 °C. After incubation for 1 h in the presence of 2% bovine serum albumin at 4 °C, oocytes were further incubated for 1 h with the anti-AQP-CD antibody (1:50 dilution). After three washes, oocytes were incubated with I-protein A for 1 h at 4 °C, and the radioactivity was measured by a scintillation counter.

Phosphoamino Acid Analysis

After wild-type AQP-CD proteins were phosphorylated in vitro for 10 min as above, the P-labeled protein bands were eluted from SDS gel using standard methodology as described (17) .

In Vivo Phosphorylation

Tissues of rat inner medulla were minced with a razor blade and suspended in Krebs-HEPES medium (130 mM NaCl, 5 mM KCl, 1 mM CaCl, 1 mM MgSO, 10 mM glucose, 25 mM Tris-HEPES buffer, pH 7.4) oxygenated with 95% O, 5% CO. After 30 min, the tissue suspensions were distributed into wells of a 24-well plate and incubated for 2 h in the presence of 0.5 mCi/ml [P]orthophosphate (DuPont NEN) at 37 °C under a 5% CO atmosphere. The samples were further incubated in the absence or the presence of 0.5 mM 8-(4-chlorophenylthio)-cAMP. The samples were immunoprecipitated with anti-AQP-CD antibody and analyzed by SDS-PAGE and autoradiography as above.


RESULTS AND DISCUSSION

As reported previously (1, 3) , the expression of wild-type AQP-CD markedly increased Pf of oocytes. We tested the effect of cAMP on Pf of AQP-CD-expressing oocytes. Fig. 1 A depicts the time course of oocyte Pf after incubation with cAMP. Wild-type AQP-CD-expressing oocytes were incubated with 8-(4-chlorophenylthio)-cAMP plus forskolin () or were directly injected with 20 pmol of cAMP at time 0 (). The responses of Pf to these maneuvers were essentially similar; Pf increased at a linear rate up to 15 min and peaked at 30 min. Fig. 1 B summarizes measurement of oocyte Pf. Pf of water-injected oocytes (control) was 21 ± 2 (S.E.) 10 cm/s. Expression of wild-type AQP-CD increased Pf 7.7-fold. Pf increased further when wild-type AQP-CD-expressing oocytes were incubated for 30 min with cAMP plus forskolin (+56%) or were directly injected with cAMP 30 min before Pf measurement (+43%). Consistent with our results, it was reported recently that incubation with cAMP increased Pf in oocytes injected with poly(A) mRNA from rat renal medulla (18) . We mutated Ser at position 256, a potential phosphorylation site for cAMP-dependent protein kinase, to Ala, Leu, Thr, Asp, or Glu. Pf of these oocytes ranged from 102 to 153 10 cm/s, indicating that the mutated AQP-CDs also possess water channel activities. However, the incubation with cAMP plus forskolin failed to increase Pf in every type of mutated AQP-CD oocyte. Moreover, the direct injection of cAMP had no effect on Pf in S256A, S256T, and S256E oocytes, suggesting that the residue at Ser-256 is required for cAMP-induced Pf stimulation.


Figure 1: Pf of oocytes. A, changes of oocyte Pf after cAMP treatments. Oocytes were injected with 4 ng of wild-type AQP-CD cRNA 48 h prior to experiments. Oocytes were incubated with cAMP plus forskolin () or were injected directly with cAMP () at time 0 as described under ``Experimental Procedures.'' After the treatments, osmotic cell swelling of the oocytes was monitored and Pf was calculated. Each point represents means ± S.E. of 8-10 oocytes. B, values of oocyte Pf. Oocytes were injected water ( Control), with wild-type AQP-CD cRNA ( Wild-type), or with mutated AQP-CD cRNAs ( S256X, Ser-256 was replaced by residue X) 48 h prior to experiments. Pf is shown as means ± S.E. cAMP + FSK, incubated for 30 min with 0.5 mM 8-(4-chlorophenylthio)-cAMP plus 50 µM forskolin; cAMP Injection, injected with 20 nl of 1 mM cAMP 30 min before experiments. The number of oocytes is shown in parentheses.



In renal collecting duct, the vasopressin binding to basolateral vasopressin V receptor triggers a series of cellular events that lead to an increase in apical water permeability. These events include cAMP production and subsequent activation of cAMP-dependent protein kinase. In the final step, it is hypothesized that AQP-CD is shuttled by exocytosis and fuses with the apical membrane (shuttle hypothesis) (19, 20) . We examined whether cAMP stimulates the exocytosis of AQP-CD in oocytes. The amount of AQP-CD protein on the oocyte membrane was quantified using an anti-AQP-CD antibody against the external domain of AQP-CD and I-protein A. First of all, I radioactivity of water-injected and AQP-CD-expressing oocytes was measured after incubation without cAMP. In three independent experiments, the mean radioactivity of water-injected oocytes was 23.3% (range, 17.9-27.3%) of that of AQP-CD-expressing oocytes. The radioactivity of water-injected oocytes was thought to be derived from a nonspecific binding of I-protein A to the oocyte membrane. This nonspecific component was subtracted from the radioactivity of AQP-CD-expressing oocytes to quantify the amount of AQP-CD on the oocyte membranes. When the radioactivity of AQP-CD-expressing oocytes after incubation without cAMP was taken as 100%, the radioactivity (the mean of three separate experiments) changed 97.8, 107.0, 103.2, and 104.1% after 7.5-, 15-, 30-, and 60-min incubation with cAMP, respectively. These results suggest that exocytosis of AQP-CD contributes little, if any, to cAMP-induced Pf stimulation in AQP-CD-expressing oocytes.

Immunoblot of wild-type AQP-CD proteins expressed in the oocytes exhibited an apparent molecular mass of a 29-kDa and a broad 35-45-kDa band as previously reported (Fig. 2, lane2). A band at 35-45-kDa was thought to be a glycosylated form of 29-kDa AQP-CD protein (1, 2, 14) . Similar amounts of AQP-CD proteins were expressed in oocytes injected with wild-type and mutated AQP-CD RNAs (Fig. 2, lanes2-7). Identical bands were detected when membrane fraction from inner medulla, where AQP-CD was most abundantly present (1, 2) , was applied (Fig. 2, lane8).


Figure 2: Western blot analysis of oocyte and inner medulla membrane proteins probed with an affinity-purified antibody against AQP-CD. Membranes were prepared from 10 oocytes injected with water ( lane1), cRNAs of wild-type ( lane2), or mutated AQP-CDs ( lanes 3-7) or were prepared from rat inner medulla ( lane8). Mutated AQP-CDs were S256A ( lane3), S256L ( lane4), S256T ( lane5), S256D ( lane6), and S256E ( lane7).



To examine whether the cAMP effect on Pf of wild-type AQP-CD-expressing oocytes is mediated by phosphorylation, wild-type AQP-CD proteins were recovered from oocyte membranes and incubated for 5-30 min with a catalytic subunit of cAMP-dependent protein kinase plus [-P]ATP (Fig. 3 A). We observed two bands with an apparent molecular mass of 29 kDa and 35-48 kDa, which corresponded to the bands identified by Western blots (Fig. 2, lane2). P incorporation into AQP-CD increased as the incubation time extended from 5 to 20 min. Analysis of Fig. 3A by densitometry showed that the density of broad 35-45-kDa bands was 3.9-5.5 times higher than of that of 29-kDa bands. In Western blot of wild-type AQP-CD protein (Fig. 2, lane2), the density of the 35-45-kDa band was similar (93%) to that of the 29-kDa band. These results suggested that glycosylated AQP-CD proteins were preferentially phosphorylated for some reason. This reason is not clear at present. The stoichiometry of phosphorylation of glycosylated and unglycosylated AQP-CD proteins may be different.


Figure 3:In vitro phosphorylation of oocyte and inner medulla membrane proteins. A, time course of phosphorylation. Membranes from wild-type AQP-CD-expressing oocytes were immunoprecipitated with an affinity-purified anti-AQP-CD antibody. The samples were incubated for 5 min ( lane1), 10 min ( lane2), 20 min ( lane3), or 30 min ( lane4) with catalytic subunit of cAMP-dependent protein kinase and [-P]ATP. Membranes from 20 oocytes were applied in each lane. B, membranes from wild-type AQP-CD-expressing oocytes ( lanes2, 9, and 10), water-injected oocytes ( lane1), mutated AQP-CD-expressing oocytes ( lanes 3-7), and inner medulla ( lane8) were analyzed. The membranes were incubated in the presence of H89, an inhibitor of cAMP-dependent protein kinase ( lane9), or in the absence of catalytic subunit of cAMP-dependent protein kinase ( lane10).



In in vitro phosphorylation studies after 10-min incubation, the pattern of labeling of rat inner medulla AQP-CD was identical to that of wild-type AQP-CD expressed in oocytes (Fig. 3 B, lanes2 and 8). By contrast, no labeling was found in any types of mutated AQP-CD proteins expressed in oocytes (Fig. 3 B, lanes 3-7). The phosphorylation of wild-type AQP-CD protein was inhibited by an inhibitor of cAMP-dependent protein kinase, H89 (15, 16) (Fig. 3 B, lane 9). No phosphorylation was achieved in the absence of the catalytic subunit of cAMP-dependent protein kinase (Fig. 3 B, lane 10). For phosphoamino acid analysis, 29-kDa phosphoproteins were eluted from SDS gel after electrophoresis of wild-type AQP-CD protein. Phosphoserine was the only amino acid detected, indicating that AQP-CD protein is phosphorylated at the serine residue (Fig. 4).


Figure 4: Phosphoamino acid analysis. P-Labeled wild-type AQP-CD proteins were eluted from SDS gel. After acid hydrolysis, the sample was mixed with cold phosphoamino acids and electrophoresed on a cellulose thin-layer chromatography plate at pH 3.5 (water/acetic acid/pyridine, 945:50:5) at 1,000 V. The positions of phosphoamino acids were visualized by ninhydrin.



We further tried to determine whether phosphorylation of AQP-CD occurs in intact tissues. Bands of phosphorylated AQP-CD protein were weakly detected by autoradiography in the control (no incubation with cAMP) (Fig. 5, lane1). We speculate that endogenous cAMP is involved in this phosphorylation. cAMP treatment enhanced the band intensity in a time-dependent manner (Fig. 5, lanes2 and 3). Thus cAMP phosphorylates AQP-CD protein of rat kidney medulla in vivo, suggesting that AQP-CD protein acts as a physiological substrate for phosphorylation by cAMP-dependent protein kinase.


Figure 5: In vivo phosphorylation. Tissues of rat inner medulla were suspended in phosphate-free medium, and the suspensions were incubated with [P]orthophosphate for 2 h. 8-(4-Chlorophenylthio)-cAMP was added to the incubation medium at a final concentration of 0.5 mM and incubated for 10 min ( lane2) or 20 min ( lane3). Alternatively, the suspension was incubated for 10 min in the absence of cAMP ( lane1). The membrane pellets were immunoprecipitated with an anti-AQP-CD antibody and analyzed by SDS-PAGE and autoradiography.



We observed that exogenous cAMP enhanced oocyte Pf in wild-type AQP-CD-expressing oocytes but not in mutated AQP-CD-expressing oocytes, which were devoid of a potential phosphorylation site for cAMP-dependent protein kinase (Ser-256). cAMP-dependent protein kinase phosphorylated wild-type AQP-CD protein in vitro at the serine residue. cAMP stimulated phosphorylation of AQP-CD protein of rat kidney medulla in vivo. The amount of AQP-CD protein expressed on the oocyte membrane was not increased by cAMP treatment. These observations suggest that the phosphorylation of AQP-CD protein at Ser-256 is responsible for Pf elevation by cAMP. We speculate that vasopressin increases Pf of in vivo collecting duct cells by phosphorylation of AQP-CD protein in addition to by the stimulation of exocytic AQP-CD insertion into apical membrane as proposed previously (19, 20) . From a physiological viewpoint, both of these mechanisms may have additive effects on the increase of collecting duct Pf in vasopressin-stimulated hydroosmotic conditions and on increasing water reabsorption. In the present study, the rate of Pf increase by cAMP was relatively slow compared with the responses of other channel proteins to cAMP (22, 23, 24) , raising the possibility that the cAMP-dependent phosphorylation may modulate the function of AQP-CD protein by another mechanism besides the direct phosphorylation at Ser-256. Further studies are necessary to determine the precise mechanism whereby phosphorylation by cAMP-dependent protein kinase increases Pf and the relative contributions of AQP-CD protein phosphorylation and the exocytic AQP-CD insertion to vasopressin-responsive Pf increase in in vivo collecting tubules.


FOOTNOTES

*
This work was supported by a grant-in-aid from the Ministry of Education, Science, and Culture, Japan and by grants from the Mitsubishi Foundation, the Salt Science Research Foundation, and Terumo Life Science Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed: Second Dept. of Internal Medicine, Tokyo Medical and Dental University, Yushima, Bunkyo-ku, Tokyo 113, Japan. Tel.: 81-3-3813-6111 (ext. 3221); Fax: 81-3-3818-7177.

The abbreviations used are: AQP-CD, aquaporin-collecting duct; MIP, eye lens major intrinsic protein; AQP-CHIP, aquaporin-channel-forming integral membrane protein; AQP 3, aquaporin 3; Pf, osmotic water permeability; PAGE, polyacrylamide gel electrophoresis.


ACKNOWLEDGEMENTS

We thank Dr. Miwako K. Homma for valuable discussions and Dr. K. Tomita for encouragement of this work.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.