14-3-3 Binding to Na+/H+ Exchanger Isoform-1 Is Associated with Serum-dependent Activation of Na+/H+ Exchange*

Stephanie Lehoux, Jun-ichi Abe, Jennifer A. Florian, and Bradford C. BerkDagger

From the Center for Cardiovascular Research and Department of Medicine, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642

Received for publication, January 17, 2001, and in revised form, February 12, 2001


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Na+/H+ exchanger isoform-1 (NHE1), the ubiquitous form of the Na+/H+ exchanger, has increased activity in hypertensive patients and in animal models of hypertension. Furthermore, NHE1 is activated in cells stimulated with growth factors. We showed previously that activation of the exchanger is dependent on phosphorylation of serine 703 (Ser(P)703) by p90 ribosomal S6 kinase (RSK). Because the NHE1 sequence at Ser(P)703 (RIGSDP) is similar to a consensus sequence (RSXSXP) specific for 14-3-3 ligands, we evaluated whether serum stimulated 14-3-3 binding to NHE1. Five different GST-NHE1 fusion proteins spanning amino acids 515-815 were phosphorylated by RSK and used as ligands in a far Western analysis; only those containing Ser(P)703 exhibited high affinity 14-3-3 binding. In PS127A cells (NHE1-overexpressing Chinese hamster fibroblasts) stimulated with 20% serum, NHE1 co-precipitation with GST-14-3-3 fusion protein increased at 5 min (5.2 ± 0.4-fold versus control; p < 0.01) and persisted at 40 min (3.9 ± 0.3-fold; p < 0.01). We confirmed that binding occurs at the RIGSDP motif using PS120 (NHE1 null) cells transfected with S703A-NHE1 or P705A-NHE1 (based on data indicating that 14-3-3 binding requires phosphoserine and +2 proline). Serum failed to stimulate association of 14-3-3 with these mutants. A GST-NHE1 fusion protein was phosphorylated by RSK and used as a ligand to assess the effect of 14-3-3 on protein phosphatase 1-mediated dephosphorylation of Ser(P)703. GST-14-3-3 limited dephosphorylation (66% of initial state at 60 min) compared with GST alone (27% of initial state; p < 0.01). The protective effect of GST-14-3-3 was lost in the GST-NHE1 P705A mutant. Finally, the base-line rate of pH recovery in acid-loaded cells was equal in unstimulated cells expressing wild-type or P705A-NHE1. However, activation of NHE1 by serum was dramatically inhibited in cells expressing P705A-NHE1 compared with wild-type (0.13 ± 0.02 versus 0.48 ± 0.06 mmol of H+/min/liter, p < 0.01). These data suggest that 14-3-3 binding to NHE1 participates in serum-stimulated exchanger activation, a new function for 14-3-3.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

NHE11 is the ubiquitous form of the Na+/H+ exchanger, responsible for regulation of intracellular pH and cell volume. NHE1 has increased activity in hypertensive patients and in animal models of hypertension, and the exchanger can be stimulated by various exogenous factors. In several examples of increased NHE1 activity, the primary change is not in NHE1 expression but in post-translational regulation of activity. In immortalized cells from hypertensive patients, enhanced NHE1 activity appears to reflect increased Vmax rather than increased NHE1 mRNA (1). Similarly, vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats (SHR) show an increased Vmax compared with their normotensive Wistar-Kyoto (WKY) counterparts but do not overexpress NHE1 mRNA or protein (2-4). Furthermore, there is no evidence for genetic linkage between the Na+/H+ exchanger gene and human hypertension (5), and no mutations have been detected in the entire coding region of NHE1 in SHR compared with WKY (6). Hence, increased NHE1 activity in hypertension is best explained by altered intracellular regulation of the exchanger. In support of this concept, phosphorylation of NHE1 by growth factors was increased in the SHR compared with the WKY (7).

NHE1 is activated in cells stimulated with numerous agonists. Epidermal growth factor, thrombin, phorbol esters, and serum were first found to stimulate Na+/H+ exchanger phosphorylation concurrently with a rise in intracellular pH (8). p160 ROCK mediates lysophosphatidic acid-induced activation of NHE1 via carboxyl-terminal phosphorylation (9). Angiotensin II activates extracellular signal-regulated kinase (ERK), which in turn stimulates NHE1 phosphorylation and activity (10, 11). In fact, ERK1/2 activation appears to be a common denominator in the process of NHE1 activation by several growth factors, since MEK1/ERK1/2 pathway inhibition with PD98059 or use of a dominant negative ERK1 construct blocks activation of the exchanger by alpha 1-adrenergic receptor agonists, serum, endothelin, platelet-derived growth factor, thrombin, and phorbol esters among others (12-16). Interestingly, in lymphoblasts from hypertensive patients, increased NHE1 activity and phosphorylation correlated with enhanced ERK1/2 activity, although there were no apparent differences in the level of tyrosine phosphorylation of ERK between cells from hypertensive and normotensive patients (17). Indeed, the relative activity of the ERK pathway in hypertension remains controversial (18, 19).

We showed previously that activation of NHE1 by serum was dependent on phosphorylation of serine 703 (Ser(P)703) by p90 ribosomal S6 kinase (RSK), which in turn was directly regulated by ERK1/2 (20). Interestingly, the NHE1 sequence at Ser(P)703 (RIGSDP) is similar to a consensus sequence (RSXSXP) specific for binding of 14-3-3. Indeed, 14-3-3 ligands share a common binding determinant that mediates their interaction with 14-3-3. First described for Raf-1, the consensus recognition motif RSXpSXP (where X designates any amino acid and pS represents phosphoserine) applies to most known binding partners of 14-3-3 (21). In the overwhelming majority of interactions documented so far, 14-3-3 necessitates a phosphorylated serine for binding. For example, mutants of Raf-1 where Ser259 or Ser621 are mutated to alanine have reduced binding to 14-3-3 (22). An arginine in the -3 or -4-position is also crucial, as is the proline at +2, although in some instances changes in the proline may be tolerated (23, 24). The phosphoserine contacts 14-3-3 by salt bridges to side chains contained within the aliphatic groove of 14-3-3. In the case of 14-3-3zeta , mutations of residues within this groove (K49E, R56E, or R127E) diminish interaction with binding partners Raf1 and Bcr (25).

Based on sequence similarity between our proposed 14-3-3 binding site in NHE1 and those identified in known 14-3-3 ligands, we hypothesized that phosphorylation of NHE1 at serine 703 would create a binding site for 14-3-3. In addition, we postulated that 14-3-3 binding would modulate NHE1 activity. In the present study, we show that 14-3-3beta associates with NHE1 in cells stimulated by serum, and using mutants of NHE1 we determined that this interaction occurs at serine 703. Functionally, binding of 14-3-3 to the exchanger was necessary for activation of NHE1 by serum, an effect that correlated with diminished dephosphorylation of Ser(P)703 observed when NHE1 is bound to 14-3-3.

    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Far Western Analysis-- Preparation of overlapping GST fusion proteins spanning amino acids 516-815, comprising the entire cytoplasmic domain of NHE1, was described previously (11). Five constructs, NHE1-(516-630), NHE1-(625-747), NHE1-(747-815), NHE1-(625-670), and NHE1-(625-714), were used (Fig. 1). GST-14-3-3 was a gift from Dr. A. J. Muslin (Washington University School of Medicine, St. Louis, MO). After transformation of GST constructs into the BL21 strain of Escherichia coli, cultures were grown to the sublog phase and induced for 3 h at 37 °C with 0.5 mM isopropyl-beta -D-thiogalactopyranoside (Sigma). Cells were collected, sonicated, and centrifuged. The supernatants were incubated with glutathione-Sepharose beads (Amersham Pharmacia Biotech) overnight at 4 °C, and bound fusion proteins were washed extensively with fusion protein lysis buffer (50 mM Tris, pH 8, 1 mM EDTA, 100 mM NaCl, 1% Triton X-100, 0.25% Nonidet P-40). Purified GST-NHE1 fusion proteins were stimulated with RSK (20 mM Tris, pH 7.4, 12 mM MgCl2, 100 mM NaCl, 5 units of p90 RSK2 (Upstate Biotechnology Biotechnology, Inc., Lake Placid, NY), 10 mM ATP) and then resolved on SDS-PAGE and transferred to nitrocellulose membranes. GST-14-3-3 fusion proteins were labeled with [gamma -32P]ATP using protein kinase A buffer (20 mM Tris, pH 7.4, 12 mM MgCl2, 100 mM NaCl, 10 µl of protein kinase A, 10 mCi of [gamma -32P]ATP), and used to probe the membranes. Alternatively, membranes containing GST-14-3-3 were probed with [gamma -32P]ATP-labeled GST-NHE1 constructs.

Cell Culture-- PS127A cells (Chinese hamster lung fibroblasts that overexpress human NHE1) and PS120 cells (Chinese hamster lung fibroblasts deficient in NHE1) were gifts of Dr. J. Pouysségur (University of Nice, France). All cells were maintained in Dulbecco's modified Eagle's medium supplemented with 100 units/ml penicillin, 100 µg/ml streptomycin, and 10% fetal calf serum (FCS). Cells were serum-starved (0% FCS) overnight prior to experiments.

Preparation of Cell Lysates-- Control PS127A and PS120 cells and cells stimulated with 20% FCS for 5-40 min, with or without PD98059 (Calbiochem), were harvested using NHE1 lysis buffer (30 mM Tris, pH 8, 10 mM NaCl, 5 mM EDTA, 10 g/liter polyoxyethylene-8-lauryl ether, 1 mM o-phenanthroline, 1 mM indoacetamide, 10 mM NaF, 5 mM Na3VO4, 10 mM sodium pyrophosphate). Cells were immediately frozen on ethanol/dry ice, and the cell lysates were then thawed on ice, scraped, sonicated, and centrifuged at 14,000 × g at 4 °C for 15 min. Supernatants were used immediately.

14-3-3 Pull-down-- Cell lysates containing 500 µg of protein were incubated overnight at 4 °C with 10 µg of GST-14-3-3 bound to glutathione-Sepharose. The beads were washed two times with 1 ml of NHE1 lysis buffer, once with 1 ml of LiCl wash buffer (500 mM LiCl, 100 mM Tris-Cl, pH 7.6, 0.1% Triton X-100, and 1 mM dithiothreitol), and two times more with 1 ml of lysis buffer before the addition of boiling Laemmli buffer. Proteins were resolved on SDS-PAGE and transferred to nitrocellulose membranes for Western analysis. Anti-NHE1 antibody was obtained from Chemicon.

Plasmid Construction and Cell Transfections-- Human NHE1 cDNA was a gift from Dr. L. Fliegel (University of Alberta, Canada). Serine 703 was mutated to alanine using polymerase chain reaction as described earlier (20). All other mutations were done using a QuikChange mutagenesis kit (Stratagene). Briefly, sense and antisense primers were designed to contain the desired mutation, indicated below by the lowercase letters, and to anneal to the same sequence on opposite strands of the plasmid. The following sense primers were used (antisense primers were the identical sequence in reverse): CGCATCGGCTCAGACgCACTGGCCTATGAGCCG (NHE1 P705A), ACCAGGCAGCGGCTGCGGgCCTACAACAGACACACGCTG (NHE1 S648A), GTCATCACCATCGACCCGGCgTCCCCGCAGTCACCCGAGTC (NHE1 S723A), ACCCGGCTTCCCCGCAGgCACCCGAGTCTGTGGAC (NHE1 S726A), TCCCCGCAGTCACCCGAGgCTGTGGACCTGGTGAATG (NHE1 S729A), and TCTCTGTTGCCTACgAGAATGTGGTAGGTGC (14-3-3 K49Q). The primers were extended with Pfu polymerase, resulting at length in the generation of mutated plasmids containing the desired nicks, and DpnI was added to the final product to digest the parental DNA. After the correct sequence was confirmed, the cDNA was subcloned into pcDNA/3.1(+) (Invitrogen Inc.) to transfect mammalian cells or into pGEX-2TK (Amersham Pharmacia Biotech) to express it as GST fusion protein.

PS120 cells were seeded at 40% confluence onto 60-mm dishes 24 h prior to transfection and transfected with 4 µg of plasmid DNA using the LipofectAMINE Plus method (Qiagen). After 3 h of incubation with the DNA-lipid complexes, the cells were refed with serum-containing medium. Two days post-transfection, the transfected cells were trypsinized and seeded onto three 100-mm dishes in medium containing 1 mg/ml neomycin. After 1 week of selection, cell colonies were picked and expanded into individual cultures. Transfected PS120 cells were maintained in Dulbecco's modified Eagle's medium, 10% FCS with 1 mg/ml neomycin, and every 2 weeks cells were subjected to acid selection to maintain high level expression of NHE1 as described previously (20). In brief, cells were exposed to 50 mM NH4Cl in TBSS (135 mM NaCl, 5 mM KCl, 1.5 mM CaCl2, 1.0 mM MgCl2, 200 mg/dl glucose, 20 mM HEPES, 20 mM Tris, pH 7.4) for 1 h followed by low NaCl/choline Cl solution buffer (130 mM choline Cl, 5 mM NaCl, 5 mM KCl, 1.5 mM CaCl2, 1.0 mM MgCl2, 200 mg/dl glucose, 20 mM HEPES, 20 mM Tris, pH 6.5) for 1 h.

PP1alpha Protection Assay-- GST-NHE1-(625-747) fusion protein was phosphorylated by p90 RSK2 in HMK buffer (20 mM Tris, pH 7.4, 12 mM MgCl2, 100 mM NaCl) at 37 °C overnight. One µg of GST-NHE1 was incubated with 40 µg of GST or 10 µg of GST-14-3-3 at 20 °C for 1 h, and then protein phosphatase 1alpha was added for 5-60 min. The phosphatase reaction was stopped by the addition of boiling Laemmli buffer. Proteins were resolved on SDS-PAGE, and phosphorylation state was evaluated by autoradiography.

Intracellular pH (pHi) Measurement-- Na+/H+ exchange was determined by ethyl isopropyl amiloride-sensitive pHi recovery following acid loading as described previously using the fluorescent pH-sensitive dye, acetoxymethyl ester of 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF-AM) (Molecular Probes, Inc., Eugene, OR). Fluorescent measurements (excitation at 490 and 450 nm with emission at 530 nm) were made with a PTI Delta scan spectrofluorometer. Briefly, cells were grown on glass coverslips and growth-arrested at 70-80% confluence by incubation in 0% FCS/Dulbecco's modified Eagle's medium for 24 h prior to use. Cells were loaded with 3 µM acetoxymethyl ester of BCECF-AM in serum-free Dulbecco's modified Eagle's medium at 37 °C for 30 min, washed twice, and incubated in a HEPES-Tris balanced salt solution (130 mM NaCl, 5 mM KCl, 1.5 mM CaCl2, 1.0 mM MgCl2, and 20 mM HEPES buffered to pH 7.4 with Tris base) for 30 min. All experiments were then performed at 25 °C. Cells were acid-loaded by incubation with Tris-buffered salt solution (130 mM NaCl, 5 mM KCl, 1.5 mM CaCl2, 1.0 mM MgCl2, 20 mM HEPES buffered with Tris base to pH 7.4) containing 20 mM NH4Cl, followed by Tris-buffered salt solution with 0 mM NH4Cl. Cell pH recovery was recorded over the next 10 min. Afterward, the nigericin/high K+ technique with 20 µM nigericin in KCl solution (130 mM KCl, 10 mM HEPES, pH 7.4-6.5) was used to calibrate the relationship between excitation ratio (F490/F450) and pHi. In serum stimulation experiments, 20% FCS was added to Tris-buffered salt solution and nigericin solutions. The rate of pHi recovery was converted to mmol of H+/min/liter of cells (JH) by multiplying by the buffering power. Buffering power was calculated from the change in cell pHi observed upon NH4Cl addition. The data were then plotted as mmol of H+/min/liter of cells (JH) versus pHi.

Statistics-- Values presented are means ± S.E. Student's t test or analysis of variance was used when appropriate. p values of <0.05 were considered statistically significant.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

GST-NHE1 Constructs That Encompass Ser703 Bind 14-3-3-- We have previously demonstrated that serum stimulates the phosphorylation of NHE1 at serine 703 (20). This serine is contained within the sequence RIGSDP, which is similar to established recognition motifs of 14-3-3 proteins. To determine whether 14-3-3 binds to NHE1, we constructed three GST fusion proteins corresponding to amino acids 516-630, 625-747, or 748-815, together comprising the entire cytoplasmic tail of the exchanger (Fig. 1). Using these GST fusion proteins in a far Western assay, we determined that 14-3-3 binds the region encompassing amino acids 625-747, whereas its association with the other two portions of the cytoplasmic tail or to GST alone was negligible (Fig. 1). Two additional GST fusion proteins spanning amino acids 625-670 or 625-714 were used to define the region that binds 14-3-3. Only GST-NHE1-(625-714) interacted with 14-3-3, indicating that 14-3-3 binds NHE1 between amino acids 670 and 714. Appropriately, this region contains serine 703 and our proposed 14-3-3 binding motif.


View larger version (20K):
[in this window]
[in a new window]
 
Fig. 1.   GST-14-3-3 binds NHE1-(625-714) and NHE1-(625-747). Five different GST-NHE1 constructs, GST-NHE1-(516-630), GST-NHE1-(625-747), GST-NHE1-(748-815), GST-NHE1-(625-670), and GST-NHE1-(625-714), spanning the entire cytoplasmic tail of the exchanger, were prepared. These constructs were resolved on SDS-PAGE and probed with labeled GST-14-3-3. Equal protein loading was confirmed by Ponceau staining (not shown). Autoradiography revealed that GST-14-3-3 only bound GST-NHE1-(625-714) and GST-NHE1-(625-747). Identical results were obtained by probing GST-14-3-3-containing membranes with labeled GST-NHE1 constructs (not shown). Hence, 14-3-3 interacts with NHE1 at residues located between amino acids 670 and 714, containing Ser703 and the proposed 14-3-3 binding motif RIGSDP.

Serum-stimulated Association of 14-3-3 with NHE1 in Cultured Cells Is Blocked by PD98059-- It is well documented that serum and growth factors stimulate NHE1 activity and phosphorylation. To confirm that 14-3-3 associates with NHE1 in cells, we stimulated PS127 fibroblasts that overexpress NHE1 with 20% FCS and immunoprecipitated NHE1 from the cell lysates. Coprecipitation of 14-3-3 with NHE1 was enhanced in serum-stimulated cells, whereas NHE1 immunoprecipitation was equivalent in all conditions (Fig. 2A). To compensate for variable quality of NHE1 antibody lots, we also verified 14-3-3 association with NHE1 using glutathione-Sepharose-bound GST-14-3-3 in a pull-down assay. Binding of NHE1 to 14-3-3 was increased 5.2 ± 0.4-fold in cells stimulated for 5 min and remained elevated (3.9 ± 0.3-fold) after 40 min (Fig. 2B). In comparison, GST-Sepharose alone did not pull down the exchanger from serum-stimulated cells.


View larger version (40K):
[in this window]
[in a new window]
 
Fig. 2.   GST-14-3-3 binding to NHE1 from serum-stimulated cells is inhibited by PD98059. PS127 cells were stimulated with 20% FCS for 0 (control) or 5-40 min, and NHE1 was immunoprecipitated (IP) from the cell lysates (A). Precipitated proteins were separated by SDS-PAGE, transferred to polyvinylidene difluoride, and immunoblotted (IB) with anti-14-3-3 or anti-NHE1 antibody. Coprecipitation of 14-3-3 with the exchanger was greater in serum-stimulated cells than in controls, whereas NHE1 precipitation was equivalent in all conditions. B, cell lysates from serum-stimulated cells were also used in a pull-down assay with GST or GST-14-3-3 bound to glutathione-Sepharose. PPT, precipitated. NHE1 from serum-stimulated cells bound GST-14-3-3 but not GST. Furthermore, 1-h pretreatment with the MEK1 inhibitor PD98059 (50 µM) inhibited serum-stimulated association of NHE1 with GST-14-3-3. Results are mean ± S.E., n = 4-5. *, p < 0.05; **, p < 0.01 versus control (0 min).

We have found that serum stimulation of cells leads to phosphorylation of serine 703 and that this phosphorylation is greatly diminished in the presence of the MEK1 inhibitor PD98059. We investigated whether MEK1 inhibition also interferes with binding of 14-3-3 to NHE1. PS127 cells were incubated with PD98059 for 1 h prior to serum stimulation. Fig. 2B confirms that 50 µM PD98059, shown to prevent agonist-stimulated ERK1/2 and RSK activation and Na+/H+ exchange (12, 14, 15, 26), completely abolished serum-stimulated association of 14-3-3 with NHE1. 1 µM PD98059 had no effect (data not shown). Based on the findings that 14-3-3 binds a region spanning amino acids 670-714, containing serine 703, and that both association of 14-3-3 with NHE1 and phosphorylation of serine 703 in serum-stimulated cells is sensitive to PD98059, we sought to confirm that Ser(P)703 lies within the 14-3-3 binding motif of NHE1.

Serum Fails to Stimulate Association of 14-3-3 with NHE1 Mutated at Serine 703 or Proline 705-- To establish that 14-3-3 binds NHE1 at serine 703, PS120 cells (which are deficient in NHE1) were stably transfected with the NHE1 S703A mutant described previously (20). Serum stimulation failed to promote interaction of 14-3-3 with NHE1 S703A, as demonstrated by a pull-down assay (Fig. 3). Hence, Ser703 plays a major role in the interaction between NHE1 and 14-3-3, and phosphorylation of this serine is likely to be a requirement for association of these two proteins.


View larger version (43K):
[in this window]
[in a new window]
 
Fig. 3.   NHE1 S703A and NHE1 P705A mutants fail to interact with GST-14-3-3. PS 120 cells were stably transfected with wild type NHE1 (NHE wt), NHE1 S703A, or NHE1 P705A (A). Cells were stimulated with 20% FCS for the indicated times and used in a GST-14-3-3 pull-down assay. Precipitated (PPT) proteins were separated by SDS-PAGE, transferred to polyvinylidene difluoride, and immunoblotted (IB) with anti-NHE1 antibody. GST-14-3-3 bound only wild type NHE1 from serum-stimulated cells. B, specificity of interaction of 14-3-3 with Ser703 and Pro705 was confirmed using PS 120 cells (NHE1 null), PS 127 cells, or PS 120 cells stably transfected with NHE1 S703A, NHE1 P705A, NHE1 S648A, NHE1 S723A, NHE1 S726A, or NHE1 S729A and stimulated for 5 min with 20% FCS. From each cell lysate, 500 µg of protein were used in a GST-14-3-3 pull-down assay, and 20 µg protein were assayed directly by Western blot using an anti-NHE1 antibody. GST-14-3-3 pulled down all NHE1 mutants from serum-stimulated cells except S703A and P705A, proving specificity of interaction of 14-3-3 with the NHE1 motif containing Ser703 and Pro705. Results are mean ± S.E., n = 4. **, p < 0.01 versus control (0 min).

Some recent publications report that 14-3-3 binds to proteins within a motif bearing not only a phosphorylated serine but also a proline in the +2-position (24). Based on those data, we postulated that mutating proline 705 to alanine would also prevent binding of 14-3-3 to the exchanger. Indeed, in PS120 cells stably transfected with NHE1 P705A, 14-3-3 could no longer pull down NHE1 despite serum stimulation (Fig. 3).

The specificity of the interaction of 14-3-3 with the motif encompassing Ser703 and Pro705 was further confirmed using stable transfectants of four other serine to alanine mutants of NHE1 in the same region of the cytoplasmic tail (amino acids 625-747) that we identified as potential kinase substrates. Fig. 3B illustrates that in cells stimulated with 20% serum for 5 min, the S648A, S723A, S726A, and S729A mutants of NHE1 were successfully pulled down by 14-3-3, unlike the S703A or P705A mutants, despite equivalent expression levels of all mutants in PS120 cells. Hence, our combined data prove that the RIGSDP sequence at amino acids 700-705 is the key motif for binding of 14-3-3 to NHE1 and that other neighboring serines are not necessary for this association.

The K49Q Mutant of 14-3-3 No Longer Binds NHE1-- 14-3-3 primarily binds ligands via a phosphorylated serine. In the 14-3-3zeta isoform, several critical amino acids lying within its ligand binding groove that make contact with phosphoserine have been identified including lysine 49, which when mutated reduces the interaction of 14-3-3zeta with its binding partners Bcr, Raf (27), and Cbl (28). Therefore, we constructed a K49Q mutant of GST-14-3-3beta and tested whether it would pull down NHE1 from serum-stimulated cells. As depicted in Fig. 4, lysine 49 is essential for binding of 14-3-3 to NHE1, since replacement of this single amino acid abolished serum-stimulated association of 14-3-3 with NHE1.


View larger version (34K):
[in this window]
[in a new window]
 
Fig. 4.   The 14-3-3 K49Q mutant does not bind NHE1. PS 127 cells were stimulated with serum for 0-40 min and lysed for use in a pull-down assay with GST-14-3-3 or GST-14-3-3 K49Q. Precipitated (PPT) proteins were resolved by SDS-PAGE, transferred to polyvinylidene difluoride, and probed with anti-NHE1 antibodies. IB, immunoblot. The single point mutation at Lys49 completely prevented interaction of 14-3-3 with NHE1. Results are mean ± S.E., n = 3. **, p < 0.01.

14-3-3 Protects NHE1 from Dephosphorylation-- 14-3-3 proteins are known to bind to phosphoserine, and one of their key roles may be prevention of dephosphorylation of the serines by phosphatases such as PP1 and PP2. PP1 has been proposed to be a NHE1 phosphatase, based on findings that okadaic acid prevents dephosphorylation of NHE1 (29). To determine the ability of 14-3-3 binding to protect the exchanger from dephosphorylation, we developed a biochemical assay in which GST-NHE1-(625-747) phosphorylated by RSK was exposed to PP1alpha for differing times in the presence of GST or GST-14-3-3. Dephosphorylation of GST-NHE1-(625-747) incubated with GST alone was marked within 5 min of PP1alpha addition and progressed steadily thereafter (to 65, 41, and 27% of initial phosphorylation at 15, 30, and 60 min, respectively; Fig. 5A). In contrast, dephosphorylation of GST-NHE1-(625-747) was significantly slower when incubated with GST-14-3-3 before exposure to PP1alpha (89, 87, and 66%, respectively of initial phosphorylation at the same times; p < 0.05 compared with GST alone; Fig. 5A). The protective effect of GST-14-3-3 was completely lost when incubated with the GST-NHE1 P705A mutant that cannot bind 14-3-3 (Fig. 5B). Similarly, the GST-14-3-3 K49Q mutant, which does not associate with NHE1, was unable to protect wild-type GST-NHE1 from dephosphorylation by PP1alpha more efficiently than GST alone (Fig. 5C). Hence, binding of 14-3-3 to NHE1 protects phosphoserine 703 from phosphatases and may contribute to maintaining the exchanger in a phosphorylated, active state.


View larger version (26K):
[in this window]
[in a new window]
 
Fig. 5.   14-3-3 protects GST-NHE1-(625-747) from dephosphorylation by PP1alpha . GST-NHE1-(625-747) or GST-NHE1-(625-747) P705A was phosphorylated by RSK with [gamma -32P]ATP and then incubated with GST or GST-14-3-3. PP1alpha was added for 0 (control) or 5-60 min. Thereafter, proteins were boiled in Laemmli buffer and separated by SDS-PAGE, and GST-NHE1 phosphorylation was quantified by autoradiography. A, incubation with GST-14-3-3 reduced dephosphorylation of GST-NHE1 by PP1alpha significantly compared with incubation with GST. B, the protective effect of GST-14-3-3 was lost on the GST-NHE1 P705A mutant, which does not bind 14-3-3. Similarly, the GST-14-3-3 K49Q mutant, which fails to interact with NHE1, could no longer protect GST-NHE1 from dephosphorylation by PP1alpha (C). Results are mean ± S.E., n = 3-5. *, p < 0.05; **, p < 0.01.

Binding of 14-3-3 Is Essential for Activation of NHE1 by Serum-- We have previously demonstrated that serine 703 plays a major role in NHE1 function, since mutation to alanine prevents serum-stimulated NHE1 activation (20). We hypothesized that association of 14-3-3 with NHE1 contributes to activation of the exchanger by serum, since phosphorylation of serine 703 correlates with enhanced exchanger activity (20) and binding of 14-3-3 to NHE1 maintains Ser(P)703 in a phosphorylated state. To test this hypothesis, we evaluated pHi recovery from acid loading in cells expressing wild type NHE1 or the NHE1 P705A mutant. Fig. 6A depicts typical pHi recovery curves of acid-loaded cells in the presence and absence of serum. PS120 cells stably expressing wild type NHE1 rapidly recovered pHi and reached pHi = 6.98 ± 0.09 by 150 s. Treatment with 20% serum accelerated pH recovery in these cells, such that pHi reached 7.30 ± 0.05 at 150 s (p < 0.01 versus untreated). PS120 cells stably expressing the NHE1 P705A mutant demonstrated pHi recovery in unstimulated cells similar to that found in cells with wild-type NHE1, reaching pHi = 6.86 ± 0.11 at 150 s. However, serum failed to stimulate exchanger activity in these cells, since pHi recovered only to 6.97 ± 0.09 at 150 s (Fig. 6B), similar to our previous observations with the NHE1 S703A mutant (20).


View larger version (28K):
[in this window]
[in a new window]
 
Fig. 6.   Serum stimulation does not accelerate pH recovery in cells transfected with the NHE1 P705A mutant. PS 120 cells stably transfected with wild type NHE1 (NHEwt) or NHE1 P705A were acid-loaded by incubation with 20 mM NH4Cl followed by 0 mM NH4Cl in 130 mM NaCl buffer at pH 7.4. pH recovery curves were plotted from the point of minimum pH (0 s). A, the rate of pH recovery was moderate in PS 120 cells transfected with wild type NHE1 under control conditions. Treatment with 20% FCS accelerated pH recovery in these cells. In contrast, the rate on pH recovery in PS 120 cells transfected with NHE1 P705A was equivalent under control and 20% FCS conditions (B). C, the rate of proton flux (JH) during pH recovery was calculated for PS 120 cells transfected with wild type NHE1 or NHE1 P705A. At pH 6.8 (D) and throughout pH recovery, only serum-stimulated cells transfected with wild type NHE1 showed increased JH. Results are mean ± S.E., n = 4-6. *, p < 0.05; **, p < 0.01.

Since serum activates NHE1 by increasing affinity of the exchanger for intracellular H+ (30), we evaluated the kinetics of H+ flux in the PS120 transfectants. As shown in Fig. 6, C and D, serum stimulation of wild-type NHE1-expressing cells stimulated the rate of H+ flux characterized by an increase in the JH from 0.22 ± 0.03 to 0.48 ± 0.05 H+/min/liter of cells at pH 6.8 (p < 0.01). Note that the JH values were lower than those we obtained previously (20), since experiments were performed at 25 °C. In contrast, JH was not increased by serum in cells expressing NHE1 P705A (0.13 ± 0.02 and 0.14 ± 0.04 H+/min/liter for cells with and without serum, respectively).

Our combined observations support a model in which agonist-induced phosphorylation of NHE1 at Ser703 creates a binding site for 14-3-3 (Fig. 7). In fact, a critical role is indicated for 14-3-3, since mutation of either amino acid required for 14-3-3 binding (Ser703 and Pro705) prevents serum stimulation of Na+/H+ exchange. It would have been interesting to assay NHE1 activity in cells transfected with a dominant negative form of 14-3-3. However, these experiments would be confounded by the fact that dominant negative 14-3-3 mutants also block activation of ERK1/2 (31), which is required for phosphorylation of NHE1 Ser703.


View larger version (21K):
[in this window]
[in a new window]
 
Fig. 7.   Schematic representation of interaction between NHE1 and 14-3-3. In unstimulated cells, 14-3-3 does not bind to NHE1. Upon serum or growth factor stimulation of cells, the MAP kinase cascade is activated, RSK phosphorylates NHE1 at Ser703, and 14-3-3 binds to the exchanger at the RIGpSDP motif. Both Ser703 and Pro705 are necessary for binding of 14-3-3 to NHE1 and for activation of the exchanger by serum, and binding of 14-3-3 and/or another Ser703/Pro705-interacting protein is essential for Na+/H+ exchange activation.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The major finding of the present study is that 14-3-3 interacts with NHE1 in serum-stimulated cells, and our data establish a new functional role for 14-3-3 proteins in the regulation of intracellular pH. We previously found that p90 RSK phosphorylated NHE1 Ser703 in response to serum and that mutation of serine 703 to alanine inhibited serum stimulation of the exchanger. The present study extends these observations and shows that 14-3-3 binds NHE1 at phospho-Ser703 and is a key determinant of exchanger activation by serum. Our results provide the first evidence for interaction of 14-3-3 with the Na+/H+ exchanger and indicate a new role for 14-3-3 proteins as regulators of intracellular pH.

While several pathways have been proposed for serum regulation of NHE1, it is clear that phosphorylation is an essential mechanism (7, 8, 20). The present study defines a critical role for 14-3-3 and serine 703 of NHE1. Previously, deletion of the carboxyl-terminal 180 amino acids of NHE1 was found to reduce agonist-induced activation of the exchanger by ~50% (32). It should be noted that removal of such a large portion of the cytoplasmic tail may have altered its conformation and modified exchanger properties, producing ambiguous results. In comparison, single point mutations of all serines in the 635-670 range did not alter the extent of growth factor-induced NHE1 activation (32). We recently found that phosphorylation of Ser703 is crucial for activation of NHE1 by serum, since a S703A mutation abolishes serum-stimulated activation of the exchanger (20). Consistent with these data, the present study shows that phosphorylation of Ser703 creates a binding site for 14-3-3. The association of 14-3-3 with the exchanger via Ser(P)703 provides a mechanistic explanation for the effects of the S703A mutation on stimulation of NHE1 activity by serum.

In addition to 14-3-3, four other NHE1-interacting proteins have been identified that may modulate exchanger activity: calmodulin (33), 70-kDa heat-shock protein (HSP70) (34), a calcineurin analogue protein (CHP) (35), and a 24-kDa protein yet to be identified (36). Recently, phosphatidylinositol 4,5-bisphosphate (37) was shown to interact with NHE1 as well. Although a role for HSP70 in the negative regulation of the exchanger remains to be demonstrated directly, its interaction with NHE1 is hindered in the presence of ATP, the depletion of which reduces NHE1 activity (34). CHP may also modulate NHE1 activity, since transient overexpression of this protein inhibits serum- and GTPase-stimulated NHE1 activity (35). In comparison, there is compelling evidence that interaction of calcium-calmodulin with NHE1 relieves the exchanger from a negative constraint (33, 38). Finally, Aharonovitz et al. (37) found that elimination of the putative phosphatidylinositol 4,5-bisphosphate-binding sites on NHE1 greatly reduced the ATP-sensitive fraction of Na+/H+ exchange and that an additional, phosphoinositide-independent mechanism also contributes to the effect of ATP on NHE1.

We suggest that the interaction of 14-3-3 with NHE1 modulates exchanger activity by two mechanisms: preventing dephosphorylation and stabilizing an active conformation. It is clear that binding of 14-3-3 to the exchanger prolongs the phosphorylation of Ser703. Our results agree with an earlier report describing increased phosphorylation state of NHE1 accompanied by enhanced exchanger activity in cells treated with okadaic acid, which inhibits PP1 and PP2 (29). The effects of okadaic acid and thrombin were additive and corresponded to phosphorylation of the same NHE1 phosphopeptide, which we expect contains Ser(P)703. Likewise, expression of SV-40 small-t antigen, which binds and inhibits protein phosphatase 2A, increased phosphorylation of NHE1 (39). In addition to this dephosphorylation, 14-3-3 probably exerts other effects such as stabilization of an active conformation, as has been demonstrated for interaction of 14-3-3 with Raf1 (40). Specifically, 14-3-3 may promote dimer formation at Ser703 (24, 28), or assembly of additional signal transduction proteins via a scaffold function (41).

Binding of 14-3-3 to NHE1 is likely to have broad implications for cell pH regulation, since 14-3-3 proteins are ubiquitously expressed and the MEK-ERK-RSK pathway is activated by many stimuli. The present study shows that MEK1 inhibition completely prevents binding of 14-3-3 to NHE1 in cells stimulated with serum. Previous reports show that inhibition of MEK1 or use of a dominant negative ERK1 construct prevents activation of NHE1 by serum, platelet-derived growth factor, angiotensin II, thrombin, phorbol esters, vasopressin, alpha 1-adrenergic receptor agonists, and H2O2 (10, 13-16, 26, 37, 42). Association with 14-3-3 probably participates in the activation of NHE1 by all stimuli acting through the MEK-ERK-RSK pathway, since RSK regulates phosphorylation of Ser703 (20), which we found here to be essential for its binding to 14-3-3.

Our laboratory has focused on mechanisms that regulate NHE1 activity, because enhanced NHE1 activity is a ubiquitous feature of hypertension in humans and several animal models, but how this occurs remains undefined. The present study suggests that alterations in 14-3-3 expression and/or function may be an important component in the pleiotropic alterations in signal transduction observed in hypertension. Increased NHE1 activity in hypertension is best explained by post-translational modification of the exchanger, and indeed phosphorylation of the exchanger is augmented in cells from hypertensive patients (43) and the SHR (7, 44). The present data suggest that increased binding of 14-3-3 to the exchanger, possibly subsequent to increased activity of kinases that phosphorylate Ser703, could contribute to activation of the exchanger in hypertension. Upstream differences in regulation of MAP kinase signaling distinguishing SHR VSMC from WKY VSMC have already been established (18, 19), and our group has characterized a 90-kDa NHE1 kinase, now proven to be RSK, which shows greater activity in SHR than WKY VSMC (10). Furthermore, another NHE1 kinase, p160 ROCK (9), has been implicated in the pathophysiology of hypertension (45). A mechanistic link between these upstream kinases and 14-3-3 is suggested by evidence that 14-3-3 interacts with Raf-1 and several MAP kinase kinases (MEKK1, MEKK2, and MEKK3), although this interaction has not been shown to influence MEKK activity (41). We suggest therefore that a change in the function of 14-3-3 proteins (increased expression and/or altered binding) that promotes increased interaction with 14-3-3 binding partners such as NHE1 (and altered binding partner activity) may be a pathophysiologic feature of hypertension.

    ACKNOWLEDGEMENTS

We thank Masatoshi Kusuhara and members of the Berk laboratory for helpful discussions.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant RO1 HL44721 (to B. C. B.) and a grant from the Canadian Institutes of Health Research (to S. L.).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.

Dagger To whom correspondence should be addressed: Center for Cardiovascular Research, Box 679, 601 Elmwood Ave., University of Rochester School of Medicine and Dentistry, Rochester, NY 14642. Tel.: 716-273-1946; Fax: 716-273-1497; E-mail: bradford_berk@urmc.rochester.edu.

Published, JBC Papers in Press, February 13, 2001, DOI 10.1074/jbc.M100410200

    ABBREVIATIONS

The abbreviations used are: NHE1, Na+/H+ exchanger isoform-1; VSMC, vascular smooth muscle cell(s); ERK, extracellular signal-regulated kinase; GST, glutathione S-transferase; MAP, mitogen-activated protein; MEK, MAP kinase/ERK kinase; SHR, spontaneously hypertensive rat; WKY, Wistar-Kyoto; RSK, ribosomal S6 kinase; FCS, fetal calf serum; PAGE, polyacrylamide gel electrophoresis.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1. Rosskopf, D., Fromter, E., and Siffert, W. (1993) J. Clin. Invest. 92, 2553-2559[Medline] [Order article via Infotrieve]
2. Lucchesi, P. A., DeRoux, N., and Berk, B. C. (1994) Hypertension 24, 734-738[Abstract]
3. Siczkowski, M., Davies, J. E., and Ng, L. L. (1994) J. Hypertens 12, 775-781[Medline] [Order article via Infotrieve]
4. LaPointe, M. S., Ye, M., Moe, O. W., Alpern, R. J., and Batlle, D. C. (1995) Kidney Int. 47, 78-87[Medline] [Order article via Infotrieve]
5. Lifton, R. P., Hunt, S. C., Williams, R. R., Pouysségur, J., and Lalouel, J. M. (1991) Hypertension 17, 8-14[Abstract]
6. Orlov, S. N., Adarichev, V. A., Devlin, A. M., Maximova, N. V., Sun, Y. L., Tremblay, J., Dominiczak, A. F., Postnov, Y. V., and Hamet, P. (2000) Biochim. Biophys. Acta 1500, 169-180[Medline] [Order article via Infotrieve]
7. Siczkowski, M., Davies, J. E., and Ng, L. L. (1995) Circ. Res. 76, 825-831[Abstract/Free Full Text]
8. Sardet, C., Counillon, L., Franchi, A., and Pouysségur, J. (1990) Science 247, 723-726[Medline] [Order article via Infotrieve]
9. Tominaga, T., Ishizaki, T., Narumiya, S., and Barber, D. L. (1998) EMBO J. 17, 4712-4722[Abstract/Free Full Text]
10. Phan, V. N., Kusuhara, M., Lucchesi, P. A., and Berk, B. C. (1997) Hypertension 29, 1265-1272[Abstract/Free Full Text]
11. Takahashi, E., Abe, J., and Berk, B. C. (1997) Circ. Res. 81, 268-273[Abstract/Free Full Text]
12. Aharonovitz, O., and Granot, Y. (1996) J. Biol. Chem. 271, 16494-16499[Abstract/Free Full Text]
13. Bianchini, L., L'Allemain, G., and Pouysségur, J. (1997) J. Biol. Chem. 272, 271-279[Abstract/Free Full Text]
14. Moor, A. N., and Fliegel, L. (1999) J. Biol. Chem. 274, 22985-22992[Abstract/Free Full Text]
15. Snabaitis, A. K., Yokoyama, H., and Avkiran, M. (2000) Circ. Res. 86, 214-220[Abstract/Free Full Text]
16. Wang, H., Silva, N. L., Lucchesi, P. A., Haworth, R., Wang, K., Michalak, M., Pelech, S., and Fliegel, L. (1997) Biochemistry 36, 9151-9158[CrossRef][Medline] [Order article via Infotrieve]
17. Sweeney, F. P., Quinn, P. A., and Ng, L. L. (1997) Metabolism 46, 297-302[CrossRef][Medline] [Order article via Infotrieve]
18. Lucchesi, P. A., Bell, J. M., Willis, L. S., Byron, K. L., Corson, M. A., and Berk, B. C. (1996) Circ. Res. 78, 962-970[Abstract/Free Full Text]
19. Kim, S., Murakami, T., Izumi, Y., Yano, M., Miura, K., Yamanaka, S., and Iwao, H. (1997) Biochem. Biophys. Res. Commun. 236, 199-204[CrossRef][Medline] [Order article via Infotrieve]
20. Takahashi, E., Abe, J., Gallis, B., Aebersold, R., Spring, D. J., Krebs, E. G., and Berk, B. C. (1999) J. Biol. Chem. 274, 20206-20214[Abstract/Free Full Text]
21. Muslin, A. J., Tanner, J. W., Allen, P. M., and Shaw, A. S. (1996) Cell 84, 889-897[Medline] [Order article via Infotrieve]
22. Michaud, N. R., Fabian, J. R., Mathes, K. D., and Morrison, D. K. (1995) Mol. Cell. Biol. 15, 3390-3397[Abstract]
23. Rittinger, K., Budman, J., Xu, J., Volinia, S., Cantley, L. C., Smerdon, S. J., Gamblin, S. J., and Yaffe, M. B. (1999) Mol. Cell 4, 153-166[Medline] [Order article via Infotrieve]
24. Yaffe, M. B., Rittinger, K., Volinia, S., Caron, P. R., Aitken, A., Leffers, H., Gamblin, S. J., Smerdon, S. J., and Cantley, L. C. (1997) Cell 91, 961-971[Medline] [Order article via Infotrieve]
25. Zhang, L., Wang, H., Liu, D., Liddington, R., and Fu, H. (1997) J. Biol. Chem. 272, 13717-13724[Abstract/Free Full Text]
26. Sabri, A., Byron, K. L., Samarel, A. M., Bell, J., and Lucchesi, P. A. (1998) Circ. Res. 82, 1053-1062[Abstract/Free Full Text]
27. Zhang, C., Baumgartner, R. A., Yamada, K., and Beaven, M. A. (1997) J. Biol. Chem. 272, 13397-13402[Abstract/Free Full Text]
28. Wang, H., Zhang, L., Liddington, R., and Fu, H. (1998) J. Biol. Chem. 273, 16297-16304[Abstract/Free Full Text]
29. Sardet, C., Fafournoux, P., and Pouysségur, J. (1991) J. Biol. Chem. 266, 19166-19171[Abstract/Free Full Text]
30. Moolenaar, W. H., Tsien, R. Y., van der Saag, P. T., and de Laat, S. W. (1983) Nature 304, 645-648[Medline] [Order article via Infotrieve]
31. Xing, H., Zhang, S., Weinheimer, C., Kovacs, A., and Muslin, A. J. (2000) EMBO J. 19, 349-358[Abstract/Free Full Text]
32. Wakabayashi, S., Bertrand, B., Shigekawa, M., Fafournoux, P., and Pouysségur, J. (1994) J. Biol. Chem. 269, 5583-5588[Abstract/Free Full Text]
33. Bertrand, B., Wakabayashi, S., Ikeda, T., Pouysségur, J., and Shigekawa, M. (1994) J. Biol. Chem. 269, 13703-13709[Abstract/Free Full Text]
34. Silva, N. L., Haworth, R. S., Singh, D., and Fliegel, L. (1995) Biochemistry 34, 10412-10420[Medline] [Order article via Infotrieve]
35. Lin, X., and Barber, D. L. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 12631-12636[Abstract/Free Full Text]
36. Goss, G., Orlowski, J., and Grinstein, S. (1996) Am. J. Physiol. 39, C1493-C1502
37. Aharonovitz, O., Zaun, H. C., Balla, T., York, J. D., Orlowski, J., and Grinstein, S. (2000) J. Cell Biol. 150, 213-224[Abstract/Free Full Text]
38. Wakabayashi, S., Bertrand, B., Ikeda, T., Pouysségur, J., and Shigekawa, M. (1994) J. Biol. Chem. 269, 13710-13715[Abstract/Free Full Text]
39. Howe, A. K., Gaillard, S., Bennett, J. S., and Rundell, K. (1998) J. Virol. 72, 9637-9644[Abstract/Free Full Text]
40. Tzivion, G., Luo, Z., and Avruch, J. (1998) Nature 394, 88-92[CrossRef][Medline] [Order article via Infotrieve]
41. Fanger, G. R., Widmann, C., Porter, A. C., Sather, S., Johnson, G. L., and Vaillancourt, R. R. (1998) J. Biol. Chem. 273, 3476-3483[Abstract/Free Full Text]
42. Kusuhara, M., Takahashi, E., Peterson, T. E., Abe, J., Ishida, M., Han, J., Ulevitch, R., and Berk, B. C. (1998) Circ. Res. 83, 824-831[Abstract/Free Full Text]
43. Rosskopf, D., Schroder, K. J., and Siffert, W. (1995) Cardiovasc. Res. 29, 254-259[CrossRef][Medline] [Order article via Infotrieve]
44. Berk, B. C., Vallega, G., Muslin, A. J., Gordon, H. M., Canessa, M., and Alexander, R. W. (1989) J. Clin. Invest. 83, 822-829[Medline] [Order article via Infotrieve]
45. Uehata, M., Ishizaki, T., Satoh, H., Ono, T., Kawahara, T., Morishita, T., Tamakawa, H., Yamagami, K., Inui, J., Maekawa, M., and Narumiya, S. (1997) Nature 389, 990-994[CrossRef][Medline] [Order article via Infotrieve]


Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.