Department of Internal Medicine, Department of Veterans Affairs Medical Center and University of Texas Southwestern Medical Center, Dallas, Texas 75235
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ABSTRACT |
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Acute hormonal modulation of NHE3 activity is partly mediated by kinases, including protein kinase C (PKC). We examined the role of NHE3 phosphorylation in regulating its activity in response to PKC activation by phorbol 12-myristate 13-acetate (PMA). In pooled NHE-deficient fibroblasts transfected with NHE3, PMA increased NHE3 activity and phosphorylation. When six potential PKC target serines were mutated, NHE3 phosphorylation was drastically reduced and PMA failed to regulate NHE3 phosphorylation or function. To examine whether NHE3 phosphorylation is sufficient for functional regulation by PKC, we exploited the heterogeneous response of NHE3 activity to PMA in individual clones of transfectants. Clones with stimulatory, inhibitory, or null responses to PMA were observed. Despite the diverse functional response, changes in NHE3 phosphorylation as revealed by tryptic phosphopeptide maps were similar in all clones. We conclude that although phosphorylation appears to be necessary, it is insufficient to mediate PKC regulation of NHE3 function and factors extrinsic to the NHE3 protein must be involved.
proximal tubule; hormones; sodium chloride transport; sodium bicarbonate transport
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INTRODUCTION |
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A SIGNIFICANT FRACTION of the transepithelial absorption of NaCl and NaHCO3 from the glomerular filtrate by the mammalian renal proximal tubule is mediated by the apical membrane Na/H exchanger (1). Regulation of this transporter by neurohormonal effectors represents a key step in extracellular fluid volume and acid-base homeostasis. A number of hormonal agonists that regulate apical membrane Na/H exchange activity generate second messengers that activate protein kinase C (PKC). One Na/H exchanger isoform that mediates proximal tubule apical Na/H exchange activity is NHE3 (2, 5). The classical paradigm of acute reversible modification of protein function by phosphorylation is an attractive model to explain the acute regulation of NHE3 by hormones acting through PKC. Although the effect of PKC activation on NaCl and NaHCO3 absorption has been examined in perfused proximal tubules (4, 13, 23) and Na/H exchange activity has been examined in apical membrane vesicles (19, 24), cultured renal cell lines (7), and fibroblasts transfected with the cloned NHE3 gene (3, 8-11, 16, 20, 27, 29), there are still considerable controversial issues. Although the bulk of the data suggests that PKC activation by phorbol esters inhibits NHE3 activity (3, 4, 8-11, 16, 20, 27, 29), some studies indicate activation of NHE3 by phorbol esters (13, 23, 24). Yip and co-workers (27) showed in PS120 NHE-null fibroblasts that PKC activation inhibits NHE3 activity without changes in NHE3 phosphorylation (27). We now demonstrate in AP-1 NHE-null fibroblasts that acute PKC activation regulates NHE3 activity and phosphorylation. In AP-1 cells, when six potential phosphoserines on NHE3 are deleted, NHE3 phosphorylation is dramatically decreased and there is no functional regulation by PKC, suggesting that NHE3 phosphorylation is necessary for NHE3 regulation. We also showed that the effect of PKC activation on NHE3 function is heterogeneous, in that inhibitory, stimulatory, and null responses could be demonstrated in individual cloned transfected cells, despite identical NHE3 phosphorylation patterns. This unequivocally proves that changes in NHE3 phosphorylation per se are insufficient to determine NHE3 activity. We postulate that the acute regulation of NHE3 activity in response to PKC activation is highly complex and involves NHE3 phosphorylation as well as factors that are extrinsic to NHE3.
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METHODS |
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Cell culture.
The full-length coding region of the rat NHE3 cDNA was tagged with six
histidines in its COOH terminus just before the stop codon by PCR, and
the fidelity of the novel construct was confirmed by sequencing. The
cDNA bearing the 6-His tag (NHE3/6His) was then cloned into the
mammalian expression plasmid pCDNA-1 (pCDNA1/NHE3/6H), stably
transfected along with pSV40Neo into Na/H exchanger-deficient AP-1
cells (Chinese hamster ovarian fibroblasts), and double selected with
G-418 and H+ survival (14). The
transfected cells (AP-1M cells) were maintained in MEM (Sigma Chemical,
St. Louis, MO) supplemented with 10% fetal bovine serum and 400 µg/ml G-418. G-418 was substituted with 100 U/ml penicillin and 100 U/ml streptomycin two passages before experimentation. Experiments were
performed on pooled transfectants (AP-1Mp) as well as individually
cloned cell lines (e.g., AP-1M1 and AP-1M2) obtained by limiting
dilution. Subconfluent monolayers were rendered quiescent by serum
removal for 14-20 h, and activation of PKC was accomplished by the
addition of phorbol 12-myristate 13-acetate (PMA; LC Laboratories,
Woburn, MA). An identical amount of DMSO [always 0.1%
(vol/vol)] was added to control cells. Additional controls were
performed using the inactive phorbol ester 4--PMA. PKC blockade was
performed by pretreating the cells for 3 h with 10 µM staurosporine
(inhibitory constant = 1 µM) before addition of PMA. Activation of
protein kinase A (PKA) was achieved by incubation with 100 µM
8-bromo-cAMP for 25 min. Ethylisopropylamiloride (EIPA) sensitivity was
tested by incubating the cells for 30 min before and during the NHE3
activity assay with EIPA at the specified concentrations.
Na/H exchanger activity assay. NHE3 activity was measured in confluent cells on glass coverslips fluorometrically (SLM, Urbana, IL) with the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein and defined as Na+-dependent intracellular pH (pHi) recovery (dpHi/dt) after acid loading with the K/H ionophore nigericin with perfusion solutions exactly as previously described (14). In studies with EIPA the Na+ concentration was lowered in the Na+-containing solution to 20 mM by isotonic replacement with choline. Buffer capacity was calculated by measuring dpHi in response to a 20 mM NH4Cl pulse at the trough pH. PMA treatment did not alter the nigericin calibration (pH = 5.52 + 0.272 × F500/450 and 5.46 + 0.285 × F500/450 for control and PMA-treated groups, respectively, where F500/450 is the ratio of fluorescence at 500 nm to that at 450 nm) or the buffer capacity (24.7 and 23.1 mM/pH unit for control and PMA-treated groups, respectively) of AP-1M cells. Because of some variations in absolute baseline values of dpHi/dt in cells over the period during the study, pooled results were expressed as percentage of controls assayed on that particular day.
In vivo phosphorylation and purification by immunoprecipitation or
Ni affinity chromatography.
NHE3 phosphorylated in vivo was purified by immunoprecipitation or Ni
affinity chromatography. After incubation in phosphate-free DMEM for 30 min, cells were labeled with phosphate-free medium containing
[32P]orthophosphate
(100-330 µCi/ml) for 90 min. After incubation with 100 nM PMA or
vehicle (DMSO) for 20 min, the cells were washed, lysed with RIPA
buffer [150 mM NaCl, 80 mM NaF, 50 mM
Tris · HCl, pH 8.0, 5 mM EDTA, 1 mM EGTA, 25 mM
sodium pyrophosphate, 1 mM sodium orthovanadate, 1% NP-40 (vol/vol),
0.5% deoxycholate (wt/vol), 0.1% SDS (wt/vol), 100 µg/ml
phenylmethylsulfonyl fluoride, 4 µg/ml leupeptin, 4 µg/ml
aprotinin, 10 µg/ml pepstatin], and the insoluble pellet was
removed by centrifugation (109,000 gmax, 50,000 rpm,
25 min, 2°C; TLX:TLA 100.3 rotor, Beckman). NHE3 was immunoprecipitated from the supernatant with use of antiserum 1314 (rabbit antisera against fusion protein of maltose-binding protein/rat
NHE3 amino acids 405-837) (14) and protein A-Sepharose beads
(Sigma Chemical), washed, and eluted in SDS buffer [5 mM Tris · HCl, pH 6.8, 10% (vol/vol) glycerol, 1%
(wt/vol) -mercaptoethanol, 0.1% (wt/vol) SDS, 0.01% (wt/vol)
bromphenol blue]. For NHE3 purification by Ni affinity
chromatography, cells were lysed in buffer
B1 [8,000 mM urea, 100 mM
Na2PO4,
10 mM Tris, pH 8.0, 30% (vol/vol) glycerol, 0.5% (vol/vol) Triton
X-100], sheared with a 25-gauge needle, and combined with an
equal volume of buffer B2 (similar to
buffer B1, but without glycerol, plus
20 mM imidazole and 10 mM 2-mercaptoethanol). The lysate was clarified
by centrifugation (109,000 gmax),
equilibrated with Ni-NTA resin (Qiagen, Chatsworth, CA) by rocking for
1 h at room temperature, and washed [8,000 mM urea,
100 mM
Na2PO4, 50 mM imidazole, 10 mM Tris, pH 8.0, 0.5% (vol/vol) Triton
X-100], and NHE3 was eluted from the resin (8,000 mM urea,
100 mM
Na2PO4, 500 mM imidazole, 10 mM Tris · HCl, pH 8.0). Purified
NHE3 was processed for SDS-PAGE, and immunoblotting was performed with antipeptide antiserum 1568 (2) by use of enhanced chemiluminescence (Amersham, Arlington Heights, IL). The
32P content of NHE3 was visualized
by autoradiography on the same filters after decay of enhanced
chemiluminescence. Both signals were quantified by densitometry, and
changes in phosphorylation were normalized to the antigenic signal. In
general, both methods of purification yielded similar results in terms
of changes in phosphorylation. Because the six-serine mutant NHE3
(NHE3/6Smut, see below) was
expressed in much lower antigenic amounts than wild-type NHE3 in AP-1
cells, we performed some studies where five to six plates of cells
expressing NHE3/6Smut were pooled
to load identical amounts of
NHE3/6Smut and wild-type NHE3 on
the same lanes on SDS-PAGE gels.
In vitro phosphorylation of NHE3.
NHE3 purified from bacterial or mammalian cells was tested as PKC
substrate in vitro. For bacteria-derived NHE3, a recombinant bacterial
fusion protein of maltose-binding protein and the cytoplasmic domain of
NHE3 (MBP/NHE3cyto) was
generated in Escherichia coli and
purified by amylose affinity chromatography. Fusion protein (1.5 µg)
was incubated with 1.5 U of purified PKC (Calbiochem, La Jolla, CA)
in reaction buffer (0.1 mM
CaCl2, 10 mM
MgCl2, 20 mM
Tris · HCl, pH 7.5, 0.02 mM ATP, 10 µCi
[
-32P]ATP,
10
7 M PMA, 4 µg
phosphatidylserine) at 22°C. Control contained all the reagents
except PKC
. The phosphorylated product was resolved by SDS-PAGE and
dried for autoradiography. For mammalian cell-expressed NHE3, AP-1M
cells were lysed with buffers B1 and
B2, cleared by centrifugation,
immobilized on Ni-NTA agarose resin as mentioned above, and partially
renatured using a linear urea gradient (wash buffer: 300 mM NaCl, pH
6.0, 20 mM Tris · HCl, 6 to 0.5 M urea). The resin
was incubated at 30°C for 25 min in reaction buffer (500 mM urea,
pH 7.5, 20 mM Tris · HCl, 300 nM NaCl,
0.25 mM CaCl2, 5 mM magnesium acetate, 0.02 mM ATP, 20 mM imidazole, 100 nM PMA, 50 µCi [
-32P]ATP, 8 µg/ml sonicated phosphatidylserine micelles) plus 3 U of purified
PKC
. After the resin was rinsed with wash buffer, NHE3 was eluted
(8,000 mM urea, 100 mM
Na2HPO4,
pH 6.0, 10 mM Tris · HCl, 500 mM imidazole) for
SDS-PAGE and tryptic phosphopeptide mapping.
Phosphoamino acid analysis. Phosphoamino acid analysis was performed by two-dimensional electrophoresis (6). Purified phosphoproteins were eluted from the acrylamide gel by 50 mM NH4HCO3, precipitated with TCA, and hydrolyzed by boiling in 6 M HCl. Equal counts per minute from control and PMA groups were spotted onto TLC plates, and 32P-labeled amino acids were resolved in a Hunter TLC electrophoresis unit (model HTLE 7000, CBS Scientific, Del Mar, CA; 1st dimension: 2.2% formic acid-7.8% acetic acid, pH 1.9; 2nd dimension: 5% acetic acid-0.5% pyridine, pH 3.5) along with cold standards (P-Ser, P-Thr, P-Tyr). Phosphoamino acids were identified by autoradiography and aligned with ninhydrin-stained standards.
Tryptic phosphopeptide mapping. The in vivo or in vitro phosphorylated NHE3 was purified by immunoprecipitation or affinity chromatography, fractionated by SDS-PAGE, and transferred to a nitrocellulose membrane. No differences were noted between the two purification methods. Membrane pieces corresponding to the immunoprecipitated NHE3 protein were localized by autoradiography, excised, and placed in 100 mM acetic acid containing 0.5% polyvinylpyrrolidone (Sigma Chemical) at 37°C for 30 min, washed, and incubated with 15 µg of N-tyrosyl-L-phenylalanine chloromethyl ketone-treated trypsin (Worthington Biochem, Freehold, NJ) in 0.05 M NH4HC03 at 37°C. Trypsin was added twice more for a total incubation of ~12 h. The sample was washed repeatedly and lyophilized until no salt residues were visible. The dried sample was resuspended in electrophoresis buffer [per liter: 25 ml formic acid (88% wt/vol), 78 ml glacial acetic acid, 897 ml deionized water, pH 1.95], and electrophoresis and chromatography were performed (6). Briefly, the sample was spotted on a cellulose TLC plate (Merck, Darmstadt, Germany), and electrophoresis was performed in a Hunter unit with the buffer described above at 1.0 kV for 30 min. Separation of the peptides in the second dimension was achieved by ascending chromatography [375:250:75:300 n-butanol-pyridine-glacial acetic acid-deionized water (vol/vol/vol/vol)]. The phosphopeptides were visualized by autoradiography.
Site-directed mutagenesis. Because all phosphorylated residues on NHE3 were serines (see RESULTS), we arbitrarily mutated six serines conforming to consensus motifs for PKC substrate to nonphosphorylatable residues. Starting from the pcDNAI/NHE3/6H expression vector, point mutations of nucleotides were introduced sequentially by a double-strand PCR method (QuickChange kit, Stratagene, La Jolla, CA). After the template was annealed with mutagenic oligonucleotides, mutated strands were generated by arithmetic PCR with Pfu DNA polymerase. The methylated nonmutated parental templates were digested with Dpn I, and the mixture was transformed into competent E. coli. cDNAs were isolated, and mutations were confirmed by direct sequencing at each step. The final six-point NHE3 mutant (pcDNAI/NHE3/6Smut) harbors the following mutations shown with the flanking amino acid sequences: S513G (RKFLS513KV), S552A (ERRGS552LA), S575A (TPRPS575TV), S661A (RKRLES661FL), S690G (KRRNS690SI), and S804G (FRLS804NK). A new cell line (AP-1Z) was generated from NHE-null AP-1 cells, as described above.
PKC isoform expression and translocation.
To measure total cellular content of PKC isoforms, cells were
solubilized in 2 ml of solution A
[20 mM Tris · HCl, pH 7.5, 1 mM EDTA, 10 mM
EGTA, 1.0 mM phenylmethylsulfonyl fluoride, 0.5 mM leupeptin, 0.15 mM
pepstatin, 1 mM dithiothreitol, 1% (vol/vol) NP-40],
homogenized, and centrifuged (109,000 gmax). Protein
from the supernatant (100 µg) and 100 µg of rat cortex were
resolved by SDS-PAGE, transferred to a nitrocellulose membrane, and
blotted with polyclonal PKC isozyme-specific antiserum (generous gift from Sloan Stribling and Yusuf Hannun, Duke University) and
subsequently with goat anti-rabbit antibody diluted as follows:
1:10,000 and 1:20,000 for PKC primary and secondary, respectively;
1:2,000 and 1:2,000 for
PKC
1 primary and secondary,
respectively; 1:2,000 and 1:2,000 for
PKC
2 primary and secondary,
respectively; 1:5,000 and 1:10,000 for PKC
primary and secondary,
respectively; 1:20,000 and 1:40,000 for PKC
primary and secondary,
respectively; 1:20,000 and 1:20,000 for PKC
primary and secondary,
respectively; 1:5,000 and 1:10,000 for PKC
primary and secondary,
respectively; and 1:10,000 and 1:20,000 for PKC
primary and
secondary, respectively. Albumin (5%) was used as blocking agent,
except for the two isoforms where 5% powdered milk was used. For the
translocation assay of PKC activation in response to PMA, cells were
incubated with 100 nM PMA or DMSO (control) for 20 min, homogenized in
solution A without NP-40, and
centrifuged (109,000 gmax), and the
supernatant was retained (cytosolic fraction). The pellet was
solubilized in buffer A and
centrifuged (109,000 gmax), and the
resultant supernatant was retained (membrane fraction). SDS-PAGE and
immunoblotting were performed.
NHE isoform mRNA. Monolayers of AP-1M cells were lysed in GTC [4 M guanidinium thiocyanate, 0.1 M 2-mercaptoethanol, 0.025 M sodium citrate, pH 7.0, 0.5% (wt/vol) N-lauroylsarcosine], and RNA was extracted with phenol-chloroform, ethanol precipitated, size fractionated by formaldehyde gel electrophoresis, and transferred to a nylon membrane. After prehybridization for 4 h at 45°C in 5× Denhardt's solution (1 mg/ml each Ficoll, polyvinylpyrrolidone, BSA), 5× SSC (0.75 M NaCl, 75 mM sodium citrate), 0.5% SDS, 50% formamide, and 0.5 mg/ml sheared salmon sperm DNA, the membrane was hybridized to hexamer-primed uniformly 32P-labeled cDNAs (NHE1: 1.9-kb BamH I fragment; NHE2: 1.85-kb Ava I fragment; NHE3: 1.2-kb Pst I fragment; NHE4: 0.2-kb Nsi I-BspE I and 0.63-kb BspE I fragments) overnight at 42°C, washed in 2× 0.1% SSC for 15 min at 20°C and then in 0.1% SSC-1% SDS for 60 min at 50°C, and exposed to film. The filters were stripped and reprobed for 28S rRNA.
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RESULTS |
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Acute PKC activation increases NHE3 activity and
phosphorylation in pooled transfectants.
PKC activation by PMA stimulated NHE3 activity in a dose- and
time-dependent fashion in the pooled transfectants (Fig.
1). For the rest of studies, 100 nM PMA was
applied for 20 min. PMA did not affect resting cell pH [7.42 ± 0.19 and 7.43 ± 0.22 (SD) for control and PMA-treated cells,
respectively] or the trough pHi at which NHE3 activity was
measured (6.39 ± 0.10 and 6.37 ± 0.11 for control and
PMA-treated cells, respectively). PMA failed to stimulate NHE3 activity
in cells pretreated with the PKC inhibitor staurosporine [6.2%
decrease in PMA-treated vs. control cells, not significant (NS)]
or when the inactive phorbol ester 4--PMA (100 nM for 20 min) was
used instead of PMA (9% decrease in 4-
-PMA-treated vs. control
cells, NS). These results suggest that the stimulation of NHE3 activity
in pooled transfectants by PMA involves activation of PKC. We next
examined whether this acute change in activity was accompanied by
changes in NHE3 phosphorylation. When NHE3 was purified from AP-1M pool
cells labeled with
32PO4
in vivo, PMA increased net phosphorylation of NHE3 by 50-150% compared with control cells (n = 8 pairs of control vs. PMA-treated cells,
P < 0.05; Fig.
2A).
Phosphoamino acid analysis revealed phosphorylation solely of serine
residues at baseline as well as in response to PMA, as shown in Fig.
2B (equal counts per minute were
loaded for control and PMA-treated cells). These studies demonstrate
that acute PKC activation simultaneously leads to increased activity
and phosphorylation of NHE3 in pooled transfectants.
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NHE3 is a direct substrate for PKC in vitro.
Because the cytoplasmic domain of NHE3 contains numerous consensus
motifs for PKC phosphorylation, we tested whether this region of the
protein is a direct substrate for PKC in vitro. Figure
3A shows
that the recombinant bacterial fusion protein MBP/NHE3cyto is phosphorylated by
PKC in vitro. In the intact cell, PKC may directly phosphorylate NHE3
and/or activate another kinase that phosphorylates NHE3. To show that
at least a component of the phosphorylation is direct, we subjected
intact whole NHE3 purified from mammalian cells to phosphorylation by
purified PKC in vitro (Fig. 3B).
Because cryptic phosphoacceptor sites may be exposed to PKC in vitro,
which may have dubious relevance in vivo, we compared the tryptic
phosphopeptide map of whole NHE3 phosphorylated in vitro with that
phosphorylated in vivo (Fig. 3B).
There were indeed in vitro sites not present in the in vivo phosphorylated protein; however, there were a number of common sites
(B, C, I, and H in Fig. 3B), which
suggest that at least a component of NHE3 is phosphorylated directly by
PKC in vivo.
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PKC activation does not regulate phosphorylation or activity in
mutant NHE3.
To examine whether changes in phosphorylation and activity are causally
related, we mutated six serines in the cytoplasmic domain of NHE3 that
are potential sites for PKC phosphorylation and expressed the mutant
NHE3 in NHE-deficient AP-1 cells. These cells were examined
simultaneously with the wild-type NHE3 controls. The baseline
expression of NHE3 activity and whole cell NHE3 antigen was decreased
proportionately in
NHE3/6Smut-expressing (AP-1Z
cells) compared with the wild-type cells (AP-1Mp; Fig.
4). We could not normalize NHE3 activity to
surface NHE3 antigen because of the inability to consistently
biotinylate surface NHE3 in AP-1 cells. There were no major qualitative
differences in staining pattern between NHE3 (AP-1M cells) and
NHE3/6Smut (AP-1Z cells) by
confocal immunocytochemistry. Baseline phosphorylation of the
NHE3/6Smut protein is drastically
reduced. Figure 4C, right, shows the
reduced but still detectable phosphorylation of
NHE3/6Smut when equal
amounts of wild-type NHE3 and
NHE3/6Smut were loaded
(65, 78, and 71% reduction in
32P-NHE3 normalized to NHE3
antigen in 3 independent experiments). On addition of PMA, although
wild-type NHE3 was activated and phosphorylated as previously
described, no significant change in phosphorylation or activity was
observed in the six-serine mutants (Fig. 4; increase in
32P-NHE3/NHE3 antigen for wild
type vs. NHE1/6Smut in 3 independent experiments: 105% increase vs. 10% decline, 52% increase
vs. 9% decline, 100% increase vs. 13% increase). These studies
showed that although the six serine mutations do not significantly
affect the baseline activity of the NHE3 protein, the lack of
phosphorylation of NHE3 renders it nonregulatable by PKC activation.
Because we observed clonal heterogeneity in the response of NHE3
activity to PMA (see below), we wanted to rule out that the lack of
response of AP-1Z cells may be due to selection of a nonresponding
clone rather than NHE3/6Smut. We
cloned AP-1Z cells by limiting dilution and examined NHE3 activity in
five clones along with the parent pooled AP-1Z cells and found no
regulation of NHE3 activity by PMA in all clones (22Na uptake in
pmol · 30 s1 · mg
protein
1 in control vs.
PMA-treated cells: pooled AP-1Z, 773 ± 104 vs. 765 ± 89;
AP-1Z1, 572 ± 103 vs. 593 ± 56; AP-1Z2, 645 ± 34 vs. 607 ± 89; AP-1Z3, 708 ± 89 vs. 781 ± 106; AP-1Z4, 498 ± 76 vs. 523 ± 86; means ± SE, n = 4 pairs for each cell type, all NS, unpaired
t-test).
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Clonal heterogeneity of the functional response of NHE3 to PMA.
The stable pooled transfectants were cloned by limiting dilution, and
individual clones were screened for NHE3's functional response to PMA.
This was performed originally with the intent to isolate maximally
responding clones for further characterization. A total of nine clones
were screened, and a serendipitous heterogeneous response was observed.
Seven of the nine clones showed comparable degrees of stimulation by
PMA (24-60% stimulation, P < 0.05 for each), with two of the nine clones showing different
responses. The results from four representative clones are summarized
in Fig. 5,
A and
B. Clone M1 showed no functional
response of NHE3 to PMA (n = 27 pairs,
NS). PMA inhibited NHE3 activity in clone M2 by 11%
(n = 28 pairs,
P = 0.006). Clones M4 and M10
represent two of the seven clones where NHE3 activity was stimulated by PMA (60% increase, n = 28 pairs,
P < 0.001; and 44% increase, n = 6 pairs,
P < 0.0001, respectively). These
effects were consistent in the four clones when repeatedly tested over
time independent of cell passage number. To test whether this disparate
response is specific for PKC, we examined the effect of PKA activation on NHE3 activity in these four clones. Figure
5C shows that PKA activation uniformly
led to inhibition of NHE3 activity in clones M1, M2, M4, and M10. These
data suggest that intrinsic differences in cellular context among these
clones confer a differential functional response of NHE3 to PKC
activation. We next exploited this clonal heterogeneity as a model to
try to study whether functional regulation is paralleled by changes in
NHE3 phosphorylation.
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Examination of differential PKC isoform expression and activation.
A possible explanation for the heterogeneous response to PMA is
differential PKC isoform expression in the different clones. Immunoblots were performed with PKC isoform-specific antisera, and the
results are shown in Fig.
6A. All
clones showed the same pattern of expression with minor quantitative
differences (Fig. 6A). To rule out
the possibility of differential activation of the isoforms, we used a
membrane translocation assay to study activation of PKC, the most
abundant isofom, by PMA in the four representative clones. Figure
6B shows that PKC
was activated to
similar degrees in all four clones. Similar translocations were seen
with PKC
and PKC
, although the signals were much weaker (data not
shown). These studies show no major differences in PKC isoform
expression or activation to account for the observed clonal heterogeneity.
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Examination of differential expression of NHE isoforms.
AP-1 cells are fibroblasts derived from Chinese hamster ovary cells
that have been randomly mutagenized and functionally selected to
exhibit no functional Na/H exchange. Therefore, AP-1M cells should
express the transfected NHE3 protein only. However, spontaneous phenotypic reversions, i.e., restoration of Na/H exchange activity, presumably from expression of native NHE genes, has been observed in
these cells (unpublished observation; C. Helmle-Kolb and H. Murer,
personal communication). Differential clonal NHE isoform expression
other than NHE3 may account for the observed heterogeneity. To test
this hypothesis, we examined the four representative clones for NHE
isoform transcripts (Fig.
7A). The
transfected NHE3 transcript is highly expressed in all four clones. The
expected transcript from the untranslated region-free transfected rat
NHE3 cDNA is ~2.6 kb. Because the pcDNA series of expression vectors
harbors SV40 intervening and polyadenylation sequences, partially
spliced slower-migrating transcripts are often observed, as evident in M pool and clone M4. The native NHE3 transcript in the positive rat
cortex control is shown as the expected 5.6-kb message. There is no
expression of NHE2 or NHE4 in all clones examined. However, faint
labeling of NHE1 was detected in M4 and M10 after prolonged exposure.
If the low-level NHE1 transcript is translated to mature surface NHE1
protein to any significant extent in M4 and M10, it may explain the
stimulatory effect of PMA on Na/H exchange activity in M4 and M10,
since NHE1 has previously been shown to be activated by PKC. To rule
out this possibility, we performed functional studies on the clones
with the competitive NHE inhibitor EIPA. A dose response of EIPA was
performed, and results from two representative clones are shown in Fig.
7B. EIPA kinetics in clone M2 (NHE3
activity inhibited by PMA) were identical to those in clone M10 (NHE3
stimulated by PMA). A single uniphasic IC50 of 6 µM characteristic of
NHE3 (10, 16) was observed for both clones with no evidence of
functional inhibition in the nanomolar range (characteristic for NHE1),
thereby excluding significant expression of NHE1 activity. For further
confirmation, we repeated the PMA experiment in the presence of 1 µM
EIPA, which completely inhibits any potential NHE1 activity. Figure
7C demonstrates that clones M2
(inhibitory) and M10 (stimulatory) exhibit the same response to PMA in
the presence and absence of 1 µM EIPA, ruling out differential
contributions from NHE isoforms as the basis for clonal heterogeneity.
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Phosphorylation and phosphopeptide mapping of NHE3 in response to
PMA in the clones.
Although identical NHE and PKC isoforms are expressed in the four
clones, differential phosphorylation of NHE3 could account for the
clonal differences. We explored whether there is a correlation between
changes in activity and phosphorylation of NHE3. As summarized in Fig.
8, regardless of whether NHE3 activity was
inhibited (M2), stimulated (M4 and M10), or not affected by PMA (M1),
NHE3 phosphorylation was increased by PMA. This study clearly
dissociates changes in NHE3 whole protein phosphorylation from changes
in NHE3 activity in response to PMA. However, the possibility remains
that although net phosphorylation increases in all four clones,
distinct phosphorylated residues may have different functional effects,
and measurement of total phosphorylation is inadequate to address this.
To examine this, we performed tryptic phosphopeptide mapping in all
four clones, and representative two-dimensional maps for M1, M2, and M4
are shown in Fig. 9. PMA induced three
novel phosphorylation sites (D, I, and K) and increased phosphorylation
in three existing sites in all four clones (E, F, and J; Fig. 9). Two
sites (C and B) showed constitutive phosphorylation. When the maps from
the four clones (4 pairs of control vs. PMA per clone) were compared, only minor quantitative differences in specific phosphoacceptor sites
but no consistent differential phosphorylation patterns could be
discerned. Hence, PMA induces phosphorylation of the same serine
residues in clones M1, M2, M4, and M10, whereas the functional response
shows an increase, a decrease, or no change in activity. This
unequivocally proves that phosphorylation of NHE3 per se is
insufficient to regulate transporter activity.
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DISCUSSION |
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There are four findings in this study: 1) PKC activation can activate or inhibit NHE3 activity. 2) PKC activation increases NHE3 phosphorylation on multiple serines. 3) NHE3 phosphorylation plays a role in regulation of NHE3 activity by PKC in the present system. 4) NHE3 phosphorylation per se is insufficient to regulate NHE3 activity by PKC. We discuss each of these in relation to the existing data.
Acute PKC activation increases HCO3 and volume absorption in the in vivo perfused rat proximal tubule (13), whereas PKC activation decreases the same parameters in the isolated perfused rabbit proximal tubule (4). Wang and Chan (23) attempted to resolve this discrepancy by demonstrating a time-dependent biphasic response to PMA of early stimulation and late inhibition of proximal tubule HCO3 and volume absorption. In the present study, dose and time dependence was demonstrated, but a biphasic response was not observed. In renal cortical apical membrane vesicles, PMA increases Na/H exchange activity (19, 24). Because current immunohistochemical, genetic, and functional evidence shows that only NHE3 is expressed and functional in proximal tubule apical membrane (2, 5, 18, 26), the data from apical membrane vesicles (19, 24) can be interpreted to reflect NHE3. In renal epithelial cell lines expressing NHE3, PMA treatment inhibits NHE3 activity (7). When rabbit NHE3 was expressed in the fibroblast-derived PS120 NHE-deficient cell line, PMA decreased the maximal reaction velocity of NHE3 without changing its affinity for Na+ or H+ (9-11, 20, 27, 29). Azarani, Kandasamy, and co-workers (3, 8) expressed NHE3 in AP-1 cells and found inhibition of its activity by a high concentration (1 µM) of PMA in pooled cells. Our finding in pooled transfectants is in contradistinction to the previous results in pooled transfectants. When we cloned our cells, we showed that PKC activation can stimulate, inhibit, or have no effect on NHE3 activity, even in clones derived from the same parental cell line. Different cellular context may explain the difference between the findings in AP-1 and PS120 cells. However, Azarani, Kandasamy, and co-workers observed inhibition in AP-1 cells. When we applied 1,000 nM PMA in our pooled transfectants, we still observed stimulation of NHE3 (data not shown). Different clonal dominance in pooled cells may explain the discrepancy between our results and those of Azarani, Kandasamy, and co-workers. The present study does not provide an explanation for the observed variability. However, it underscores the complexity and the cell context dependence of the functional response of NHE3 to PKC activation, which is consistent with the variable results in the literature.
The cytoplasmic domain of NHEs contains numerous putative
phosphorylation sites for multiple kinases. NHE1 and NHE3 have been shown to be phosphoproteins (9, 14, 17, 27). An increase in NHE3
phosphoserines is now noted in response to PKC activation. Fourteen of
the 40 serines in the predicted cytoplasmic domain of rat NHE3 conform
to motifs for potential phosphoacceptor sites for various kinases. It
is not surprising to find a complex tryptic phosphopeptide map. There
are a total of 11 major tryptic phosphopeptide fragments. Of these,
phosphate content increased in six in response to PKC activation, and
with prolonged exposure, other minor fragments could also be
identified. The present data do not identify which phosphopeptide
corresponds to which specific serine. It is difficult to show that a
protein is a direct substrate for a specific kinase in vivo. The direct
in vitro phosphorylation of the NHE3 cytoplasmic domain by PKC and
the presence of common phosphoacceptor sites from the in vivo and in
vitro phoshorylated protein suggest that PKC likely directly
phosphorylates NHE3 in vivo. Sites present only in the in vitro
reaction may reflect exposure of cryptic sites under the partial
denaturing conditions of the lipid micelle environment. Sites
present only in the in vivo reaction may represent participation of
other kinases activated by PMA. In fact, the complexity of the in vivo
map suggests that PKC likely activates other kinases that phosphorylate
NHE3. A major discrepancy exists between our results and that of Yip
and co-workers (27), who reported that there were multiple
phosphorylated sites on rabbit NHE3 when expressed in PS120 cells but
that PMA addition did not increase NHE3 phosphorylation. Although Yip
and co-workers studied rabbit rather than rat NHE3, the difference
likely resides in the host cell rather than the NHE3 protein. It is
conceivable that phosphorylation of NHE3 by PKC depends on other
cellular factors that are absent in PS120 cells. The lack of
cAMP-induced inhibition of NHE3 in PS120 cells compared with AP-1 cells
was shown by Yun and co-workers (28) to be due to lack of specific regulatory proteins.
We next addressed whether NHE3 phosphorylation is important for its
functional regulation. Commensurate changes in phosphorylation and
activity have been described for NHE1 and NHE3 (9, 14, 17). Cytoplasmic
truncation of NHE1 or NHE3 harboring all phosphorylation sites rendered
the transporter largely constitutive (21, 22, 29). However, gross
alterations in protein structure obliterate more than just
phosphorylation sites. Smaller internal deletions of the NHE1
cytoplasmic domain eliminated phosphorylation only partially but
abolished regulation by growth factors (21, 22). Point mutations of
serines in the cytoplasmic domain of NHE3 should theoretically perturb
protein structure to a lesser extent than truncations. For unclear
reasons, baseline protein expression is about five times lower in the
six-point mutant. It is possible that the mutations affect NHE3 protein
half-life. However, activity per antigen in
NHE3/6Smut is not significantly
altered, indicating that the mutations likely did not grossly affect
baseline transport function. We acknowledge that we do not have a
quantitative measure of the relative distribution of plasma membrane
vs. intracellular NHE3, although qualitatively the distributions of
wild-type NHE3 and NHE3/6Smut
appeared comparable. Although not abolished, phosphorylation of
NHE3/6Smut was reduced by
65-80%. The key finding is that, in
NHE3/6Smut, activation of PKC did
not induce changes in NHE3 activity or phosphorylation. Although
unlikely, a possibility remains that there is a small degree of
inhibition of NHE3/6Smut activity
by PMA, and the low baseline activity precluded its detection. The
finding of simultaneous abolition of PMA-induced regulation of function
and phosphorylation supports the necessity of phosphorylation for
functional regulation. However, it is in contradistinction to the
results of Yip and co-workers (27), who found changes in NHE3 activity
without changes in phosphorylation. One possible explanation is that
PMA modulates NHE3 activity via phosphorylation-dependent and
phosphorylation-independent mechanisms. One can speculate that some
degree of NHE3 phosphorylation is required for the functional
regulation. Although direct comparison is not possible because of
species difference, the phosphopeptide map of rabbit NHE3 in PS120
cells (27) revealed much more phosphorylated sites than that of rat
NHE3 in AP-1 cells under control conditions. If there are
phosphorylation-dependent and phosphorylation-independent mechanisms in
mediating the functional regulation of NHE3, it is conceivable that
there is sufficient NHE3 phosphorylation of rabbit NHE3 in PS120 cells
at baseline to permit phosphorylation-independent mechanisms to
regulate NHE3 activity on addition of PMA. Phosphorylation was
demonstrated to be necessary for regulation of NHE3 by PKA by Kurashima
and co-workers (9) (Ser-605 and Ser-634) and Zhao and co-workers (30)
(Ser-552 and Ser-605). Ser-552 was included in the
NHE3/6Smut, although the role of
individual serines has not been established for PKC. Interestingly,
acute hyperosmolar stress (osmolarity = 150 mM mannitol, 1 min)
suppressed the activity of
NHE3/6Smut by 45%
(n = 5, P < 0.05), indicating intact
regulation of NHE3 activity in the absence of changes in
phosphorylation. This is in agreement with Nath and co-workers (15),
who found that NHE3 protein with completely cytoplasmic truncations was
inhibited by hyperosmolarity.
If NHE3 phosphorylation is necessary, is it sufficient to regulate activity? This is difficult to prove in the intact cell, inasmuch as one cannot exclude simultaneous contributions from other cellular factors. In the present study we used the serendipitous clonal heterogeneity in phenotype to look for correlation between phosphorylation and activity. The basis for the differential response likely did not reside in differences in PKC. Because NHE1 is stimulated by PKC activation, expression of native NHE1 in clones M4 and M10 could possibly account for the stimulation observed. Indeed, these two clones did appear to express very low levels of NHE1 transcript. However, the EIPA pharmacokinetics and the effect of PMA on NHE activity in the presence of 1 µM EIPA unequivocally proved that NHE3 was the sole isoform expressed and regulated in our cells. The possibility remains that the disparate NHE3 phosphorylation pattern may explain the heterogenous functional response. We then demonstrated that total NHE3 protein and site-specific NHE3 phosphorylation were similar in all clones. The dissociation of phosphorylation and NHE3 activity therefore rules out phosphorylation as a sole mechanism of regulation of activity. This finding is in agreement with that of Yip and co-workers (27). The present data indicate indisputably that factors extrinsic to NHE3 must be involved in its regulation by PKC. The NHE regulatory factor family of proteins interacts with NHE3 and is functionally important for PKA's ability to regulate NHE3 (25, 28). Although the present findings are compatible with cofactors mediating regulation of NHE3 by PKC, the roles of the NHE regulatory factor proteins in the PKC effect remain to be determined. PKC activation also leads to an increase in NHE1 activity and phosphorylation (17). Deletion of the domain that contains all phosphorylated residues only partially abolished the regulation, whereas deletion of a more proximal portion completely abrogated the stimulation of NHE1 by growth factors (21, 22). A cofactor recently identified that binds to this region is the calcineurin homologous protein (12), which downregulates the ability of serum to activate NHE1. We propose a paradigm for regulation of NHE3 by PKC that involves phosphorylation of NHE3 cytoplasmic domain as prerequisite but by itself insufficient to alter transporter function. Only in conjunction with NHE3 interacting with other cofactors does PKC phosphorylation of NHE3 lead to modification of transporter activity.
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ACKNOWLEDGEMENTS |
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The authors acknowledge the valuable technical expertise of Ladonna A. Crowder. The authors are also indebted to Sloan Stribling and Yusef Hannun for providing the PKC isoform-specific antisera, Melanie Cobb and David Russell for discussions and helpful advice, and Michel Baum and Robert Alpern for careful reading of the manuscript.
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FOOTNOTES |
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These studies were supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-48482-01, the Research Service of the Department of Veterans Affairs, and the National Kidney Foundation of Texas.
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: O. W. Moe, Dept. of Internal Medicine, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75235-8856 (E-mail: omoe{at}mednet.swmed.edu).
Received 10 November 1998; accepted in final form 17 February 1999.
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