Ca2+-dependent inhibition of NHE3 requires PKC{alpha} which binds to E3KARP to decrease surface NHE3 containing plasma membrane complexes

Whaseon Lee-Kwon,1,* Jae Ho Kim,1,2,* Jung Woong Choi,1 Kazuya Kawano,1 Boyoung Cha,1 Darlene A. Dartt,3 Driss Zoukhri,3 and Mark Donowitz1

1Department of Medicine, Gastrointestinal Division, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205; 2Division of Physiology, Pusan National University, Pusan, Republic of Korea; and 3The Schepens Eye Research Institute, and Harvard Medical School, Boston, Massachusetts 02114

Submitted 13 January 2003 ; accepted in final form 8 August 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The intestinal brush border (BB) Na+/H+ exchanger isoform 3 (NHE3) is acutely inhibited by elevation in the concentration of free intracellular Ca2+ ([Ca2+]i) by the cholinergic agonist carbachol and Ca2+ ionophores in a protein kinase C (PKC)-dependent manner. We previously showed that elevating [Ca2+]i with ionomycin rapidly inhibited NHE3 activity and decreased the amount of NHE3 on the plasma membrane in a manner that depended on the presence of the PDZ domain-containing protein E3KARP (NHE3 kinase A regulatory protein, also called NHERF2). The current studies were performed in PS120 fibroblasts (NHE-null cell line) stably transfected with NHE3 and E3KARP to probe the mechanism of PKC involvement in Ca2+ regulation of NHE3. Pretreatment with the general PKC inhibitor, GF109203X prevented ionomycin inhibition of NHE3 without altering basal NHE3 activity. Similarly, the Ca2+-mediated inhibition of NHE3 activity was blocked after pretreatment with the conventional PKC inhibitor Gö-6976 and a specific PKC{alpha} pseudosubstrate-derived inhibitor peptide. [Ca2+]i elevation caused translocation of PKC{alpha} from cytosol to membrane. PKC{alpha} bound to the PDZ1 domain of GST-E3KARP in vitro in a Ca2+-dependent manner. PKC{alpha} and E3KARP coimmunoprecipitated from cell lysates; this occurred to a lesser extent at basal [Ca2+]i and was increased with ionomycin exposure. Biotinylation studies demonstrated that [Ca2+]i elevation induced oligomerization of NHE3 in total lysates and decreased the amount of plasma membrane NHE3. Treatment with PKC inhibitors did not affect the oligomerization of NHE3 but did prevent the decrease in surface amount of NHE3. These results suggest that PKC{alpha} is not necessary for the Ca2+-dependent formation of the NHE3 plasma membrane complex, although it is necessary for decreasing the membrane amounts of NHE3, probably by stimulating NHE3 endocytosis.

Na absorption; PDZ domains; signal complex


WE HAVE PREVIOUSLY DEMONSTRATED that elevation of free intracellular Ca2+ ([Ca2+]i) rapidly inhibits the mammalian small intestinal neutral NaCl-absorptive process and its component brush border (BB) Na+/H+ exchanger NHE3 (7, 11-13, 33). Neutral NaCl absorption is important for digestive physiology, explaining small intestinal Na absorption between meals and the increase in the ileal Na absorption that occurs after meals. It is also important for the pathophysiology of diarrhea because it is the major Na-absorptive process inhibited in most diarrheal diseases. Elevating [Ca2+]i by the cholinergic agonist carbachol and Ca2+ ionophores causes similar inhibition in ileal NaCl absorption (7, 12, 33). The inhibition of NHE3 activity by carbachol is associated with a decrease in the amount of BB NHE3, implicating changes in trafficking in its regulation by Ca2+ (Li XH and Donowitz M, unpublished observations). Protein kinase C (PKC) activity is necessary for the ileal inhibition of NaCl absorption caused by carbachol and Ca2+ ionophores and is associated with translocation to and activation of PKC in the ileal BB (7, 12, 33). However, it is still unknown how PKC plays a role in the Ca2+-dependent inhibition of ileal NaCl absorption and which PKC isoform is involved.

It is becoming clear that kinase regulation of NHE3 requires physical association of NHE3 with multiple proteins, including PDZ domain proteins (18, 50). In previous studies to examine how elevated [Ca2+]i regulates NHE3, we used a NHE-null cell model, the PS120 fibroblast, which is derived from Chinese hamster lung cells (30). These cells, in addition, lack the two-PDZ domain-containing protein E3KARP, which binds NHE3 and is required for elevated [Ca2+]i inhibition of NHE3 (50). E3KARP or another related two-PDZ domain protein, Na+/H+ exchanger regulatory factor (NHERF; also called NHERF1), had previously been shown to be necessary to allow cAMP to regulate NHE3 in PS120 cells and in the renal proximal tubule OK cell line (50). E3KARP, bound to NHE3 at basal Ca2+, forms a complex when Ca2+ is elevated that also includes {alpha}-actinin-4 (18). One explanation of the specificity of E3KARP in the Ca2+ regulation of NHE3 was that E3KARP but not NHERF bound {alpha}-actinin-4 (18). The elevated Ca2+ inhibition of NHE3 appears to have two steps: 1) formation of an NHE3/E3KARP/{alpha}-actinin-4 complex, and 2) complex internalization, with complex formation preceding internalization (18).

The current studies used PS120 cells stably expressing NHE3 and E3KARP to demonstrate a role for PKC in these effects of elevated [Ca2+]i, to identify the PKC isoform involved, and to probe the mechanism by which PKC regulates NHE3. PKC is a family of homologous serine and threonine kinases. The eleven isozymes of PKC have been divided into three groups on the basis of their calcium and phospholipid requirements for activation: classic or conventional PKC (cPKC), including {alpha}, {beta}I, {beta}II, and {gamma}, which are calcium and phospholipid dependent; new PKC (nPKC), including {delta}, {epsilon}, {eta}, {theta}, and µ, which are calcium independent and phospholipid dependent, and atypical PKC ({alpha}PKC), including {xi} and {lambda}, which are calcium and phospholipid independent (28, 41). Fibroblasts contain at least {alpha}, {delta}, and {epsilon} isoforms (2, 37). In this study, PKC{alpha} is identified as the isoform involved in NHE3 regulation and its role shown to be in NHE3 complex internalization.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Materials. pGEX4T-1 was from Amersham Pharmacia Biotech; ionomycin was from Sigma; NHS-SS-biotin and avidinagarose were from Pierce; immobilized protein A-Sepharose beads were from Pharmacia Biotech. Monoclonal anti-vesicular stomatitis virus G protein (VSVG) antibody P5D4 was kindly provided by Dr. D. Louvard (Curie Institute, Paris, France). Monoclonal anti-E3KARP antibody was kindly provided by Dr. Pann-Ghill Suh (Pohang University of Science and Technology, Republic of Korea), and polyclonal anti-E3KARP antibody was provided by Dr. C. Yun (JHUSOM; current address: GI Division, Emory University School of Medicine, Atlanta, GA). Monoclonal anti-PKC{alpha} antibodies were from Transduction Laboratories. BL-21 cells were from Stratagene. PKC inhibitors, GF109203X, and Gö-6976 were from Biomol Research Laboratories, A synthetic peptide of a palmitylated pseudosubstrate sequence of PKC{alpha} (PALMPKC{alpha} pseudosubstrate inhibitor [PALM-PKC{alpha} aa 15-28:K (PAM) DVANRFSRKGALRQ]) was provided by Darlene Dartt and Dris Zoukhri (Harvard Medical School, Cambridge, MA).

Cell culture. PS120/E3V/E3KARP (E3V refers to NHE3 epitope tagged on the COOH terminus with VSVG) has been previously described (17). Fibroblasts were grown in Dulbecco's modified Eagle's medium supplemented with 25 mM NaHCO3, 10 mM HEPES, 50 units/ml penicillin, 50 µg/ml streptomycin, and 10% fetal bovine serum in a 5% CO2-95% O2 humidified incubator at 37°C. Where appropriate, acid killing and hygromycin (600 units/ml) selections were applied to maintain high Na+/H+ exchange activity and expression of E3KARP, as described previously (21).

Measurement of Na+/H+ exchange. Na+/H+ exchange in PS120/E3V/E3KARP cells was determined fluorometrically using the intracellular pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) with cells grown to 60-70% confluency on glass coverslips, as described previously, including consideration of buffering capacity (21). The cells were loaded with the acetoxymethyl ester of BCECF (BCECF-AM; 5 µM) in Na+ medium (130 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgSO4, 1 mM NaH2PO4, 25 mM glucose, and 20 mM HEPES, pH 7.4) for 20 min and then washed with TMA+ medium (130 mM tetramethylammonium chloride, 5 mM KCl, 2 mM CaCl2, 1 mM MgSO4, 1 mM NaH2PO4, 25 mM glucose, and 20 mM HEPES, pH 7.4) to remove the extracellular dye; the coverslip was then mounted at an angle of 60° in a 100-µl fluorometer cuvette designed for perfusion and thermostated at 37°C. The cells were pulsed with 40 mM NH4Cl in TMA+ medium for 3 min, followed by TMA+ medium, which resulted in the acidification of the cells. Na+ medium was then added, which induced alkalinization of cells. Na+/H+ exchange (H+ efflux) was calculated as described previously as the product of sodium-dependent change in intracellular pH (pHi) times the buffering capacity at each pHi. Kinetic analyses used a nonlinear regression data analysis program (Origin) that allowed fitting of data to a general allosteric model described by the Hill equation, with estimates for Vmax and K'[H+]i (21) and their respective distributions (SE). When GF109203X, Gö-6976, and PKC peptide inhibitors were studied, cells were pre-treated with inhibitors for 15 min during the dye loading and during the prepulse period.

Glutathione S-transferase fusion proteins and binding assays. A series of glutathione S-transferase (GST) fusion proteins that include full-length NHERF and E3KARP and the various domains of E3KARP were prepared for this study, as described previously (18), by subcloning into the pGEX4T-1 vector. The GST-tagged fusion proteins, including the E3KARP first PDZ domain (P1, aa 12-92), the second PDZ domain (P2, aa 155-231), the COOH-terminal domain (C, aa 231-327), and the second PDZ domain plus the internal sequence between P1 and P2 (LnP2, aa 92-231), were expressed individually in Escherichia coli BL-21 cells. Purification of the GST fusion proteins was conducted as described previously with glutathione Sepharose (18).

In vitro binding of PKC{alpha} with GST-E3KARP fragments (2 µg each) was performed in buffer A (20 mM Tris · HCl, pH 7.4, 1 mM EGTA, 5 mM MgCl2, 1 mM EDTA, 150 mM NaCl, 0.1% Triton X-100). Free Ca2+ was set at 1 µM with 0.9 mM CaCl2 added to buffer A. PKC{alpha} purified from baculovirus-infected Sf9 cells was kindly provided by Dr. Sung Ho Ryu (Pohang University of Science and Technology, South Korea). An aliquot (0.1 µg) of purified PKC{alpha} was added to buffer A and reacted with immobilized GST-E3KARP fragments in the presence of 1 µM free Ca2+ for 1 h at 4°C. The resultant beads were washed three times with buffer A, and the amounts of PKC{alpha}-bound to GST-E3KARP fragments were determined by Western blot analysis using anti-PKC{alpha} antibody.

Immunoprecipitation and immunoblotting. Coimmunoprecipitation experiments were performed using cell lysates from PS120 cells expressing NHE3V and E3KARP in the absence or presence of ionomycin. Cell lysates were prepared in cell lysis buffer [20 mM Tris-Cl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 5 mM MgCl2, and 1% Triton X-100 with a mixture of protease inhibitors (Sigma) (nominally Ca2+ free)]. After a brief centrifugation for 5 min at 10, 000 g, the supernatants were subjected to protein assay. Aliquots of lysates (1 mg) were incubated with the anti-E3KARP antibody for 2 h at 4°C, followed by the addition of 35 µl of 50% protein A-Sepharose beads and gentle mixing on a rotator at 4°C overnight. After being washed five times with the wash buffer (50 mM Tris · HCl, pH 7.5, 150 mM NaCl, and 1% Triton X-100), the bound proteins were eluted and analyzed by SDS-PAGE and immunoblotted with the anti-PKC{alpha} or E3KARP antibodies.

Cell surface biotinylation. The surface amount of NHE3 was measured by cell surface biotinylation as described (18). PS120 cells grown in 10-cm petri dishes were treated with either agonist or vehicle at 37°C and then were rinsed extensively with phosphate-buffered saline (PBS) followed by borate buffer (154 mM NaCl, 7.2 mM KCl, 1.8 mM CaCl2, and 10 mM H3BO3, pH 9.0) at 4°C. Cells then were incubated for 40 min with 1.5 mg of NHS-SS-biotin in borate buffer. Unbound NHS-SS-biotin was quenched with Tris buffer (120 mM NaCl and 20 mM Tris, pH 7.4; N+ buffer). Cells were then rinsed with PBS and lysed in 1 ml of 150 mM NaCl, 3 mM KCl, 5 mM EDTA trisodium, 3 mM EGTA, 1% Triton X-100, and 60 mM HEPES, pH 7.4, and sonicated for 20 s. The lysates were agitated at 4°C for 30 min and spun to remove insoluble cell debris. Protein content in the supernatant was measured by the method of Bradford, and equal amounts of cell lysate were incubated with streptavidinagarose at 4°C. The remaining supernatant was retained as the intracellular fraction. The streptavidin-agarose beads were washed repeatedly in N+ buffer and boiled in Laemmli sample buffer. The total, surface, and intracellular fractions were resolved by SDS-PAGE with boiling/{beta}-mercaptoethanol. Separated proteins were transferred to nitrocellulose membrane and probed with monoclonal anti-VSVG antibody P5D4 (hybridoma culture medium at 1:5 dilution). Bands were visualized using enhanced chemiluminescence and quantified using densitometric analysis, as described (18).

Membrane PKC{alpha} and phosphoconventional PKC. PS120/E3V/E3KARP cells were incubated with 2 µM ionomycin (in Na medium) and compared with solvent control. Cells were then harvested on ice. The cells were washed twice with cold PBS, resuspended in homogenization buffer [20 nM Tris · HCl, pH 8.0, 2 mM EDTA, 2 mM dithiothreitol, 1 mM vanadate, 50 mM NaF, 5 mM sodium pyrophosphate (NaPPi), and 10 mM {beta}-glycerophosphate with protease inhibitors from Sigma], and sonicated three times for 5 s each and then centrifuged at 1,000 g for 10 min at 4°C. Supernatants were collected and centrifuged in a TL-100.3 rotor at 40,000 rpm for 40 min. The high-speed pellet was designated as the membrane fraction and was solubilized in homogenization buffer, whereas the supernatant was designated as the cytoplasmic fraction. Protein concentrations were determined according to the Bradford method using reagents from Bio-Rad. The membrane pellet was resuspended in an equal volume of homogenizing buffer. Proteins (20 µg) of total membrane and cytosol were mixed 1:5 with 5x Laemmli's buffer. Samples were boiled for 5 min and subjected to Western analysis with monoclonal anti-PKC{alpha} antibodies for immunodetection.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Elevated [Ca2+]i-induced NHE3 inhibition is PKC dependent. Previous studies have shown that elevation of [Ca2+]i by ionomycin and thapsigargin in PS120/E3V/E3KARP cells inhibit NHE3 activity with similar magnitude (18). We examined whether PKC activity was necessary for the ionomycin-induced inhibition of NHE3. As shown in Fig. 1, exposure to ionomycin (2 µM) for 10 min decreased NHE3 transport activity by 30-40% by decreasing the Vmax and increasing the K'[H+]i, as described (18). To demonstrate the involvement of PKC in the Ca2+/NHE3 signaling pathway, NHE3 transporter activity was examined after pretreatment with the general PKC inhibitor, GF109203X (38) (Fig. 1). Pretreatment with GF109203X for 15 min prevented the ionomycin-induced inhibition of NHE3 Vmax. Treatment with GF109203X alone had no effect on NHE3 activity. These results demonstrate that PKC is necessary for elevated [Ca2+]i inhibition of NHE3 activity.



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Fig. 1. Ionomycin-induced inhibition of Na+/H+ exchanger (NHE3) activity is PKC dependent. PS120 cells were rendered quiescent by serum removal for at least 4 h, and ionomycin (2 µM) was added for 10 min before NHE3 activity was assayed fluorometrically (BCECF) as Na+-dependent intracellular pH (pHi) recovery after an acid load. Cell was exposed to GF109203X (2 µM) for 15 min before Ca2+ elevation. Elevation of Ca2+ by ionomycin inhibits NHE3 activity. Pretreatment with GF109203X prevented the ionomycin-induced inhibition of NHE3 activity, and GF109203X did not alter basal NHE3 activity. The data represent 1 example of 3 similar experiments.

 

PKC{alpha} is the isoform involved in elevated [Ca2+]i-induced NHE3 inhibition. Which isoform(s) of PKC was involved in the ionomycin-induced inhibition of NHE3 activity was further characterized. Of the classes of PKC, only conventional isoforms are Ca2+ dependent. The effect of Gö-6976, a specific inhibitor of conventional PKCs (24), and a synthetic peptide of a palmitoylated peptide derived from the pseudosubstrate sequence of PKC{alpha} (PALM-PKC{alpha} pseudosubstrate inhibitor) (14, 35, 53-55), which is a PKC{alpha} inhibitor, were studied. NHE3 transport activity was measured after pretreatment with Gö-6976 and the PALM-PKC{alpha} pseudosubstrate inhibitor. As shown in Fig. 2A, pretreatment with 1 uM of Gö-6976 for 15 min prevented ionomycin-induced inhibition of NHE3 activity. The lack of ionomycin effect on K'[H+]i in this series of studies is interpreted as experimental variability. Cells treated with Gö-6976 alone had no effect on NHE3 activity. Similar results were obtained with pretreatment with the PALM-PKC{alpha} pseudosubstrate inhibitor (100 nM) with no effect on basal Na+/H+ exchange but prevention of ionomycin-induced inhibition (Fig. 2B). All PKC inhibitors caused similar magnitude of reversal of [Ca2+]i inhibition of NHE3 activity. These data, including the magnitude of inhibition of elevated [Ca2+]i regulation of NHE3, indicate that PKC{alpha} is probably the single PKC isoform involved in Ca2+ inhibition of NHE3 activity.



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Fig. 2. PKC{alpha} is necessary for ionomycin-induced NHE3 inhibition. PS120 cells were pretreated (15 min) with the conventional PKC inhibitor Gö-6976 (A) and PALM-PKC{alpha} pseudosubstrate-derived inhibitor peptide (B). Both prevented the effect of ionomycin (2 µM) to inhibit NHE3. A: Gö-6976 (1 µM) prevented the ionomycin-induced inhibition of NHE3 activity without altering basal NHE3 activity. NHE3 activity is shown as the rate of Na+-dependent cell pH recovery from an acid load. Results of 1 experiment that was repeated 3 times are shown. B: PALM-PKC{alpha} pseudosubstrate-derived inhibitor peptide (100 nM) prevented ionomycin inhibition without altering basal NHE3 activity. Initial Na/H exchange rates (dpHi/s) are shown for the effect of ionomycin and PALM-PKC{alpha} pseudosubstrate-derived inhibitor peptide. Results are shown as means ± SE of 3 similar experiments.

 

PKC{alpha} binds to E3KARP in a Ca2+-dependent manner. To understand how PKC{alpha} is involved in elevated [Ca2+]i inhibition of NHE3, it was determined whether PKC{alpha} binds to either E3KARP (needed for Ca2+-related NHE3 inhibition) or NHERF (not needed for Ca2+-related NHE3 inhibition), and whether the binding is Ca2+ dependent. The interaction between NHERF or E3KARP and PKC{alpha} was examined by in vitro pulldown experiments using GST fusion proteins encoding full-length E3KARP and NHERF. Binding assays were performed in which GST fusion proteins of E3KARP and NHERF were incubated with an aliquot (0.1 µg) of purified PKC{alpha} (Fig. 3). In the absence of calcium, PKC{alpha} did not bind to GST or GST-NHERF but did bind to GST-E3KARP. The amount of PKC binding to GST-E3KARP was increased when free calcium was elevated to 1 µM. Note GST-NHERF also bound PKC{alpha} at 1 µM [Ca2+]i, but the binding was less than to E3KARP for the same amount of fusion protein. The data presented in Fig. 3 show that E3KARP binds PKC{alpha} and their interaction is increased by elevation in [Ca2+]i.



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Fig. 3. PKC{alpha} binds E3KARP in vitro in a calcium-dependent manner. Full-length glutathione S-transferase (GST)-NHE3 kinase A regulatory protein (NHERF) and GST-E3KARP were immobilized on GST beads. An aliquot (0.1 µg) of recombinant PKC{alpha} was added to immobilized GST alone (lanes 2 and 5), GST-NHERF (lanes 3 and 6), and GST-E3KARP (lanes 4 and 7) in the absence or presence of 1 µM free calcium. The bound proteins were resolved by SDS-PAGE and transferred to immobilon membrane, and the amounts of PKC{alpha} were detected by Western blot analysis using anti-PKC{alpha} antibody. Input used for the experiment is shown in lane 1. Fusion protein (2 µg) was used. Lanes are numbered from left.

 

PKC{alpha} binds the PDZ1 domain of E3KARP. To determine which region of E3KARP interacts with PKC{alpha}, a series of GST fusion proteins that encode PDZ1, PDZ2, lnPDZ2, and COOH terminus of E3KARP were constructed (Fig. 4A). Assays in which GST fusion proteins were incubated with an aliquot (0.1 µg) of purified PKC{alpha} showed that the domain PKC{alpha} bound maximally was the PDZ1 domain of E3KARP, with much less binding to PDZ2. PKC{alpha} did not bind to the E3KARP COOH terminus or lnPDZ2 (Fig. 4B).



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Fig. 4. PKC{alpha} binds to the PDZ1 domain of E3KARP. A: linear diagram of E3KARP shows the amino acids of the domains studied in B. B: series of immobilized GST fusion proteins (2 µg) that encoded different domains of E3KARP, including PDZ1, PDZ2, extended PDZ2 (LnP2), and the COOH terminus, were prepared. For the binding assays, aliquots (0.1 µg) of purified PKC{alpha} were added to immobilized GST fusion proteins and the bound proteins were resolved on SDS-PAGE. The amounts of PKC{alpha}-bound to GST-E3KARP domains were measured by Western blot analysis using anti-PKC{alpha} antibody.

 

E3KARP coprecipitates PKC{alpha} in a Ca2+-dependent manner. The previous experiments indicated that E3KARP bound to PKC{alpha}, predominantly via its PDZ1 domain. To determine whether this association occurs in intact cells, immunoprecipitation was performed, starting with PS120 cell lysate made from cells exposed to elevated [Ca2+]i/controls. As shown in Fig. 5, PKC{alpha} was coimmunoprecipitated with anti-E3KARP antibody. In addition, the in vivo results demonstrated that the association between PKC{alpha} and E3KARP was dependent upon elevation of [Ca2+]i. The amount of PKC{alpha} coprecipitated with E3KARP was increased by elevated [Ca2+]i (Fig. 5). Thus the in vivo association of E3KARP and PKC{alpha} is calcium dependent, in agreement with the data presented in the in vitro binding assay (Fig. 3). Of note, the elevated Ca2+ conditions were only present in intact tissue exposed to ionomycin, and upon lysis, all tissues were in nominally Ca2+-free conditions. This suggests that the NHE3/E3KARP complexes formed in vivo with elevated Ca2+ do not reverse rapidly in vitro when Ca2+ is lowered.



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Fig. 5. Elevation of [Ca2+]i increases the coprecipitation of PKC{alpha} by E3KARP. PS120/E3KARP/E3V cells were treated with ionomycin (2 µM)/control for 10 min, lysed, and immunoprecipitated (IP) with anti-E3KARP antibody or preimmune serum. The immune complexes were resolved by SDS-PAGE, and the precipitated proteins were probed with anti-PKC{alpha} or anti-E3KARP antibodies, the latter to show the extent of immunoprecipitation. Preimmune serum was only exposed to lysates from ionomycin exposed cells. A representative study, which was repeated 3 times with similar results, is shown.

 

Elevation of [Ca2+]i induces subcellular redistribution of PKC{alpha}. To assess whether the elevation of [Ca2+]i affected membrane PKC, as has been shown to occur with many examples of PKC activation (7, 45), the amounts of PKC{alpha} in membrane fractions were measured by Western analysis. Cells were treated with ionomycin for 2 min. Membrane and cytosol fractions were prepared by ultracentrifugation, and the amounts of PKC{alpha} were determined (Fig. 6A). Compared with unstimulated cells (Fig. 6A), treatment for 2 min with ionomycin increased membrane PKC{alpha} (Fig. 6, A and B). Although no loading control of another protein was simultaneously examined, that statistically significant changes occurred suggests that random differences in loading did not explain the finding. These data show that elevation of [Ca2+]i induces a rapid increase of membrane PKC{alpha} amount in PS120/E3V/E3KARP cells.



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Fig. 6. Elevation of [Ca2+]i redistributes PKC{alpha} to membranes of PS120/E3V/E3KARP cells. Cells were treated with ionomycin (2 µM) or vehicle at 37°C for 2 min and then separated into membrane and cytosol. A: PKC{alpha} amount was determined with anti-PKC{alpha} antibody after 2 min of ionomycin (2 µM) exposure in vitro. Membrane and cytosol fractions were resolved by SDS-PAGE and probed with anti-PKC{alpha} antibody. A representative experiment is shown. B: quantification by scanning densitometry for the membrane fraction in A normalized to the nonionomycin control. Data are means ± SE of 3 experiments. *P < 0.05 compared with control (ionomycin).

 

Decrease in the amount of surface NHE3 by [Ca2+]i elevation is PKC{alpha} dependent, but NHE3 oligomerization is PKC independent. Ca2+ inhibition of NHE3 in PS120/E3V/E3KARP is thought to involve two steps; complex formation and decrease in surface NHE3 (18). To determine which step or steps were PKC dependent, we used cell surface biotinylation. PS120/E3V/E3KARP cells were exposed to elevated [Ca2+]i for 10 min with and without pretreatment (15 min) with two PKC inhibitors, GF109203X and Gö-6976. Cells were then placed at 4°C, and cell surface biotinylation was performed with NHS-SS-biotin and total and surface NHE3 was determined (Fig. 7). Total NHE3 (Fig. 7, left) is used to illustrate the effect of the PKC inhibitors on NHE3 oligomerization. Under basal conditions, NHE3 is mostly in monomers and a size approximately double that of the monomer (dimers), but with elevated Ca2+ there is less NHE3 monomer and more dimer and larger sizes. Under basal conditions total NHE3 is distributed: monomer, 40 ± 10%,; dimer, 55 ± 82; oligomers, 5 ± 7%. After Ca2+ elevation, NHE3 is distributed: monomer 25 ± 7%; dimer, 60 ± 10%; oligomers 15 ± 10%. The dimers and oligomers are assumed to represent only NHE3 but could represent NHE3 in larger complexes with other proteins. There was no change in this Ca2+ related oligomerization of total NHE3 by pretreatment with either PKC inhibitor when the same amount of protein was used for immunoprecipitation.



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Fig. 7. Pretreatment with PKC inhibitors does not alter distribution of NHE3 among oligomer, dimer, and monomer but prevents the decrease in surface amount of NHE3. Ionomycin (2 µM, 10 min, 37°C) or vehicle-pretreated cells, some pretreated with GF109203X (2 µM, 15 min) or Gö-6976 (1 µM, 15 min), were surface labeled with biotin after temperature was lowered to 4°C. Surface proteins were retrieved from the cell by streptavidin precipitation. NHE3 protein abundance was quantitated by scanning densitometry of Western blots using NHE3 immunoblotted with anti-vesicular stomatitis virus G protein (VSVG). Total (left) and surface (right) NHE3 were examined. This is a representative experiment that was repeated 3 times with similar results. Molecular mass standards are shown at right.

 

Surface NHE3 is shown in Fig. 7, right. Ca2+ elevated for 10 min was associated with a decrease in surface NHE3 as reported (18) (surface NHE3 15 ± 6% of total under basal condition and 5 ± 10% of total after Ca2+ elevation). The PKC inhibitors largely prevented the Ca2+-induced decrease in surface amount of NHE3 (surface NHE3 after GF 11 ± 8% of total and after 60 min 13 ± 7% of total, both of which were not significantly different from the basal levels). Thus PKC is involved in NHE3 internalization and not in NHE3 complex formation. The greater Ca2+-induced decrease in the amount of surface NHE3 than the decrease in transport (compare Figs. 1 and 2) in this study differs from our previous report (18) in which the magnitude of the changes was more similar. Possible explanations as to why there are differences between surface NHE3 and amount and activity include the possibility that the molecules of NHE3 have different activities based on the complex in which they are present. This is totally speculative for Ca2+ regulation, although this was suggested for NHE3 in renal proximal tubule brush border on the basis of megalin association (5, 6).


    DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
These studies demonstrate a role for PKC{alpha} in elevated [Ca2+]i regulation of NHE3. We previously reported that PKC was an intermediate in elevated Ca2+-induced inhibition of ileal neutral NaCl absorption and BB Na+/H+ exchange (12, 30, 41). In this ileal model, elevated [Ca2+]i was associated with translocation of PKC amount and activity to the BB, and inhibiting PKC with the PKC inhibitor H7 prevented elevated [Ca2+]i from decreasing neutral NaCl absorption (7). The current studies in PS120 fibroblasts demonstrated similar Ca2+-dependent inhibition of NHE3, which was PKC dependent. This is consistent with similar regulation being involved in ileal Na absorptive cells, which contain endogenous NHE3 and E3KARP (50), and in PS120 fibroblasts, which express only exogenous E3KARP and NHE3 (50). The fact that E3KARP is necessary for this Ca2+ regulation of NHE3 has been demonstrated in PS120 cells. The advances in the studies presented here in PS120 cells are mechanistic, although the mechanisms have not yet been shown to apply to the BB of intestinal Na-absorptive epithelial cells.

A single PKC isoform is probably involved in Ca2+ regulation of NHE3 on the basis of similarity in inhibition of the Ca2+ effect on NHE3 activity of the general PKC{alpha} GF109203X inhibitor and the specific PKC{alpha} inhibitor. The PKC isoform involved in NHE3 regulation was PKC{alpha} based on pharmacological (inhibitor) and biochemical studies. The Ca2+ inhibition was reversed at least 70% by the general PKC inhibitor GF109203X, the conventional PKC inhibitor Gö-6976, and a palmitoylated pseudosubstrate-derived PKC{alpha} inhibitor peptide, which is specific for PKC{alpha} in intact cell studies (14, 35, 52-55). The GF109203X data indicated that PKC was involved. Because a Ca2+-dependent effect was being studied, it appeared likely that a conventional PKC would be involved because only the conventional class of PKCs are Ca2+ dependent (28, 41). Note, the specific PKC isoforms that are present in PS120 fibroblasts have not been defined. However, the PKC isoforms in two other fibroblast lines (AP-1 cells, a Chinese hamster ovarian cell derivative, and NIH 3T3 cells) were shown to include PKC{alpha}, {delta}, and {epsilon} (2, 37). Thus the only conventional PKC isoform likely to be involved in Ca2+ regulation of NHE3 in fibroblasts was the {alpha} isoform. The involvement of PKC{alpha} was supported by inhibition of Ca2+ effects on NHE3 by the conventional PKC inhibitor Gö-6976. The involvement of PKC{alpha} was strongly suggested by the use of the palmitoylated PKC{alpha} peptide inhibitor. These pharmacological results were supported by demonstration of an increased amount of membrane PKC{alpha} that occurred at the earliest time point studied (2 min) in the PS120 cells after [Ca2+]i elevation.

The fact that PKC{alpha} was the involved isoform was used to begin further understanding mechanisms of NHE3 regulation by elevated [Ca2+]i. PKC{alpha} is the only conventional PKC isoform to have a PDZ binding domain on its COOH terminus (QSAV) (30). Thus we postulated that PKC{alpha} might bind to E3KARP and with elevated [Ca2+]i, PKC{alpha} might enter the E3KARPNHE3-containing complex that we previously showed also includes {alpha}-actinin-4 (18). The present studies showed that with elevated Ca2+, there was increased coprecipitation of PKC{alpha} by E3KARP. There was some basal association of E3KARP with PKC{alpha} with increased in vivo coprecipitation of PKC{alpha} by E3KARP in the presence of 1 µM [Ca2+]i.

Because we showed that in the absence of E3KARP no increased NHE3 oligomerization occurred (18), we suggest E3KARP is central to elevated [Ca2+]i NHE3 complex formation. The fact that PKC{alpha} binds to PDZ1 of E3KARP but not PDZ2, whereas NHE3 and {alpha}-actinin-4 (the latter previously shown to be involved in Ca2+ inhibition of NHE3 and to be in an NHE3/E3KARP complex) bind to PDZ2 and not PDZ1 (18, 49), indicates that PKC{alpha} and either NHE3 or {alpha}-actinin-4 could associate with the same E3KARP molecule. It is possible that E3KARP multimerizes either with other E3KARP molecules or with other PDZ domain-containing proteins in the ileal BB to include NHE3, {alpha}-actinin-4, and PKC{alpha} in the same complex, if not on the same E3KARP molecule. Further studies will be required to define the stoichiometry of the components of this complex and to identify which other proteins are in this Ca2+-dependent NHE3, E3KARP, {alpha}-actinin-4, PKC{alpha}-containing complex. Given that the size of the NHE3-containing complexes, as determined by sucrose density gradient centrifugation, on the surface of NHE3 containing PS120, Caco-2, and OK cells is ~400 to ~900 kDa under basal [Ca2+]i conditions and that the size of NHE3 complexes increase with elevated [Ca2+]i (Ref. 3 and Li X and Donowitz M, unpublished), the full picture of which proteins form this complex with elevated [Ca2+]i remains incomplete.

We previously reported that E3KARP is necessary for Ca2+ inhibition of NHE3, whereas NHERF is not able to play this role (18). This specificity of E3KARP appears to be due to the fact that E3KARP is necessary for forming the NHE3-containing complexes by binding to {alpha}-actinin-4, which we showed is necessary for Ca2+ inhibition of NHE3. In contrast, specificity does not clearly relate to E3KARP binding to PKC{alpha}. As shown in Fig. 3, at basal [Ca2+]i only E3KARP and not NHERF binds PKC{alpha}, whereas at 1 µM [Ca2+]i both E3KARP and NHERF bind PKC{alpha}, although E3KARP binds a larger amount of PKC{alpha} than NHERF. Whether intracellular conditions are such that with elevated [Ca2+]i, PKC{alpha} binds NHERF in addition to E3KARP is not known. Thus understanding of the specificity of the E3KARP/PKC{alpha} interaction in NHE3 regulation compared with that of the other BB PDZ domain-containing proteins remains incomplete but is unlikely to be a major factor.

The role of PKC{alpha} in NHE3 regulation has been partially defined in these studies. Elevated [Ca2+]i inhibits NHE3 by affecting two steps: 1) formation of plasma membrane NHE3-containing complexes in which NHE3 appears to multimerize, and 2) internalization. Elevated [Ca2+]i decreases the amount of surface and increases the amount of intracellular NHE3, an effect prevented by PKC inhibitors (Fig. 7). The step PKC{alpha} is involved in does not appear to be complex formation. Rather, it is in the decrease in surface NHE3. What this PKC-dependent step consists of is not yet established but is likely to be due to increased endocytosis, with decreased exocytosis possible but less likely. The rapid time course is consistent with the fact that at least part of this effect involves increased endocytosis. PKC has previously been shown to stimulate endocytosis, and less commonly to stimulate exocytosis, with the {alpha} and {epsilon} isoforms most commonly involved (4, 10, 16, 20, 25, 27, 32, 36, 46). The forms of PKC stimulated endocytosis includes clathrin-dependent, clathrin-independent, and fluid-phase endocytosis (3, 10, 32, 36). PKC affects endocytosis of multiple plasma membrane proteins, including receptors and transport proteins, although not all plasma membrane proteins are affected. Moreover, PKC has been shown to phosphorylate multiple components of the endocytosis machinery (8, 9, 23, 31, 34). In previous studies, phorbol esters also affected endocytosis via non-PKC-dependent mechanisms, making it necessary to consider more direct demonstration of a PKC role in aspects of endocytosis (1). In addition, regulation of NHE3 by endocytosis in response particularly to elevated cAMP has previously been elegantly demonstrated (17b). The new observation here is that elevated Ca2+ acting by PKC stimulates endocytosis of an NHE3 complex that includes E3KARP, {alpha}-actinin-4, and PKC{alpha} and is dependent on E3KARP, {alpha}-actinin-4, and PKC{alpha} (18). Whether NHE3 or other complex components undergo PKC-dependent endocytosis that exceeds that of other endocytosed plasma membrane proteins will be of interest to determine. This is especially true compared with other E3KARP-binding proteins, which are not part of this complex, which must be defined to understand how specifically Ca2+ regulates Na absorption in these cells.

The substrate PKC phosphorylates to allow the decrease in surface NHE3 has also not been determined. Although NHE3 contains PKC phosphorylation consensus sequences, it has not yet been clearly shown to have increased phosphorylation of these sites with elevated [Ca2+]i. Certainly, the role of an increase in NHE3 phosphorylation in Ca2+ inhibition/endocytosis of NHE3 is not established. Moe et al. (44) previously showed that phorbol ester exposure increases NHE3 phosphorylation in AP-1 fibroblasts. However, there was no correlation between changes in phosphorylation of NHE3 and whether a specific clone of NHE3 responded to phorbol ester with elevated, decreased, or nonchanged NHE3 activity (44). AP-1 cells do not express E3KARP (Ref. 48 and Donowitz M, Cha B, and Yun C, unpublished observations). In addition, in non-E3KARP-containing PS120/E3V cells, phorbol ester did not alter NHE3 phosphorylation, determined by two-dimensional phosphopeptide mapping (47). Whether the phosphorylation status of NHE3 in response to elevated [Ca2+]i is involved with its regulation when E3KARP is present is unknown. Because PKC also phosphorylates multiple components of the endocytosis machinery, including dynamin, synaptojanin, AP2, and clathrin (8, 9, 23, 31, 39), and the fact that there are putative PKC phosphorylation sites in other components of the NHE3 complex, including {alpha}-actinin-4 and E3KARP (36), the substrate(s) PKC{alpha} phosphorylates that is necessary for NHE3 internalization remains unknown. The fact that PKC can stimulate endocytosis independently of phosphorylation of the protein taken up has been emphasized recently (1).

The nature of the NHE3 complexes, including what appears to be NHE3 multimers under basal conditions, which increase with elevated Ca2+, is not fully understood. Fafournoux, Noel, and Pouyssegur showed in 1994 (15) that NHE1 and NHE3 appeared as both monomers and dimers on SDS/reducing gels. The amount of monomer was decreased and that of dimer increased with crosslinkers. This initially suggested that NHEs, including NHE3, might exist as complexes or multimers under basal conditions. Our results for NHE3 under basal conditions are similar, although with a larger percent of total NHE3 appearing as dimers and oligomers (monomer 40 ± 6%; dimer 55 ± 8%; oligomer 5 ± 7%, n = 3). In addition, NHE3 has been shown to scaffold several Ca2+-binding proteins, including calmodulin, calcineurin homologous protein, and megalin (5, 6, 22, 29, 39, 40, 46), further suggesting that it could be involved in Ca2+-related complex formation. Nonetheless, in spite of seeing what appears to be NHE3 complex formation by confocal microscopy (18) and by surface biotinylation studies, the nature of the complexes are uncertain and attempts to isolate the complexes are underway. Definition of the meaning of NHE3 multimerization is complicated by the fact that boiling samples in Laemmli's solution before running the samples on a gel causes changes similar to the Ca2+ effect, decreasing monomeric and increasing dimeric and multimeric NHE3 (data not shown). Nonetheless, the fact that when all samples were similarly boiled, Ca2+-exposed tissue still has decreased monomeric and increased dimeric and multimeric NHE3 is most consistent with Ca2+ exposure, causing NHE3 complex formation.

NHE3-containing complexes scaffolded by E3KARP or NHERF have now been reported for cAMP and Ca2+ regulation of NHE3 (15, 19, 42, 43). The complexes involved in cAMP regulation of NHE3 in fibroblasts and OK cells (opossum kidney proximal tubule cell line) appear to accept either NHERF or E3KARP, seem static over time, and also involve ezrin, PKAII, and actin. In OK cells, although over time cAMP leads to a decrease in surface NHE3 (17b, 51), there have been no other reported changes in the nature of the NHE3-E3KARP-containing plasma membrane complexes. The complexes involved in Ca2+ regulation are different in that they are dynamic with {alpha}-actinin-4 and PKC{alpha} joining the complex with elevated Ca2+, and all complex components appear together in the cytosol over time with Ca2+ elevation (Ref. 18 and data not shown). An interesting, unresolved question is whether the complexes involving E3KARP and NHE3 are the same in both cAMP and Ca2+ regulation of NHE3. In the past, cAMP and Ca2+ regulation of NHE3 have been shown to be at least additive in PS120 cells (Cha BY, Yun C, and Donowitz M, unpublished observations). We hypothesize that NHE3 spontaneously exists in separate complexes that take part in different aspects of its regulation.

Regulation of NHE3 by elevated [Ca2+]i almost certainly occurs by multiple, time-dependent changes in trafficking and intracellular handling of NHE3. Until now, only the initial complex formation and internalization have been demonstrated, although multiple molecules come together for that regulation. Changes in rates of NHE3 exocytosis, half-life, distribution in lysosomes/proteosomes, and binding to regulatory proteins in the NHE3/E3KARP complex are all predicted to be part of regulation of NHE3 activity. Defining which of these processes are affected by PKC{alpha} and its short- and long-term role in NHE3 regulation will be important to understand Ca2+ regulation of NHE3 in normal physiology and disease.


    DISCLOSURES
 
These studies were supported in part by National Institute of Diabetes and Digestive and Kidney Diseases Grants RO1-DK-26523, PO1-DK-44484, and T32-DK-07632, and The Hopkins Center for Epithelial Disorders and by a grant (02-PJ1-PG3-20706-0004) from the Korea Health 21 R&D Project, Ministry of Health and Welfare, Republic of Korea.

Current addresses: J. W. Choi, Div. of Molecular and Life Sciences, Pohang University of Science and Technology, Pohang, South Korea; K. Kawano, Dept. of Environmental Biochemistry and Toxicology, University of Shizuoka, Shizuoka, Japan; W. Lee-Kwon, Div. of Nephrology, University of Maryland School of Medicine, Baltimore, MD.


    ACKNOWLEDGMENTS
 
We acknowledge the expert secretarial assistance of H. McCann.


    FOOTNOTES
 

Address for reprint requests and other correspondence: M. Donowitz, Johns Hopkins Univ. School of Medicine, 925 Ross Research Bldg., 720 Rutland Ave., Baltimore, MD 21205-2195.

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.

* W. Lee-Kwon and J. H. Kim contributed equally to this work. Back


    REFERENCES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
1. Aballay A, Stahl PD, and Mayorga LS. Phorbol ester promotes endocytosis by activating a factor involved in endosomal fusion. J Cell Sci 112: 2549-2557, 1999.[Abstract/Free Full Text]

2. Acs P, Beheshti M, Szallasi Z, Li L, Yuspa SH, and Blumberg PM. Effect of a tyrosine 155 to phenylalanine mutation of protein kinase C delta on the proliferative and tumorigenic properties of NHE3 fibroblasts. Carcinogenesis 21: 887-891, 2000.[Abstract/Free Full Text]

3. Akhter S, Kovbasnjuk O, Li X, Cavet M, Noel J, Arpin M, Hubbard AL, and Donowitz M. Na+/H+ exchanger 3 is in large centrally located complexes on the brush border of proximal tubule-derived OK cells. Am J Physiol Cell Physiol 283: C927-C940, 2002.[Abstract/Free Full Text]

4. Backer JM and King GL. Regulation of receptor-mediated endocytosis by phorbol esters. Biochem Pharmacol 41: 1267-1277, 1991.[ISI][Medline]

5. Biemesderfer D, DeGray B, and Aronson PS. Active (9.6.s) and inactive (21 s) oligomers of NHE3 in microdomains of the renal brush border. J Biol Chem 276: 10161-10167, 2001.[Abstract/Free Full Text]

6. Biemesderfer D, Nagy T, DeGray B, and Aronson PS. Specific association of megalin and the Na+/H+ exchanger isoform NHE3 in the proximal tubule. J Biol Chem 274: 17518-17524, 1999.[Abstract/Free Full Text]

7. Cohen ME, Wesolek J, McCullen J, Rys-Sikora K, Pandol S, Rood RP, Sharp GW, and Donowitz M. Carbachol and elevated Ca2+-induced translocation of functionally active protein kinase C to the brush border of rabbit ileal Na+ absorbing cells. J Clin Invest 88: 855-863, 1991.[ISI][Medline]

8. Cousin MA, Tan TC, and Robinson PJ. Protein phosphorylation is required for endocytosis in nerve terminals: potential role for the dephosphins dynamin I and synaptojanin, but not AP180 or amphiphysin. J Neurochem 76: 105-116, 2001.[ISI][Medline]

9. Daniels GM and Amara SG. Regulated trafficking of the human dopamine transporter. Clathrin-mediated internalization and lysosomal degradation in response to phorbol esters. J Biol Chem 274: 35794-35801, 1999.[Abstract/Free Full Text]

10. Defazio G, Nico B, Trojano M, Ribatti D, Giorelli M, Ricchiuti F, Martino D, Roncali L, and Livrea P. Inhibition of protein kinase C counteracts TNF{alpha}-induced intercellular adhesion molecule 1 expression and fluid phase endocytosis on brain microvascular cells. Brain Res 863: 245-248, 2000.[ISI][Medline]

11. Donowitz M. Calcium in the control of active intestinal Na and Cl transport and involvement in neurohumoral action. Am J Physiol Gastrointest Liver Physiol 245: G165-G177, 1983.[Abstract/Free Full Text]

12. Donowitz M, Cohen ME, Gould M, and Sharp GW. Elevated intracellular Ca2+ acts through protein kinase C to regulate rabbit ileal NaCl absorption: evidence for sequential control by Ca2+/calmodulin and protein kinase C. J Clin Invest 83: 1953-1962, 1989.[ISI][Medline]

13. Donowitz M, Fogel R, Battisti L, and Asarkof N. Calcium content in rabbit ileum is increased by the neurohumoral secretagogues serotonin, carbachol, substance P and neurotensin. Life Sci 31: 1929-1937, 1982.[ISI][Medline]

14. Eichholtz T, deBont DB, de Widt J, Liskamp RM, and Ploegh HL. A myristoylated pseudosubstrate peptide, a novel protein kinase C inhibitor. J Biol Chem 268: 1982-1986, 1993.[Abstract/Free Full Text]

15. Fafournoux P, Noel J, and Pouyssegur J. Evidence that Na+/H+ exchanger isoforms NHE1 and NHE3 exist as stable dimmers in membranes with a high degree of specificity for homodimers. J Biol Chem 269: 2589-2596, 1994.[Abstract/Free Full Text]

16. Holm PK, Eker P, Sandvig K, and van Deurs B. Phorbol myristate acetate selectively stimulates apical endocytosis via protein kinase C in polarized MDCK cells. Exp Cell Res 217: 157-168, 1995.[ISI][Medline]

17. Hoogerwerf WA, Tsao S, Levine SA, Montgomery JLM, Yun CHC, Yip JW, Cohen M, Lazenby AJ, Tse CM, and Donowitz M. NHE2 and NHE3 are human and rabbit intestinal brush border proteins. Am J Physiol Gastrointest Liver Physiol 270: G29-G41, 1996.[Abstract/Free Full Text]

17. Hu MC, Fan L, Crowder LA, Karim-Jimenez Z, Murer H, and Moe OW. Dopamine acutely stimulates Na+/H+ exchanger (NHE3) endocytosis via clathrin-coated vesicles: dependence on protein kinase A-mediated NHE3 phosphorylation. J Biol Chem 276: 26906-26915, 2001.[Abstract/Free Full Text]

18. Kim JH, Lee-Kwon W, Park JB, Ryu SH, Yun CH, and Donowitz M. Ca2+-dependent inhibition of Na+/H+ exchanger 3 requires a NHE3/E3KARP/{alpha}-actinin-4 complex for oligomerization and endocytosis. J Biol Chem 277: 23714-23724, 2002.[Abstract/Free Full Text]

19. Lamprecht G, Weinman EJ, and Yun CH. The role of NHERF and E3KARP in the cAMP-mediated inhibition of NHE3. J Biol Chem 273: 29972-29978, 1998.[Abstract/Free Full Text]

20. Le TL, Joseph SR, Yap AS, and Stow JL. Protein kinase C regulates endocytosis and recycling of E-cadherin. Am J Physiol Cell Physiol 283: C489-C499, 2002.[Abstract/Free Full Text]

21. Levine SA, Nath SK, Yun CH, Yip JW, Montrose M, Donowitz M, and Tse CM. Separate C-terminal domains of the epithelial specific brush border Na+/H+ exchanger isoform NHE3 are involved in stimulation and inhibition by protein kinases/growth factors. J Biol Chem 270: 13716-13725, 1995.[Abstract/Free Full Text]

22. Lin X and Barber DL. A calcineurin homologous protein inhibits GTPase-stimulated Na/H exchange. Proc Natl Acad Sci USA 93: 12631-12636, 1996.[Abstract/Free Full Text]

23. Liu JP, Powell KA, Sudhof TC, and Robinson PJ. Dynamin IisaCa2+ sensitive phospholipid-binding protein with very high affinity for protein kinase C. J Biol Chem 269: 21043-21050, 1994.[Abstract/Free Full Text]

24. Martiny-Baron G, Kazanietz MG, Mischak H, Blumberg PM, Kochs G, Hug H, Marme D, and Schachtele C. Selective inhibition of protein kinase C isozymes by the indolocarbazole Go6976. J Biol Chem 268: 9194-9197, 1993.[Abstract/Free Full Text]

25. Menne C, Sorensen TM, Siersma V, von Essen M, Odum N, and Geisler C. Endo- and exocytic rate constants for spontaneous and protein kinase C activation in cell receptor cycling. Eur J Immunol 22: 616-626, 2002.

27. Mostov KE. Regulation of protein traffic in polarized epithelial cells. Histol Histopathol 10: 423-431, 1995.[ISI][Medline]

28. Newton AC. Protein kinase C: structure, function and regulation. J Biol Chem 270: 28495-28498, 1995.[Free Full Text]

29. Pang T, Su X, Wakabayashi S, and Shigekawa M. Calcineurin homologous protein as an essential cofactor for Na+/H+ exchangers. J Biol Chem 276: 17367-17372, 2001.[Abstract/Free Full Text]

30. Pouyssegur J, Sardet C, Franchi A, L'Allemain G, and Paris S. A specific mutation abolishing Na+/H+ antiport activity in human fibroblasts precludes growth at neutral and acidic pH. Proc Natl Acad Sci USA 81: 4833-4837, 1984.[Abstract]

31. Powell KA, Valova VA, Malladi CS, Jensen ON, Larsen MR, and Robinson P. Phosphorylation of dynamin I on Ser-795 by protein kinase C blocks its association with phospholipids. J Biol Chem 275: 11610-11617, 2000.[Abstract/Free Full Text]

32. Reinhardt J, Kosch M, Lerner M, Bertram H, Lemke D, and Oberleithner H. Stimulation of protein kinase C pathway mediates endocytosis of human non-gastric H+/K+/ATPase, ATP1AL1. Am J Physiol Renal Physiol 283: F335-F343, 2002.[Abstract/Free Full Text]

33. Reinlib L, Mikkelsen R, Zahniser D, Dharmsathaphorn K, and Donowitz M. Carbachol-induced cytosolic free Ca2+ increases in T84 colonic cells seen by microfluorimetry. Am J Physiol Gastrointest Liver Physiol 257: G950-G960, 1989.[Abstract/Free Full Text]

34. Slepnev VI, Ochoa GC, Butler MH, Grabs D, and De Camilli P. Role of phosphorylation in regulation of the assembly of endocytic coat complexes. Science 281: 821-824, 1998.[Abstract/Free Full Text]

35. Sohn UD, Zoukhri D, Dartt D, Sergheraert C, Harnett KM, Behar J, and Biancani P. Different protein kinase C isozymes mediate lower esophageal sphincter tone phasic contraction of esophageal circular smooth muscle. Mol Pharmacol 51: 462-470, 1997.[Abstract/Free Full Text]

36. Song JC, Hrnjez BJ, Farokhzad OC, and Matthews JB. PKC-epsilon regulates basolateral endocytosis in human T84 intestinal epithelial of F-actin and MARCKS. Am J Physiol Cell Physiol 277: C1239-C1249, 1999.[Abstract/Free Full Text]

37. Thiam K, Loing E, Zoukhri D, Rommens C, Hodges R, Dartt D, Verwaerde C, Auriault C, Gras-Masse H, and Sergheraert C. Direct evidence of cytoplasmic delivery of PKC-alpha,-epsilon-zeta pseudosubstrate lipopeptides: study of their implication in induction of apoptosis. FEBS Lett 459: 285-290, 1999.[ISI][Medline]

38. Toullec D, Pianetti P, Coste H, Bellevergue P, Grand-Perret T, Ajakane M, Baudet V, Boissin P, Boursier E, and Loriolle F. The bisindolylmaleimide GF 109203X is a potent and selective inhibitor of protein kinase C. J Biol Chem 266: 15771-15781, 1991.[Abstract/Free Full Text]

39. Wakabayashi S, Bertrand B, Ikeda T, Pouyssegur J, and Shigekawa M. Mutation of calmodulin-binding site renders the Na+/H+ exchanger (NHE1) highly H+ sensitive and Ca2+ regulation-defective. J Biol Chem 269: 13710-13715, 1994.[Abstract/Free Full Text]

40. Wakabayashi S, Ikeda T, Iwamoto T, Pouyssegur J, and Shigekawa M. Calmodulin-binding autoinhibitory domain controls pH-sensing in the Na+/H+ exchanger NHE1 through sequence-specific interaction. Biochemistry 36: 12854-12861, 1997.[ISI][Medline]

41. Webb BLJ, Hirst SJ, and Giembycz MA. Protein kinase C isoenzymes: a review of their structure, regulation and role in regulating airways smooth muscle tone and mitogen. Br J Pharmacol 130: 1433-1452, 2000.[Abstract/Free Full Text]

42. Weinman EJ, Steplock D, Donowitz M, and Shenolikar S. NHERF associations with sodium-hydrogen exchanger isoform (NHE3) and ezrin are essential for cAMP-mediated phosphorylation and inhibition of NHE3. Biochemistry 39: 6123-6129, 2000.[ISI][Medline]

43. Weinman EJ, Steplock D, Wade JB, and Shenolikar S. Ezrin binding domain-deficient NHERF attenuates cAMP-mediated inhibition of Na+/H+ exchange in OK cells. Am J Physiol Renal Physiol 281: F374-F380, 2001.[Abstract/Free Full Text]

44. Wiederkekr MR, Zhao H, and Moe OW. Acute regulation of Na/H exchanger NHE3 activity by protein kinase C: role of phosphorylation. Am J Physiol Cell Physiol 276: C1205-C1217, 1999.[Abstract/Free Full Text]

45. Wooten MW and Wrenn RW. Redistribution of phospholipid/calcium-dependent protein kinase and altered phosphorylation of its soluble and particulate substrate proteins in phorbol ester-treated rat pancreatic acini. Cancer Res 45: 3912-3917, 1985.[Abstract]

46. Wright EM, Hirsch JR, Loo DD, and Zampighi GA. Regulation of Na/glucose co-transporters. J Exp Biol 200: 287-293, 1997.[Abstract/Free Full Text]

47. Yip JW, Ko WH, Viberti G, Huganir RL, Donowitz M, and Tse CM. Regulation of the epithelial brush border Na+/H+ exchanger isoform 3 stably expressed in fibroblasts by fibroblast growth factor and phorbol esters is not through changes in phosphorylation of the exchanger. J Biol Chem 272: 18473-18480, 1997.[Abstract/Free Full Text]

48. Yun CC, Chen Y, and Lang F. Glucocorticoid activation of Na+/H+ exchanger isoform 3 revisited. The role of SGK1 and NHERF2. J Biol Chem 277: 7676-7683, 2002.[Abstract/Free Full Text]

49. Yun CH, Lamprecht G, Forster DV, and Sidor A. NHE3 kinase A regulatory protein E3KARP binds the epithelial brush border exchanger NHE3 and the cytoskeletal protein ezrin. J Biol Chem 273: 25856-25863, 1998.[Abstract/Free Full Text]

50. Yun CH, Oh S, Zizak M, Steplock D, Tsao S, Tse CM, Weinman EJ, and Donowitz M. Cyclic AMP mediated inhibition of the epithelial brush border Na+/H+ exchanger, NHE3, requires an associated regulatory protein. Proc Natl Acad Sci USA 94: 3010-3015, 1997.[Abstract/Free Full Text]

51. Zhao H, Wiederkehr MR, Fan L, Collazo RL, Crowder LA, and Moe OW. Acute inhibition of Na/H exchanger NHE3 by cAMP. Role of protein kinase NHE3 phosphoserines 552 and 605. J Biol Chem 274: 3978-3987, 1999.[Abstract/Free Full Text]

52. Zizak M, Lamprecht G, Steplock D, Tariq N, Shenolikar S, Donowitz M, Yun CHC, and Weinman EJ. cAMP-induced phosphorylation and inhibition of Na+/H+ exchanger (NHE3) is dependent on the presence but not the phosphorylation of NHERF. J Biol Chem 274: 24753-24758, 1999.[Abstract/Free Full Text]

53. Zoukhri D, Hodges RR, Sergheraert C, and Dartt DA. Lacrimal gland functions are differentially controlled by protein kinase C isoforms. Ann NY Acad Sci 842: 217-220, 1998.[Free Full Text]

54. Zoukhri D, Hodges RR, Sergheraert C, and Dartt DA. Protein kinase C isoforms differentially control lacrimal gland functions. Adv Exp Med Biol 438: 181-186, 1998.[ISI][Medline]

55. Zoukhri D, Hodges RR, Sergheraert C, Toker A, and Dartt DA. Lacrimal gland PKC isoforms are differentially involved in agonist-induced protein secretion. Am J Physiol Cell Physiol 272: C263-C269, 1997.[Abstract/Free Full Text]