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J. Biol. Chem., Vol. 280, Issue 16, 16272-16277, April 22, 2005

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The 14-3-3 Protein Translates the NA⁺,K⁺-ATPase ₁-Subunit Phosphorylation Signal into Binding and Activation of Phosphoinositide 3-Kinase during Endocytosis^*

Riad Efendiev, Zongpei Chen, Rafael T. Krmar, Sabine Uhles¶, Adrian I. Katz||, Carlos H. Pedemonte, and Alejandro M. Bertorello**

From the Department of Medicine, Atherosclerosis Research Unit, Membrane Signaling Networks, and ^¶Department of Molecular Medicine, Karolinska Institutet, Karolinska University Hospital-Solna, S-171 76 Stockholm, Sweden, Department of Pharmacological and Pharmaceutical Sciences, College of Pharmacy, University of Houston, Houston, Texas 77204, and ^||Department of Medicine, University of Chicago, Chicago, Illinois 60637

Received for publication, January 14, 2005 , and in revised form, February 11, 2005.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Clathrin-dependent endocytosis of Na⁺,K⁺-ATPase molecules in response to G protein-coupled receptor signals is triggered by phosphorylation of the -subunit and the binding of phosphoinositide 3-kinase. In this study, we describe a molecular mechanism linking phosphorylation of Na⁺,K⁺-ATPase -subunit to binding and activation of phosphoinositide 3-kinase. Co-immunoprecipitation studies, as well as experiments using confocal microscopy, revealed that dopamine favored the association of 14-3-3 protein with the basolateral plasma membrane and its co-localization with the Na⁺,K⁺-ATPase -subunit. The functional relevance of this interaction was established in opossum kidney cells expressing a 14-3-3 dominant negative mutant, where dopamine failed to decrease Na⁺,K⁺-ATPase activity and to promote its endocytosis. The phosphorylated Ser-18 residue within the -subunit N terminus is critical for 14-3-3 binding. Activation of phosphoinositide 3-kinase by dopamine during Na⁺,K⁺-ATPase endocytosis requires the binding of the kinase to a proline-rich domain within the -subunit, and this effect was blocked by the presence of a 14-3-3 dominant negative mutant. Thus, the 14-3-3 protein represents a critical linking mechanism for recruiting phosphoinositide 3-kinase to the site of Na⁺,K⁺-ATPase endocytosis.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Regulation of Na⁺,K⁺-ATPase activity by hormones within the renal tubule epithelial cells provides control for transepithelial sodium transport and thereby urinary sodium excretion during salt loading or deprivation (1). During its regulation, the Na⁺,K⁺-ATPase molecule shuttles between the plasma membrane and intracellular compartments in response to hormones (2). Thus, a balance between the increase or decrease in the number of copies within the plasma membrane determines the cell Na⁺,K⁺-ATPase activity. The responses to different agonists regulating the intracellular distribution of Na⁺,K⁺-ATPase are tissue-specific (3), and in renal tubule cells, the direction of the physiological response (increase versus decrease) in response to hormones that influence urinary sodium excretion appears to depend largely on the concentration of intracellular sodium (4, 5).

A complex intracellular signaling network is responsible for the removal or insertion of new molecules within the plasma membrane. In the rodent renal epithelia, phosphorylation of Na⁺,K⁺-ATPase ₁-subunits (at Ser-18) that are present at the plasma membrane is necessary for dopamine (DA)¹-induced endocytosis (6–8) and (at Ser-11) for parathyroid hormone-induced endocytosis (9). In contrast, recruitment of new molecules to the plasma membrane in response to angiotensin II (10) or serotonin (11) requires the phosphorylation of Na⁺,K⁺-ATPase molecules (Ser-11/Ser-18) present within endosomes. DA-mediated phosphorylation of the ₁-subunit and Na⁺,K⁺-ATPase endocytosis requires activation of the PKC- isoform, whereas serotonin- and angiotensin II-dependent increase in Na⁺,K⁺-ATPase activity requires activation of the PKC- isoform (11, 12). Nonetheless, phosphorylation in either circumstance does not change the catalytic properties of the enzyme (7, 13), but constitutes a triggering factor essential for initiating their recruitment into clathrin vesicles and traffic to their final destination.

Endocytosis of Na⁺,K⁺-ATPase molecules in response to DA in renal epithelial cells is initiated by adaptor protein-2 binding to the Na⁺,K⁺-ATPase ₁-subunit Tyr-537 residue (14) and clathrin recruitment (15). Endocytosis and binding of adaptor protein-2 to the ₁-subunit requires the activation of phosphoinositide 3-kinase (PI 3-kinase) (16, 17). This process is triggered by phosphorylation of the Na⁺,K⁺-ATPase ₁-subunit and binding of the PI 3-kinase (class I_A) p85 regulatory subunit (SH3 domain) to a proline-rich domain (PRD) motif within the ₁-subunit N terminus upstream of the PKC phosphorylation site (17). The mechanism that translates phosphorylation of the ₁-subunit Ser-18 residue into binding of PI 3-kinase to the PRD is not yet understood.

Structural studies using analogous comparisons between the Na⁺,K⁺-ATPase ₁-subunit and the tertiary structure of the skeletal muscle sarcoplasmic reticulum Ca²⁺-ATPase ₁-subunit (they share several common structural/functional features) revealed a structurally exposed PRD motif on the molecule N-domain (18, 19). This observation may suggest that phosphorylation of the ₁-subunit at Ser-18 might not be required for inducing a structural conformation of the Na⁺,K⁺-ATPase ₁-subunit N terminus to make the PRD accessible for its binding to the PI 3-kinase but rather to facilitate its interaction with a linker that would make, instead, the PI 3-kinase available to the PRD motif.

The 14-3-3 proteins represent an important model of scaffolding proteins turning serine-phosphorylated residues within target proteins into recruitment modules linking the components of diverse cellular signaling networks during a functional response (20–22). Because 14-3-3 proteins are targeted primarily to phosphorylated serine residues and because of their ability to interact with the PI 3-kinase (23–25), we hypothesized that 14-3-3 proteins may provide the linking mechanisms guiding the PI 3-kinase to the PRD within the ₁-subunit N terminus after it has been phosphorylated at the Ser-18 residue, thus initiating endocytosis of the subunits.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Reagents—The 14-3-3 -isoform wild type and negative mutant (208–255) were kindly provided by Dr. T.-A. Sato (Columbia University, New York, NY). The Na⁺,K⁺-ATPase antibody used for immunoprecipitation was a gift of Dr. Mercer (Washington University, Saint Louis, MO), and the antibody used for Western blots was a gift of Dr. M. Caplan (Yale University, New Haven, CT). A monoclonal antibody against PI 3-kinase was purchased from Transduction Laboratories, and a polyclonal antibody (Z-8) was from Santa Cruz Biotechnology. The antibody against 14-3-3 (K-19) was purchased from Santa Cruz Biotechnology. Fluorescent-labeled antibodies (Alexa dyes) were purchased from Molecular Probes.

Cell Culture and Transfection—Experiments were performed in OK cells expressing stably the rodent Na⁺,K⁺-ATPase ₁-subunit wild type (13) or carrying a green fluorescent tag in its ₁-subunit, as described previously (14). OK cells stably transfected with the rodent wild-type ₁-subunit were transiently transfected with plasmids bearing either 14-3-3 wild type or a deletion mutant (26). The cells at 90% confluence in 10-cm dishes were exposed for 24 h to the mixture of 25 µl of LipofectAMINE 2000 and 10 µg of plasmid, preincubated according to Invitrogen protocol. Mutations in the PKC phosphorylation site (S11A and S18A), amino acid deletions, and stable expression of the Na⁺,K⁺-ATPase ₁-subunit were performed as described previously (8).

Preparation of Proximal Tubule Cells—PCT cells were obtained as described previously (27) from Sprague-Dawley rats weighing 150–200 g. Briefly, homogenates from the kidney outermost cortex were minced and incubated with 0.7 mg/ml collagenase A (Roche Diagnostics GmbH, Manheim, Germany) in 10 ml of Dulbecco's modified Eagle's medium, (Invitrogen). The incubation lasted for 20 min at 37 °C, and the solution was continuously exposed to 95% O₂/5% CO₂ during this period. After pouring the material through graded sieves, the cell suspension consisting mostly of PCT cells was washed (four times in Dulbecco's modified Eagle's medium) and finally resuspended in Hanks' medium (Invitrogen) to yield a protein concentration of 1–3 mg/ml. The experiments were performed immediately after preparation.

Preparation of Basolateral Membranes—Basolateral membranes were obtained from PCT cells using a Percoll (Amersham Biosciences AB, Uppsala, Sweden) gradient centrifugation as described previously (28). In brief, postnuclear supernatants were centrifuged at 20,000 x g for 20 min at 4 °C. The yellow layer obtained was resuspended in homogenization buffer and further centrifuged at 48,000 x g for 30 min at 4 °C. The pellet was resuspended in 1 ml of homogenization buffer, and 0.2 ml of Percoll was added. The suspension was gently mixed and centrifuged at 48,000 x g for 30 min at 4 °C.

Biotin Labeling—Transiently transfected cells after 24 h were transferred to Hanks' solution for 30 min and then incubated for 5 min with 1 µM DA or vehicle at room temp. Incubation was stopped on ice, and the medium was changed to ice-cold 10 mM Tris-HCl (pH 7.5), 2 mM CaCl₂, 150 mM NaCl, 1.5 mg/ml sulfo-N-hydroxysuccinimidobiotin, the cells were incubated for 1 h at 4 °C. Following biotin labeling, the cells were scraped into immunoprecipitation buffer (20 mM Tris, 2 mM EDTA, 2 mM EGTA, 30 mM sodium pyrophosphate, pH 7.3) containing a protease inhibitor mixture, frozen in liquid nitrogen, thawed rapidly, probe-sonicated twice on ice-water bath, and frozen-thawed again. The cell suspension was centrifuged at 14,000 x g at 4 °C for 5 min. Supernatants were transferred to clean tubes, and 1% Triton X-100 and 0.2% SDS were added. Anti-₁ monoclonal antibody was added and incubated for 1 h at 4 °C with end-over-end shaking and then protein A/G-agarose, pre-washed three times with PBS and once with immunoprecipitation buffer containing 1% Triton X-100, was added and incubated overnight. The pellet was washed three times with this buffer containing 1% Triton X-100 and 0.1% SDS, once with 50 mM Tris-HCl (pH 7.4), and finally resuspended in Laemmli sample buffer. Electrophoresis, Western blot using extravidin, and densitometric analysis were performed as described previously (13).

Determination of Na⁺,K⁺-ATPase Activity—Na⁺,K⁺-ATPase activity was determined from the ouabain-inhibitable ⁸⁶Rb⁺-transport. To assess the effect of DA, cells were preincubated at room temperature with 5 µM monensin (Sigma) for 30 min, as described by Seri et al. (5), and then with 1 µM DA (5 min) before assay. Measurements of Na⁺,K⁺-ATPase-mediated ⁸⁶Rb⁺ transport were performed as described previously (13), and Na⁺,K⁺-ATPase activity was expressed as nmol of Rb⁺/mg of protein/min.

Determination of PI 3-Kinase Activity—After preincubation with DA under different conditions, the cells were transferred to 1.5-ml Eppendorff tubes in the cold, homogenized in 400 µl of lysis buffer (137 mM NaCl, 2.7 mM KCl, 1 mM MgCl₂, 0.5 mM Na₃VO₄, 1% Triton X-100, 10% v/v glycerol, 20 mM Tris-HCl, 10 µg/ml leupeptin, 0.2 mM PMSF, 10 mM NaF, 10 mg/ml aprotinin) and solubilized by passing through needles (27-gauge x -inch) 20 times and by stroking with a pestle for 30 s. After centrifugation at 800 x g for 10 min, the supernatant was collected, and 500 µg of protein (in 1 ml) was incubated with an antibody against the Na⁺,K⁺-ATPase -subunit (gift of Dr. Mercer) by end-over-end rotating overnight. The next morning, 40 µl of protein A/G-agarose beads (Pharmacia Corp.) were added and incubated for 2 h by end-over-end rotating. The immune complex was washed three times with lysis buffer, two times with washing buffer 2 (137 mM NaCl, 0.1 M Tris-HCl, 10 µg/ml leupeptin, 0.2 mM PMSF, 10 mM NaF, 10 mg/ml aprotinin), once with washing buffer 3 (0.15 M NaCl, 1 mM EDTA, 10 mM Tris-HCl, 10 µg/ml leupeptin, 0.2 mM PMSF, 10 mM NaF, 10 mg/ml aprotinin), once with washing buffer 4 (20 mM Hepes, 1 mM dithiothreitol, 5 mM MgCl₂, 10 µg/ml leupeptin, 0.2 mM PMSF, 10 mM NaF, 10 mg/ml aprotinin), and then resuspended in 20 µl of kinase assay buffer (20 mM -glycerophosphate, 5 mM Na-pyrophosphate, 30 mM NaCl, 1 mM dithiothreitol). The PI 3-K activity in the immunoprecipitate was assessed directly on the protein A/G-agarose beads as described previously (29). The reaction was initiated by the addition of 30 µl of reaction mixture (20 µl of buffer (12.5 µM ATP, 7.6 mM MgCl₂,20mM -glycerophosphate, 5 mM Na-pyrophosphate, 30 mM NaCl, 1 mM dithiothreitol, 4µCi [-³²P]ATP) plus 40 µg of lipids (Avanti Biochemicals, Birmingham, AL) in 10 µl of cholate buffer (10 mg sodium cholate/1 ml kinase assay buffer)). The pellets were incubated for 15 min at room temperature, and the reaction was terminated by the sequential addition of 20 µl of HCl and 160 µl of chloroform/methanol (1:1, v/v) with vigorous vortexing. After centrifugation at 14,000 rpm for 5 min, 80 µl of the lower phase was spotted on aluminum-backed silica gel thin layer chromatography plates (Merck). The lipids were resolved by chromatography in methanol/CHCl₃/ammonia/H₂O (47:34:12.5:6.5). The dot corresponding to phosphatidylinositol 3-phosphate was analyzed by autoradiography and quantitated using phosphorimaging.

Immunoprecipitation—OK cells grown in Petri dishes (10 cm) were incubated in Hanks' medium for 30 min at 25 °C prior to incubation in the presence or absence of 1 µM DA for the times indicated above at 23 °C. Thereafter, incubation solutions were replaced by immunoprecipitation buffer (in mM, 100 NaCl, 50 Tris-HCl, 2 EGTA, 1 PMSF, 5 mg/ml protease inhibitors (aprotinin, leupeptin, antipain), and 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. Immunoprecipitation of the Na⁺,K⁺-ATPase ₁-subunit, PI 3-kinase, and 14-3-3 was performed as described previously (13). In brief, aliquots (500 µg of protein) were incubated overnight at 4 °C with 70 µl of a Na⁺,K⁺-ATPase antibody (gift from Dr. Mercer), 6 µg of a polyclonal antibody raised against the PI 3-kinase p85 subunit (Z-8, Santa Cruz Biotechnology), or with 5 µg of 14-3-3 antibody (K-19, Santa Cruz Biotechnology) and the simultaneous addition of excess protein A-Sepharose beads (Amersham Biosciences AB, Uppsala, Sweden). Protein content was determined according to Bradford (30). Samples were analyzed by SDS-PAGE using the Laemmli buffer system (31). Proteins were transferred to polyvinylidene difluoride membranes (Hybond-P, Amersham Biosciences AB), and Western blots were performed using an antibody against the Na⁺,K⁺-ATPase ₁-subunit and developed with an ECL Plus (Amersham Biosciences AB) detection kit.

Microscopy—These experiments were performed using OK cells stably transfected with the Na⁺,K⁺-ATPase ₁-subunit carrying a green fluorescent protein tag in the NH₂ terminus. The presence of the tag does not affect the intrinsic properties of the enzyme or its regulation by dopamine signals (14, 32). OK cells were incubated in the presence or absence of 1 µM DA for 2 min at 23 °C. Incubation was terminated by fixation of the cells with 4% formaldehyde in PBS for 10 min at room temperature. After rinsing twice with PBS, the cells were transferred to acetone (–20 °C) for 5 min and then quenched with PBS (containing 1% bovine serum albumin) for 30 min. Staining with primary (14-3-3 (1: 100)) and secondary fluorescent-labeled antibody (1:100) was performed at room temperature for 1 h. After rinsing with PBS, the coverslips were mounted (SlowFade Light, Molecular Probes, Eugene, OR) and examined using a confocal microscope (Leica TCS SP2, Leica Lasertechnik GmbH, Heidelberg, Germany).

Statistics—Comparison between two experimental groups was made with the non-paired Student's t test. p < 0.05 was considered significant. In all figures, bars indicate mean ± S.D.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Phosphorylation of the Na⁺,K⁺-ATPase ₁-subunit in response to G protein-coupled receptor (DA) stimulation is central for organizing, in time and space, a variety of signals that ultimately will lead to the endocytosis of Na⁺,K⁺-ATPase molecules. Among those signals, binding (to a proline-rich motif within the Na⁺,K⁺-ATPase ₁-subunit) and activation of PI 3-kinase activity are of utmost relevance for recruiting recognition molecules (adaptins) and clathrin formation (15, 17), as well as recruiting dynamin for clathrin vesicle fission (33). Phosphorylation of the Na⁺,K⁺-ATPase ₁-subunit at Ser-18 is critical for PI 3-kinase binding, but the molecular mechanisms of this interaction remain unknown. The fact that 14-3-3 proteins can form dimers (20), thereby creating interactive modules potentially linking serine-phosphorylated residues with diverse signaling molecules (including PI 3-kinase (23–25)), prompted us to study the possibility that their association with the Na⁺,K⁺-ATPase ₁-subunit may guide the PI 3-kinase regulatory subunit (p85) to the proline-rich domain upstream of the Ser-18 phosphorylation site and regulate Na⁺,K⁺-ATPase activity and subunit endocytosis.

Because there are numerous 14-3-3 isoforms, we examined their presence in PCT cells isolated from rat kidney by Western blotting using an antibody that recognizes all of the isoenzymes (34). The 14-3-3 protein was present in PCT cells, and high speed centrifugation of PCT cell homogenate revealed that most of the protein is present in the cytosol (Fig. 1A). Although a negligible amount was associated with the basolateral membrane fraction, the presence of DA induced a time-dependent increase in the abundance of 14-3-3 within this compartment (Fig. 1B). Additionally, confocal images of OK cells revealed the presence of 14-3-3 mostly in the cytosol in non-treated cells (V: vehicle) (Fig. 1C), whereas in the presence of DA, there is an increased 14-3-3 immunofluorescence associated with the plasma membrane (arrows) (Fig. 1C, D (dopamine)). Furthermore, support for a direct association of 14-3-3 with the Na⁺,K⁺-ATPase molecules at the plasma membrane in response to DA signals was obtained in co-immunoprecipitation assays. DA treatment is associated with increased Na⁺,K⁺-ATPase immunoreactivity in the material immunoprecipitated with a 14-3-3 protein antibody (Fig. 2A, left panel), and in the reciprocal experiment, increased 14-3-3 is observed in the material immunoprecipitated with a Na⁺,K⁺-ATPase antibody (Fig. 2A, right panel). These results strongly suggest that, in response to DA signals, the 14-3-3 becomes associated with basolateral plasma membrane, where it binds to the phosphorylated Ser-18 residue in the Na⁺,K⁺-ATPase ₁-subunit.

View larger version (69K): [in this window] [in a new window]   FIG. 1. Presence of 14-3-3 in PCT cells. A, homogenates (H), cytosol (C), total membranes (M), and basolateral membranes (BLM) prepared from PCT cells were separated on SDS-PAGE (31), and Western blot analysis was performed using a 14-3-3 antibody (1:500). Equal amounts of protein (10 µg) were analyzed in each lane. B, PCT cells were incubated with 1 µM DA at 23 °C for different periods of time, and the presence of 14-3-3 in basolateral membranes was determined by Western blot. Quantitative analysis of four experiments performed independently is presented. Inset, representative Western blot of 10 µg of BLM analyzed in each lane. C, confocal images of 14-3-3 (arrows) in fixed OK cells previously treated with (D) or without (V) 1 µM DA for 2 min at 23 °C. The experiment was repeated twice, and several cells were analyzed in each experiment.

View larger version (26K): [in this window] [in a new window]   FIG. 2. A, interaction of 14-3-3 with the Na+,K+-ATPase. Homogenates (500 mg) from PCT cells previously incubated in the presence (DA) or absence (V, vehicle) of 1 µM DA for 2 min at 23 °C were incubated with either a Na+,K+-ATPase (right panel) or 14-3-3 (left panel) (5 µg) antibody. The immunoprecipitated (IP) material was analyzed by Western blot (WB) with either Na+,K+-ATPase (1:1000) or 14-3-3 (1:500) antibody. A representative Western blot of five experiments is shown. B, co-immunoprecipitation of the Na+,K+-ATPase with 14-3-3 protein was performed exactly as described above for A in OK cells stably transfected with the Na+,K+-ATPase bearing the mutation Ser11 Ala (S11A), Ser18 Ala (S18A), or deletion of the first 26 amino acids (26) in the 1-subunit, as described by Chibalin et al. (8). Upper panel, representative Western blot. Lower panel, quantitative analysis of two experiments. WT, wild type.

Although the experimental data obtained strongly suggested a structural interaction between the 14-3-3 and the Na⁺,K⁺-ATPase -subunit, the following experiments were designed to examine whether such interaction also had functional relevance, i.e. if it is necessary for the regulation of Na⁺,K⁺-ATPase activity and the removal of active units from the plasma membrane in response to DA. OK cells were transiently transfected with wild type 14-3-3 or a deletion mutant (208–255) of this protein lacking essential amino acids (in bold; Lys-49, Arg-59, Arg-127, Tyr-128, Trp-228, Asn-173, Lys-120, Asn-224, Leu-216, Ile-217, Leu-220) within the phosphopeptide-interactive domain (26, 35). DA decreased Na⁺,K⁺-ATPase activity in non-transfected cells, mock-transfected cells, and in OK cells transfected with the wild type 14-3-3, whereas it failed to do so in OK cells transiently expressing the 14-3-3 mutant (Fig. 3A). Moreover, in OK cells expressing the mutant form of 14-3-3, DA failed to promote the endocytosis of Na⁺,K⁺-ATPase molecules (Fig. 3B). These results further suggest a functional interaction between 14-3-3 protein and the Na⁺,K⁺-ATPase ₁-subunit. Although the 14-3-3 mutant employed may have limited its ability to interact with the phosphorylated Na⁺,K⁺-ATPase ₁-subunit, we cannot exclude the possibility that this mutant did also affect its recognition motif with other proteins involved in the Na⁺,K⁺-ATPase regulatory process, such as the PI 3-kinase.

View larger version (29K): [in this window] [in a new window]   FIG. 3. DA-dependent inhibition of Na+,K+-ATPase activity and subunit endocytosis requires 14-3-3 proteins. A, Na+,K+-ATPase activity (% of control) in OK cells transiently expressing the wild type (14-3-3 (WT)) or a deletion mutant (14-3-3 (M)) of 14-3-3. Non-transfected (NT) and mock (Lipofectamine-treated) OK cells are also shown. Each bar represents the mean of three experiments performed in triplicate. B,Na+,K+-ATPase abundance at the plasma membrane was examined by cell surface biotinylation in transiently transfected OK cells as described in A. OK cells were incubated with 1 µM DA for 2 min at 23 °C. A representative Western blot of three experiments is shown.

View larger version (18K): [in this window] [in a new window]   FIG. 4. A, interaction between PI 3-kinase and 14-3-3 protein. Homogenates (500 mg) from PCT cells that were previously incubated in the presence (DA) or absence (V, vehicle) of 1 µM DA for 2 min at 23 °C were incubated with a 14-3-3 (5 µg) antibody. The immunoprecipitated (IP) material was analyzed by Western blot (WB) with a PI 3-kinase (1:1250) antibody. B, association of PI 3-kinase with the Na+,K+-ATPase 1-subunit in OK cells transiently expressing the wild type (14-3-3 (WT)) or deletion mutant (14-3-3 (M)) of 14-3-3. Non-transfected (NT) and mock (Lipofectamine-treated) OK cells are also shown. Left panel, representative Western blot. Right panel, quantitation of three experiments.

View larger version (24K): [in this window] [in a new window]   FIG. 5. PI 3-kinase activity was determined in the material immunoprecipitated with a Na+,K+-ATPase -subunit antibody from cells transfected with the vector (mock) or wild type (14-3-3 (WT)) or a deletion mutant (14-3-3 (M)) of 14-3-3. Representative experiment (upper panel) and the quantitative analysis of three independent experiments is depicted (lower panel).

These results provide the identity of a new signaling partner, the 14-3-3 protein, that interacts with the Na⁺,K⁺-ATPase molecule at the plasma membrane (Fig. 6). This association requires phosphorylation of the Na⁺,K⁺-ATPase ₁-subunit N terminus, and it permits the binding of PI 3-kinase to a prolinerich domain located upstream of the phosphorylation site. Thus, 14-3-3 is an essential part of the signaling network regulating Na⁺,K⁺-ATPase activity and endocytosis in response to G protein-coupled receptor ligands, a process that is essential for controlling kidney tubule Na⁺,K⁺-ATPase activity in response to natriuretic hormones.

View larger version (32K): [in this window] [in a new window]   FIG. 6. Schematic representation of the molecular organization of different signaling molecules during Na+,K+-ATPase 1-subunit endocytosis in response to G protein-coupled receptor signals. The N terminus of the 1-subunit represents a scaffolding organizing the assembly of signaling molecules necessary for Na+,K+-ATPase endocytosis. The signal is initiated by phosphorylation of the Ser-18 residue (KKS18KK) followed by binding of 14-3-3 and locking of PI 3-kinase (via its SH3 domain) with the proline-rich domain (PP78PTTP). Binding and activation of PI 3-kinase (PI 3-k) serves several purposes; e.g. production of phosphatidylinositol 3-phosphate is necessary for increasing the affinity of adaptor protein-2 (µ2) subunit to a tyrosine-rich motif (Y537LEL in the 1-subunit); and recruiting dynamin, which is necessary for fission of clathrin-coated pits at the site of Na+,K+-ATPase endocytosis.

	FOOTNOTES

^** To whom correspondence should be addressed: King Gustaf V Research Institute, Karolinska University Hospital-Solna, M1:01, 171 76 Stockholm, Sweden. Tel.: 46-8-5177-9746; Fax: 46-8-31-12-98; E-mail: alejandro.bertorello{at}medks.ki.se.

¹ The abbreviations used are: DA, dopamine; PKC, protein kinase C; PI, phosphoinositide; PCT, proximal convoluted tubules; OK, opossum kidney; PRD, proline-rich domain; PBS, phosphate-buffered saline; PMSF, phenylmethylsulfonyl fluoride.

	ACKNOWLEDGMENTS

 
We thank Drs. M. Guthridge, I. B. Leibiger, and J. Arguello for fruitful discussions. We are also thankful to Dr. T-A. Sato for providing the 14-3-3 cDNAs.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 

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