The 14-3-3 Protein Translates the NA+,K+-ATPase α1-Subunit Phosphorylation Signal into Binding and Activation of Phosphoinositide 3-Kinase during Endocytosis*

Clathrin-dependent endocytosis of Na+,K+-ATPase molecules in response to G protein-coupled receptor signals is triggered by phosphorylation of the α-subunit and the binding of phosphoinositide 3-kinase. In this study, we describe a molecular mechanism linking phosphorylation of Na+,K+-ATPase α-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 α-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 α-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 α-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.

Regulation of Na ϩ ,K ϩ -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 ϩ ,K ϩ -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 ϩ ,K ϩ -ATPase activity. The responses to different agonists regulating the intracellular distribution of Na ϩ ,K ϩ -ATPase are tissue-specific (3), and in renal tubule cells, the direction of the physiological response (increase versus de-crease) 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 ϩ ,K ϩ -ATPase ␣ 1 -subunits (at  that are present at the plasma membrane is necessary for dopamine (DA) 1 -induced endocytosis (6 -8) and (at Ser-11) for parathyroid hormoneinduced 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 ϩ ,K ϩ -ATPase molecules (Ser-11/Ser-18) present within endosomes. DA-mediated phosphorylation of the ␣ 1 -subunit and Na ϩ ,K ϩ -ATPase endocytosis requires activation of the PKC-isoform, whereas serotonin-and angiotensin II-dependent increase in Na ϩ ,K ϩ -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 ϩ ,K ϩ -ATPase molecules in response to DA in renal epithelial cells is initiated by adaptor protein-2 binding to the Na ϩ ,K ϩ -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 ϩ ,K ϩ -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 ϩ ,K ϩ -ATPase ␣ 1 -subunit and the tertiary structure of the skeletal muscle sarcoplasmic reticulum Ca 2ϩ -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 ϩ ,K ϩ -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)(24)(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
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 ϩ ,K ϩ -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 ϩ ,K ϩ -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 l of LipofectAMINE 2000 and 10 g 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 ϩ ,K ϩ -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% O 2 /5% CO 2 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 ϳ1-3 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 ϫ g for 20 min at 4°C. The yellow layer obtained was resuspended in homogenization buffer and further centrifuged at 48,000 ϫ 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 ϫ 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 M 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 CaCl 2 , 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 ϫ 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).
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 M 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 ϩ ,K ϩ -ATPase ␣ 1 -subunit, PI 3-kinase, and 14-3-3 was performed as described previously (13). In brief, aliquots (500 g of protein) were incubated overnight at 4°C with 70 l of a Na ϩ ,K ϩ -ATPase antibody (gift from Dr. Mercer), 6 g of a polyclonal antibody raised against the PI 3-kinase p85␣ subunit (Z-8, Santa Cruz Biotechnology), or with 5 g 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 ϩ ,K ϩ -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 ϩ ,K ϩ -ATPase ␣ 1 -subunit carrying a green fluorescent protein tag in the NH 2 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 M 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 (Ϫ20°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 Ͻ 0.05 was considered significant. In all figures, bars indicate mean Ϯ S.D.

RESULTS AND DISCUSSION
Phosphorylation of the Na ϩ ,K ϩ -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 ϩ ,K ϩ -ATPase molecules. Among those signals, binding (to a proline-rich motif within the Na ϩ ,K ϩ -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 ϩ ,K ϩ -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 ϩ ,K ϩ -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 ϩ ,K ϩ -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 ϩ ,K ϩ -ATPase molecules at the plasma membrane in response to DA signals was obtained in co-immunoprecipitation assays. DA treatment is associated with increased Na ϩ ,K ϩ -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 ϩ ,K ϩ -ATPase antibody ( Fig. 2A, right panel). These results strongly suggest that, in response to DA signals, the 14-3-3 becomes associated with  2. A, interaction of 14-3-3 with the Na ϩ ,K ϩ -ATPase. Homogenates (500 mg) from PCT cells previously incubated in the presence (DA) or absence (V, vehicle) of 1 M DA for 2 min at 23°C were incubated with either a Na ϩ ,K ϩ -ATPase (right panel) or 14-3-3 (left panel) (5 g) antibody. The immunoprecipitated (IP) material was analyzed by Western blot (WB) with either Na ϩ ,K ϩ -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 ϩ ,K ϩ -ATPase with 14-3-3 protein was performed exactly as described above for A in OK cells stably transfected with the Na ϩ ,K ϩ -ATPase bearing the mutation Ser 11 3 Ala (S11A), Ser 18 3 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. basolateral plasma membrane, where it binds to the phosphorylated Ser-18 residue in the Na ϩ ,K ϩ -ATPase ␣ 1 -subunit. Although 14-3-3 binds to proteins at a consensus motif (RSX-pSXP), where pS is a phosphorylated serine critical for triggering its binding (35), this protein has been reported to bind to other sites as well. Several PKC phosphorylation sites have been described within the Na ϩ ,K ϩ -ATPase ␣ 1 -subunit in vitro (36). Among these, Ser-18 (within the rodent ␣ 1 -subunit) had proven to be relevant for controlling removal of Na ϩ , K ϩ -ATPase from the plasma membrane in response to DA in intact cells (8). To examine whether this phosphorylation site is also responsible for the interaction of 14-3-3 with the Na ϩ ,K ϩ -ATPase ␣ 1 -subunit, we examined this interaction in OK cells that were stably transfected with the wild type ␣ 1 -subunit, the S11A (another PKC phosphorylation site) or S18A mutant, and with the ␣ 1 -subunit lacking the first 26 amino acids of the N terminus. DA increased the association of 14-3-3 with the Na ϩ ,K ϩ -ATPase in cells expressing the wild type but not the S18A mutant (Fig. 2B). The interaction between 14-3-3 and the Na ϩ ,K ϩ -ATPase was also absent in cells expressing the Na ϩ ,K ϩ -ATPase carrying a deletion (amino acids 1-26) within the N terminus that includes the Ser-18 residue. Mutation in another adjacent PKC phosphorylation site (S11) did not affect the interaction between Na ϩ ,K ϩ -ATPase and 14-3-3 triggered by DA. These results indicate that Ser-18, within the rat Na ϩ ,K ϩ -ATPase ␣ 1 -subunit, is a likely binding site for 14-3-3 in response to DA. A weak interaction between these proteins can also be noticed in non-stimulated cells, and this association was not entirely absent in cells expressing the ␣ 1 -subunit carrying the S18A mutation or the 1-26 deletion, suggesting the possibility that other 14-3-3 binding sites might be present within the Na ϩ ,K ϩ -ATPase ␣ 1 -subunit, which, however, are not under DA control.
Although the experimental data obtained strongly suggested a structural interaction between the 14-3-3 and the Na ϩ ,K ϩ -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 ϩ ,K ϩ -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 ϩ ,K ϩ -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 ϩ ,K ϩ -ATPase molecules (Fig. 3B). These results further suggest a functional interaction between 14-3-3 protein and the Na ϩ ,K ϩ -ATPase ␣ 1 -subunit. Although the 14-3-3 mutant employed may have limited its ability to interact with the phosphorylated Na ϩ ,K ϩ -ATPase ␣ 1 -subunit, we cannot exclude the possibility that this mutant did also affect its recognition motif with other proteins involved in the Na ϩ ,K ϩ -ATPase regulatory process, such as the PI 3-kinase.
It has been demonstrated in several cell systems that the 14-3-3 protein binds to PI 3-kinase (23)(24)(25) and thereby affects various physiological functions. Therefore, OK cells were incubated with or without DA, and the presence of PI 3-kinase (p85␣-subunit) was examined in the immunoprecipitated material with a 14-3-3 antibody (Fig. 4A). Indeed, cells incubated with DA demonstrated a significant increase in PI 3-kinase associated with 14-3-3 compared with vehicle-treated cells. To establish whether the interaction of PI 3-kinase with the FIG. 3. DA-dependent inhibition of Na ؉ ,K ؉ -ATPase activity and subunit endocytosis requires 14-3-3 proteins. A, Na ϩ ,K ϩ -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 ϩ ,K ϩ -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 M DA for 2 min at 23°C. A representative Western blot of three experiments is shown .   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 M DA for 2 min at 23°C were incubated with a 14-3-3 (5 g) 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 ϩ ,K ϩ -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. Na ϩ ,K ϩ -ATPase requires 14-3-3, we next examined their colocalization in response to DA in OK cells transiently expressing the 14-3-3 wild type or mutant (Fig. 4B). In the presence of DA, there was an increased co-localization of the Na ϩ ,K ϩ -ATPase ␣-subunit and PI 3-kinase in non-transfected and mock-transfected cells and in cells transfected with the wild type 14-3-3 but not in cells transiently expressing the 14-3-3 mutant. These data strongly indicate that the 14-3-3 protein represents a linker between the phosphorylated Na ϩ ,K ϩ -ATPase ␣-subunit and binding of PI 3-kinase to its proline-rich domain within the N terminus. In addition, transient expression of the 14-3-3 negative mutant also prevented the increase in PI 3-kinase activity associated with the binding of the Na ϩ ,K ϩ -ATPase ␣-subunit to the p85␣ regulatory subunit (Fig.   5). We do not know yet the nature of the interaction between 14-3-3 and the PI 3-kinase. It is possible that such interaction may involve either of the kinase subunits (p85␣ or p110) or an alternative regulatory protein.
Binding of 14-3-3 to the Na ϩ ,K ϩ -ATPase ␣-subunit may not be limited to providing an anchor for the PI 3-kinase. Thus, 14-3-3 binding to phosphorylated serine residues modulates the action of protein phosphatases upon those residues with direct functional consequences in the physiological responses. For example, dephosphorylation of NHE1 and human ether-ago-go-related gene K ϩ channel was protected by the binding of 14-3-3, thereby resulting in stimulation of NHE1 activity and prolonged human ether-a-go-go-related gene K ϩ channel activation by adrenergic stimulation (37), as well as dephosphorylation of Bcl2 antagonist of cell death and regulation of its pro-apoptotic function (38). Because phosphorylation of the Na ϩ ,K ϩ -ATPase ␣ 1 -subunit is essential for triggering its endocytosis, it is likely that the action of 14-3-3, in addition to facilitating the binding of PI 3-kinase, may also protect Ser-18 from being dephosphorylated by the action of protein phosphatases. Indeed, we previously reported (15) that Na ϩ ,K ϩ -ATPase endocytosis was not blocked by dephosphorylation of the ␣ 1 -subunit but rather through an increase in inositol hexakisphosphate, which prevents the association of adaptor proteins with the Na ϩ ,K ϩ -ATPase. Thus, the presence of 14-3-3 protecting the Ser-18 phosphorylation site could prevent the phosphatase activity associated with the PP2A that constitutively binds to the Na ϩ ,K ϩ -ATPase ␣ 1 -subunit (39).
These results provide the identity of a new signaling partner, the 14-3-3 protein, that interacts with the Na ϩ ,K ϩ -ATPase molecule at the plasma membrane (Fig. 6). This association requires phosphorylation of the Na ϩ ,K ϩ -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 ϩ ,K ϩ -ATPase activity and endocytosis in response to G protein-coupled receptor ligands, a process that is essential for controlling kidney tubule Na ϩ ,K ϩ -ATPase activity in response to natriuretic hormones.