AQP2 is a substrate for endogenous PP2B activity within an inner medullary AKAP-signaling complex

Inho Jo1,2,*, Donald T. Ward1,3,*, Michelle A. Baum1, John D. Scott4, Vincent M. Coghlan5, Timothy G. Hammond6, and H. William Harris1,7

1 Division of Nephrology, Children's Hospital, Harvard Medical School, Boston, Massachusetts 02115; 2 Department of Biomedical Sciences, National Institute of Health, Seoul, Korea; 3 School of Biological Sciences, University of Manchester, Manchester M13 9PT, United Kingdom; 4 Vollum Institute, Howard Hughes Medical Institute, and 5 Neurological Sciences Institute, Oregon Health Sciences University, Portland, Oregon 97201; 6 Tulane University Medical Center and Veterans Affairs Medical Center, New Orleans, Louisiana 70112; and 7 Maine Medical Center Research Institute, Portland, Maine 04102


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We have demonstrated that inner medullary collecting duct (IMCD) heavy endosomes purified from rat kidney IMCD contain the type II protein kinase A (PKA) regulatory subunit (RII), protein phosphatase (PP)2B, PKCzeta , and an RII-binding protein (relative molecular mass ~90 kDa) representing a putative A kinase anchoring protein (AKAP). Affinity chromatography of detergent-solubilized endosomes on cAMP-agarose permits recovery of a protein complex consisting of the 90-kDa AKAP, RII, PP2B, and PKCzeta . With the use of small-particle flow cytometry, RII and PKCzeta were localized to an identical population of endosomes, suggesting that these proteins are components of an endosomal multiprotein complex. 32P-labeled aquaporin-2 (AQP2) present in these PKA-phosphorylated endosomes was dephosphorylated in vitro by either addition of exogenous PP2B or by an endogenous endosomal phosphatase that was inhibited by the PP2B inhibitors EDTA and the cyclophilin-cyclosporin A complex. We conclude that IMCD heavy endosomes possess an AKAP multiprotein-signaling complex similar to that described previously in hippocampal neurons. This signaling complex potentially mediates the phosphorylation of AQP2 to regulate its trafficking into the IMCD apical membrane. In addition, the PP2B component of the AKAP-signaling complex could also dephosphorylate AQP2 in vivo.

kidney; water channel; adenosine 3',5'-cyclic monophosphate; protein phosphatase 2B; aquaporin-2; A kinase anchoring protein


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

TRANSEPITHELIAL WATER REABSORPTION across renal collecting duct cells is stimulated by arginine vasopressin (AVP). AVP acts by increasing the number of aquaporin-2 (AQP2) water channels in the apical membrane of inner medullary collecting ducts (IMCDs), thus increasing its permeability to water (reviewed in Refs. 19 and 27). Before AVP stimulation, AQP2 resides primarily in subapical membrane vesicles, and the subsequent stimulation increases the insertion of these AQP2-containing vesicles into the apical membrane itself. It is known that AVP binding to the V2 receptor stimulates cAMP formation and that the insertion of AQP2 vesicles into the apical membrane involves phosphorylation of AQP2 by cAMP-dependent protein kinase A (PKA). A present area of active investigation is how the activated PKA interacts with its AQP2 substrate and whether it is specifically targeted to it. In addition, although a number of studies have investigated PKA-mediated AQP2 phosphorylation, little is known regarding the dephosphorylation of AQP2.

The COOH-terminal region of AQP2 contains a single putative phosphorylation site for PKA (Ser256) (12, 26). In LLC-PK1 cells transfected with wild-type AQP2, addition of AVP and forskolin induces insertion of AQP2-containing vesicles into the cell membrane. However, when such expression studies are performed with an AQP2 mutant possessing an alanine instead of Ser256, this AQP2 insertion response is abolished (16). Studies using both purified AQP2-containing vesicles (22) and Xenopus laevis oocytes (21) suggest that PKA-mediated phosphorylation of AQP2 does not alter its intrinsic water permeability but rather may be a key event permitting AQP2 access to the plasma membrane. Recently, Klussmann et al. (20) have provided evidence for the involvement of A kinase anchoring proteins (AKAPs) in the translocation of AQP2 from intracellular vesicles to the IMCD membrane. Although these data demonstrate that compartmentalization of PKA by AKAPs is necessary for AQP2 translocation, questions remain regarding the structure of this PKA-AKAP complex and the location of the intracellular compartment to which it is bound.

The subcellular trafficking of vesicles has been studied extensively in neurons, which are also polarized and may be related to epithelial cells (17). The postsynaptic densities of hippocampal neurons have been demonstrated to contain a multiprotein-signaling complex consisting of a 79-kDa A kinase anchoring protein (150 kDa in mice) (7) associated with a number of signaling molecules, including the PKA regulatory subunit, protein phosphatase 2B (PP2B or calcineurin), and PKC (reviewed in Ref. 8). This hippocampal neuron-signaling complex is postulated to provide the molecular specificity for cycles of PKA-mediated phosphorylation and dephosphorylation of membrane protein components, including ion channels. In mice, a novel AKAP family has also been cloned that localizes to the apical membranes of renal proximal tubule cells (AKAP-KL) (9).

Given the postulated evolutionary and functional relationships between polarized neurons and epithelial cells (17), we investigated whether rat IMCD heavy endosomes, which we have previously shown to be apically derived and AQP2 containing (14, 22), also contain such an AKAP complex, because such a mechanism could help explain how AQP2 trafficking is regulated in response to AVP. Furthermore, given the presence of PP2B in the neuronal AKAP complex, we investigated whether it is also expressed in IMCD cells and whether it can dephosphorylate AQP2 previously phosphorylated by PKA.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. Various items were obtained from the following sources: [gamma -32P]ATP (10 Ci/mmol) was from NEN Life Science Products, Sepharose 4B was from Pharmacia (Uppsala, Sweden), polyvinylidene fluoride membrane was from MSI (Westborough, MA), and SDS, acrylamide, beta -mercaptoethanol, and ammonium persulfate were from Bio-Rad. All other reagents were from either sources described previously (22) or Sigma (St. Louis, MO). Male Sprague-Dawley rats (200-250 g) were purchased from Charles River Laboratories (Cambridge, MA).

Purification of IMCD endosomes. Endosomes were prepared from rat kidney inner medullary papillae as described previously (14). Briefly, papillae were homogenized in buffer A (300 mM mannitol and 12 mM HEPES, titrated to pH 7.6 with Tris) and centrifuged at 2,500 g to remove nuclei and unlysed cells. The postnuclear supernatant was centrifuged at 20,000 g for 20 min, and the new supernatant and the lightly packed upper layer of the pellet were collected. This postmitochondrial supernatant was then recentrifuged at 48,000 g for 30 min, and the resulting pellet was resuspended in buffer A and fractionated on a self-forming Percoll gradient (18% Percoll by weight). After centrifugation for 30 min at 48,000 g, the bottom one-third of the gradient was collected and dispersed in an eightfold larger volume of buffer B [(in mM) 300 mannitol, 100 KCl, 5 MgSO4, and 5 HEPES, adjusted to pH 7.6 with Tris] and left on ice for 15 min. This vesicle suspension was then recentrifuged at 48,000 g for 30 min, and the resulting membrane pellet was resuspended in buffer B. After centrifugation at 5,000 g for 15 min, the endosomes appeared as a pearly white layer overlaying a darker membrane pellet. These vesicles, previously characterized as apical endosomes, were either used immediately or stored at -80°C until use. Endosomal protein content was determined by the method of Bradford (3).

Immunoblotting. Western blot analysis of IMCD proteins was performed as described previously (15) using the following antisera: anti-PKCzeta and anti-PKCdelta rabbit polyclonal (Life Technologies, Gaithersburg, MD), and, anti-RII goat polyclonal and anti-PP2B (catalytic subunit) mouse monoclonal antibodies (Upstate Biotechnology, Lake Placid, NY).

Immunohistochemistry. Rats were perfusion fixed as described previously (25), using freshly prepared 4% paraformaldehyde followed by sucrose cryoprotection. Tissue samples were then embedded in OCT compound (Miles, Elkart, IN), snap-frozen in 2-methylbutane in liquid N2, and stored at -70°C until further use. Immunohistochemistry was then performed as previously described (24) using frozen sections (4 µm) cut from the tip of individual rat kidney inner medullae and stained with various antisera. Counterstaining was performed with methyl green (Fisher, Pittsburgh, PA).

32P-RII overlay. Rat tissue samples were subjected to SDS-PAGE electrophoresis and transferred to a nitrocellulose membrane that was incubated in buffer containing 1% (wt/vol) bovine serum albumin and recombinant RII protein, which was phosphorylated using [gamma -32P]ATP as described previously (7). The membrane was incubated in the resulting 32P-RII probe for 14 h at 4°C in the presence or absence of the blocking peptide Ht31 (4). The membrane was then washed free of unbound probe, and the presence of bound 32P-RII was determined by autoradiography.

Affinity chromatography using cAMP-agarose. IMCD heavy endosomes were solubilized on ice for 2 h in hypotonic buffer [(mM) 10 HEPES (pH 7.9), 1.5 MgCl2, 10 KCl, 1 polymethylsulfonyl fluoride, 0.5 dithiothreitol, 1 benzamidine, and 0.01 IBMX] containing 0.5% (vol/vol) Nonidet P-40 and centrifuged at 15,000 g for 15 min. The detergent-soluble supernatant was mixed with cAMP-agarose equilibrated in hypotonic buffer containing 0.1% Nonidet P-40. After being mixed by rotation at 4°C for 14 h, the cAMP-agarose pellet was washed four times in hypotonic buffer and then analyzed for its protein content by SDS-PAGE and immunoblotting (6).

Localization of dual antibody labels by small-particle flow cytometry. Freshly prepared endosomes were incubated in 1:1 normal donkey serum (clarified by centrifugation at 180,000 g for 20 min) for 1 h at room temperature, washed, and then incubated in primary antibodies produced in goats or rabbits at 4°C for 14 h. The endosomes were washed again and incubated in fluorescently conjugated anti-donkey secondary antibody (1:100 dilutions) for 2 h at room temperature. After a final wash, endosomes were analyzed by small-particle flow cytometry (14). Secondary antibodies (Jackson Immunochemicals, Bar Harbor, ME) were donkey anti-goat cyanine-5 (for anti-RII) and donkey anti-rabbit cyanine-3 (for anti-AQP2 and anti-PKCzeta ). Cyanine-3 was excited with 100 mW of 488-nm blue argon ion laser light, and cyanine-5 was excited with 100 mW of 647-nm ruby-red krypton laser light. The fluorescence of each of the 2,000 individual particles was collected in photomultipliers beyond a 575 ± 26- or 675 ± 20-nm (cyanine-3 or cyanine-5, respectively) band-pass filter. Flow cytometry files were collected and analyzed using Becton Dickinson Cell Quest flow cytometry software.

[32P] labeling and immunoprecipitation of AQP2. Endosomes were phosphorylated using the catalytic subunit of PKA in the presence of [gamma -32P]ATP, as described previously (22). The 32P-labeled endosomes were then subjected to various treatments during incubation at 37°C in buffer containing (in mM) 20 Tris (pH 7.0), 50 KCl, 3 Mg2+, 0.1 Ca2+, 0.5 Ni2+, and 0.1 dithiothreitol as well as 3,900 U PKI. The resulting [32P]AQP2 was then collected by immunoprecipitation and assayed for its 32P content. Briefly, after 32P labeling, endosomes were solubilized using 1% (vol/vol) Triton X-100 (1 h on ice) and centrifuged at 14,000 g for 30 min. The supernatant was combined with rabbit anti-AQP2 antiserum coupled to Sepharose 4B and incubated with continuous mixing at 4°C for 14 h. The immunoprecipitate was pelleted by centrifugation, washed three times in phosphate-buffered saline, and denatured with Laemmli buffer. [32P]AQP2 was then resolved by SDS-PAGE, detected by autoradiography, and quantified by densitometry.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Purified IMCD heavy endosomes possess a 90-kDa AKAP, RII, PP2B, and PKCzeta . With the use of specific antisera, Fig. 1 shows that rat inner medulla contains a 55-kDa RII species and the 18-kDa PP2B beta -subunit (Fig. 1, A and B). Sands et al. (25) have reported previously that purified rat IMCD heavy endosomes also possess PKCzeta . Because these three proteins represent the basic components of an AKAP-signaling complex present in hippocampal neurons (10, 18), we sought to determine whether IMCD heavy endosomes also possess a similar AKAP-signaling complex. As detected using a 32P-RII overlay assay shown in Fig. 1, AQP2 endosomes also possess AKAP(s). To demonstrate that the RII probe is binding specifically to endogenous AKAP, we used the anchoring inhibitor peptide Ht31 to ablate its binding (Fig. 1C, lane 6). Ht31 represents the peptide sequence within AKAP that is responsible for RII binding (8). We have assigned the single most prominent band a molecular mass of ~90 kDa based on data from multiple experiments (n = 12). However, it should be noted that the band is broad and in some experiments appears as a doublet that included a smaller band of an ~75-kDa relative molecular mass (Fig. 1C). More complete characterization will be required to determine whether these multiple AKAP bands observed on 32P-RII overlay blots result from either proteolysis of a larger AKAP protein or to posttranslational modification of an AKAP(s). Alternatively, these multiple bands may derive from two independent species of RII-binding protein. The data shown in Fig. 1 are consistent with and extend those reported by Klussman et al. (20).


View larger version (43K):
[in this window]
[in a new window]
 
Fig. 1.   Presence of type II protein kinase A (PKA) regulatory subunit (RII), protein phosphatase (PP)2B, and a putative A kinase anchoring protein (AKAP) in rat inner medulla (IM). A: rat IM homogenate (40 µg protein) contains a single 55-kDa anti-RII immunoreactive band that comigrates on immunoblots with a similar band present in rat brain (Br), demonstrating that rat IM contains type II PKA. B: immunoblot showing that total rat kidney homogenate (Kid), total IM homogenate, and IM endosomes (End) each contains a PP2B-immunoreactive band that comigrates with neuronal PP2B. C: autoradiograph showing the binding of [32P]-labeled RII to proteins from rat brain (lanes 1 and 4), IM membranes (lanes 2 and 5), and inner medullary collecting duct (IMCD) endosomes (lanes 3 and 6; 40 µg protein/lane) resolved by SDS-PAGE and electrophoretically transferred to a membrane. Incubation of the membrane with 32P-RII was performed in the absence (lanes 1-3) or presence (lanes 4-6) of Ht31 anchoring inhibitor peptide. When the 32P-RII probe binds to a protein and the binding is inhibited by Ht31, the association indicates the presence of a putative AKAP (4). The rat brain sample exhibits numerous specific associations and is overexposed due to the large quantities of RII-binding proteins compared with kidney. In contrast, purified aquaporin-2 (AQP2)-containing endosomes contain 2 specific RII-binding proteins (right). Note that the binding of the 32P-RII probe to a 55-kDa protein is present in all 3 tissues and was not affected by the presence of Ht31. This binding results from dimerization of the 32P-RII probe with RII in the sample. Left: positions of the molecular mass markers. Results are representative of a minimum of 4 separate experiments.

To verify that RII, PP2B, and PKCzeta colocalized within collecting duct cells, we immunostained sections of rat inner medulla using antisera specific for each of the proteins. As shown in Fig. 2, rat IMCDs exhibited diffuse immunoreactivity to each of the antibodies tested with the exception of anti-PP2B antibody, which strongly stained the apical region of IMCD cells (Fig. 2C).


View larger version (53K):
[in this window]
[in a new window]
 
Fig. 2.   Rat inner medullary collecting ducts contain RII, PP2B, and PKCzeta . Representative light microscopy immunohistochemical stains show that IMCDs contain RII (A), PP2B (B), and PKCzeta (C) and that the antibody staining for each protein includes immunoreactivity in the region of the apical membrane. Specific antibody binding is indicated by the rose-colored reaction product (magnification: 2,500).

IMCD heavy endosomes possess a multiprotein phosphorylation complex similar to that found in neurons. With the employment of a methodology similar to that used previously to demonstrate the existence of an AKAP-signaling complex in neurons (7), detergent-solubilized IMCD heavy endosomes were mixed with cAMP-agarose, with the resulting cAMP-agarose-bound proteins identified by an anti-RII immunoblot or by a 32P-RII overlay assay. As shown in Fig. 3A, cAMP-agarose retained both RII and the 90-kDa putative AKAP, substantially clearing these proteins from a mixture of detergent-soluble endosomal proteins.


View larger version (39K):
[in this window]
[in a new window]
 
Fig. 3.   Absorption of AKAP-containing multiprotein complex from IMCD endosomes using cAMP-agarose. A: cAMP-binding proteins and their associated proteins were affinity precipitated from Nonidet (N)P-40-solubilized purified AQP2 containing IMCD endosomes using cAMP-agarose. The cAMP-agarose precipitate was pelleted by centrifugation and solubilized in SDS-Laemmli buffer. The pellet (P) and supernatant (S) were assayed for their RII content by immunoblotting (lanes 1 and 2) and for their AKAP content by 32P-RII overlay assay (lanes 3-6). The cAMP-agarose successfully precipitated almost all of the RII and the putative 90-kDa AKAP broad band contained in IMCD endosomes. B: a multiprotein complex was subsequently purified from NP-40-solubilized AQP2 endosomes by affinity chromatography using cAMP-agarose. Equal amounts of the cAMP-agarose precipitate were loaded onto consecutive lanes of an SDS-PAGE gel, and the proteins were electrophoretically transferred to a membrane. Individual lanes of the membrane were then separated, and each was either immunoblotted against a specific antiserum (lanes 1 and 4-7) or incubated in 32P-RII in the absence (lane 2) or presence (lane 3) of Ht31 blocking peptide and developed by autoradiography. As shown here, the cAMP-agarose precipitate contains RII (lane 1), a 90-kDa AKAP (lane 2), PP2B (lane 4), and PKCzeta (lane 5) but not PKCdelta (lane 6) or AQP2 (lane 7). Left: positions of the molecular mass markers. Results are from a minimum of 4 independent experiments.

By employing affinity chromatography using cAMP-agarose, we then examined whether any endosomal proteins can be coabsorbed with RII and the 90-kDa putative AKAP and, if so, whether they represent other components of the hippocampal AKAP complex such as PP2B and PKC. As shown in Fig. 3B, the resulting cAMP-agarose precipitate contained RII (lane 1), a 90-kDa AKAP (lane 2), PP2B (lane 4), and PKCzeta (lane 5). Anti-PKCdelta (lane 6) and anti-AQP2 (lane 7) antisera failed to immunodetect either protein in this complex despite these proteins being abundantly present in purified IMCD heavy endosomes (25).

Colocalization of RII with AQP2 and PKCzeta in the same IMCD endosomal population. To further test the hypothesis that RII and PKCzeta are associated together within a putative AKAP complex present in IMCD heavy endosomes, small-particle flow cytometry was utilized to examine whether anti-RII and anti-PKCzeta antibodies colocalize to the same population of heavy endosomes. Rat IMCD heavy endosomes, shown previously to contain abundant AQP2 (14), were incubated with antisera against RII and PKCzeta , and their presence was determined with species-specific secondary antibodies coupled to either of the fluorophores cyanine-3 or cyanine-5. Perfusion of the rats with fluorescein-dextran (F-dextran) before kidney harvest results in the uptake of the concentrated F-dextran by endocytic vesicles retrieved from the apical membrane of the IMCD as previously described (14). Therefore, through the measurement of fluorescence at the three different emission wavelengths corresponding to excitation of fluorescein, cyanine-3, and cyanine-5, it is possible to demonstrate whether a single F-dextran-containing endosome possesses one or two specific proteins bound to its cytoplasmic surface.

Exposure of purified IMCD endosomes to both secondary antisera conjugated with either cyanine-3 or cyanine-5 yielded an overall low level of cyanine-3- or -5-fluorescent endosomes (Fig. 4A). This control demonstrates there is little nonspecific fluorophore binding in the absence of primary antibodies. As an additional control, the same endosomes were incubated with an irrelevant control rabbit polyclonal antiserum that also produced little (5%) highly fluorescent cyanine-3- and -5-binding endosomes (Fig. 4B). In contrast, preincubation of F-dextran-containing endosomes with the combination of rabbit anti-PKCzeta (x-axis) and goat anti-RII (y-axis) antisera produced a significant number (40%) of highly fluorescent endosomes. In a similar manner, coincubation of endosomes with rabbit anti-AQP2 (x-axis) and goat anti-RII (y-axis) also produced a group (27%) of F-dextran-containing endosomes containing bound anti-AQP2 and RII antibodies (Fig. 4D). Taken together, these data suggest that components of an AKAP complex are present on the surface of F-dextran-containing endocytic vesicles. It is not possible to perform similar flow cytometry studies on vesicles immunoisolated using anti-AQP2 antiserum (23) because of an inability to elute the resulting bound vesicles without loss of the F-dextran fluorophore that is essential for flow cytometry detection, as shown in Fig. 4 (data not shown).


View larger version (52K):
[in this window]
[in a new window]
 
Fig. 4.   Colocalization of RII with AQP2 and PKCzeta in the same IMCD endosomal population. Two-dimensional plots show the colocalization of various antisera on fluorescein-dextran (F-dextran)-containing rat IMCD endosomes as detected by small-particle flow cytometry. Each diagram shows 2,000 individual endosomes containing F-dextran entrapped within their lumens as a frequency histogram displayed on an arbitrary log scale. The x-axis displays binding of donkey anti-rabbit cyanine-3 (cy-3), the y-axis shows binding of donkey anti-goat cyanine-5 (cy-5), and the origin of each of the axes is marked by a zero. In A, there is little nonspecific binding of secondary antisera to endosomes in the absence of primary antibodies. On the basis of these data, an arbitrary value equal to 40 times basal fluorescence in either cyanine-3 or -5 was established as the threshold gating of signal shown in the box in the top right corner. Addition of an irrelevant rabbit antiserum yields a similar result, albeit with larger background fluorescence (5%). In contrast, incubation with rabbit anti-PKCzeta (x-axis) and goat anti-RII (y-axis) antisera yields 40% of particles that are highly cofluorescent for both cyanine-3 and -5 (C, top right quadrant). Thus because both antibodies bind to endosomes, this indicates colocalization of the two proteins in the same population of F-dextran-containing endosomes. In D (top right quadrant), there is colocalization of AQP2 (x-axis) and RII (y-axis) in the same endosomes (27% highly fluorescent). It is noteworthy that vesicles or membranes not containing entrapped F-dextran are not visualized by this method. Results are from F-dextran fluorescent vesicles derived from aliquots of a single vesicle preparation per experiment that was performed a total of 5 times.

AQP2 is dephosphorylated by endogenous PP2B activity and by exogenous PP2B in vitro. Previously, we have shown that the AQP2 present in IMCD heavy endosomes is phosphorylated by endogenous PKA activity (22). To test whether this endogenous AQP2 is also a substrate for exogenous PP2B in vitro, endosomes were 32P labeled using the catalytic subunit of PKA and incubated at 37°C for various intervals in the presence or absence of an exogenous PP2B catalytic subunit. AQP2 was then immunoprecipitated from the endosomes, and the 32P content of AQP2 was determined by SDS-PAGE and autoradiography. As shown in Fig. 5, addition of the PP2B catalytic subunit increased the rate of [32P]AQP2 dephosphorylation.


View larger version (66K):
[in this window]
[in a new window]
 
Fig. 5.   Dephosphorylation of [32P]AQP2 in IMCD endosomes is increased by exogenous PP2B. 32P-labeled AQP2-containing endosomes were incubated at 37°C for either 5 (lane 1, control), 15 (lanes 2 and 5), 30 (lanes 3 and 6), or 60 min (lanes 4 and 7) with either no additions (lanes 1-4) or in the presence of 0.1 µg/µl PP2B (lanes 5-7). [32P]AQP2 was then immunoprecipitated from each of the samples as described in METHODS and detected by autoradiography. Left: positions of the nonglycosylated (29-kDa) and glycosylated (35- to 50-kDa) AQP2 species.

To examine whether the endogenous phosphatase activity that dephosphorylates AQP2 in vitro is reduced by addition of inhibitors of PP2B, the 32P-labeled endosomes were subjected to various incubations in the presence or absence of PP2B inhibitors. Incubation of 32P-labeled endosomes at 37°C resulted in a time-dependent reduction in the 32P content of AQP2 (Fig. 6, lanes 1-4). Coincubation with the cyclophilin-cyclosporin A complex inhibited this dephosphorylation by 50% (±13 SD, n = 6, lane 5). Exposure of endosomes to EDTA completely abolished AQP2 dephosphorylation (lane 6). AQP2 dephosphorylation was also inhibited by incubating the endosomes on ice (lane 7), suggesting that the phosphatase responsible is sensitive to temperature and divalent cation chelation as well as the cyclophilin-cyclosporin A complex. To demonstrate that incubation on ice does not permanently inactivate this phosphatase, another aliquot of 32P-labeled endosomes was incubated on ice and then at 37°C to demonstrate that AQP2 dephosphorylation still occurred (lane 8).


View larger version (61K):
[in this window]
[in a new window]
 
Fig. 6.   Endogenous PP2B mediates [32P]AQP2 dephosphorylation in IMCD endosomes. 32P-labeled AQP2-containing endosomes were incubated at 37°C for either 5 (lane 1, control), 30 (lane 2), 60 (lane 3), or 120 min (lanes 4-6) with either no additions (lanes 1-4) or in the presence of 1 mM cyclophilin-10 µM cyclosporin A complex (CysA; lane 5) or 5 mM EDTA (lane 6). Alternatively, the 32P-labeled endosomes were incubated for 60 min on ice (lane 7) or for 60 min on ice followed by 60 min at 37°C (lane 8). [32P]AQP2 was then detected as in Fig. 5.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The phosphorylation of AQP2 by PKA is a crucial signal in the AVP-elicited water reabsorption pathway. The trafficking of AQP2 vesicles into the IMCD apical membrane is regulated by PKA activity (reviewed in Ref. 27) and AKAPs (19), although the exact subcellular location of the PKA responsible for AQP2 phosphorylation has not been determined. Studies of the postsynaptic densities of hippocampal neurons have revealed the existence of a signaling complex containing RII, PP2B, and PKC that is bound to a 79-kDa AKAP. This multiprotein complex permits the regulation of membrane receptors and ion transporters, such as AMPA/kainate receptors, Ca2+ channels, or N-methyl-D-aspartate receptors, via protein phosphorylation by PKA or PKC or via dephosphorylation by PP2B (11).

The data reported here show that a purified population of IMCD heavy endosomes also contains the various protein components of a similar AKAP complex. In previous work, these IMCD heavy endosomes have been shown to 1) be of apical origin, 2) possess abundant AQP2 protein by immunoblotting analysis, and 3) exhibit a very large mercury-sensitive osmotic water permeability as determined by using stop-flow fluorimetry (14, 22). A combination of Western blotting and immunocytochemistry suggests that AKAP complex constituents, including RII, PP2B and PKCzeta , are present in IMCD tubules.

Studies using a 32P-RII probe demonstrate its binding to a protein band/doublet of 75-to 90-kDa approximate molecular mass in purified IMCD heavy endosomes (Fig. 3). The 75- to 90-kDa putative AKAP protein(s) reported on here awaits further characterization to determine whether it is a novel member of the AKAP family or represents a member of the AKAP-KL (expressed in kidney and lung) family of proteins, as reported by Dong et al. (9). Although AKAP-KL has been localized to the apical membranes of murine proximal tubules, it has not been reported as being expressed in IMCD.

Affinity chromatography using cAMP-agarose demonstrated that RII, PP2B, PKCzeta , and a single 90-kDa RII-binding protein (putative AKAP) were all retained on the cAMP matrix whereas PKCdelta and AQP2 were not absorbed. Data reported here suggest the presence of a multiprotein AKAP-signaling complex, similar to that described in neurons, in rat AQP2 apical endosomes. Because renal AQP2 expression is upregulated in dehydration and after chronic AVP treatment, it would be interesting to know whether these conditions also alter the relative expression of these AKAP complex proteins.

To demonstrate that these proteins are actually associated with apical endosomes rather than being present in contaminating membranes in some portion of the purified IMCD heavy endosomal preparation, we performed three-color, small-particle flow cytometry to colocalize constituents of a putative AKAP complex on the surface of apical endosomes containing entrapped F-dextran. The studies demonstrate that binding of both RII and PKCzeta is colocalized on the cytoplasmic surface of F-dextran-containing vesicles, providing further evidence for the presence of an AKAP complex in apically derived IMCD endosomes. A significant number of these endosomes also contain immunoreactive AQP2 protein.

The data displayed in Figs. 1-4 provide the first evidence for the presence of an AKAP complex on the endocytic arm of AQP2-containing vesicles in IMCD cells. In contrast, studies by Klussmann et al. (20) provide strong support for the importance of AKAPs in the corresponding exocytic arm of AQP2 vesicle insertion into the IMCD apical membrane. Thus the presence of AKAPs in AQP2-containing vesicles may be similar to the situation in neurons, where the AKAP-signaling complex is a dynamic structure with successive phases of protein association and disassociation. It is possible that the AKAP-containing signaling complex described here would possess all the kinases and phosphatases necessary to potentially initiate and terminate the AVP-mediated insertion and removal of AQP2 water channels in the IMCD apical membrane. A similar mechanism might also mediate the function of other membrane transporter proteins. In this regard, ROMK1 channels expressed in Xenopus laevis oocytes are insensitive to forskolin treatment, but when ROMK1 is coexpressed with the 79-kDa AKAP, it exhibits cAMP-dependent activation (1).

We reported previously (22) that AQP2 can be both phosphorylated by an endogenous cAMP-dependent protein kinase and dephosphorylated by an endogenous phosphatase present in IMCD heavy endosomes. Data presented here extend these studies and show that endosomal [32P]AQP2 is dephosphorylated by an endogenous phosphatase that is inhibitable by EDTA or the cyclophilin-cyclosporin A complex. Moreover, AQP2 dephosphorylation also occurred after addition of the exogenous PP2B catalytic subunit (Fig. 5). Together, these data indicate that PKA-phosphorylated AQP2 is an in vitro substrate for PP2B and suggest that PP2B may also be present on the cytoplasmic surfaces of IMCD heavy endosomes in vivo. However, additional work is necessary to demonstrate whether AQP2 dephosphorylation will promote its retrieval from the apical IMCD membrane. Moreover, the incomplete inhibition of endogenous AQP2 dephosphorylation by the cyclophilin-cyclosporin A complex might also suggest the presence of another divalent cation-dependent phosphatase in IMCD heavy endosomes.

The functional role of PKCzeta in the endosomal signaling complex is not understood. In this regard, rat AQP2 contains a PKC phosphorylation consensus sequence at Ser226 (Ser231 in human AQP2). Although there is evidence to suggest that PKC activation may have an inhibitory effect on AVP-induced water reabsorption in collecting ducts (2), it is not known whether this effect is caused by the PKC-mediated phosphorylation of AQP2 as occurs with AQP4 (13). Alternatively, PKC may act indirectly, perhaps promoting retrieval of AQP2 endosomes in a process similar to that described for glucose transporters expressed in oocytes (28). The evidence linking PKC to water reabsorption is presently based on the use of phorbol esters (1,2 diacylglycerol analogs) that are unable to activate the atypical PKCzeta , whereas PKCzeta is instead known to be activated via phosphatidylinositol 3-kinase. Thus, if the PKCzeta present within this AKAP-signaling complex does play a role in water reabsorption, then the role could be to modulate the cAMP-mediated ADH response by a phosphatidylinositol 3-kinase-mediated hormonal or ionic signal.


    ACKNOWLEDGEMENTS

We thank Wendy Campbell and Edmund Benes for assistance in the flow cytometry experiments and Dr. Daniela Riccardi for a critical reading of the manuscript.


    FOOTNOTES

* I. Jo and D. T. Ward contributed equally to this work.

This work was supported in part by National Institutes of Health Grants DK-38874 (H. W. Harris) and GM-48231 (J. D. Scott).

Address for reprint requests and other correspondence: D. T. Ward, G38 Stopford Bldg., Univ. of Manchester, Oxford Rd., Manchester M13 9PT. UK (E-mail: d.ward{at}man.ac.uk).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 13 September 2000; accepted in final form 3 July 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Ali, S, Chen X, Lu M, Xu JZ, Lerea KM, Hebert SC, and Wang WH. The A kinase anchoring protein is required for mediating the effect of protein kinase A on ROMK1 channels. Proc Natl Acad Sci USA 95: 10274-10278, 1998[Abstract/Free Full Text].

2.   Ando, Y, Jacobson HR, and Breyer MD. Phorbol myristate acetate, dioctanoylglycerol, and phosphatidic acid inhibit the hydroosmotic effect of vasopressin on rabbit cortical collecting tubule. J Clin Invest 80: 590-593, 1987[ISI][Medline].

3.   Bradford, MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-254, 1976[ISI][Medline].

4.   Carr, DW, Hausken ZE, Fraser ID, Stofko-Hahn RE, and Scott JD. Association of the type II cAMP-dependent protein kinase with a human thyroid RII-anchoring protein. Cloning and characterization of the RII-binding domain. J Biol Chem 267: 13376-13382, 1992[Abstract/Free Full Text].

5.   Carr, DW, Stofko-Hahn RE, Fraser ID, Cone RD, and Scott JD. Localization of the cAMP-dependent protein kinase to the postsynaptic densities by A-kinase anchoring proteins. Characterization of AKAP 79. J Biol Chem 67: 16816-16823, 1992.

6.   Coghlan, VM, Langeberg LK, Fernandez A, Lamb NJ, and Scott JD. Cloning and characterization of AKAP 95, a nuclear protein that associates with the regulatory subunit of type II cAMP-dependent protein kinase. J Biol Chem 269: 7658-7665, 1994[Abstract/Free Full Text].

7.   Coghlan, VM, Perrino BA, Howard M, Langeberg LK, Hicks JB, Gallatin WM, and Scott JD. Association of protein kinase A and protein phosphatase 2B with a common anchoring protein. Science 267: 108-111, 1995[ISI][Medline].

8.   Colledge, M, and Scott JD. AKAPs: from structure to function. Trends Cell Biol 9: 216-221, 1999[ISI][Medline].

9.   Dong, F, Felsmesser M, Casadevall A, and Rubin CS. Molecular characterization of a cDNA that encodes six isoforms of a novel murine A kinase anchor protein. J Biol Chem 273: 6533-6541, 1998[Abstract/Free Full Text].

10.   Faux, MC, Rollins EN, Edwards AS, Langeberg LK, Newton AC, and Scott JD. Mechanism of A-kinase-anchoring protein 79 (AKAP79) and protein kinase C interaction. Biochem J 343: 443-452, 1999[ISI][Medline].

11.   Fraser, DC, and Scott JD. Modulation of ion channels: a "current" view of AKAPs. Neuron 23: 423-426, 1999[ISI][Medline].

12.   Fushimi, K, Uchida S, Hara Y, Hirata Y, Marumo F, and Sasaki S. Cloning and expression of apical membrane water channel of rat kidney collecting tubule. Nature 361: 549-552, 1993[ISI][Medline].

13.   Han, Z, Wax MB, and Patil RV. Regulation of aquaporin-4 water channels by phorbol ester-dependent protein phosphorylation. J Biol Chem 273: 6001-6004, 1998[Abstract/Free Full Text].

14.   Harris, HW, Zeidel ML, Jo I, and Hammond TG. Characterization of purified endosomes containing the antidiuretic hormone sensitive water channel from rat renal papilla. J Biol Chem 269: 11993-12000, 1994[Abstract/Free Full Text].

15.   Jo, I, Nielsen S, and Harris HW. The 17 kDa band identified by multiple anti-aquaporin 2 antisera in rat kidney medulla is a histone. Biochim Biophys Acta 1324: 91-101, 1997[ISI][Medline].

16.   Katsura, T, Gustafson CE, Ausiello DA, and Brown D. Protein kinase A phosphorylation is involved in regulated exocytosis of aquaporin-2 in transfected LLC-PK1 cells. Am J Physiol Renal Physiol 272: F817-F822, 1997[Abstract].

17.   Kelly, RB. Secretion: a question of endosomes. Nature 364: 487-488, 1993[ISI][Medline].

18.   Klauck, TM, Faux MC, Labudda K, Langeberg LK, Jaken S, and Scott JD. Coordination of three signaling enzymes by AKAP79, a mammalian scaffold protein. Science 271: 1589-1592, 1996[Abstract].

19.   Klussmann, E, Maric K, and Rosenthal W. The mechanisms of aquaporin control in the renal collecting duct. Rev Physiol Biochem Pharmacol 141: 33-95, 2000[ISI][Medline].

20.   Klussmann, E, Maric K, Wiesner B, Beyermann M, and Rosenthal W. Protein kinase A anchoring proteins are required for vasopressin-mediated translocation of aquaporin-2 into cell membranes of renal principal cells. J Biol Chem 274: 4934-4938, 1999[Abstract/Free Full Text].

21.   Kurahara, M, Fushimi K, Terada Y, Bai L, Marumo F, and Sasaki S. cAMP-dependent phosphorylation stimulates water permeability of aquaporin-collecting duct water channel protein expressed in Xenopus oocytes. J Biol Chem 270: 10384-10387, 1995[Abstract/Free Full Text].

22.   Lande, MB, Jo I, Zeidel ML, Somers M, and Harris HW. Phosphorylation of aquaporin-2 does not alter the membrane permeability of rat papillary water channel-containing vesicles. J Biol Chem 271: 5552-5557, 1996[Abstract/Free Full Text].

23.   Marples, D, Schroer TA, Ahrens N, Taylor A, Knepper MA, and Nielsen S. Dynein and dynactin colocalize with AQP2 water channels in intracellular vesicles from kidney collecting duct. Am J Physiol Renal Physiol 274: F384-F394, 1998[Abstract/Free Full Text].

24.   Sands, JM, Flores F, Kato A, Baum MA, Brown ED, Ward DT, Hebert SC, and Harris HW. Vasopressin-elicited water and urea permeabilities are altered in IMCD in hypercalcemic rats. Am J Physiol Renal Physiol 274: F978-F985, 1998[Abstract/Free Full Text].

25.   Sands, JM, Naruse M, Baum M, Jo I, Hebert SC, Brown EM, and Harris HW. An extracellular calcium/polyvalent cation-sensing receptor (CaR) localized to endosomes containing aquaporin 2 water channels modulates vasopressin-elicited water permeability in rat kidney inner medullary collecting duct. J Clin Invest 99: 1399-1405, 1997[Abstract/Free Full Text].

26.   Sasaki, S, Fushimi K, Saito H, Saito F, Uchida S, Ishibashi K, Kuwahara M, Ikeuchi T, Inui K, Nakajima K, Watanabe TX, and Marumo F. Cloning, characterization and chromosomal mapping of human aquaporin of collecting duct. J Clin Invest 93: 1250-1256, 1994[ISI][Medline].

27.   Ward, DT, Hammond TG, and Harris HW. Modulation of vasopressin-elicited water transport by trafficking of aquaporin2-containing vesicles. Annu Rev Physiol 61: 683-697, 1999[ISI][Medline].

28.   Wright, EM, Hirsch JR, Loo DD, and Zampighi GA. Regulation of Na+/glucose cotransporters. J Exp Biol 200: 287-293, 1997[Abstract/Free Full Text].


Am J Physiol Renal Fluid Electrolyte Physiol 281(5):F958-F965
0363-6127/01 $5.00 Copyright © 2001 the American Physiological Society