Characterization of Protein Kinase A-mediated Phosphorylation of Ezrin in Gastric Parietal Cell Activation*

Gastric ezrin was initially identified as a phosphoprotein associated with parietal cell activation. To explore the nature of ezrin phosphorylation, proteins from resting and secreting gastric glands were subjected to two-dimensional SDS-PAGE. Histamine triggers acid secretion and a series of acidic isoforms of ezrin on two-dimensional SDS-PAGE. Mass spectrometric analysis of these acidic ezrin spots induced by stimulation suggests that Ser 66 is phosphorylated. To determine whether Ser 66 is a substrate of protein kinase A (PKA), recombinant proteins of ezrin, both wild type and S66A mutant, were incubated with the catalytic subunit of PKA and [ 32 P]ATP. Incorporation of 32 P into wild type but not the mutant ezrin verified that Ser 66 is a substrate of PKA. In addition, expression of S66A mutant ezrin in cultured parietal cells attenuates the dilation of apical vacuolar membrane associated with stimulation by histamine, indicating that PKA-mediated phosphorylation of ezrin is necessary for acid secretion. In fact, expression of phospho-rylation-like S66D mutant in parietal cells mimics hista-mine-stimulated apical vacuole remodeling. Further examination of H,K-ATPase distribution

Ezrin is an actin-binding protein of the ezrin/radixin/moesin (ERM) 1 family of cytoskeleton membrane linker proteins (1). Within the gastric epithelium, ezrin has been localized exclusively to parietal cells and primarily to the apical canalicular membrane of these cells (e.g. Refs. 2 and 3). Because of its cytolocalization and observed stimulation-dependent phosphorylation, an implied role for ezrin was suggested in the apical surface membrane remodeling associated with parietal cell activation via the protein kinase A pathway. Phosphorylation of ezrin has also been shown to be associated with surface membrane remodeling of A431 cells stimulated by epidermal growth factor, although activation in this case was via protein tyrosine kinase (4,5). Our previous studies showed that gastric ezrin is co-distributed with the ␤-actin isoform in vivo (6) and preferentially bound to the ␤-actin isoform in vitro (7). However, it is still not clear how ezrin is involved in the membrane cytoskeletal dynamics triggered by histamine stimulation.
Using fluorescence resonance energy transfer monitored by fluorescence lifetime imaging microscopy and chemotaxis assays (8), it has been shown that protein kinase C-mediated phosphorylation of CD44 and ezrin modulates the interaction between these two proteins in vivo and that this phosphorylation was critical for CD44-directed cell motility, suggesting that phosphorylation of ezrin and its accessory proteins provides means to regulate the membrane cytoskeletal dynamics in response to stimulation. Whereas protein kinase C-mediated phosphorylation of CD44 was mapped to Ser 291 , the nature of ezrin phosphorylation is not characterized.
Phosphorylation has been proposed to regulate ERM activation, since phosphorylation of ERM proteins correlates with their cytoskeletal association, whereas dephosphorylation of ezrin is parallel to its liberation from actin-based cytoskeleton (e.g. Refs. 9 and 10). Ezrin is phosphorylated on tyrosine residues upon growth factor stimulation (11)(12)(13). In response to epidermal growth factor, ezrin phosphorylation on tyrosines 145 and 353 is concomitant with an increase in dimer formation, suggesting a causal relationship between phosphorylation and oligomerization (14,15). However, mutations of these tyrosines into phenylalanines does not alter ezrin localization in microvilli, and production of this mutated ezrin does not affect cell morphology (13). Thus, it has been proposed that tyrosine phosphorylation of ezrin may serve in signal transduction rather than mediating its cytoskeletal association. This notion was supported by the experiments in which phosphorylation of tyrosine 353 was found to signal cell survival during epithelial differentiation (16).
A phosphothreonine residue, originally identified in moesin (17), is localized in a conserved COOH-terminal region of ERM proteins (Thr 567 in ezrin, Thr 564 in radixin, and Thr 558 in moesin). Using phosphospecific antibodies, this phosphorylated residue was detected in ezrin, radixin, and moesin from a variety of cells and tissues, and phosphorylated ERM proteins were shown to be present in actin-rich membrane structures (18 -21). Two kinases, protein kinase C and Rho kinase, and two phosphatases, myosin phosphatase and PP2C, were found in different systems to regulate the phosphorylation status of the conserved C-terminal threonine in ERM proteins (19,(21)(22)(23). The primary consequence of phosphorylating the COOHterminal threonine is thought to regulate ezrin activity. Using an overlay assay, phosphorylation of Thr 564 in the radixin COOH-terminal domain impaired its association with the NH 2terminal domain (19). Similarly, a T558D mutation of moesin, which mimics the phosphorylated state, was shown to alter the intra-and intermolecular interactions of ezrin (24). From the crystal structure, it appears that the phosphorylation of moesin Thr 558 weakens the N/C-ERMAD interaction due to both electrostatic and steric effects (25). The phosphorylation of an isolated COOH-terminal fragment of ERM proteins does not affect its association with F-actin (19,24). However, expression of Thr 3 Asp mutant forms of ezrin or moesin potentiates the formation of microvilli-like dorsal projections by growth factors (20,26), whereas transfection of the nonphosphorylatable T558A moesin inhibits RhoA-induced formation of these structures (20,27).
To explore the nature of ezrin phosphorylation associated with histamine-mediated stimulation of acid secretion, we carried out mass spectrometric analyses of ezrin phosphorylated in vivo and in vitro to identify a novel phosphorylation site on ezrin. Our studies show that Ser 66 is a substrate of cAMP-dependent protein kinase and suggest that phosphorylation of Ser 66 by PKA is critical for parietal cell activation. Given the fact that nonphosphorylatable ezrin prevents parietal cell activation, we proposed that PKA-mediated phosphorylation of ezrin provides a link between the activation of PKA to apical membrane cytoskeletal remodeling associated with acid secretion in parietal cells.

EXPERIMENTAL PROCEDURES
Reagents-[ 14 C]Aminopyrine and [ 32 P]ATP were obtained from PerkinElmer Life Sciences. Monoclonal antibody (JL-18) against rGFP was purchased from Clontech (Palo Alto, CA), whereas ezrin antibody 4A5 was produced and described by Hanzel et al. (28). Antibody 2G11 against the ␤-subunit of HK-ATPase was described by Chow et al. (29). Monoclonal antibody against phosphoserine and all chemicals were ordered from Sigma. Rhodamine-coupled phalloidin was purchased from Molecular Probes, Inc. (Eugene, OR). LipofectAMINE 2000 was obtained from Invitrogen.
Isolation of Gastric Glands and Aminopyrine Uptake Assay-Gastric glands were isolated from New Zealand White rabbits as modified by Yao et al. (30). Briefly, the rabbit stomach was perfused under high pressure with PBS (2.25 mM K 2 HPO 4 , 6 mM Na 2 HPO 4 , 1.75 mM NaH 2 PO 4 , and 136 mM NaCl) containing 1 mM CaCl 2 and 1 mM MgSO 4 . The gastric mucosa was scraped from the smooth muscle layer, minced, and then washed twice with minimal essential medium (MEM) buffered with 20 mM HEPES, pH 7.4 (HEPES-MEM). The minced mucosa was then digested at 37°C for ϳ30 min in a minimal amount (ϳ20 ml) of HEPES-MEM containing 15 mg of collagenase (Sigma) and 20 mg each of bovine serum albumin (Sigma). Intact gastric glands were collected from the digestion mixture for 10 -15 min and then washed three times in HEPES-MEM. In all subsequent gland experiments (aminopyrine uptake assay and two-dimensional SDS-PAGE analysis), glands were resuspended at 5% cytocrit (v/v) in the appropriate buffer for final assay.
Stimulation of rabbit gastric glands was quantified using the aminopyrine (AP) uptake assay as modified by Yao et al. (30). Briefly, glands were loaded with [ 14 C]aminopyrine followed by treatment with 100 M cimetidine. Stimulation of glands was achieved with 50 M IBMX and 100 M histamine. Gland preparations were incubated for different time intervals at 37°C with shaking (ϳ160 oscillation/min) followed by a brief spin to separate the glands from supernatant. The gland pellets were dried and weighed, and aliquots of both the supernatant and pellet were counted in a Beckman liquid scintillation counter. These data were used to calculate the AP accumulation ratio (ratio of intracellular to extracellular AP concentration) as described (30). To calculate the stimulation index of AP uptake, the data are expressed as a -fold level of the resting control from each individual time points.
Extract Preparation and Two-dimensional Electrophoresis-Gastric glands were either maintained in resting or stimulated states for appropriate intervals as described (6). At the end of treatment, the glands were pelleted and solubilized with lysis buffer (7 M urea, 2 M thiourea, 4% CHAPS, 2% dithiothreitol, 2% IPG buffer (pH 4 -7) (Amersham Biosciences), 1 mM benzamidine, 2 g/ml pepstatin A, 20 g/ml leupeptin, 10 g/ml aprotinin, 1 mM sodium vanadate, 1 M microcystin-LR, 1.5 mM EDTA, and 1.5 mM EGTA). Glandular cell proteins were extracted for 1 h at room temperature followed by centrifugation at 100,000 ϫ g for 20 min. ReadyStrips (pH 3-10) were loaded with 200 g of whole-cell extract for analytical two-dimensional gels and allowed to rehydrate for 18 h at room temperature. A gradient of 300 -3500 V was applied to the strips followed by constant 3500 V, with focusing complete after 80,000 V-h. Prior to the second dimension, strips were incubated (15 min) in equilibration buffer (6 M urea, 2% SDS, 0.375 M Tris (pH 8.8), 30% glycerol), first with 65 mM dithiothreitol (reductive) and second with 243 mM iodoacetamide (alkylating). Equilibrated strips were inserted onto gradient SDS-PAGE gels (8 ϫ 10 cm; 6 -16%) followed by electrophoresis. Coomassie Blue-stained gels were preserved by air drying between sheets of moistened cellophane.
To probe for the nature of ezrin phosphorylation, aliquots of gastric glands were treated with cimetidine and histamine plus IBMX for different time intervals and harvested for two-dimensional SDS-PAGE. The proteins separated by two-dimensional SDS-PAGE gel were then transferred onto a nitrocellulose membrane probed with an anti-phosphoserine antibody (Sigma). Blots were developed using ECL, and the spot intensities were quantified using a Typhoon PhosphorImager (Amersham Biosciences).
Preparation of Samples for Mass Spectrometry-Excised two-dimensional protein spots were destained, chopped into small fragments with a razor blade, and subjected to digestion by modified porcine trypsin (50 -100 ng/digestion; Promega, Madison, WI) according to Zhou et al. (31). Peptides were recovered by three extractions of the digestion mixture with 50% acetonitrile plus 5% trifluoroacetic acid and desalted and concentrated using C 18 ZipTips (Millipore Corp., Bedford, MA), eluting peptides in 50% (v/v) acetonitrile/water. All supernatants were pooled and concentrated to 5 l in a Speedvac and brought back up to 25 l in 50% acetonitrile, 5% trifluoroacetic acid. The peptide mix was stored at 20°C until analysis.
Matrix-assisted Laser Desorption Time-of-flight (MALDI-TOF) Mass Spectrometric Identification of Phosphopeptides of Ezrin-Aliquots of unseparated tryptic digests were co-crystallized with cyano-4-hydroxycinnamic acid and analyzed using a MALDI delayed extraction reflectron TOF instrument (Bi-flex; Bruker-Daltons, Framingham, MA) equipped with a nitrogen laser. Measurements were performed in a positive ionization mode. All MALDI spectra were externally calibrated using a standard peptide mixture (Sigma).
Data base interrogations based on experimentally determined peptide masses were carried out using mass spectrometry (MS)-Fit, and PSD data interrogation was performed using MS-Tag; both software programs were developed in the University of California San Francisco MS Facility and are available on the World Wide Web at prospector. ucsf.edu. Both the National Center for Biotechnology Information protein data base and Swiss-Prot data base were searched. Search parameters included the putative protein molecular weight and a peptide mass tolerance of 100 -200 parts/million.
In Vitro Phosphorylation of Ezrin by PKA-Both the GST-wild type ezrin and GST-S66A ezrin proteins were expressed in Escherichia coli BL21 (pLys), and the purification of the GST fusion proteins was carried out using glutathione-Sepharose beads (Sigma) as described (32). The fusion proteins bound to glutathione-Sepharose beads were suspended in phosphorylation buffer prior to use.
To verify whether Ser 66 is a substrate for PKA, 5 g of purified GST-ezrin fusion protein, both wild type and S66A mutant, were incubated with 10 units of the catalytic subunit of PKA (New England Biolabs) in phosphorylation buffer containing 20 mM HEPES, pH 7.5, 10 mM MgCl 2 , 5 mM EGTA, 1 mM dithiothreitol, 10 nM ATP, and 5 Ci of [␥-32 P]ATP (PerkinElmer Life Sciences) in a total volume of 30 l. Kinase reaction was carried out at room temperature for 15 min and terminated by the addition of 10 l of 4ϫ SDS-PAGE sample buffer and separated by 6ϳ16% gradient SDS-PAGE. The gel was stained with Coomassie Brilliant Blue, dried, and quantified by a PhosphorImager (Amersham Biosciences).
Cell Culture and Transfection-Primary cultures of gastric parietal cells from rabbit stomach were produced and maintained as described (31). Separate cultures of parietal cells were transfected with plasmids encoding GFP-tagged wild type ezrin and two mutant forms of ezrin (S66A and S66D) using LipofectAMINE 2000 (Invitrogen) according to the manufacturer's instructions. Briefly, 1 g of DNA was incubated in 600 l of Opti-MEM (antibiotics-free) while 6 l of LipofectAMINE 2000 was added and left at room temperature for 25 min. The cultured parietal cells (ϳ3% cytocrit; 6-well plates) were washed once with Opti-MEM. The DNA-lipid mix was added to the plates and incubated for 4 h, followed by replacement of 1.5 ml of medium B. The transfected cells were then maintained in culture at 37°C until use for protein expression, partition, immunoprecipitation, or immunofluorescence.
For analyzing the exogenous protein expression, transfected parietal cells were harvested 30 -36 h after transfection and lysed in 1ϫ SDS-PAGE sample buffer. To analyze the relative level of ezrin associated with cytoskeleton, transfected cells were raised with PHEM buffer (100 mM Pipes, 20 mM Hepes, pH 6.9, 5 mM EGTA, 2 mM MgCl 2 , and 4 M glycerol) twice followed by incubation of PHEM buffer containing a proteinase inhibitor mixture (pepstatin-A, leupeptin, aprotinin, and chymostatin; final concentration 5 g/ml for each inhibitor) plus 0.1% Triton X-100 for 1 min at room temperature to allow cytosolic proteins to be released into extracellular medium (33). The extracted cells were then harvested from a Petri dish and centrifuged at 1,500 ϫ g for 5 min. The resulting pellets, designated as insoluble materials, were solubilized in 1ϫ SDS-PAGE sample buffer, whereas the supernatants, called the soluble fraction, were concentrated with 5% trichloroacetic acid followed by reconstitution in 1ϫ SDS-PAGE sample buffer as described (30).
For immunoprecipitation of GFP-ezrin proteins, GFP-ezrin-transfected parietal cells were stimulated with histamine plus IBMX for 20 min as described (31). The cells were then harvested and lysed in 1.5 ml of Tris-buffered saline (20 mM Tris-Cl, pH 7.4, 150 mM NaCl, 2 mM EGTA, 0.1% Triton X-100) containing a proteinase inhibitor mixture (pepstatin A, leupeptin, aprotinin, and chymostatin; final concentration 5 g/ml for each inhibitors) and phosphatase inhibitor okadaic acid (10 nM). The cell lysates were clarified by using an Eppendorf centrifuge at 13,000 rpm for 10 min. The resulting supernatants were then incubated with 15 g of GFP monoclonal antibody (JL-18) at room temperature for 2 h followed by the addition of 10 l of protein A/G beads for an additional 1 h (Pierce). The beads were collected and washed with Tris-buffered saline before boiling in SDS-PAGE sample buffer. Immunoprecipitates were then fractionated by SDS-PAGE, and proteins were transferred onto nitrocellulose membrane for Western blotting analyses. The blot was first labeled with ezrin antibody 4A5 to verify the efficiency of GFP immunoprecipitation. The blot was then stripped with SDS-PAGE sample buffer at 55°C for 20 min followed by validation of serine phosphorylation on GFP-ezrin using a phosphoserine antibody.
Immunofluorescence Microscopy-For cytolocalization of exogenously expressed ezrin, cultured parietal cells were transfected with GFP, wild type GFP-ezrin, and GFP-ezrin mutants (S66A and S66D) and maintained in MEM for 30 -36 h. Some cultures were treated with 100 M cimetidine to maintain a resting state; others were treated with the secretory stimulants 100 M histamine plus 50 M IBMX in the presence of SCH28080, a proton pump inhibitor (34). Treated cells were then fixed with 2% formaldehyde for 10 min and washed three times with PBS followed by permeabilization in 0.1% Triton X-100 for 5 min. Prior to application of primary antibody, the fixed and permeabilized cells were blocked with 0.5% bovine serum albumin in phosphatebuffered saline followed by incubation of primary antibodies against ezrin (4A5) or GFP. The endogenous and exogenous ezrin proteins were labeled by a fluorescein isothiocyanate-conjugated goat anti-mouse antibody and counterstained with rhodamine-coupled phalloidin to visualize filamentous actin. Coverslips were supported on slides by grease pencil markings and mounted in Vectorshield (Vector). Images were taken on a Zeiss Axiovert 200 fluorescence microscope using a 63 ϫ 1.3 numerical aperture PlanApo objective. Figures were constructed using Adobe Photoshop.
Confocal Microscopy-Immunostained parietal cells were examined under a laser-scanning confocal microscope LSM510 NLO (Carl Zeiss) scan head mounted transversely to an inverted microscope (Axiovert 200; Carl Zeiss) with a 40 ϫ 1.0 numerical aperture PlanApo objective. Single images were collected by an average of 10 scans at a scan rate of 1 s/scan. Optical section series were collected with a spacing of 0.4 m in the z axis through the ϳ12-m thickness of the cultured parietal cells. The images from double labeling were simultaneously collected using a dichroic filter set with Zeiss image processing software (LSM 5; Carl Zeiss). Digital data were exported into Adobe Photoshop for presentation.
Measurement of the Diameter of the Apical Vacuoles-The diameter of phalloidin-stained apical vacuoles was measured using LSM 5 software (Carl Zeiss) and a Zeiss Axiovert 200 fluorescent microscope calibrated with a stage micrometer. When suitable vacuoles were identified, the image was enlarged 3-fold to facilitate accurate placement of a computer-generated cursor over the vacuoles outlined by phalloidin staining. Vacuole diameter was calculated as the mean of the major and minor axis of the F-actin-outlined vacuolar staining.
Western Blot-Samples were subjected to SDS-PAGE on 6 -16% gradient gel and transferred onto nitrocellulose membrane. In some cases, samples were first separated by isoelectric focusing followed by SDS-PAGE on 6 -16% gradient gel and subsequently transferred onto nitrocellulose membrane. Proteins were probed by appropriate primary antibodies and detected using ECL (Pierce). The band intensity was then quantified using a PhosphorImager (Amersham Biosciences).

Identification of Phosphoezrin Associated with Parietal Cell
Secretion-Separation of ezrin by two-dimensional gel electrophoresis is shown in Fig. 1A for resting and stimulated gastric glands. The Coomassie Brilliant Blue-stained gel shows that stimulation with histamine plus IBMX resulted in a shift of ezrin spots to more acidic pH, consistent with the stimulationdependent phosphorylation of ezrin reported previously (e.g. Ref. 10). Stimulation through the histamine/cAMP pathway induced a reduction in the relative intensity of the major alkaline spot (0; estimated pI of 6.7) and a shift toward a series of more acidic isoforms 1, 2, and 3, an estimated pI varying from 6.6 to 6.4, respectively. Treatment with H89, a relatively selective inhibitor of PKA, prevented such shifts in ezrin isotypes on two-dimensional SDS-PAGE (not shown), verifying that the phosphorylation of ezrin is downstream from the activation of protein kinase A.
Histamine stimulation induced three acidic ezrin spots separating with a distance equivalent to ϳ0.1 pH unit, which reflects multiple phosphorylation sites involved. The labeling of 1, 2, and 3 represents the number of phosphomodification sites on ezrin proteins based on the shift of pI. Over the years, monoclonal antibodies against phosphoamino acids (e.g. serine, theronine, and tyrosine) have been generated as useful probes to detect phosphoprotein and the nature of the phosphorylated amino acid. To test if any of these acidic spots are related to phosphoserine induced by histamine stimulation, we carried out Western blotting using two-dimensional SDS-PAGE of separated ezrin spots. As shown in Fig. 1B, phosphoserine antibody selectively reacts strongly with acidic spot 3 of ezrin from the stimulated preparation, although there was also very minor reactivity with spot 2. Since an isoelectric shift is characteristic of protein phosphorylation, the acidic phosphoserinepositive spots suggest that phosphorylation occurred on a serine residue of ezrin.
To evaluate the stimulus-induced ezrin phosphorylation in relation to activation of acid secretion, we used gastric glands to measure the time course of ezrin phosphorylation judged by a phosphoserine antibody, and in a parallel set of glands we measured acid secretion by the AP uptake assay (Fig. 1C). A typical two-dimensional Western blot used to quantify the extent of ezrin phosphorylation is shown as Fig. 1B, demonstrating that the phosphoserine antibody rather selectively reacts with acidic ezrin spots (2 and 3). For each time point after stimulation with histamine, the summed intensity of the acidic, phosphoserine-positive, ezrin spots was compared with the resting control, normalized by ezrin spot intensity, and plotted in Fig. 1C (solid circles). The acid stimulation index, defined as AP stimulated /AP resting, is also plotted on the same scale (Fig. 1C, open circles). The relative acid secretory response reached a maximal level after about 15 min of stimulation and was sustained for the 35 min of measurement. On the other hand, the index of protein phosphorylation peaked earlier, at about 7.5 min, and slowly returned to near resting level by 35 min. The temporal profiles of AP uptake and ezrin protein phosphorylation are consistent with reports in the literature (e.g. Ref. 10) and suggest that the phosphorylation event may be required for parietal cell activation.
To identify the phosphoamino acids associated with histamine stimulation, the acidic ezrin isotype spots (e.g. 1-3 from the histamine-stimulated samples) were removed from Coomassie Brilliant Blue-stained two-dimensional gel, combined, and subjected to in-gel digestion with trypsin. The corresponding regions of two-dimensional gel from resting samples were used as control. The resulting phosphopeptides were identified by peptide mass fingerprinting using MALDI-TOF mass spectrometry. These multiple spots represent the same ezrin protein but with different modifications. The tryptic peptides recovered from mass spectrometric analysis indicate that Thr 36 , Ser 66 , and Tyr 191 are possible substrates accounting for ezrin phosphorylation in parietal cell activation stimulated by histamine.
Ser 66 Is a Substrate of Protein Kinase A-Early analysis of the amino acid sequence of ezrin suggested three potential PKA phosphorylation sites, such as Ser 66 (11). Our mass spectrometric analysis also pointed to the possibility of Ser 66 phosphorylation in response to histamine stimulation. To test whether Ser 66 is a substrate of PKA, we performed in vitro phosphorylation on recombinant GST-ezrin fusion proteins, including both wild type ezrin and a mutant ezrin in which serine 66 was replaced by alanine (S66A). Both GST fusion proteins, wild type and S66A mutant ezrin, migrate at about the predicted 105 kDa as shown in Fig. 2A. Incubation of the fusion proteins with [ 32 P]ATP and the catalytic subunit of PKA resulted in the incorporation of 32 P into wild type but not S66A mutant ezrin (Fig. 2B). This PKA-mediated phosphorylation is specific, since incubation of ezrin with [ 32 P]ATP in the absence of the kinase resulted in no detectable incorporation of radioactivity into the wild type protein. Thus, Ser 66 of ezrin is probably a substrate for PKA.
To verify whether Ser 66 of ezrin is phosphorylated in response to histamine stimulation, we transfected GFP-ezrin, both wild type and nonphosphorylatable S66A mutant, into cultured parietal cells followed by stimulation and immunoprecipitation of GFP-ezrin fusion proteins from the stimulated cells. GFP antibody absorbed a small portion of exogenously expressed GFP-ezrin proteins, but not endogenous ezrin, from the parietal cell lysates as labeled by ezrin antibody 4A5 (Fig.  2C, ezrin blot). However, anti-phosphoserine antibody only marks wild type GFP-ezrin and not mutant S66A ezrin from stimulated parietal cells, indicating that Ser 66 is responsible for histamine-stimulated phosphorylation on serine. Thus, we conclude that Ser 66 of ezrin is involved in histamine stimulation of parietal cells.
Exogenously Expressed GFP-Ezrin Is Primarily Associated with Cytoskeleton-To evaluate the efficacy of exogenous ezrin expression, cultured parietal cells were transfected with a GFP-tagged wild type ezrin plasmid. Western blotting analysis carried out using transfected cells showed that exogenously expressed ezrin protein was about twice the level of endogenous ezrin in cultured parietal cells (Fig. 3A). Assuming a transfection efficiency of about 45-50%, the actual expression level of GFP-ezrin in positively transfected cells is about 4-fold higher than that of endogenous protein. To determine whether there were any major changes in the behavior of exogenously expressed GFP-ezrin, we measured the partitioning of endogenous ezrin and exogenously expressed GFP-ezrin into the Triton X-100-soluble fraction compared with the insoluble "cytoskeletal" fraction based on the Western blotting analyses. In the case of transfected cells, only GFPezrin content was measured. As summarized in Fig. 3, 71.7 Ϯ 3.3% of endogenous ezrin resides in the Triton X-100-insoluble fraction, consistent with previous reports (e.g. Refs. 31 and 35).
Partitioning of wild type GFP-ezrin is similar to endogenous ezrin; 68.5 Ϯ 3.7% of wild type GFP-ezrin is associated with the Triton X-100-insoluble fraction.
To probe for the potential role of Ser 66 phosphorylation in promoting the association of ezrin with the cytoskeleton, we generated two mutant ezrin plasmids that encode mutant proteins mimicking nonphosphorylatable Ser 66 (S66A) and permanently phosphorylated ezrin (S66D), respectively. As shown in Fig. 3B, both mutant ezrin proteins have a distribution pattern   FIG. 2. Serine 66 of ezrin is a substrate of PKA. For A and B, bacterially expressed GST-ezrin fusion proteins, both wild type and mutant S66A, were purified on glutathione-Sepharose beads and phosphorylated in vitro using [ 32 P]ATP and purified catalytic subunit of PKA as described under "Experimental Procedures." Samples were separated by 6 -16% gradient SDS-PAGE gel. A, Coomassie Brilliant Blue-stained gel of samples of wild type GST-ezrin without PKA added (Ezrin only), wild GST-ezrin plus PKA (Ezrin ϩ PKA), and mutant S66A GST-ezrin plus PKA (S66A ϩ PKA). Note that roughly equivalent amounts of GST-ezrin protein was present in the three reactions. B, the same gel was dried gel and subsequently incubated with x-ray film. Note that in the presence of PKA there was dramatic incorporation of 32 P into wild type, but not mutant, ezrin protein. C, the cultured parietal cells transfected with GFP-ezrin (wild type and S66A) were collected 36 h post-transfection for immunoprecipitation as outlined under "Experimental Procedures." Equivalent amounts of proteins from the lysates and nonbinding fraction were applied to SDS-PAGE. Following separation by 6 -16% gradient SDS-PAGE and subsequent transblotting onto nitrocellulose membrane, the blot was first probed with an anti-ezrin antibody 4A5 and developed with an ECL kit (Pierce). Because GFP adds about 25 kDa in mass to the fusion proteins, GFP-tagged ezrin migrates as upper bands (arrow), whereas endogenous ezrin protein migrates as lower bands (arrowhead). After ezrin probing, the blot was stripped and reprobed with a monoclonal antibody reacting with phosphoserine and visualized by ECL. The phosphoserine antibody recognizes wild type GFP-ezrin but not nonphosphorylatable GFP-ezrin. Note that endogenous ezrin from stimulated parietal cells was also labeled by the phosphoserine antibody.

FIG. 3. Exogenously expressed GFP-ezrin protein behaves like endogenous ezrin.
A, samples from both mock-transfected and GFPezrin-transfected parietal cells were prepared and separated on SDS-PAGE, blotted to nitrocellulose, and probed by an ezrin antibody 4A5. Note that the ezrin antibody recognizes both endogenous (80-kDa) and exogenously expressed GFP-ezrin (105-kDa) proteins. B, cultures of parietal cells were either mock-transfected (endogenous) or transfected with one of several GFP-ezrin constructs, including wild type ezrin, S66A substitution mutant, and S66D substitution mutant. 30 -36 h post-transfection, the cells were extracted with 0.1% Triton X-100 solution and separated into soluble (s) and insoluble (i) fractions as described under "Experimental Procedures." Equivalent amounts of proteins from the soluble and insoluble fractions were applied to SDS-PAGE. Following separation on 6 -16% gradient SDS-PAGE and transblotted onto nitrocellulose membrane, the blots were probed with an anti-ezrin antibody 4A5 and developed with an ECL kit (Pierce). GFP-tagged ezrin migrates around 105 kDa, whereas endogenous protein situates around 80 kDa on SDS-PAGE gel, it was then possible to distinguish endogenous protein and exogenously expressed ezrin as exemplified in A. The signals were quantified using a PhosphorImager with values expressed as a percentage of total (soluble ϩ insoluble). The error bars represent S.E.; n ϭ 3 preparations. similar to the wild type. These results indicate that exogenously expressed GFP-ezrin bears biochemical characteristics similar to endogenous protein.
Localization of GFP-Ezrin to the Apical Membrane Independent of Ser 66 -The subcellular localization of the exogenously expressed GFP-ezrin constructs was compared with that of endogenous ezrin by fluorescence microscopy (Fig. 4A). Control cultured parietal cells were double stained for endogenous ezrin using an ezrin antibody (green) and for F-actin using phalloidin (red). The transfected cells were double-stained for GFP-ezrin using a monoclonal GFP antibody (green) and double-stained for F-actin using phalloidin (red). Fig. 4A shows optical sections from control and transfected cells, all maintained in the nonsecreting state. Similar to what has been noted in earlier studies, endogenous ezrin in control cells is localized to the plasma membranes, most prominently to the apical membrane vacuoles that have been sequestered into the cell interior and somewhat more sparsely to the basolateral membrane that surrounds the cells. The ezrin signal is relatively co-localized with F-actin (Fig. 4, a, aЈ, and aЉ). The distribution of the signal for all three GFP-ezrin constructs (wild type, S66A, and S66D) was similar to that of endogenous ezrin (i.e. primarily associated with apical membrane vacuoles and to a lesser extent with basolateral membrane) (Fig. 4A, b,  c, and d). The distribution of F-actin was also not altered by the transfections (Fig. 4A, bЈ, cЈ, and dЈ). These data demonstrate that transfected GFP-ezrin is targeted to the same loci as endogenous ezrin and that phosphorylation of Ser 66 is not responsible for targeting of ezrin to actin-based cytoskeleton at the apical plasma membrane.
There was, however, at least one striking difference in the morphology of nonsecreting parietal cells transfected with the Ser 66 mutants. Cells expressing mutant S66D, a mutant pro-tein mimicking phosphorylated ezrin, have dilated apical vacuole membranes, characteristic of secreting parietal cells, although no stimulant was added. As shown in Table I, the average diameter of apical vacuoles in S66D-expressed cells (7.2 Ϯ 0.5 m) is about twice as big as those of cells expressing wild type GFP-ezrin (3.5 Ϯ 0.3 m) or S66A mutant ezrin (3.4 Ϯ 0.3 m). These data suggest that phosphorylation of Ser 66 is not required for apical localization of ezrin protein but may be involved in apical membrane cytoskeletal remodeling.
Phosphorylation of Ser 66 Is Required for Apical Membrane Dynamic-Stimulation of parietal cells by histamine results in dramatic expansion of the apical canalicular plasma membrane due to insertion of H,K-ATPase-containing vesicular membranes and subsequent proton pumping into the canalicular space. In cultured parietal cells, the same membrane transformations occur, but, because the apical canalicular membrane has been incorporated into the vacuolar forms, stimulation results in dilation of apical membrane vacuoles as active HCl and water transport occur (34). Because of this swelling, stimulated parietal cells are considerably larger in diameter than their resting counterparts (34). Since the expression of S66D mutant ezrin effected relatively dilated apical membrane vacuoles, we tested whether phosphorylation of Ser 66 is critical for apical cytoskeletal remodeling. To this end, we assessed the effects of stimulation on parietal cells transfected with wild type, S66A, and S66D ezrin tagged with GFP. Fig. 4B shows optical sections taken from parietal cells treated with the secretagogues histamine plus IBMX and probed for F actin and for ezrin (using either anti-ezrin or anti-GFP antibodies, similar to the protocol used in Fig. 4A). As for resting parietal cells, GFP-ezrin and F-actin are primarily co-localized to the same regions in secreting cells. For all conditions, F-actin labeling (Fig. 4B, aЈ-dЈ) outlines the dilated apical membrane in addi-

FIG. 4. Exogenous expression of GFP-ezrin targets to the apical membrane of parietal cells and is involved in apical membrane cytoskeletal dynamics.
This triple montage represents confocal images collected from resting and stimulated gastric parietal cells doubly stained for ezrin (green), F-actin (red), and their merged images. A, localization of GFP-ezrin in resting parietal cells (a, aЈ, and aЉ). Endogenous ezrin is mainly located to the apical plasma membrane of parietal cells, which was seen as rings, in a pattern suggestive of the apical plasma membrane invaginations that form the intracellular canaliculi. Expression of GFP-ezrin is mainly located to the apical membrane of parietal cells, which is superimposed to that of F-actin distribution (red). This becomes readily evident when the two images are merged (yellow). Mutant S66A and S66D ezrin proteins are expressed and located to the apical membrane, similar to that of wild type. Note that cells expressing S66D have dilated apical membrane vacuoles. Bar, 20 m. B, nonphosphorylatable mutant ezrin S66A blocks apical membrane dilation stimulated by histamine. Stimulation induces remodeling of the apical membrane seen as a dilation of apical vacuoles in mock-transfected cells (a, aЈ, and aЉ). Expression of wild type GFP-ezrin outlines the dilated apical membrane. However, mutant S66A ezrin (but not S66D) attenuates the dilation of apical vacuoles, suggesting that PKA-mediated phosphorylation of Ser 66 is required for apical membrane dynamics associated with an activation of acid secretion. Bar, 20 m. tion to some basolateral surface staining. In the case of secreting cells transfected with wild type GFP-ezrin or S66D mutant GFP-ezrin, the dilated apical canalicular vacuoles occupy most of the cytoplasmic space (e.g. Fig. 4B, a and aЈ). Thus, GFPezrin remains associated with the actin cytoskeleton at the apical plasma membrane of secreting parietal cells, with the S66D mutant making no apparent difference in targeting.
However, as shown in Fig. 4B (d and dЈ), mutations in Ser 66 did make a significant difference in the morphological and physiological responses to stimulation. Whereas stimulation of parietal cells expressing wild type or S66D mutant ezrin produced a characteristic vacuolar swelling in response to stimulation, cells expressing S66A ezrin displayed a morphology in which dilation of apical vacuoles is less apparent. Previous studies revealed that diameter of vacuoles can be used as a reporter for parietal cell secretory activity (34). Thus, we surveyed approximately 75 cells from resting and stimulated populations transfected with either wild type or mutant ezrin constructs. Vacuolar measurements summarized in Table I show that stimulation dramatically extends vacuole diameter to 15.7 Ϯ 1.1 and 16.3 Ϯ 1.0 m in wild type and S66Dexpressing cells, respectively. However, the average vacuole diameter of S66A-transfected, stimulated cells was only 6.3 Ϯ 0.5 m. Thus, expression of S66A mutant ezrin greatly attenuates secretion-dependent dilation of the vacuoles, suggesting that phosphorylation of ezrin Ser 66 is essential for dynamic remodeling of the apical cytoskeleton of parietal cells associated with the stimulation.
Phosphorylation of Ser 66 Is Important for Parietal Cell Activation-A large part of parietal cell activation involves translocation and insertion of H,K-ATPase-containing vesicular membranes into the apical membrane for subsequent proton pumping into canalicular space (e.g. Refs. 3 and 36). Several earlier studies established the importance of the actin-based cytoskeleton (e.g. Ref. 37) and the integrity of ezrin (30) in parietal cell activation. Given the observed morphological responses to the mutant forms, it was of interest to ascertain the function of the phosphorylation of ezrin Ser 66 on the translocation and/or insertion of H,K-ATPase from cytoplasmic vesicles to the apical membrane. Accordingly, we examined the distribution of H,K-ATPase in cultured parietal cells expressing wild type, S66A, and S66D mutant ezrin proteins. Fig. 5 shows optical sections taken from secreting parietal cells si-multaneously probed with fluorescein-coupled H,K-ATPase antibody (2G11; Fig. 5, a, b, e, and c) and rhodamine-conjugated phalloidin (aЈ, bЈ, cЈ). The F-actin distribution is similar to that shown in Fig. 4, outlining the apical membrane vacuoles and the basolateral membrane surface. In resting cells, H,K-ATPase staining is distributed throughout the cytoplasm as previously documented, although we also noted some accumulation on the apical membrane vacuoles (e.g. Ref. 34; Fig. 5d). Stimulation of cells transfected with wild type ezrin revealed a diminution of H,K-ATPase in the cytoplasmic compartment concomitant with an enrichment in the apical membrane of vacuoles (Fig. 5e), indicating that stimulation-induced translocation of H,K-ATPase occurs in cells expressing exogenous ezrin. Overlapping images demonstrate a superimposition of the F-actin and H,K-ATPase probes in the apical membrane of parietal cells, verifying the apical trafficking of H,K-ATPase (yellow, Fig. 5eЉ). However, for cells expressing S66A mutant ezrin, stimulation failed to mobilize H,K-ATPase to the apical membrane, since the staining remains in cytoplasm (Fig. 5b), suggesting that phosphorylation of Ser 66 might be involved in the recruitment of H,K-ATPase. Indeed, examination of parietal cells expressing S66D ezrin show that the mutant does facilitate the stimulation-induced translocation of H,K-ATPase. We therefore conclude that PKA-mediated phosphorylation of ezrin Ser 66 is important for proton pump translocation essential for parietal cell activation. DISCUSSION Ezrin, a founding member of the membrane-cytoskeleton linker family of ERM proteins, has been implicated in a variety of dynamic cellular functions such as determination of cell shape, cell adhesion, motility, and survival (e.g. Ref. 1). Gastric ezrin was identified by virtue of its phosphorylation correlated with histamine-stimulated gastric acid secretion in parietal cells (10). Despite numerous investigations on the structurefunctional interrelationships, the PKA-mediated phosphorylation on ezrin has not been well characterized. Here we provide the first evidence that ezrin is substrate of PKA in vivo and in vitro. Moreover, we have mapped an important PKA-mediated phosphorylation site on ezrin to Ser 66 . Furthermore, our studies show that heterologously expressed ezrin linked to GFP is targeted to the apical membrane of cultured parietal cells, similar to endogenous ezrin. However, mutations of Ser 66 on ezrin alter apical membrane dynamics associated with histamine stimulation, suggesting that phosphorylation of Ser 66 on ezrin is required for proton pump mobilization and polarized secretion in gastric parietal cells. A major role for the actin cytoskeleton in the secretory processes of parietal cells has been inferred from studies using actin disruptors that disorganize actin filaments and act to inhibit acid secretion (37). Highly organized microfilaments are typical features of microvilli at the apical membrane within the parietal cell canaliculus. In going from rest to the secreting state, there are major changes at the apical canalicular surface, including elongation of microvilli. Interestingly, as the parietal cell returns to the resting state after withdrawal of stimulants, microfilament ultrastructural changes become apparent as a disorganization of actin filaments along with collapse of the apical canalicular surface (36,38). These morphological studies indicate that reversible actin-based cytoskeletal dynamics are tightly linked to the secretory cycle in parietal cells.
Phosphorylation of C-terminal ezrin (e.g. Thr 567 ) has been shown to regulate its association with the actin cytoskeleton (39). This observation was supported by the finding that phosphorylation of the homologous threonine 558 in moesin is required for F-actin binding in vitro (21,40). Using mutant ezrin T567D, mimicking the phosphorylated protein, Matsui et al. a Diameters of apical vacuoles were measured as an index for apical membrane extension associated with acid secretion. Data were obtained from resting and stimulated parietal cells in which apical vacuoles were in the same focal plane. These measurements were made from three different preparations in which more than 80 cells from each category were examined. In resting cells, measurements were carried out on 2-5 vacuoles/cell, whereas 1-3 vacuoles/cell were scaled stimulated preparation. Data are expressed as mean Ϯ S.E. b p Ͻ 0.05; the diameter of S66D was compared with that of wild type ezrin-expressing resting cells. c p Ͻ 0.05; the diameter of S66A was compared with that of wild type ezrin-expressing secreting cells.
showed that ezrin phosphorylation alters intramolecular interactions. T567D ezrin is a strongly morphogenic variant that triggers the formation of wide lamellipodia, extensive membrane ruffles, and microvilli-rich projections when overexpressed, indicating that T567D ezrin promotes actin cytoskeletal dynamics. Our studies revealed no evidence that Thr 567 is phosphorylated and involved in parietal cell activation. It is possible that an upstream kinase responsible for Thr 567 phosphorylation is not related to the parietal cell activation cascade. Our present studies, however, do show that phosphorylation of ezrin is dynamic and correlated with histamine-stimulated parietal cell secretion. Alteration of Ser 66 phosphorylation did not change ezrin association with the cytoskeleton but did modulate the activity of apical membrane dilation in response to stimulation, consistent with the notion that the C-terminal domain of ezrin is responsible for actin binding, whereas its N-terminal region is responsible for the association of ezrin with other proteins proximal to the plasma membrane. To search for ERM binding partners potentially involved in membrane association, Reczek et al. (41) used GST-ezrin as an affinity matrix to isolate a 50-kDa phosphoprotein named EBP50 from human placental cell lysates. These authors further showed that the N-terminal ezrin binds to the C-terminal PDZ domain of EBP50 (42). However, an initial search for EBP50 in gastric parietal cells was negative. 2 Thus, it is likely that ezrin binds to a functional homologue of EBP50, which mediates the association of ezrin with the apical plasma membrane of parietal cells. Since parietal cell activation involves translocation of H,K-ATPase from cytoplasm to the apical plasma membrane, it would be of great interest to illustrate how mutant ezrin S66A blocks the apical membrane dynamics and H,K-ATPase translocation process. Since the translocation of H,K-ATPase onto the apical membrane involves multiple steps, including the possible trafficking over actin filaments, docking to secretory sites, insertion of the pump into the apical membrane, and perhaps maintenance of the pump in apical membrane during active secretion, it will be important to distinguish precisely where the phosphorylation of Ser 66 participates.
Our studies show that histamine-stimulated incorporation of phosphate onto ezrin peaks in about 8 min and then declines. Interestingly, acid secretion reaches its maximum about 15 min after the stimulation, suggesting that phosphorylation of serine is an early event of the activation process. In fact, expression of ezrin mutant S66D, which mimics phosphorylated Ser 66 , results in phenotypes of partially stimulated parietal cells, suggesting that phosphorylation of Ser 66 alone is not sufficient for maximal activation. It is likely that a parallel pathway distant from the Ser 66 phosphorylation cascade is required for complete activation. Alternatively, phosphorylation of ezrin in other residues may be synergistic for the activation process. In fact, we have noticed other suggestive phosphorylation sites including Ser 366 and Ser 412 in some of our preparations. In any event, further characterization of ezrin phosphorylation associated with histamine stimulation will provide detailed structure-function relationships of the role of ezrin in parietal cell secretion.
Taken together, the present work reveals that ezrin is phosphorylated by PKA on Ser 66 and that this PKA-induced phosphorylation is essential for parietal cell activation. Finally, we show that nonphosphorylatable ezrin blocks translocation of H,K-ATPase to the apical membrane. We propose that phosphorylation of ezrin links proton pump trafficking to apical membrane-cytoskeletal dynamics required for polarized secretion in epithelial cells. 2 X. Cao, E. Chen, and X. Yao, unpublished results.

FIG. 5. Exogenous expression of nonphosphorylatable GFP-ezrin diminishes H,K-ATPase trafficking.
A, this pair of triple montage represents confocal images collected from resting and stimulated gastric parietal cells doubly stained for H,K-ATPase (green), ezrin (red), and their merged images. As described in the legend to Fig. 4B, stimulation induces remodeling of the apical membrane due to the fusion of H,K-ATPasecontaining vesicles, which was readily apparent by a co-localization of H,K-ATPase with ezrin at the periphery of the expanded apical membrane vacuoles (b, bЈ, and bЉ). In contrast, H,K-ATPase was seen throughout the cytoplasm of resting parietal cells with some localization to the apical membrane vacuoles, where ezrin is primarily localized (a, aЈ, aЉ). B, this set of triple montage represents confocal images collected from histamine-stimulated parietal cells, expressing exogenous ezrin (wild type, S66A, and S66D), doubly stained for H,K-ATPase (green), F-actin (red), and their merged results. Stimulation of parietal cells expressing exogenous ezrin triggers mobilization of H,K-ATPase (c) to the apical membrane vacuoles, which is evident by superimposition of H,K-ATPase staining onto that of apical F-actin staining (cЈ and cЉ). However, mutant ezrin (S66A but not S66D) attenuates the dilation of apical vacuoles and prevents the translocation of H,K-ATPase from cytoplasm to the apical membrane vacuoles (d, dЈ, and dЉ), suggesting that PKA-mediated phosphorylation of Ser 66 is necessary for mobilization of the proton pump. Note that histamine stimulation induces remodeling of apical membrane and H,K-ATPase translocation in S66D-expressing cells (e, eЈ, and eЉ). Bar, 20 m.