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Originally published In Press as doi:10.1074/jbc.M703481200 on September 25, 2007

J. Biol. Chem., Vol. 282, Issue 46, 33879-33887, November 16, 2007
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Phosphorylation of PDZ1 Domain Attenuates NHERF-1 Binding to Cellular Targets*Formula

James W. Voltz{ddagger}1, Matthew Brush{ddagger}, Suzanne Sikes{ddagger}, Deborah Steplock§, Edward J. Weinman§, and Shirish Shenolikar{ddagger}2

From the {ddagger}Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710 and the §Department of Medicine, University of Maryland School of Medicine, Department of Veterans Affairs, Baltimore, Maryland 21201

Received for publication, April 26, 2007 , and in revised form, August 31, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
NHERF-1 (Na+-H+ exchanger regulatory factor 1, also known as EBP50 ezrin-binding protein of 50 kDa) is a phosphoprotein that assembles multiprotein complexes via two PDZ domains and a C-terminal ezrin-binding domain. Current work utilized metabolic labeling in cultured cells expressing wild type GFP-NHERF-1 to define the physiological importance of NHERF-1 phosphorylation. Treatment of cells with phosphatase inhibitors calyculin A and okadaic acid enhanced NHERF-1 phosphorylation and inhibited its dimerization. Eliminating C-terminal serines abolished the modulation of NHERF-1 dimerization by phosphatase inhibitors and identified the phosphorylation of the PDZ1 domain that attenuated its binding to physiological targets, including beta2-adrenergic receptor, platelet-derived growth factor receptor, cystic fibrosis transmembrane conductance regulator, and sodium-phosphate cotransporter type IIa. The major covalent modification of PDZ1 was mapped to serine 77. Confocal microscopy of cultured cells suggested key roles for PDZ1 and ERM-binding domain in localizing NHERF-1 at the cell surface. The substitution S77A eliminated PDZ1 phosphorylation and increased NHERF-1 localization at the cell periphery. In contrast, S77D reduced NHERF-1 colocalization with cortical actin cytoskeleton. These data suggested that serine 77 phosphorylation played key role in modulating NHERF-1 association with plasma membrane targets and identified a novel mechanism by which PDZ1 phosphorylation may transduce hormonal signals to regulate the function of membrane proteins in epithelial tissues.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
NHERF-1 (Na+-H+ exchanger regulatory factor isoform 1) was isolated as a phosphoprotein required for cAMP-mediated inhibition of renal Na+/H+ exchanger isoform 3 (NHE3)3 (1). NHE3 bound PDZ2 (2, 3), the second of two PDZ domains in NHERF-1 (4, 5). The C terminus of NHERF-1 bound the ERM (ezrin/radixin/moesin/merlin) family of actin-binding proteins, linking NHE3 to actin cytoskeleton (5, 6). Through the function of ezrin as an AKAP (protein kinase A-anchoring protein), this complex also recruited PKA (7) to promote NHE3 phosphorylation (2) and inhibit sodium-hydrogen exchange. Subsequent studies suggested a stable association of NHERF-1 with NHE3 in cells, and the contribution of NHERF-1 phosphorylation to hormonal control of NHE3 remains unknown.

Subsequent studies identified additional NHERF-1 targets (9).beta2-Adrenergic receptor (beta2AR) bound NHERF-1 in agonist-dependent manner and competed with NHE3 for NHERF-1 binding and paradoxically activate NHE3 in some tissues (10). Moreover, GRK-5 catalyzed the phosphorylation of beta2AR within the C-terminal PDZ-binding motif to attenuate NHERF-1 binding and direct receptor internalization and degradation in lysosomes (11). These studies not only suggested a potential competition among PDZ targets for NHERF-1 binding but also pointed to a dynamic association of NHERF-1 controlled by ligand binding or receptor phosphorylation. However, no role for NHERF-1 phosphorylation has been established in the internalization and recycling of this or other G protein-coupled receptors (GPCRs) (12).

NHERF-1 dictated the apical expression and hormonal control of CFTR (13-15), by formation of a CFTR/NHERF-1/ezrin/PKA complex analogous to that reported for NHE3. Unlike beta2-AR, CFTR docked with both PDZ1 and PDZ2, and thus, a single NHERF-1 molecule could assemble a CFTR dimer (16, 17) to enhance chloride transport (17). Conversely, PKC phosphorylation of human NHERF-1 (serine 162) attenuated CFTR binding to PDZ2 (18) and inhibited transport (15). These studies provided the first hints that NHERF-1 phosphorylation may regulate the assembly and function of cellular NHERF complexes. However, serine 162 is not conserved in NHERF-1 from other species, and the physiological relevance of serine-162 phosphorylation remains to be established.

Ligand-induced receptor dimerization and mitogenic signaling by the platelet-derived growth factor receptor (PDGFR) is enhanced by NHERF-1 binding (19), and a complex of PDGFR with NHERF-1 and ERM proteins may regulate actin cytoskeleton and cell migration (20). Because PDGFR showed a preference for PDZ1, it was postulated that a NHERF-1 homodimer facilitated PDGFR signaling. However, later studies showed that PDGFR, like CFTR, could bind both PDZ1 and PDZ2 (21), suggesting that a monomeric NHERF-1 may also stabilize the ligand-bound PDGFR dimer. However, PDGFR signaling was unexpectedly enhanced rather than impaired in NHERF-1 null MEFs. This suggested quite a different role for NHERF-1, namely to promote the assembly of a complex containing PDGFR and PTEN, a phosphoinositide phosphatase that attenuates mitogenic signaling (20). Because PTEN also showed a preferential binding to PDZ1, it still remains unclear whether monomeric or dimeric NHERF-1 regulates PDGFR signaling.

It is noteworthy that binding of many PDZ1 targets, including beta2-AR and PDGFR, enhanced NHERF-1 dimerization in vitro (19). Fractionation of tissue extracts and coimmunoprecipitation studies (22, 23) also hinted at the existence of NHERF-1 dimers. In this regard, a number of studies have shown that phosphorylation of NHERF-1 by GRK-6A, Cdk-2, and PKC in vitro modulated NHERF-1 dimerization (23-25). Structure-function studies of monomeric NHERF-1 also noted an intramolecular interaction of a C-terminal PDZ motif at the NHERF-1 terminus with PDZ2 (18, 21) that required either the engagement of ezrin (21) or PKC phosphorylation of C-terminal serines (339 and 340), previously implicated in the enhancement of NHERF-1 dimerization (25) to promote activation of the "head-to-tail" monomer and the binding of PDZ1 (21) and PDZ2 targets (18, 21). This suggested that mechanisms that activate the NHERF-1 monomer may also promote its dimerization. On the other hand, NHERF-1 phosphorylation at a major C-terminal site (serine 289) that also enhanced dimerization appears to be constitutive (23) and totally dispensable for hormonal regulation of NHE3 (2). Finally, mass spectrometry of rat NHERF-1 expressed in HEK293 cells identified additional phosphorylations whose physiological role still remains to be investigated.

The current studies focused on the analysis of NHERF-1 phosphorylation in cells with the goal of identifying covalent modifications that regulate the assembly of cellular complexes. Metabolic labeling of cells treated with the phosphatase inhibitors, okadaic acid, and calyculin A established that C-terminal phosphorylation of NHERF-1 inhibited its dimerization. Elimination of three major C-terminal sites identified a phosphorylation of PDZ1 domain of NHERF-1. Biochemical studies showed that the phosphorylation, which occurred at serine 77, attenuated NHERF-1 binding to several PDZ1 targets. Altered localization of mutant NHERF-1, specifically S77A and S77D, suggested a key role for association of PDZ1 targets for NHERF-1 localization at the plasma membrane and suggested a novel mechanism for hormonal regulation of NHERF-1-associated membrane processes in polarized epithelial cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
NHERF-1 Expression Plasmids—Human NHERF-1 cDNA was excised from plasmid encoding HA-tagged human NHERF-1 (26) and inserted in-frame into pEGFP-C2 (Clontech). GFP-D1 was generated by digesting GFP-NHERF-1 with SacI, the cDNA encoding amino acids 1-150 was religated into pEGFP-C2, and its orientation was verified by direct sequencing in the Duke Comprehensive Cancer Center DNA sequencing facility. GFP-D2 was generated by digesting GFP-NHERF-1 with SacI and KpnI and inserted into pEGFP-C3 to yield GFP fused to amino acids, 151-358, of NHERF-1. For {Delta}ERM, site-directed mutagenesis was used to introduce stop codon at amino acids 331. NHERF-1 cDNAs with the individual PDZ-binding sequences, PNGYGF, modified to HNGAGA (the P to H substitution was fortuitous), were cloned into pET30a and pEGFR-C1. Serines 279, 289, and 301 were substituted with alanines using PCR-based site-directed mutagenesis (Stratagene QuikChange® II site-directed mutagenesis kit). Plasmids encoding hexahistidine-tagged NHERF-1, D1, and D2 were previously described (22).

Transfection of Cultured Cells—COS7, HEK293, and NIH3T3 were maintained in Dulbecco's modified Eagle's medium (DMEM; Invitrogen) containing 10% (v/v) fetal bovine serum (HyClone) at 37 °C in 5% CO2 and 95% air. Transfections of plasmid DNA (1 µg) were performed in 6-well plates (Falcon) using 5 µl of Lipofectamine (Invitrogen) in serum-free DMEM. After 3 h, the cells were restored to DMEM containing 10% (v/v) fetal bovine serum.

Cell Treatment and Immunoblotting—Twelve to sixteen hours following transfection, the cells were treated for 20 min with okadaic acid (OA) or calyculin A (CalyA) obtained from Alexis. The cells were washed with phosphate-buffered saline and lysed at 4 °C in radioimmune precipitation assay buffer containing protease inhibitors (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.5% (w/v) deoxycholic acid, 0.1% (w/v) sodium dodecyl sulfate, 1% (w/v) Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, and 1 mM benzamidine). The cell lysates were subjected to SDS-PAGE on 10% (w/v) polyacrylamide gels and electrophoretically transferred to polyvinylidene difluoride membranes. The membranes blocked in 50 mM Tris-HCl, pH 7.5, containing 150 mM NaCl, 0.1% Tween 20 and 4% (w/v) dried milk were incubated overnight with antibodies at 4 °C, and immune complexes were detected by Western Lightning Chemiluminescence Reagent Plus (PerkinElmer Life Sciences).

Metabolic Labeling with [32P]Orthophosphate—The cells (106/ml) were incubated in 6-well plates containing DMEM lacking phosphate with 10% (v/v) fetal bovine serum. After 1 h at 37 °C, the cells were incubated with phosphate-free medium containing 500 µCi/ml of [32P]orthophosphate (PerkinElmer Life Sciences) for 2 h. Following exposure to phosphatase inhibitors (see above), the cells were washed with phosphate-buffered saline and lysed in radioimmune precipitation assay buffer containing protease inhibitors. The cell lysates were incubated with anti-GFP polyclonal antibody (Clontech) for 1 h at 4 °C, and immune complexes were isolated using 1:1 mixture of protein A-agarose (Bio-Rad) and protein G-agarose (Sigma). The immunoprecipitates were washed with 20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 1% (v/v) Nonidet P-40 and subjected to SDS-PAGE. The gels were analyzed by autoradiography (Kodak MR x-ray film) or phosphorimaging. Parallel gels were immunoblotted for protein expression using anti-GFP antibodies (Chemicon; 1:5000 dilution).

Expression of Recombinant Proteins—Plasmids encoding glutathione S-transferase (GST) fused to C termini of beta2AR and PDGFR (provided by R. A. Hall, Emory University), CFTR (J. Biber, University of Zurich), Npt2a (A. Gupta, University of Maryland and S. M Gisler, University of Zurich), NHE3 (O. Moe, University of Texas Southwestern Medical Center), and N-terminal ERM domain of merlin (D. Gutmann, Washington University) were transformed into Escherichia coli BL-21, and bacteria were grown in 500 ml of LB medium at 37 °C until A600 between 0.6 and 0.8. Protein expression was induced by isopropyl beta-D-thiogalactopyranoside (0.1 mM) for 3 h. The bacteria were lysed in 50 mM Tris-HCl, pH 7.5, containing 1 mM EGTA, 1 mM EDTA, 1 mM dithiothreitol, 0.1% (v/v) Nonidet P-40, 1 mM benzamidine, and 1 mM phenylmethylsulfonyl fluoride, and the lysates were incubated with glutathione-Sepharose for 30 min at 4 °C. After extensive washing, the bound proteins were eluted with 50 mM Tris-HCl, pH 8.0, containing 150 mM NaCl, 20 mM glutathione, 1 mM dithiothreitol, and 0.1% (v/v) Nonidet P-40 at 4 °C for 15 min. Eluates, dialyzed against 50 mM Tris-HCl, pH 7.5, containing 1 mM EDTA and 0.1% (v/v) 2-mercaptoethanol, were stored at 4 °C. The protein concentration was estimated using Bio-Rad protein assay (27).


Figure 1
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  		FIGURE 1.
NHERF-1 proteins analyzed. The schematic shows NHERF-1 proteins fused at N terminus to either hexahistidine (Hexahis, filled circle) or GFP (shaded box) analyzed in this study. WT NHERF-1 is shown with two PDZ domains (PDZ1, hatched box; PDZ2, dotted box). The C-terminal 28 amino acids bound ERM proteins and are shown as stippled box. {Delta}ERM lacked the C-terminal 28 amino acids. In {Delta}PDZ1 and {Delta}PDZ2, core hydrophobic residues in the carboxylate-binding loop, GYGF, were substituted with alanines. The mutagenesis resulted in two additional substitutions that converted PNGYGF to NHGAGA. AAA represented full-length NHERF-1 in which serines 279, 289, and 301 were substituted with alanines.

 
NHERF-1 Dimerization—Cells transfected with plasmids encoding GFP-NHERF-1 proteins were lysed with 50 mM Tris-HCl, pH 7.5, containing 0.1% (v/v) Nonidet P-40 and 50 mM NaCl, and cell debris was removed at 1,000 x g for 30 min. Purified His-NHERF-1 proteins were incubated with cell lysates for 1 h at 4 °C before the addition of NTA-agarose (Sigma) for 30 min at 4 °C. The beads were washed with buffer containing 150 mM NaCl, and the bound proteins were eluted with SDS sample buffer and subjected to SDS-PAGE. Western immunoblotting with anti-hexahistidine antibody (Sigma) and anti-GFP antibody monitored the association of His- and GFP-NHERF-1.

NHERF-1 Binding by Cellular Targets—Lysates containing GFP-NHERF-1 were incubated with increasing amounts of recombinant GST fusion proteins for 30 min at 4 °C. The mixtures were incubated with glutathione-Sepharose (Amersham Biosciences) for 1 h, and the beads were washed extensively prior to elution with SDS sample buffer and SDS-PAGE and immunoblotting with anti-GFP.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Activity of GFP-NHERF-1—Prior studies used untagged (28) and His-NHERF-1 (29) to transduce cAMP signals that inhibited NHE3 activity in PS120/NHE3V cells. Because GFP represented a larger (approximately 27 kDa) protein fusion that could potentially compromise NHERF-1 function, we analyzed the ability of GFP-NHERF-1 (expressed at various levels) to mediate cAMP inhibition of NHE3 in PS120-NHE3V cells. NHE3 activity in PS120-NHE3V cells expressing the highest levels of GFP-NHERF-1 was inhibited 40-50% by 10 µM Fsk. (supplemental Fig. S1). Comparison at different protein expression levels suggested that GFP-NHERF-1 was functionally indistinguishable from untagged or His-NHERF-1 (Fig. 1). Treatment of PS120/NHE3V with GFP-NHERF-1 with 100 nM CalyA, a cell-permeable phosphatase inhibitor, achieved similar inhibition of NHE3 (supplemental Fig. S1). These studies showed that PKA activation or phosphatase inhibition resulted in a similar decrease in NHE3 activity. The combined effects of CalyA and Fsk were similar to either agent alone, suggesting a common mechanism for NHE3 inhibition.

NHERF-1 Dimerization in Cells—Prior studies used okadaic acid to inhibit NHERF-1 dimerization in cells (22). Because okadaic acid displayed significant cytotoxicity in some cells, we re-evaluated NHERF-1 dimerization by coexpressing HA-NHERF-1 and His-NHERF-1 in PS120 cells (Fig. 2) treated with either vehicle or 100 nM CalyA. Although equal amounts of His-NHERF-1 bound NTA-agarose from control and CalyA-treated cells, HA-NHERF-1 cosedimented with His-NHERF-1 was greatly reduced (75-80%) by 100 nM CalyA. No detectable HA-NHERF-1 bound NTA-agarose when cells did not also express His-NHERF-1. Comparison of cells expressing His- and HA-{Delta}PDZ1/2 showed that the mutant protein dimerized effectively, and its dimerization was also inhibited by CalyA. This suggested that NHERF-1 dimerization and modulation by phosphatase inhibitors did not require functional PDZ domains.

To identify NHERF-1 domains required for CalyA-regulated dimerization, we modified the above assay. Lysates of cells expressing WT GFP-NHERF-1 were incubated with defined quantities of recombinant His-NHERF-1, and GFP-NHERF-1 was sedimented using NTA-agarose (Fig. 3). WT GFP-NHERF-1 expressed in COS-7 cells (or HeLa cells; data not shown) migrated as two bands on SDS-PAGE (Fig. 3A). The upper band represented phosphorylated NHERF-1 as it was completely eliminated by incubation of NHERF-1 complexes bound to NTA-agarose with calf intestinal phosphatase (or bacteriophage {lambda} phosphatase; data not shown). Pulldowns with full-length or truncated (D1 and D2) His-NHERF-1 showed that they preferentially bound the lower unphosphorylated or less phosphorylated form of GFP-NHERF-1. These data suggested that NHERF-1 dimerization in cells was mediated by both D1 and D2 regions and was inhibited by phosphorylation. Moreover, GFP-NHERF-1 binding by all three baits was inhibited 75-80% by CalyA.


Figure 2
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  		FIGURE 2.
Homodimerization of NHERF-1 in PS120 cells. PS120 cells expressed either WT NHERF-1 or {Delta}PDZ1/2 were expressed as both HA- and His-tagged proteins. The cells were incubated in the absence (-) or presence (+) of 100 nM CalyA for 20 min. Following cell lysis, His proteins were sedimented using NTA-Sepharose ("Materials and Methods"). The bound NHERF-1 proteins were then subjected to SDS-PAGE and analyzed by immunoblotting (IB) with both anti-HA and anti-His antibodies. A representative experiment from three independent studies is shown.

 


Figure 3
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  		FIGURE 3.
Structural requirements for NHERF-1 dimerization. COS7 cells expressing WT GFP-NHERF-1 were incubated in the absence (-) or presence (+) of 100 nM CalyA for 20 min. The cell lysates were incubated with the indicated recombinant His-NHERF-1 polypeptide. Following sedimentation with NTA-Sepharose, the bound GFP-NHERF-1 was detected by immunoblotting (IB) with anti-GFP antibody. A shows the binding of GFP-NHERF-1 by His-NHERF-1 (FL), D1, and D2. Representative immunoblot from more than eight independent experiments is shown. B shows the sedimentation of GFP-AAA containing alanine substitutions in place of serines 279, 289, and 301 by WT His-NHERF-1.

 
Prior studies identified three major phosphorylated serines, 279, 289, and 301, near the C terminus of NHERF-1 that modulated dimerization (24, 26). We expressed GFP-AAA, a fulllength NHERF-1 with alanines substituted in place of serines 279, 289, and 301. Compared with WT-NHERF-1, GFP-AAA migrated as a single polypeptide, suggesting that phosphorylation of one or more of these C-terminal serines generated the slower migrating form of NHERF-1. In pulldowns, GFP-AAA bound His-NHERF-1 (Fig. 3B), but treatment of cells with CalyA resulted in little or no diminution of GFP-AAA bound by His-NHERF-1. Thus, inhibition of NHERF-1 dimerization was mediated by CalyA-induced phosphorylation of some or all of these C-terminal serines.

Calyculin A Induced PDZ1 Phosphorylation—For direct analysis of CalyA-induced phosphorylation, COS7 cells expressing GFP-NHERF-1 were metabolically labeled with [32P]orthophosphate. GFP-NHERF-1 was immunoprecipitated using anti-GFP and immunoprecipitates subjected to SDS-PAGE and autoradiography (Fig. 4A) or phosphorimaging. As previously seen in HEK293 and PS120 cells (23, 30), full-length NHERF-1 is extensively phosphorylated in COS7 cells under basal conditions. NHERF-1 phosphorylation was further increased 1.5-2-fold in cells treated with CalyA with maximum 32P-labeling occurring between 10 and 100 nM CalyA. Substitution of alanines in place of serines 279, 289, and 301 eliminated the basal phosphorylation of GFP-AAA. However, when cells were exposed to 100 nM CalyA, the phosphorylation of GFP-AAA was readily visible. GFP-D2 was highly phosphorylated in untreated cells, and its phosphorylation was moderately increased when cells were treated with 100 nM CalyA, consistent with the presence of the C-terminal serines 279, 289, and 301. By comparison, GFP-D1 showed no detectable basal phosphorylation but was phosphorylated only in response to 100 nM CalyA.

OA differs from CalyA in the inhibition of type I and type II serine/threonine phosphatases in being a more potent inhibitor of type 2 phosphatases. Metabolic labeling of COS7 cells treated with increasing concentrations of OA showed that GFP-D1 phosphorylation was enhanced between 1 and 10 µM OA (Fig. 4B) to a level similar to 100 nM CalyA. The data suggested that GFP-D1 phosphorylation was most likely reversed by type I protein serine/threonine phosphatase that required OA concentrations higher than 1 µM for effective inhibition. Because 100 nM CalyA lacked the cytotoxicity seen with micromolar concentrations of OA, subsequent analyses of GFP-D1 phosphorylation utilized cells treated with CalyA.

Calyculin A-stimulated Phosphorylation Sites in PDZ1—NHERF-1 is a phosphoprotein, whereas its structural homologue, NHERF-2, is not. There are 6 serines and threonines in NHERF-1 D1 region, and some within the PDZ1 domain are also conserved in NHERF-2 (Fig. 5A). We substituted alanines in place of serines 2, 46, 77, and 143 and threonines 71 and 95, whereas serine 143 was eliminated by the C-terminal truncation, {Delta}140 (Fig. 5A). Mutant GFP-D1 proteins were expressed in COS7 cells metabolically labeled with [32P]orthophosphate in the presence of 100 nM CalyA. Immunoblotting with anti-GFP established equivalent expression of all GFP-D1 peptides. Autoradiography of anti-GFP immunoprecipitates showed that eliminating serines 2 and 46 and threonine 71 had no significant effect on the in vivo labeling of GFP-D1 (Fig. 5B). By comparison, T95A and {Delta}140 consistently showed 10-20% reduced phosphorylation compared with WT GFP-D1. Most strikingly, the in vivo phosphorylation of S77A was reduced 75-85%, identifying serine 77, uniquely present in PDZ1 of NHERF-1, as the major site of CalyA-induced phosphorylation.


Figure 4
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  		FIGURE 4.
Phosphorylation of NHERF-1 in COS7 cells. A shows the phosphorylation of WT GFP-NHERF-1, GFP-AAA, GFP-D1, and GFP-D2 immunoprecipitated (IP) using anti-GFP antibody from lysates of COS7 cells metabolically labeled with [32P]orthophosphate as described under "Materials and Methods." The cells were treated with increasing concentrations of CalyA. B shows the phosphorylation of GFP-D1 in cells treated with 100 nM CalyA and increasing concentrations of OA. Representative autoradiographs from more than six independent experiments are shown.

 


Figure 5
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  		FIGURE 5.
Identification of serine 77 as the major calyculin A-induced phosphorylation site in PDZ1. A shows the alignment of primary sequences of PDZ1 domains from NHERF-1, a known phosphoprotein, and NHERF-2, which is not phosphorylated in cells. Serines and threonines conserved in both proteins are shown in bold and underlined. Serine 77 is uniquely present in NHERF-1 and is highlighted as an enlarged bold letter. The schematic shows the location of serines in GFP-D1, and {Delta}140 shows the C-terminal truncation that eliminated serine 143. B shows the immunoprecipitates of GFP proteins with individual serines substituted with alanines. The lower panel shows the anti-GFP immunoblot (IB) of anti-GFP immunoprecipitates from COS7 cells treated with 100 nM CalyA. The upper panel shows the corresponding autoradiograph. A representative figure from six independent experiments is shown.

 
Phosphorylation of Serine 77 Attenuates Binding of PDZ1 Targets—To assess the function of CalyA-induced phosphorylation, GFP-D1 was sedimented from lysates of cells treated with CalyA using recombinant GST-betaAR encompassing the C terminus of beta2-adrenergic receptor, a known PDZ-I target. Increasing amounts of GFP-D1 were sedimented by 0.1-10 µg of GST-betaAR from control COS7 lysates (Fig. 6A). Treatment of cells with 10-100 nM CalyA reduced GFP-D1 binding by GST-betaAR in a dose-dependent manner. The inhibition was most readily visible at 0.5 and 0.1 µg of bait protein with 100 nM CalyA inhibiting GFP-D1 binding by 70 and 90%, respectively (Fig. 6B). CalyA also inhibited GFP-D1 binding by other PDZ1 targets, GST-PDGFR and GST-CFTR (Fig. 6C). No detectable sedimentation of GFP-D1 was observed with GST alone in the presence or absence of CalyA. Essentially identical results were obtained for the binding of full-length GFP-NHERF-1 by all three PDZ1 targets (data not shown).

Bone loss resulting from phosphate wasting in mice lacking a functional NHERF-1 gene established the renal sodium-phosphate cotransporter, Npt2a, as a physiological target of NHERF-1 (32). Pulldowns with GST-Npt2a containing the C terminus of Npt2a established its binding to full-length GFP-NHERF-1, which was significantly inhibited by the treatment of cells with 100 nM CalyA (Fig. 6D). In several independent experiments, 100 nM CalyA resulted in 45-50% inhibition of GFP-NHERF-1 binding by GST-Npt2a.


Figure 6
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  		FIGURE 6.
Calyculin A-induced phosphorylation inhibits PDZ1 binding. A shows a representative immunoblot (IB) demonstrating the sedimentation of GFP-D1 from lysates of COS7 cells treated with increasing concentrations of calyculin A by GST-beta2AR, encompassing the C terminus of beta2AR (µg of purified protein). B shows the dose-dependent inhibition of GFP-NHERF-1 binding to GST-betaAR (0.1 and 0.5 µg) by calyculin A. C shows calyculin A-mediated inhibition of GFP-D1 binding to GST-betaAR, GST-PDGFR, and GST-CFTR, representing the C termini of three known NHERF-1 targets. D shows calyculin A-mediated inhibition of binding of full-length GFP-NHERF-1 by GST-Npt2a, encompassing C terminus of sodium-phosphate cotransporter type IIa, a physiological target of NHERF-1 in the mammalian kidney. The left panel shows a representative immunoblot for binding of GFP-NHERF-1 by GST-Npt2a, and the right panel shows the quantitation of four independent experiments with standard errors.

 


Figure 7
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  		FIGURE 7.
Importance of Serine 77 phosphorylation in calyculin A inhibition of PDZ1 target binding. A shows representative immunoblot (IB) of GFP-D1 sedimented from COS7 cell lysates by GST-betaAR (0.1 and 2.5 µg). Binding of WT GFP-D1, S46A, T71A, and S77A is shown. B shows the three-dimensional structure of NHERF-1 PDZ1 domain. The core carboxylate binding motif, GYGF, is shown in blue. Serine 46, which diminished basal binding to GST-betaAR, is shown in red. Other potential in vivo phosphorylation sites, serine 77, threonine 71, and threonine 95 are shown in green. The major site for calyculin A-induced phosphorylation identified in this study, serine 77, is highlighted by a red circle.

 
To identify the residues in PDZ1 that transduced the inhibitory effects of CalyA, we undertook pulldowns from COS7 lysates containing WT and mutant GFP-D1 lacking individual serines and threonines using GST-betaAR as bait (Fig. 7A). With 0.1 and 2.5 µg of the bait protein, we noted dose-dependent sedimentation of WT GFP-D1, which was inhibited 90 and 65%, respectively, by 100 nM CalyA. S46A showed a dramatic reduction in basal binding to GST-betaAR, potentially disrupting the critical carboxylate loop required for binding of target C termini. The residual binding was still sensitive to CalyA and virtually eliminated in cells treated with 100 nM CalyA. Like WT GFP-D1, CalyA inhibited T71A binding to GST-betaAR. However, GST-betaAR binding to S77A was not inhibited by CalyA, most clearly seen with 2.5 µg of bait. This suggested that phosphorylation of serine 77 located on the PDZ1 {alpha}-helix involved in target binding (Fig. 7B) mediated the inhibitory effect of CalyA.


Figure 8
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  		FIGURE 8.
Confocal microscopy of COS7 cells expressing GFP-NHERF-1. A, COS7 cells transfected with cDNA encoding WT GFP-NHERF-1 were fixed as described under "Materials and Methods" and subjected to laser scanning microscopy. GFP fluorescence is shown in green, actin cytoskeleton was stained with rhodamine-phalloidin (red), and the nucleus was stained with the DNA-binding dye, 4',6'-diamino-2-phenylindole (DAPI, blue). The top row shows a representative cell (in black-and-white) showing images at the individual channels, whereas the bottom row shows color images of GFP alone, an overlay of F-actin and nuclear staining and overlay of all three channels. B shows the overlays of rhodamine-phalloidin and GFP fluorescence of cells expressing WT GFP-NHERF-1, {Delta}ERM, {Delta}PDZ1, and {Delta}PDZ2. C shows representative the three color images for GFP-S77D and GFP-S77A. In all above panels, an asterisk marks the nucleus, and arrows point to areas of cell periphery where rhodamine-phalloidin and/or GFP are concentrated.

 
GFP-NHERF-1 Localization in Cells—Analysis of subcellular distribution of GFP-NHERF-1 in COS7 cells (Fig. 8) showed that WT NHERF-1 was predominantly cytosolic with some protein colocalizing with actin cytoskeleton stained by rhodamine-phalloidin at the cell periphery (white arrows in Fig. 8A). Most importantly, WT GFP-NHERF-1 was largely excluded from the nucleus and stained with 4',6'-diamino-2-phenylindole (asterisk in Fig. 8A). Deletion of C-terminal ERM-binding domain reduced the concentration of GFP-{Delta}ERM at the cell periphery and increased its entry into the nucleus (asterisk in Fig. 8B). GFP-{Delta}PDZ1 that lacked the core GLGF sequence required for binding of PDZ1 targets (33) also showed a reduced concentration at the cell periphery and increased entry into the nucleus. In contrast, {Delta}PDZ2 lacking a functional PDZ2 domain was essentially identical to WT GFP-NHERF-1 and was largely excluded from the nucleus. Essentially identical localization of GFP-NHERF-1 was seen in HEK293 and NIH3T3 cells, although lower expression of GFP proteins in these cells yielded a weaker signal (data not shown).

Pulldowns using GST-betaAR, a PDZ1 target (10), and GST-NHE3, a PDZ2 target (6), established that these proteins bound WT GFP-NHERF-1. By contrast, GST-betaAR and GST-NHE3 failed to bind {Delta}PDZ1 and {Delta}PDZ2, respectively. All three proteins bound GST-merlin, demonstrating the presence of a functional ERM-binding domain (data not shown).

Remarkably, COS7 cells expressing the GFP-NHERF-1 mutant, S77A, which abolished PDZ1 phosphorylation, showed enhanced peripheral localization compared with WT NHERF-1 (Fig. 8C). In contrast, S77D that mimicked phosphorylation showed reduced localization at the cell periphery, displaying little overlap with rhodamine-phalloidin. Neither S77A nor S77D showed significant nuclear localization. Thus, unlike {Delta}PDZ1, S77D did not represent a complete loss of function of PDZ1, which may be necessary for NHERF-1 redistribution to cell nucleus, but reflected altered affinity for PDZ1 targets, most of which are located at the plasma membrane.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In Vivo Phosphorylation of NHERF-1—Mass spectrometry of rat NHERF-1 expressed in HEK293 cells identified six phosphoserines and threonines located either in the PDZ1 domain or near the C terminus (25). The majority of the protein-bound phosphate was on serine 287 (serine 289 in human NHERF-1) and was unresponsive to cell stimuli (23). The constitutive serine 289 phosphorylation is catalyzed by GRK6A, which binds NHERF-1 PDZ1 and possibly PDZ2 domains (23). WT NHERF-1 expressed in COS7 (or HEK293, NIH3T3, and HeLa; not shown) migrated on SDS-PAGE as two polypeptides with the upper band representing highly phosphorylated NHERF-1 and thus eliminated by dephosphorylation of His-NHERF-1 bound to NTA-agarose or GFP-NHERF-1 in anti-GFP immunoprecipitates (data not shown) with nonspecific phosphatases. In contrast, {Delta}PDZ1/2, which lacked functional PDZ domains, migrated as a single polypeptide, suggesting that PDZ domains recruited protein kinase(s) that generates the slower migrating NHERF-1. Although GRK6A and PKC, which is recruited to NHERF-1 via RACK1, the receptor for activated C-kinase (REF), both bind NHERF-1 PDZ1, the actions of these kinases promote rather than inhibit NHERF-1 dimerization. In this regard, our studies showed that His-NHERF-1 preferentially bound the faster migrating or less phosphorylated NHERF-1, favoring phosphorylations by a kinase like Cdc2 that inhibit dimerization (24). Consistent with this notion, substitutions of alanines in place of serines 279 and 301 as well as the constitutive site at serine 289 yielded GFP-AAA that migrated as a single species, whose association with His-NHERF-1 was no longer inhibited by phosphatase inhibitors. Hence, the more slowly migrating, phosphorylated NHERF-1 that failed to bind His-NHERF-1 may be generated by the combined actions of PDZ-associated kinases like GRK6A or PKC and a Cdc2-like kinase, whose phosphorylations must be functionally dominant to reduce NHERF-1 dimerization in CalyA-treated cells.

Metabolic labeling revealed the phosphorylation of GFP-AAA only in the presence of phosphatase inhibitors, suggesting a rapidly turned over or transient phosphorylation. GFP-D1 phosphorylation was similarly stimulated by CalyA and was mapped to serine 77. Interestingly, mass spectrometry of rat NHERF-1 had identified inconsistent phosphorylation of serine 77 and threonine 71. Although the substitution S77A diminished in vivo phosphorylation of GFP-D1 by more than 80%, T71A had no effect and thus appeared not to be phosphorylated in COS7 cells. Instead, T95A, which reduced GFP-D1 radiolabeling by approximately 15%, accounted for the remaining protein-bound phosphate.

Serine 77 is located on the surface of the {alpha}2-helix of PDZ1. Histidine 72 and arginine 80 on this helix make critical contacts with side chains of amino acids at the -2 position and leucine 0 to tether the C termini of PDZ-I targets. The introduction of a negative charge in this vicinity might be predicted to reduce affinity for PDZ targets. Consistent with this notion, functional studies showed that phosphorylation of serine 77 elicited by CalyA reduced the binding of recombinant peptides representing known PDZ1 targets, specifically beta2AR, PDGFR, CFTR, and Npt2a.

Modulation of NHERF-1 Dimerization—At low protein concentrations, NHERF-1 is monomer, whereas at the higher concentrations seen in renal and other epithelial tissues, it is largely dimeric. Recent studies suggest that monomeric NHERF-1 represents an inactive form formed by the intramolecular interaction of the NHERF-1 C terminus with PDZ2 domain (21) that prevents recruitment of PDZ2 and possibly some PDZ1 targets (18, 21). However, monomeric NHERF-1 can be activated by the binding of ezrin (21) or by phosphorylation of serines 339 and 340 by PKC (18). Interestingly, PKC phosphorylation at these sites (serines 337 and 338 in rat NHERF-1) also promoted dimerization (25), which may in part result from the enhanced binding of PDZ1 targets that also promotes the recruitment of PDZ2 targets to the activated NHERF-1 monomer. Because many PDZ1 ligands, including beta2AR and PDGFR, promoted NHERF-1 dimerization (19), we speculate that interactions of ezrin and PDZ1 targets possibly combined with the constitutive phosphorylation of serine 289 promotes the formation of NHERF-1 dimers to enlarge and stabilize multiprotein complexes at the apical membranes. In support of this hypothesis, NHERF-1, which is highly concentrated at apical membranes of the mouse kidney (32) and intestine (34), was more diffusely distributed in intestine of ezrin null mice. Moreover, basal or constitutive NHERF-1 phosphorylation was dramatically reduced in these mutant mice (35).

It is also possible that even in the context of the NHERF-1 dimer, some complexes like the dimeric CFTR (17), CFTR and NHE3 (36), or PDGFR and PTEN (20) are assembled by binding to PDZ1 and PDZ2 within the same NHERF-1 molecule, whereas others, like the ligand-bound PDGFR dimer (19), CFTR and beta2AR (37), PTEN and PDGFR (20), and PTH1R and phospholipase-Cbeta (38, 39) may bind adjacent PDZ1 domains on the dimeric NHERF-1. In this regard, the phosphorylation of PDZ1 at serine 77 may preferentially attenuate the recruitment of some PDZ1 targets and dissociate selected NHERF-1 complexes in response to hormones and other physiological stimuli.

Cellular Targeting of NHERF-1—NHERF-1 was first purified from rabbit renal brush border membranes (1) and required mild detergents for its efficient extraction. Immunohistochemistry of mouse and human renal proximal tubules provided further evidence for a high NHERF-1 concentration at apical membranes (32). Finally, more than two-thirds of known NHERF-1 targets represent PDZ1-binding proteins that reside in the plasma membrane or the underlying cytoskeleton (9). By comparison, the few nuclear targets show a preferred binding to PDZ2. Thus, GFP-NHERF-1 expressed in COS7, HEK293, and NIH3T3 cells showed similar diffuse cytosolic distribution with some concentration at or near the plasma membrane, most readily seen in COS7 cells, which expressed higher levels of GFP-NHERF-1. Most notably, WT GFP-NHERF-1 was largely excluded from the nucleus. Deletion of the C-terminal ERM-binding region, {Delta}ERM, or the mutation, {Delta}PDZ1, that abolished the binding of PDZ1 targets, dramatically reduced plasma membrane localization of these mutant NHERF-1 proteins, allowing their nuclear entry. This pointed to the combined actions of PDZ1 targets and ezrin in localizing NHERF-1 outside the nucleus or concentrating NHERF-1 at the cell periphery. Remarkably, the mutant S77A showed enhanced localization at the cell periphery in COS7 cells, whereas the concentration of S77D, which mimicked PDZ1 phosphorylation and attenuated the binding of cellular targets at the cell periphery, was reduced. These data suggested that serine 77 phosphorylation did not represent a complete loss of PDZ1 function but modified the affinity of NHERF-1 to regulate the trafficking and/or activity for the many membrane targets.

Impact on Hormone Signaling—A major physiological defect in the NHERF-1 null mice was the loss of bone density-associated excessive renal phosphate wasting (32). Mechanistic studies demonstrated reduced abundance of Npt2a in apical membranes of the mutant mice and identified Npt2a as a physiological target of NHERF-1 in the mammalian kidney. Moreover, renal phosphate transport in NHERF-1 mutant mice was unresponsive to PTH and alterations in dietary phosphate (8, 40), and both defects were corrected by adenoviral re-expression of NHERF-1 (8).

PTH signals via PTH1R to activate PKC and PKA and down-regulate Npt2a in the mammalian kidney. Our studies suggested that PKC but not PKA (supplemental Fig. S2) phosphorylated PDZ1 in vitro, and this phosphorylation could be eliminated by the substitution, S77A (31). Most importantly, the analysis of primary renal proximal tubule cells from NHERF-1 null mice expressing WT NHERF-1, S77D, and S77A using adenovirus showed that PTH signals promote NHERF-1 phosphorylation at serine 77 to dissociate it from Npt2a. This in turn increases Npt2a internalization to inhibit sodium-phosphate transport (31). In summary, current studies provided compelling biochemical evidence for a regulated assembly of cellular complexes by transient and reversible phosphorylation of PDZ1 and suggested that cell mechanisms potentially acting through NHERF-1 phosphorylation may control both the assembly and disassembly of membrane complexes in epithelial tissues.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grant DK55881 (to S. S. and E. J. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2. Back

1 Present address: NIEHS, Laboratory for Epithelial Biology, Research Triangle Park, NC 27709. Back

2 To whom correspondence should be addressed: Dept. of Pharmacology and Cancer Biology, Duke University Medical Center, LSRC C315, Research Dr., Durham, NC 27710. Tel.: 919-613-8460; Fax: 919-681-9567; E-mail: Sheno001{at}mc.duke.edu.

3 The abbreviations used are: NHE3, Na+/H+ exchanger isoform 3; GFP, green fluorescent protein; WT, wild type; PKA, cAMP-dependent protein kinase; beta2AR, beta2-adrenergic receptor; GPCR, G protein-coupled receptor; PDGFR, platelet-derived growth factor receptor; PKC, protein kinase C; HA, hemagglutinin; DMEM, Dulbecco's modified Eagle's medium; OA, okadaic acid; CalyA, calyculin A; GST, glutathione S-transferase; NTA, nitrilotriacetic acid; PTH, parathyroid hormone; CFTR, cystic fibrosis transmembrane conductance regulator. Back


   ACKNOWLEDGMENTS
 
We thank Drs. Biber, Gisler, Gupta, Guttman, Hall, and Moe for providing key reagents.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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