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Originally published In Press as doi:10.1074/jbc.M205173200 on August 29, 2002

J. Biol. Chem., Vol. 277, Issue 46, 44108-44114, November 15, 2002
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Relevance of Dopamine Signals Anchoring Dynamin-2 to the Plasma Membrane during Na+,K+-ATPase Endocytosis*

Riad EfendievDagger §, Guillermo A. YudowskiDagger , Jean Zwiller, Barbara LeibigerDagger , Adrian I. Katz||, Per-Olof BerggrenDagger , Carlos H. Pedemonte§, Ingo B. LeibigerDagger , and Alejandro M. BertorelloDagger **

From the Dagger  Department of Molecular Medicine, The Rolf Luft Center for Diabetes Research, Karolinska Institutet, Karolinska Hospital, S-171 76 Stockholm, Sweden, the § Department of Pharmacological and Pharmaceutical Sciences, College of Pharmacy, University of Houston, Houston, Texas 77204,  INSERM Unité 338, 67084 Strasbourg, France, and the || Department of Medicine, University of Chicago, Chicago, Illinois 60637

Received for publication, May 27, 2002, and in revised form, August 21, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Clathrin-dependent endocytosis of Na+,K+-ATPase in response to dopamine regulates its catalytic activity in intact cells. Because fission of clathrin-coated pits requires dynamin, we examined the mechanisms by which dopamine receptor signals promote dynamin-2 recruitment and assembly at the site of Na+,K+-ATPase endocytosis. Western blotting revealed that dopamine increased the association of dynamin-2 with the plasma membrane and with phosphatidylinositol 3-kinase. Dopamine inhibited Na+,K+-ATPase activity in OK cells and in those overexpressing wild type dynamin-2 but not in cells expressing a dominant-negative mutant. Dephosphorylation of dynamin is important for its assembly. Dopamine increased protein phosphatase 2A activity and dephosphorylated dynamin-2. In cells expressing a dominant-negative mutant of protein phosphatase 2A, dopamine failed to dephosphorylate dynamin-2 and to reduce Na+,K+-ATPase activity. Dynamin-2 is phosphorylated at Ser848, and expression of the S848A mutant significantly blocked the inhibitory effect of dopamine. These results demonstrate a distinct signaling network originating from the dopamine receptor that regulates the state of dynamin-2 phosphorylation and that promotes its location (by interaction with phosphatidylinositol 3-kinase) at the site of Na+,K+-ATPase endocytosis.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Motion of integral membrane proteins from and to the plasma membrane in response to G protein-coupled receptor (GPCR)1 signals is a complex process that requires selectivity as well as spatial and temporal organization. Although the traffic in both directions appears to be different in nature, it shares several mechanistic features as follows: (a) cargo recognition, (b) mechanisms for membrane budding, (c) formation of clathrin-coated pits, and (d) fission and release of the clathrin vesicles (for review, see Refs. 1 and 2). The complexity of these processes could be anticipated from the fact that only a small number of molecules traffic in response to a given receptor signal and that this signal selects a specific protein to be internalized among many within the plasma membrane.

Clathrin-dependent endocytosis of Na+,K+-ATPase molecules in response to dopamine (DA) in renal epithelial cells has proven to be an interesting model for studying the organization of signaling networks during endocytosis of integral membrane proteins in response to a given GPCR. The Na+,K+-ATPase is exclusively located at the basolateral domain of renal epithelial cells (3, 4), and its function in the renal tubules provides the force for the vectorial transport of sodium and of the water that follows iso-osmotically. DA, an intrarenal natriuretic hormone (5-7), inhibits renal tubule Na+,K+-ATPase activity, thereby contributing to the mechanisms that increase urinary sodium excretion (8). At the cellular level, inhibition of Na+,K+-ATPase activity is mediated by removal of active molecules from the plasma membrane and their transport into early and late endosomes via a clathrin-coated vesicle-dependent mechanism (9-11). PKC-zeta -dependent phosphorylation of Na+,K+-ATPase alpha -subunit constitutes a triggering signal for endocytosis (11, 12) by facilitating the activation of phosphatidylinositol 3-kinase (PI 3-kinase) (13, 14). This effect involves the interaction of PI 3-kinase (p85alpha -SH3 domain) with a proline-rich domain (PRD) located upstream of the phosphorylated residue (Ser18) within the Na+,K+-ATPase alpha -subunit (14). Activation of PI 3-kinase is necessary for binding of adaptor protein-2 (AP-2) to the Na+,K+-ATPase alpha -subunit and recruitment of clathrin (14, 15). Although we begin to better understand the organization of signals initiating the traffic of Na+,K+-ATPase molecules (i.e. selection of the cargo and AP-2/clathrin recruitment), the mechanisms by which DA receptor signals promote the fission of clathrin vesicles remain unclear.

Release of clathrin-coated vesicles during plasma membrane endocytosis as well as the fission of vesicles derived from intracellular organelles requires the coordinated action of many proteins, among them dynamin (16, 17). In addition, dynamin is a necessary factor regulating compensatory endocytosis following neurotransmitter release within the nerve terminal (18). Multiple protein-protein interactions via specific domains are likely to be responsible for the assembly of dynamin at the neck of invaginated membrane pits. For example, the PRD within its carboxyl terminus interacts with amphiphysin and enables dynamin to anchor with adaptor proteins and clathrin (19). Moreover, its PRD binds with high affinity to SH3 domains present in phospholipase Cgamma , growth factor receptor-bound protein (Grb2), and the regulatory subunit of PI 3-kinase (20). In addition, the PRD has been suggested to act as a positive regulator of intramolecule assembly and GTPase activity (21). The role of GTP hydrolysis, relevant for dynamin assembly, is still a matter of debate (22). Because dynamin present in the cytosol is phosphorylated, a role for post-translational modifications (such as Ser/Thr as well as Tyr phosphorylation) in regulating its assembly and location has also been proposed (23-25). In vitro studies demonstrated that dynamin-1 (dyn1) is phosphorylated in a PKC-dependent manner and that dephosphorylation by calcineurin, associated with calcium-induced depolarization in the nerve terminal (26), represents a calcium sensor for vesicle endocytosis (18).

Dynamin exists in three isoforms and several splice variants (27, 28). The dyn1 isoform is localized in neuronal cells, whereas dynamin-2 (dyn2) is ubiquitously distributed, and dynamin-3 is mainly present in the testes and to a lesser extent in lung and neurons (29). Recent studies (30) performed in polarized Madin-Darby canine kidney cells and transiently expressing different dynamin isoforms have indicated that dyn1 is mainly involved in endocytic processes originated at the apical domain of the cell, whereas dyn2 is responsible for endocytosis occurring at the basolateral domain of the cell. These observations suggest a role for the dyn2 isoform during Na+,K+-ATPase endocytosis, not only for being an isoform present in the kidney epithelial cells but also for being responsible for basolateral membrane endocytosis (site where the Na+,K+-ATPase is located in renal proximal tubule cells).

The purpose of this study has been to examine the relevance of dyn2 during receptor-mediated endocytosis of Na+,K+-ATPase molecules in renal epithelial cells, and to identify the possible cellular signals originated from GPCRs responsible for providing spatial and temporal organization during recruitment of dyn2 molecules to the site of Na+,K+-ATPase endocytosis in these cells.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The experimental protocols were carried out in isolated PCT from rat kidney (9) or in a cell line derived from opossum proximal tubules (OK cells) which expresses the DA receptor and utilizes the same signaling pathways as PCT during regulation of Na+,K+-ATPase activity and endocytosis (31). For consistency with previous observations, all the experiments were performed at 23 °C. Although reactions occur faster at 37 °C than at 23 °C and low temperatures may halt intracellular protein recycling and trafficking, no mechanistic differences were observed between the two conditions.

Reagents and Antibodies-- Immunoprecipitation of dyn2 was performed with a polyclonal antibody (Hudy-1) that recognizes both dyn1 and dyn2 isoforms (Upstate Biotechnology, Inc.); monoclonal antibody against PI 3-kinase and the PP2A antibody were from Transduction Laboratories (Lexington, KY); polyclonal antibody against GFP and fluorescent-labeled antibodies (anti-mouse and anti-rabbit) with either Oregon Green, Texas Red, Alexa Fluor 546 and Alexa Fluor 633 were from Molecular Probes (Eugene, OR). DA was purchased from Sigma, and Hanks' balanced salt solution (HBSS) and LipofectAMINE were from Invitrogen. The antibody against dyn2 isoform as well as the dyn2 cDNA were generously provided by Dr. M. A. Minivan (Mayo Clinic, Rochester, MN). Antibodies against phosphoserine or phosphothreonine residues were purchased from Sigma. The PP2A mutants were kindly provided by Dr. B. A. Hemmings (Friedrich Miescher Institut, Basel, Switzerland). All other reagents were of highest grade available.

Plasmids-- Site-directed mutagenesis was performed on pCR3.Dyn2 or pCR3.GFP-dyn2 by employing the QuikChange mutagenesis kit (Stratagene). The mutants were generated by exchanging nucleotides as follows: K44A (AAG versus GCC), S848A (AGC versus GCC), S848D (AGC versus GAC), and S848E (AGC versus GAG). The GFP-tagged dyn2 construct, pCR3.GFP-dyn2, was generated by first introducing an NruI site upstream from the translation start site and subsequent in-frame insertion of GFP0 cDNA. The GFP0 cDNA, which lacks the stop codon, was obtained from pB.CMV.GFP0 (32). To obtain the plasmid pCMV.GFP-Na+,K+-ATPase, we first introduced an NruI site into the 5'-untranslated region of the alpha -subunit of the rat Na+,K+-ATPase in pCMV ouabain (Pharmingen). The GFP0 cDNA was inserted in-frame in pCMV ouabain-NruI following digestion with NruI and ClaI. All constructs were verified by DNA sequence analysis.

Cell Culture and Transfection-- OK cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, penicillin/streptomycin (100 IU/ml and 100 µg/ml respectively), and 2 mM glutamine in a 5% CO2 incubator at 37 °C. Transfection of OK cells was performed by lipofection using LipofectAMINE (Invitrogen). Expression constructs (15 µg) were transferred into 1 × 105 cells according to the manufacturer's instructions. Following transfection (16-24 h), cells were transferred to 24-well plates and cultured in Dulbecco's modified Eagle's medium supplemented as described above at 37 °C for 6-18 h. All experiments were performed 1-2 days after transfection. Transfection efficiency was monitored by staining the cells with a GFP antibody (varies between 50 and 70%). The efficiency was comparable to the one previously reported (33) by using various dynamin DNAs reported in OK cells. Transfection with the active and dominant-negative mutants of PP2A was performed as described above. Selection of stable clones expressing the GFP-tagged rat Na+,K+-ATPase alpha -subunit was performed as described previously (34).

Determination of Na+,K+-ATPase Activity-- Enzyme activity was determined in intact OK cells incubated in the presence or absence of DA. Na+,K+-ATPase activity was expressed as the rate of 86Rb+ transport per mg of protein for 1 min (34).

Immunoprecipitation-- OK cells or isolated PCT cells were incubated in the presence or absence of DA for different times. Thereafter, the medium was replaced by immunoprecipitation buffer (in mM, 100 NaCl, 50 Tris-HCl, 2 EGTA, 1 phenylmethylsulfonyl fluoride, 5 mg/ml protease inhibitors (aprotinin, leupeptin, and antipain), 1% Triton X-100 (pH 7.5)), and the samples were transferred to ice. The cells were disrupted by homogenization with a motor pestle homogenizer (Kimble-Kontes, Vineland, NJ). Equal aliquots (500 µg of protein/1 ml) were incubated overnight at 4 °C with 5 µg of a polyclonal antibody raised against a shared epitope of dyn2 and dyn2, Hudy-1 (Upstate Biotechnology, Inc., Lake Placid, NY), and the simultaneous addition of excess protein A-Sepharose beads (Amersham Biosciences). Samples were analyzed by SDS-PAGE using the Laemmli buffer system (35). Proteins were transferred to polyvinylidene difluoride membranes (Immobilon-P, Millipore, Bedford, MA). Western blots were developed with an ECL Plus detection kit (Amersham Biosciences). Protein content was determined according to Bradford (36).

Confocal Microscopy of Fixed Cells-- OK cells were fixed in 4% formaldehyde/PBS for 10 min at room temperature. After rinsing with PBS, the cells were transferred to acetone (-20 °C) for 5 min and then quenched with bovine serum albumin (1% in PBS) for 30 min (31). Staining with Hudy-1 or PI 3-kinase antibodies was performed at room temperature for 1 h. Thereafter, coverslips were mounted (SlowFade Light, Molecular Probes, Eugene, OR) and examined using a confocal laser scanning microscope (Leica TCS SP2, Leica Lasertechnik GmbH, Heidelberg, Germany). The confocal microscope was equipped with an Ar/Kr laser and a double dichroic mirror and a 63× lens (Leica HCX PL APO 63×/1.20-0.17, UV) lens.

Determination of Protein Phosphatase Activity-- After incubation with or without DA for different periods, the samples were transferred to ice, centrifuged, and resuspended in homogenization buffer. Protein phosphatase activity was determined in total cell extracts or in extracts subjected to a chromatographic separation after a brief centrifugation (10,000 × g, 15 min). Proteins remaining in the supernatant were applied onto a MonoQ ion-exchange chromatography column and separated by fast protein liquid chromatography (Amersham Biosciences). The column was eluted by a NaCl gradient (0-0.4 M in a 20 mM triethanolamine HCl buffer, pH 7.0, containing 0.1 mM EGTA and 10% glycerol) at a flow rate of 0.8 ml/min in 0.8-ml fractions. Protein phosphatase activity was assayed by measuring the release of 32Pi from [32P]phosphohistone, as described previously (37).

Calcineurin activity was assayed using the Quantizyme assay system (Biomol) by measuring the dephosphorylation of the phospho-RII regulatory subunit of cAMP-dependent protein kinase. Measurements were carried out for 30 min at 30 °C in a medium (50 µl final) containing 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 6 mM MgCl2, 0.5 mM CaCl2, 0.5 mM dithiothreitol, 0.025% Nonidet P-40, 0.25 µM calmodulin, and 0.15 mM phosphopeptide RII substrate. Detection of inorganic phosphate released from RII phosphopeptide was performed using a chromogenic assay based on the Malachite Green method (38).

Miscellaneous-- Isolation of basolateral membrane was performed in confluent OK cells and in freshly isolated PCT cells as described previously (10).

Statistics-- Comparison between two experimental groups was made with the nonpaired Student's t test. p < 0.05 was considered significant.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Clathrin-dependent Endocytosis of Na+,K+-ATPase Molecules Requires Dynamin-2-- We have used an antibody (Hudy-1) that recognizes a shared epitope between dyn1 and dyn2 to examine dyn2 association with basolateral membranes (BLM) prepared from rat proximal tubule (PCT) cells and from OK cells. Dyn2 immunoreactivity was observed in BLM from PCT and OK cells (Fig. 1A); its abundance in BLM was higher in DA-treated cells (% of control, PCT cells, 167 ± 14, n = 4; OK cells, 206 ± 26, n = 3). In contrast, the abundance of GLUT-2, a glucose transporter that does not change its distribution in response to DA (9) and is located exclusively at the BLM of kidney proximal tubules, did not change significantly after DA treatment (% of control, PCT cells, 117 ± 14, n = 4; OK cells, 119 ± 13, n = 3).


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  		Fig. 1.   Dynamin associates with plasma membranes in response to DA. A, PCT and OK cells were incubated with 1 µM DA or vehicle (V, Hanks' medium) for 2.5 min at 23 °C, homogenized, and BLM prepared as described under "Materials and Methods." Samples were analyzed by SDS-PAGE and Western blot using a dyn2 or a GLUT-2 antibody. The data are representative of three experiments performed independently. B, upper panel, comparable expression of GFP-tagged dyn2 using a GFP antibody: lane 1, mock-transfected; lane 2, dyn2-wt; lane 3, dyn2-K44A. Lower panel, DA-dependent regulation of Na+,K+-ATPase activity and endocytosis requires dynamin. OK cells were transiently transfected with dyn2 wild type (Dyn-wt), with a dominant negative dyn2 mutant (dyn-DN), or treated with LipofectAMINE (mock) under identical conditions and incubated with 1 µM DA or vehicle (V, Hanks' medium) for 2.5 min at 23 °C. Na+,K+-ATPase activity was determined as the rate of ouabain-sensitive rubidium transport. Bars correspond to the mean ± S.E. of four experiments performed in triplicate. *, p < 0.05; ns, not significant.

The importance of dyn2 for the process of Na+,K+-ATPase endocytosis was further examined in OK cells transiently expressing a dyn2 wild type or a dominant-negative mutant (K44A) of dyn2. Because decreased Na+,K+-ATPase activity is a reflection of endocytosis of active molecules (11), we examined the ability of DA to regulate Na+,K+-ATPase activity in cells expressing the different forms of dyn2. The dyn2 wild type and K44A mutants were comparably expressed (Fig. 1B, upper panel). Although DA decreased Na+,K+-ATPase activity in mock-transfected OK cells (only exposed to LipofectAMINE) and in OK cells overexpressing the wild type dyn2, it failed to induce a significant decrease in Na+,K+-ATPase activity in OK cells expressing the dominant-negative mutant (Fig. 1B). Noteworthy, the failure of DA to decrease Na+,K+-ATPase activity and to promote endocytosis of active units cannot be attributed to deficient DA receptor signaling, as the Na+,K+-ATPase alpha -subunit was phosphorylated by DA to the same extent in all groups (not shown). Basal Na+,K+-ATPase activity was not affected by overexpressing the wild type dyn2 nor by expressing any of the mutants. The long half-life of Na+,K+-ATPase (considered to be between 36 and 48 h) and the low turnover rate (activity and endocytosis) of this enzyme in cell lines (housekeeping function) may have contributed to the lack of any effect, and only when the endocytic process was accelerated by treatment with dopamine the presence of the mutants becomes rate-limiting.

Dopamine Dephosphorylates Dynamin-2 during Na+,K+-ATPase Endocytosis by Activating PP2A-- The state of dynamin phosphorylation/dephosphorylation is considered to be of importance for its recruitment to membrane (27, 39). Therefore, we initially established whether DA increases protein phosphatase activity, and whether this effect was associated with inhibition of Na+,K+-ATPase activity and endocytosis of molecules. In PCT cells DA treatment was associated with an increase in total protein phosphatase activity (Fig. 2A). Incubation with DA induced a rapid (within 30 s) increase in protein phosphatase activity (maximal ~20%), and at 2.5 min the phosphatase activity returned to control levels. PP2B activity did not increase in response to DA during this period (not shown). To examine whether PP1 or PP2A was activated by DA and to obtain an estimate of the magnitude of its activation, samples from vehicle- and DA-treated cells were applied to a MonoQ ion-exchange chromatography column and eluted by a NaCl gradient (Fig. 2B). The highest DA-induced phosphatase activity (% of control, 50 ± 4, n = 3) was observed with the first peak of activity, which was eluted at ~0.21 M NaCl. Interestingly, this is the fraction where purified PP2A elutes under similar chromatographic conditions (40). Moreover, we found that okadaic acid at a concentration as low as 1 nM produced 50% inhibition of protein phosphatase activity in this peak (Fig. 2C), supporting the conclusion that PP2A (and not PP1) was the most likely phosphatase present in the DA-stimulated peak. In Western blot analysis we found that PP2A was indeed present in lysates of renal tubule cells and OK cells (Fig. 2D).


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  		Fig. 2.   DA increases PP2A activity in renal epithelial cells. A, kinetics of histone phosphatase activity was examined in PCT cells incubated with 1 µM DA for 0.5, 1, 2.5, and 5 min at 23 °C as described under "Materials and Methods." Protein phosphatase (PPase) activity is expressed as percent of control, and each point represents the mean ± S.E. of five independent experiments. *, p < 0.01. B, chromatographic separation of homogenates of vehicle-treated (squares) or DA-treated (1 µM for 30 s at 23 °C) PCT cells (circles). The protein phosphatase activity was determined in fractions eluted from a MonoQ/fast protein liquid chromatographic column, as described under "Materials and Methods," and is expressed as cpm/30 µl. Right ordinates indicate A280 (OD280) (continuous line) and NaCl gradient applied (dashed line). The experiment was repeated on three separate occasions. C, protein phosphatase activity was determined in the first peak of activity eluted at ~0.21 M NaCl corresponding to dopamine-stimulated PCT cells in the presence of various concentrations of okadaic acid. Results are expressed as % of control. D, the presence of PP2A immunoreactivity was examined in lysates from A431 (positive control, 20 µg) and cytosol from PCT (25 µg) and OK (25 µg) cells by Western blot using an anti-PP2A antibody. E, Na+,K+-ATPase activity (rate of ouabain-sensitive rubidium transport) in non-transfected, PP2A wild type- (PP2A-Wt) or PP2A dominant negative mutant (PP2A-DN)-transfected OK cells, incubated with 1 µM DA for 2.5 min at 23 °C (DA) or vehicle (V). Bars represent the mean ± S.E. of three independent experiments performed in triplicate. *, p < 0.05; n.s., not significant. F, state of dyn2 phosphorylation was determined in OK cells transfected and treated as described in E. Dyn was immunoprecipitated using Hudy-I antibody, and Western blots were performed on the immunoprecipitate using an antibody against phosphoserine residues. Experiments were repeated three times.

We next examined whether activation of PP2A was functionally associated with down-regulation of Na+,K+-ATPase activity. The experiments were carried out in OK cells transiently expressing either the wild type- or a dominant-negative mutant Leu199 right-arrow Pro (L199P) form of PP2A (41). DA significantly inhibited Na+,K+-ATPase activity in non-transfected cells, LipofectAMINE-treated cells, and in cells transfected with the wild type PP2A, whereas it failed to induce a significant inhibition of enzyme activity in OK cells transfected with the dominant-negative mutant of PP2A (Fig. 2E). Moreover, by using the same protocol, we observed that DA dephosphorylates dyn2 in non-transfected cells, and in cells transfected with the wild type PP2A, it failed to do so in OK cells transfected with the dominant-negative mutant of PP2A (Fig. 2F). Western blot analysis performed with an antibody raised against phosphorylated serine residues of immunoprecipitated dyn2 revealed that DA reduces the state of phosphorylation (Fig. 3A), whereas a similar procedure using an anti-phosphorylated threonine antibody did not demonstrate any immunoreactivity (not shown). The effect of DA was present in both OK cells and in cells isolated from rat renal proximal tubules, and it occurred as early as within 1 min after incubation with DA (Fig. 3B).


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  		Fig. 3.   DA-dependent inhibition of Na+,K+-ATPase activity requires dephosphorylation of dyn2. A, the state of dyn2 phosphorylation was examined using a phosphoserine antibody (P-Ser) in material immunoprecipitated with a dynamin antibody from OK and PCT cells previously treated with 1 µM DA or vehicle (V, Hanks' medium) for 1 min at 23 °C. Control Western blots (WB) were performed with anti-dynamin antibody (lower panel). B, the state of dyn2 phosphorylation was examined in OK (closed circles) and PCT (open circles) cells incubated with 1 µM DA for 1, 2.5, and 5 min at 23 °C. Each point represents the mean ± S.E. of five experiments. *, p < 0.05. C, the presence of dyn2 was examined in material immunoprecipitated with a phosphoserine antibody from OK cells by Western blot using a specific antibody against dyn2. D, upper panel, comparable expression of GFP-tagged dyn2 using a GFP antibody: lane 1, mock-transfected; lane 2, dyn2-wt; lane 3, dyn2-S848A. Lower panel, dyn2 phosphorylation in response to 1 µM DA at 23 °C for 2.5 min was examined in OK cells expressing the S848A mutant (dyn2 S848A) and wild type dyn2 (dyn2-wt) using the same strategy as described in A. E, Na+,K+-ATPase activity was determined in mock-transfected OK cells (LipofectAMINE-treated) and in cells expressing wild type dyn2 (dyn2-wt) or the S848A mutant. Each bar represents the mean ± S.E. of three independent experiments performed in triplicate. *, p < 0.05; n.s., non significant. F, upper panel, comparable expression of GFP-tagged dyn2 using a GFP antibody: lane 1, non-transfected; lane 2, dyn2-wt; lane 3, dyn2-S848D; lane 4, dyn2-S848E. Lower panel, OK cells expressing the constitutively phosphorylated form (S848E and S848D) of dyn2 were incubated with 1 µM DA for 5 min at 23 °C or vehicle (Hanks' medium). Na+,K+-ATPase activity (rate of rubidium transport) is expressed as percentage of inhibition. Each bar represents the mean ± S.E. of three experiments performed independently and in triplicate determinations. * p < 0.05. NT, non-transfected; WT, wild type dyn2.

Further support for constitutive serine phosphorylation of the dyn2 molecules was obtained under non-stimulated conditions in immunoprecipitates of OK cells obtained with an antibody against phosphorylated serine residues (Fig. 3C), where a protein with a molecular mass of ~100 kDa was recognized by a specific dyn2 antibody.

Although dyn1 is a good substrate for protein kinases (24), we also identified a highly consensus site (Ser848) for protein kinase C (PKC) phosphorylation in the dyn2 molecule. To evaluate whether there was a causal relationship between dyn2 dephosphorylation and reduction in Na+,K+-ATPase activity elicited by DA, several mutations were introduced in the dyn2 molecule in which the Ser848 was replaced by either alanine (S848A) or by negatively charged residues (S848E and S848D). Expression of these dynamin cDNAs was comparable in each experiment (Fig. 3D, upper panel, and Fig. 3F, upper panel). Substitution of Ser848 by Ala significantly reduced dyn2 phosphorylation and consequently the response to DA (Fig. 3D) in cells transiently transfected with this mutant. Na+,K+-ATPase activity in response to DA was also studied in OK cells transiently expressing these mutants. Expression of the wild type dyn2 did not affect the action of DA, whereas transient expression of the dyn2 S848A mutant prevented the inhibitory effect of DA on Na+,K+-ATPase activity (Fig. 3E). Expression of dyn2 S848E and S848D mutants significantly reduced the inhibitory effect of DA (Fig. 3F).

Phosphatidylinositol 3-Kinase Recruits Dynamin-2 to the Site of Endocytosis-- The mechanism(s) that directs dyn2 to the site of Na+,K+-ATPase endocytosis is not known. Phosphorylated dyn2 is known to interact with many proteins as part of an "endocytic network," among them the PI 3-kinase p85alpha subunit (39), and such interaction has been shown to occur in vitro (42), but no reports have demonstrated to date its existence in intact cells. Because DA favors the interaction of PI 3-kinase with the Na+,K+-ATPase alpha -subunit (14), we next examined whether an active PI 3-kinase (that interacts with Na+,K+-ATPase molecules) might represent a possible anchor signal by interacting in vivo with dyn2. The association of Na+,K+-ATPase with PI 3-kinase and dyn2 was initially studied in OK cells stably expressing Na+,K+-ATPase alpha -subunit bearing a GFP tag at the amino terminus (Fig. 4). Stable expression of this construct did not inactivate the Na+,K+-ATPase, as its catalytic activity was the same in OK cells expressing wild type Na+,K+-ATPase alpha -subunit or the GFP-tagged form (GFP-NK-alpha ). The fusion of GFP to the Na+,K+-ATPase molecule did not affect its regulation either (15). Triple fluorophore analysis of fixed OK cells using GFP-NK-alpha , Alexa Fluor 546 for PI 3-kinase, and Alexa Fluor 633 for dyn2 under microscopy was performed (Fig. 4). Although the majority of GFP-NK-alpha was localized to the plasma membrane, PI 3-kinase and dyn2 were mainly localized in the cytosol. There was no degree of colocalization between the three structures in clusters of vehicle-treated cells (Fig. 4A). In contrast, in cells that have been exposed to DA, colocalization between the three fluorophores was observed in selected areas of the plasma membrane, evidenced as white staining and indicated by the arrow in both the merge image and the enlarged inset (Fig. 4B).


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  		Fig. 4.   Simultaneous detection of Na+,K+-ATPase, PI 3-kinase, and dyn2 molecules in response to DA. After treatment with DA (B) (1 µM; 2 min at room temperature) or its vehicle (A), OK cells expressing stably the Na+,K+-ATPase alpha -subunit carrying GFP were fixed and incubated with antibodies against PI 3-kinase (Santa Cruz Biotechnology, polyclonal) and against dyn2 (Upstate Biotechnology, Inc., monoclonal). Secondary labeled antibodies (dilution 1:100) against PI 3-kinase (Alexa Fluor 546) and dyn2 (Alexa Fluor 633) were used. Cells were analyzed by laser scanning confocal microscopy at ×63. Images are representative of multiple cell analyses of two experiments performed independently.

We further evaluated whether the association of PI 3-kinase and dyn2 was affected by the level of dyn2 phosphorylation. For this purpose we tagged the dyn2 molecules with GFP. OK cells transiently expressing the GFP-tagged dyn2 responded to DA similarly to cells transiently expressing the non-tagged form (not shown). OK cells were transiently transfected with the wild type GFP-dyn2 or GFP-dyn2 bearing the different mutations, in the site bearing GTPase activity (K44A) or in the phosphorylation site (S848A, S848E, and S848D). The PI 3-kinase was immunoprecipitated with a p85alpha subunit antibody, and the association with GFP-dyn2 was examined using a GFP antibody and thereby excluding the possible association of PI 3-kinase with endogenous dyn2 (lacking GFP). As expected, the non-transfected cells do not show any GFP immunoreactivity (Fig. 5, upper panel). All constructs tested interacted under non-stimulated conditions with the p85alpha subunit. In cells transfected with the wild type dynamin, there is a significant increase in GFP immunoreactivity in the DA-treated cell. The increased colocalization of GFP-dyn2 with the PI 3-kinase was absent in cells expressing different mutations in the phosphorylation site. Interestingly, the K44A mutant that lacks GTPase activity also failed to increase in the PI 3-kinase immunoprecipitated in response to DA. Quantitation of several experiments indicated that the presence of a mutation within the Ser-848 phosphorylation site significantly reduces the ability of dyn2 to associate with the PI 3-kinase (Fig. 5, lower panel).


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  		Fig. 5.   Colocalization of dyn2 and PI 3-kinase. GFP-Dyn2 was identified (using an antibody against GFP) by Western blot in the material immunoprecipitated with a p85alpha antibody. Experiments were performed in nontransfected OK cells (NT) and in OK cells transiently transfected with the wild type GFP- dyn2 (WT) and the mutant forms (K44A, S848A, S848D, and S484E). Cells were incubated with 1 µM DA (DA) or vehicle (V) for 2.5 min at 23 °C. Upper panel, representative Western blot. Lower panel, the ratio between GFP-dyn2 and immunoprecipitated p85alpha was established in vehicle- and DA-treated cells, and the percentual changes between these two groups are expressed as relative abundance. Results are from 3 to 4 independent experiments. p < 0.05 (all mutants versus WT).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

This study demonstrates that in renal epithelial cells clathrin-dependent endocytosis of Na+,K+-ATPase molecules in response to DA receptor signals requires the action of dyn2. It also demonstrates that activation of PP2A in response to DA promotes the dephosphorylation of dyn2 (Ser848) and its recruitment to the basolateral membrane where it is required for endocytosis of Na+,K+-ATPase molecules. Recruitment of dyn2 to the site of Na+,K+-ATPase endocytosis appears to be mediated, at least in part, by its interaction with class IA PI 3-kinase.

The role of dyn2 during endocytosis triggered by activation of membrane receptors as well as the signaling network responsible for dyn2 assembly at the site of cargo endocytosis have not been clearly defined. In particular, it is not clear whether that process occurs by default or if it requires a defined receptor signal. In renal epithelial cells the Na+,K+-ATPase activity varies in response to numerous hormones (43-45). These events do not represent direct modifications of Na+,K+-ATPase intrinsic catalytic activity but are the result of changes in the number of enzyme units present within the plasma membrane (11, 46-48). One such hormone, DA, reduces Na+,K+-ATPase activity by increasing the rate of endocytosis (via clathrin vesicles) of active molecules from the plasma membrane. We therefore utilized this information as a basis to study the role and regulation of dyn2 during endocytosis in renal epithelial cells in response to a prototype G protein-coupled receptor (dopamine) signal. We found that dyn2 was essential for the regulation of Na+,K+-ATPase activity and endocytosis in response to DA. This was evident in experiments performed in OK cells that have been transiently transfected with a dominant negative mutant of dyn2 that lacks GTPase activity. Whereas transient expression of dyn2 mutants could result in different receptor signaling responses, however, an impaired DA receptor signal in this study could be ruled out by the fact that Na+,K+-ATPase alpha -subunit phosphorylation in response to DA remained unaffected by the presence of the mutant.

The majority of dynamin molecules present in the cell cytosol are in a phosphorylated form (24, 27), and dephosphorylation represents a regulatory factor that favors dyn2 assembly at the neck of the clathrin-coated pit (27). Most studies have indicated that dyn1 is a substrate for PKC. Phosphorylation of dyn1 occurs at Ser795, and this effect also blocks its association to phospholipids (39). Using specific dyn2 and phosphoserine antibodies, our results suggest that dyn2 is also a good phosphosubstrate, most likely for PKC. Transient expression of dyn2 carrying a mutation of the putative phosphorylation site (S848A) produced a blunted response to DA on Na+,K+-ATPase activity. Contrary to what was anticipated, the S848A dyn2 behaved as a negative acting mutant suggesting that transient dephosphorylation is more critical for dyn2 action than a persistent dephosphorylation-like state. It is conceivable that a permanent "dephosphorylated" state may have resulted in abnormal cellular orientation/localization of dyn2. Alternatively, because dyn2 is present in the cytosol in a phosphorylated state, a permanent dephosphorylated dyn2 (S848A mutation) could have led to enhanced self-association and thereby reduced ability of dyn2 molecules to interact with the plasma membrane upon stimulation by GPCR. dyn2 mutants in which the serine residue was replaced by negatively charged residues were also examined. These mutations turned dyn2 into constitutively "phosphorylated" forms (unable to become a target for protein phosphatases), and as expected the inhibitory effect of DA on Na+,K+-ATPase activity was significantly attenuated, further suggesting that transient dynamin dephosphorylation was necessary for its action during Na+,K+-ATPase endocytosis.

Experiments demonstrating the interaction of p85alpha subunit of PI 3-kinase with dynamin have been performed in vitro (39, 42). Our data further indicate that such interaction also occurs in intact cells and is enhanced in response to GPCR signals. Both dephospho- and phospho-dynamin are capable of interacting with the PI 3-kinase p85alpha -SH3 in vitro (39). Because in our studies using OK cells transiently expressing the dyn2 mutants (S848A, S848D, and S848E) behave as negative regulator of Na+,K+-ATPase endocytosis/activity and they do bind to PI 3-kinase p85alpha as efficiently as the dyn2 wild type, we can speculate that dynamin dephosphorylation in intact cells is necessary for its self-assembly at the neck of the coated pit, and possibly for its interaction with other intracellular partners during Na+,K+-ATPase endocytosis. Similarly, the dyn2-K44A mutant also failed to increase the PI 3-kinase immunoprecipitate in response to DA, suggesting that indeed the ability of dynamin to hydrolyze GTP is necessary for self-assembly and probably not for interacting with PI 3-kinase at the membrane interface.

Dephosphorylation of dyn2 is likely to be needed for assembly at the neck of clathrin-coated pits containing Na+,K+-ATPase, whereas its interaction with the p85alpha regulatory subunit of PI 3-kinase may represent the anchor signal to the appropriate location in the cell where Na+,K+-ATPase molecules need to be internalized. Because DA fails to promote the binding of dyn2 to PI 3-kinase in cells expressing Na+,K+-ATPase alpha -subunit mutants (lacking the binding site for PI 3-kinase, not shown), it is likely that such an interaction promoted by DA requires an activated PI 3-kinase. DA-induced activation of PI 3-kinase requires its interaction with a PRD present in the Na+,K+-ATPase alpha -subunit. It is therefore tempting to speculate that an activated PI 3-kinase/Na+,K+-ATPase (p85alpha /alpha -subunit) complex may help recruiting dyn2 to the site of Na+,K+-ATPase endocytosis. Further support for such a protein complex was also obtained in fixed OK cells expressing the GFP-NK-alpha where these three molecules colocalize at the plasma membrane of DA-treated cells.

DA-dependent dephosphorylation of dyn2 is associated with increased protein phosphatase activity. Previous reports (49, 50) indicating that dopamine regulates protein phosphatase activity in renal proximal tubules were conflicting. Our experiments indicate that the effect of DA is selective for PP2A and, more importantly, that it occurred as early as 30 s after incubation with the agonist and preceding the maximal dephosphorylation of dyn2 occurring at 1 min (Fig. 2B). A direct association between changes in PP2A activity and regulation of Na+,K+-ATPase activity/endocytosis was established using a PP2A dominant negative mutant. In OK cells transiently expressing a PP2A mutant that has impaired catalytic activity (41), DA failed to decrease Na+,K+-ATPase activity.

In summary, our data suggest that clathrin-dependent endocytosis of Na+,K+-ATPase molecules in response to DA receptor signals is a complex process that results from the activation of distinct intracellular signaling pathways. This includes stimulation of PKC-zeta (12) and protein phosphatase 2A activity. Activation of PKC-zeta (leading to phosphorylation of Ser-18 within the alpha -subunit amino terminus) is necessary for cargo (Na+,K+-ATPase) selection and activation of PI 3-kinase leading to AP-2/clathrin recruitment, whereas activation of PP2A appears to be necessary for dyn2 dephosphorylation and self-assembly but not for its binding to activated PI 3-kinase (Fig. 6). It is likely that a dynamic transition between phospho- and dephospho-states regulates dyn2 self-assembly and the interaction with other partners of the endocytic network, and thereby its availability at the plasma membrane interface during endocytosis.


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  		Fig. 6.   Schematic representation of the interaction of dyn2 with the Na+,K+-ATPase during endocytosis. PI 3-kinase provides the bridge between dyn2 and the Na+,K+-ATPase during its endocytosis. Clathrin-dependent endocytosis of Na+,K+-ATPase molecules in response to DA requires simultaneous activation of protein kinases (Dopamine signals, 1) and protein phosphatases (Dopamine signals, 1'). Phosphorylation of the Na+,K+-ATPase alpha -subunit represents a cargo selection signal (activation of PI 3-kinase, AP-2 binding, and clathrin recruitment). An activated PI 3-kinase bound to the Na+,K+-ATPase recruits dyn2 to site of endocytosis. Dephosphorylation of dyn2 (occurring by the action of PP2A) does not affect its binding to PI 3-kinase, but it is necessary for its self- association and possibly for its interaction with other unknown partners of the endocytic network in response to DA.


    ACKNOWLEDGEMENTS

The technical work of Marie Odile Revel is greatly appreciated. We thank B. A. Hemmings and M. A. MacNiven for the generous gift of reagents and T. Moede for useful discussions.

    FOOTNOTES

* This work was supported in part by Swedish Research Council Grant 10860, the Swedish Heart and Lung Foundation, Novo Nordisk Fond, National Institutes of Health Grant DK 53460, and American Heart Association, Texas Affiliate, Grant 0050801.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.

** To whom correspondence should be addressed: Dept. of Molecular Medicine, L3, Karolinska Hospital, 171 76 Stockholm, Sweden. Tel.: 46-8-5177-5727; Fax: 46-8-5177-9450; E-mail: alejandro.bertorello@molmed.ki.se.

Published, JBC Papers in Press, August 29, 2002, DOI 10.1074/jbc.M205173200

    ABBREVIATIONS

The abbreviations used are: GPCR, G protein-coupled receptors; dyn1, dynamin-1; dyn2, dynamin-2; DA, dopamine; PP2A, protein phosphatase 2A; PI 3-kinase, phosphatidylinositol 3-kinase; AP-2, adaptor protein 2; PCT, proximal convoluted tubule cells; GFP, green fluorescent protein; PBS, phosphate-buffered saline; BLM, basolateral membranes; wt, wild type; PKC, protein kinase C.

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