Spatial Control of Proton Pump H,K-ATPase Docking at the Apical Membrane by Phosphorylation-coupled Ezrin-Syntaxin 3 Interaction

The digestive function of the stomach depends on acidification of the gastric lumen. Acid secretion into the lumen is triggered by activation of a cAMP-dependent protein kinase (PKA) cascade, which ultimately results in the insertion of gastric H,K-ATPases into the apical plasma membranes of parietal cells. A coupling protein is ezrin whose phosphorylation at Ser-66 by PKA is required for parietal cell activation. However, little is known regarding the molecular mechanism(s) by which ezrin operates in gastric acid secretion. Here we show that phosphorylation of Ser-66 induces a conformational change of ezrin that enables its association with syntaxin 3 (Stx3) and provides a spatial cue for H,K-ATPase trafficking. This conformation-dependent association is specific for Stx3, and the binding interface is mapped to the N-terminal region. Biochemical analyses show that inhibition of ezrin phosphorylation at Ser-66 prevents ezrin-Stx3 association and insertion of H,K-ATPase into the apical plasma membrane of parietal cells. Using atomic force microscopic analyses, our study revealed that phosphorylation of Ser-66 induces unfolding of ezrin molecule to allow Stx3 binding to its N terminus. Given the essential role of Stx3 in polarized secretion, our study presents the first evidence in which phosphorylation-induced conformational rearrangement of the ezrin molecule provides a spatial cue for polarized membrane trafficking in epithelial cells.     

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 Ser66 is phosphorylated. To determine whether Ser66 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 [32P]ATP. Incorporation of 32P into wild type but not the mutant ezrin verified that Ser66 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 phosphorylation-like S66D mutant in parietal cells mimics histamine-stimulated apical vacuole remodeling. Further examination of H,K-ATPase distribution revealed a blockade of stimulation-induced proton pump mobilization in S66A but not S66D ezrin-expressing parietal cells. These data suggest that PKA-mediated phosphorylation of ezrin plays an important role in mediating the remodeling of the apical membrane cytoskeleton associated with acid secretion in parietal cells.     

Cell Polarity Kinase MST4 Cooperates with cAMP-dependent Kinase to Orchestrate Histamine-stimulated Acid Secretion in Gastric Parietal Cells

The digestive function of the stomach depends on acidification of the gastric lumen. Acid secretion into the lumen is triggered by activation of the PKA cascade, which ultimately results in the insertion of gastric H,K-ATPases into the apical plasma membranes of parietal cells. A coupling protein is ezrin, whose phosphorylation at Ser-66 by PKA is required for parietal cell activation. However, little is known regarding the molecular mechanism(s) by which this signaling pathway operates in gastric acid secretion. Here we show that PKA cooperates with MST4 to orchestrate histamine-elicited acid secretion by phosphorylating ezrin at Ser-66 and Thr-567. Histamine stimulation activates PKA, which phosphorylates MST4 at Thr-178 and then promotes MST4 kinase activity. Interestingly, activated MST4 then phosphorylates ezrin prephosphorylated by PKA. Importantly, MST4 is important for acid secretion in parietal cells because either suppression of MST4 or overexpression of non-phosphorylatable MST4 prevents the apical membrane reorganization and proton pump translocation elicited by histamine stimulation. In addition, overexpressing MST4 phosphorylation-deficient ezrin results in an inhibition of gastric acid secretion. Taken together, these results define a novel molecular mechanism linking the PKA-MST4-ezrin signaling cascade to polarized epithelial secretion in gastric parietal cells.     

PALS1 specifies the localization of ezrin to the apical membrane of gastric parietal cells

The ERM (ezrin/radixin/moesin) proteins provide a regulated linkage between membrane proteins and the cortical cytoskeleton and also participate in signal transduction pathways. Ezrin is localized to the apical membrane of parietal cells and couples the protein kinase A activation cascade to regulated HCl secretion in gastric parietal cells. Here, we show that the integrity of ezrin is essential for parietal cell activation and provide the first evidence that ezrin interacts with PALS1, an evolutionarily conserved PDZ and SH3 domain-containing protein. Our biochemical study verifies that ezrin binds to PALS1 via its N terminus and is co-localized with PALS1 to the apical membrane of gastric parietal cells. Furthermore, our study shows that PALS1 is essential for the apical localization of ezrin, as either suppression of PALS1 protein accumulation or deletion of the PALS1-binding domain of ezrin eliminated the apical localization of ezrin. Finally, our study demonstrates the essential role of ezrin-PALS1 interaction in the apical membrane remodeling associated with parietal cell secretion. Taken together, these results define a novel molecular mechanism linking ezrin to the conserved apical polarity complexes and their roles in polarized epithelial secretion of gastric parietal cells.     

The secretion-stimulated 80K phosphoprotein of parietal cells is ezrin, and has properties of a membrane cytoskeletal linker in the induced apical microvilli.

Stimulation of gastric acid secretion in parietal cells involves the translocation of the proton pump (H,K-ATPase) from cytoplasmic tubulovesicles to the apical membrane to form long, F-actin-containing, microvilli. Following secretion, the pump is endocytosed back into tubulovesicles. The parietal cell therefore offers a system for the study of regulated membrane recycling, with temporally separated endocytic and exocytic steps. During cAMP-mediated stimulation, an 80 kDa peripheral membrane protein becomes phosphorylated on serine residues. This protein is a major component, together with actin and the pump, of the isolated apical membrane from stimulated cells, but not the resting tubulovesicular membrane. Here we show that the gastric 80 kDa phosphoprotein is closely related or identical to ezrin, a protein whose phosphorylation on serine and tyrosine residues was recently implicated in the induction by growth factors of cell surface structures on cultured cells [Bretscher, A. (1989) J. Cell Biol., 108, 921-930]. Light and electron microscopy reveal that ezrin is associated with the actin filaments of the microvilli of stimulated cells, but not with the filaments in the terminal web. In addition, a significant amount of ezrin is present in the basolateral membrane infoldings of both resting and stimulated cells. Extraction studies show that ezrin is a cytoskeletal protein in unstimulated and stimulated cells, and its association with the cytoskeleton is more stable in stimulated cells. These studies indicate that ezrin is a membrane cytoskeletal linker that may play a key role in the control of the assembly of secretory apical microvilli in parietal cells and ultimately in the regulation of acid secretion. Taken together with the earlier studies, we suggest that ezrin might be a general substrate for kinases involved in the regulation of actin-containing cell surface structures.     

SAP 97 is a potential candidate for basolateral fixation of ezrin in parietal cells

 Acid secretion in gastric parietal cells is preceded by a dramatic increase in surface area of the apical membrane compartment, due to fusion of the H⁺/K⁺-ATPase-containing tubulovesicles. The resulting canaliculi must be fixed for a period of minutes by cytoskeletal elements to sustain acid secretion. Using immunofluorescence microscopy, the cytoskeletal linker molecule, ezrin, localizes to the apical canalicular membrane of parietal cells. Antibodies against ezrin precipitate H⁺/K⁺-ATPase and β-actin. In addition to its apical localization, ezrin is found to be colocalized at the basolateral compartment with synapse-associated protein (SAP) 97. Immunoprecipitation confirms a direct binding of SAP 97 and ezrin. We conclude that ezrin is fixed to the basolateral compartment by SAP 97. Upon stimulation of acid secretion, ezrin moves to the apical surface where it might stabilize the canalicular microvilli by connecting to β-actin and H⁺/K⁺-ATPase, thereby sustaining acid secretion. Accepted: 14 January 1999     

Polarized distribution of IQGAP proteins in gastric parietal cells and their roles in regulated epithelial cell secretion

Actin cytoskeleton plays an important role in the establishment of epithelial cell polarity. Cdc42, a member of Rho GTPase family, modulates actin dynamics via its regulators, such as IQGAP proteins. Gastric parietal cells are polarized epithelial cells in which regulated acid secretion occurs in the apical membrane upon stimulation. We have previously shown that actin isoforms are polarized to different membrane domains and that the integrity of the actin cytoskeleton is essential for acid secretion. Herein, we show that Cdc42 is preferentially distributed to the apical membrane of gastric parietal cells. In addition, we revealed that two Cdc42 regulators, IQGAP1 and IQGAP2, are present in gastric parietal cells. Interestingly, IQGAP2 is polarized to the apical membrane of the parietal cells, whereas IQGAP1 is mainly distributed to the basolateral membrane. An IQGAP peptide that competes with full-length IQGAP proteins for Cdc42-binding in vitro also inhibits acid secretion in streptolysin-O-permeabilized gastric glands. Furthermore, this peptide disrupts the association of IQGAP and Cdc42 with the apical actin cytoskeleton and prevents the apical membrane remodeling upon stimulation. We propose that IQGAP2 forms a link that associates Cdc42 with the apical cytoskeleton and thus allows for activation of polarized secretion in gastric parietal cells.     

Helicobacter pylori induces gastric epithelial cell invasion in a c-Met and type IV secretion system-dependent manner

Helicobacter pylori interacts with gastric epithelial cells, activating signaling pathways important for carcinogenesis. In this study we examined the role of H. pylori on cell invasion and the molecular mechanisms underlying this process. The relevance of H. pylori cag pathogenicity island-encoded type IV secretion system (T4SS), CagA, and VacA for cell invasion was also investigated. We found that H. pylori induces AGS cell invasion in collagen type I and in Matrigel invasion assays. H. pylori-induced cell invasion requires the direct contact between bacteria and cancer cells. H. pylori-mediated cell invasion was dependent on the activation of the c-Met receptor and on increased MMP-2 and MMP-9 activity. The abrogation of the c-Met receptor using the specific NK4 inhibitor or the silencing of c-Met expression with small interference RNA suppressed both cell invasion and MMP activity. Studies with different H. pylori strains revealed that cell invasion, c-Met tyrosine phosphorylation, and increased MMP-2 and MMP-9 activity were all dependent on the presence of a functional bacterial T4SS, but not on VacA cytotoxicity. Our findings demonstrate that H. pylori strains with a functional T4SS stimulate gastric epithelial cell invasion through a c-Met-dependent signaling pathway that comprises an increase in MMP-2 and MMP-9 activity.     

Phospho-regulated ACAP4-Ezrin Interaction Is Essential for Histamine-stimulated Parietal Cell Secretion

The ezrin-radixin-moesin proteins provide a regulated linkage between membrane proteins and the cortical cytoskeleton and also participate in signal transduction pathways. Ezrin is localized to the apical membrane of parietal cells and couples the protein kinase A activation cascade to the regulated HCl secretion. Our recent proteomic study revealed a protein complex of ezrin-ACAP4-ARF6 essential for volatile membrane remodeling (Fang, Z., Miao, Y., Ding, X., Deng, H., Liu, S., Wang, F., Zhou, R., Watson, C., Fu, C., Hu, Q., Lillard, J. W., Jr., Powell, M., Chen, Y., Forte, J. G., and Yao, X. (2006) Mol. Cell Proteomics 5, 1437–1449). However, knowledge of whether ACAP4 physically interacts with ezrin and how their interaction is integrated into membrane-cytoskeletal remodeling has remained elusive. Here we provide the first evidence that ezrin interacts with ACAP4 in a protein kinase A-mediated phosphorylation-dependent manner through the N-terminal 400 amino acids of ACAP4. ACAP4 locates in the cytoplasmic membrane in resting parietal cells but translocates to the apical plasma membrane upon histamine stimulation. ACAP4 was precipitated with ezrin from secreting but not resting parietal cell lysates, suggesting a phospho-regulated interaction. Indeed, this interaction is abolished by phosphatase treatment and validated by an in vitro reconstitution assay using phospho-mimicking ezrin^S66D. Importantly, ezrin specifies the apical distribution of ACAP4 in secreting parietal cells because either suppression of ezrin or overexpression of non-phosphorylatable ezrin prevents the apical localization of ACAP4. In addition, overexpressing GTPase-activating protein-deficient ACAP4 results in an inhibition of apical membrane-cytoskeletal remodeling and gastric acid secretion. Taken together, these results define a novel molecular mechanism linking ACAP4-ezrin interaction to polarized epithelial secretion.     

Polarized distribution of actin isoforms in gastric parietal cells.

The actin genes encode several structurally similar, but perhaps functionally different, protein isoforms that mediate contractile function in muscle cells and determine the morphology and motility in nonmuscle cells. To reveal the isoform profile in the gastric monomeric actin pool, we purified actin from the cytosol of gastric epithelial cells by DNase I affinity chromatography followed by two-dimensional gel electrophoresis. Actin isoforms were identified by Western blotting with a monoclonal antibody against all actin isoforms and two isoform-specific antibodies against cytoplasmic beta-actin and gamma-actin. Densitometry revealed a ratio for beta-actin/gamma-actin that equaled 0.73 +/- 0.09 in the cytosol. To assess the distribution of actin isoforms in gastric glandular cells in relation to ezrin, a putative membrane-cytoskeleton linker, we carried out double immunofluorescence using actin-isoform-specific antibodies and ezrin antibody. Immunostaining confirmed that ezrin resides mainly in canaliculi and apical plasma membrane of parietal cells. Staining for the beta-actin isoform was intense along the entire gland lumen and within the canaliculi of parietal cells, thus predominantly near the apical membrane of all gastric epithelial cells, although lower levels of beta-actin were also identified near the basolateral membrane. The gamma-actin isoform was distributed heavily near the basolateral membrane of parietal cells, with much less intense staining of parietal cell canaliculi and no staining of apical membranes. Within parietal cells, the cellular localization of beta-actin, but not gamma-actin, isoform superimposed onto that of ezrin. In a search for a possible selective interaction between actin isoforms and ezrin, we carried out immunoprecipitation experiments on gastric membrane extracts in which substantial amounts of actin were co-eluted with ezrin from an anti-ezrin affinity column. The ratio of beta-actin/gamma-actin in the immunoprecipitate (beta/gamma = 2.14 +/- 0.32) was significantly greater than that found in the cytosolic fraction. In summary, we have shown that beta- and gamma-actin isoforms are differentially distributed in gastric parietal cells. Furthermore, our data suggest a preferential, but not exclusive, interaction between beta-actin and ezrin in gastric parietal cells. Finally, our results suggest that the beta- and gamma-actin-based cytoskeleton networks might function separately in response to the stimulation of acid secretion.     

Helicobacter pylori and hormones.

Helicobacter pylori affects gastric acid secretion via several mechanisms. One of these is by changing gastric regulatory physiology. The infection elevates plasma gastrin levels and decreases gastric mucosal expression of the inhibitory peptide somatostatin. These changes may be due to products of H. pylori itself or inflammatory cytokines released in H. pylori infection: acid secretion is inhibited less by a low intra-gastric pH, infusions of cholecystokinin and gastric distention in infected persons. Eradication of H. pylori rapidly decreases basal acid secretion and gastrin-releasing, peptide-stimulated acid secretion. There are now reports that maximally-stimulated acid secretion, a measure of the parietal cell mass, falls significantly six and 12 months after eradication of H. pylori from duodenal ulcer patients. This might be due to withdrawal of the trophic effect of gastrin. However H. pylori can also decrease gastric acid secretion, both through the mechanisms described in Dr. Cave''s paper and by causing gastric mucosal atrophy with loss of parietal cells. The net effect on acid presumably depends on which mechanism predominates. The processes involved may be crucial determinants of clinical outcome. For example, infection with little atrophy and high acid secretion is associated with duodenal ulcers, while infection with atrophy and low acid secretion increases the risk of gastric cancer of the intestinal-type.     

PKA-mediated protein phosphorylation protects ezrin from calpain I cleavage

Ezrin is localized to the apical membrane of parietal cells and couples the cAMP-dependent protein kinase (PKA) activation cascade to the regulated HCl secretion in gastric parietal cells. Our recent studies demonstrate the functional relevance of PKA-mediated phosphorylation of ezrin in parietal cell secretion [R. Zhou, X. Cao, C. Watson, Y. Miao, Z. Guo, J.G. Forte, X. Yao, Characterization of protein kinase A-mediated phosphorylation of ezrin in gastric parietal cell activation, J. Biol. Chem. 278 (2003) 35651]. Here we show that activation of PKA protects ezrin from calpain I-mediated proteolysis without alteration of calpain I activation and fodrin breakdown. To determine whether phosphorylation of Ser66 by PKA affects the insensitivity to the calpain I-mediated cleavage, recombinant proteins of ezrin, both wild type and S66A/D mutants, were incubated with the purified calpain I. Indeed, phosphorylation-like S66D mutant ezrin is resistant to calpain I-mediated proteolysis while wild type and S66A mutant were sensitive. In fact, expression of phosphorylation-like S66D, but not S66A, mutant in parietal cells confers its resistance to calpain I-mediated proteolysis. Taken together, these results indicate that phosphorylation of ezrin by PKA modulates its sensitivity to calpain I cleavage.     

Ezrin oligomers are the membrane-bound dormant form in gastric parietal cells

Ezrin is a member of ezrin, radixin, moesin (ERM) protein family that links F-actin to membranes. The NH₂- and COOH-terminal association domains of ERM proteins, known respectively as N-ERMAD and C-ERMAD, participate in interactions with membrane proteins and F-actin, and intramolecular and intermolecular interactions within and among ERM proteins. In gastric parietal cells, ezrin is heavily represented on the apical membrane and is associated with cell activation. Ezrin-ezrin interactions are presumably involved in functional regulation of ezrin and thus became a subject of our study. Fluorescence resonance energy transfer (FRET) was examined with cyan fluorescent protein (CFP)- and yellow fluorescent protein (YFP)-tagged ezrin incorporated into HeLa cells and primary cultures of parietal cells. Constructs included YFP at the NH₂ terminus of ezrin (YFP-Ez), CFP at the COOH terminus of ezrin (Ez-CFP), and double-labeled ezrin (N-YFP-ezrin-CFP-C). FRET was probed using fluorescence microscopy and spectrofluorometry. Evidence of ezrin oligomer formation was found using FRET in cells coexpressing Ez-CFP and YFP-Ez and by performing coimmunoprecipitation of endogenous ezrin with fluorescent protein-tagged ezrin. Thus intermolecular NH₂- and COOH-terminal association domain (N-C) binding in vivo is consistent with the findings of earlier in vitro studies. After the ezrin oligomers were separated from monomers, FRET was observed in both forms, indicating intramolecular and intermolecular N-C binding. When the distribution of native ezrin as oligomers vs. monomers was examined in resting and maximally stimulated parietal cells, a shift of ezrin oligomers to the monomeric form was correlated with stimulation, suggesting that ezrin oligomers are the membrane-bound dormant form in gastric parietal cells. fluorescence resonance energy transfer; acid secretion; radixin; moesin; cytoskeleton; ERM family     

Interaction of Helicobacter pylori with host cells: function of secreted and translocated molecules

Secreted proteins are of general interest from the perspective of bacteria-host interaction. The gastric bacterial pathogen Helicobacter pylori uses a set of secreted and translocated proteins--including outer membrane adhesins, secreted extracellular enzymes and translocated effector proteins--to adapt to its extraordinary habitat, the gastric mucosa. Two major virulence factors of H. pylori are the vacuolating cytotoxin (VacA) and the cag type-IV secretion system and its translocated effector protein, cytotoxin-associated antigen A (CagA). VacA targets not only epithelial cells, but also cells of the immune system and induces immunosuppression. CagA has been shown to interact with a growing set of eucaryotic signaling molecules in phosphorylation-dependent and -independent ways.     

The Helicobacter pylori CagA protein induces tyrosine dephosphorylation of ezrin

Helicobacter pylori is one of the most wide-spread bacterial pathogens and infects the human stomach to cause diseases, such as gastritis, gastric ulceration, and gastric cancer. A major virulence determinant is the H. pylori CagA protein (encoded by the cytotoxin-associated gene A) which is translocated from the bacteria into the cytoplasm of host cells by a type IV secretion system. In the host cell, CagA is phosphorylated on tyrosine residues and induces rearrangements of the actin cytoskeleton. We have previously shown that tyrosine-phosphorylated CagA inhibits the catalytic activity of Src family kinases and induces tyrosine dephosphorylation of several host cell proteins. Here, we identified one of these proteins as ezrin by a combination of preparative gel electrophoresis, two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS). Specific pharmacological inhibition of Src family kinases also induces ezrin dephosphorylation. Therefore, ezrin dephosphorylation appears to be induced by CagA-mediated Src inactivation. Ezrin is the founding member of the ezrin-radixin-moesin (ERM) family of proteins which are signalling integrators at the cell cortex. Since ezrin is a component of microvilli and a linker protein between actin filaments and membrane proteins, this observation has important implications for H. pylori pathogenesis and might also help to explain the development of gastric cancer.     

Acid secretion and membrane reorganization in single gastric parietal cell in primary culture

A digitally-enhanced videomicroscopy study of rabbit gastric parietal cells in primary culture was performed using alternate observations with differential interference contrast and fluorescence optics of cells mounted and perfused on a temperature-controlled microscope stage. The effect of histamine, a physiological effector of acid secretion, was followed. Isolated parietal cells possess an internal apical vacuole, which kept the cell in a pseudopolarized state. This apical vacuole is a site of acid secretion. This was demonstrated by the direct visualization of the uptake of the fluorescent weak base 9-amino acridine and of the concomitant enormous swelling of the acid vacuole which reached an estimated size of 3-7 times the normal cell volume. This morphological change of shape and acidification of apical vacuoles was fully reversible and cells could respond to successive stimulations. A quantitative study of these events provided a value of the acid accumulation index for each single cell in response to histamine. Individual cell response varied within a factor of 7. The cellular localization of the proton pump complex responsible for acid secretion and of the major components of the secretory microvilli, actin and ezrin, a histamine-dependent phosphorylation target of protein kinase A, were detected by indirect immunofluorescence microscopy in resting and stimulated cells. Both actin and ezrin colocalized at the apical vacuole membrane in resting and stimulated cells, whereas the proton pump shifted from an intracytoplasmic pool to the apical vacuole membrane upon stimulation.     

Acid secretion-associated translocation of KCNJ15 in gastric parietal cells

Potassium ions are required for gastric acid secretion. Several potassium channels have been implicated in providing K(+) at the apical membrane of parietal cells. In examining the mRNA expression levels between gastric mucosa and liver tissue, KCNJ15 stood out as the most highly specific K(+) channel in the gastric mucosa. Western blot analysis confirmed that KCNJ15 is abundant in the stomach. Immunofluorescence staining of isolated gastric glands indicated that KCNJ15 was expressed in parietal cells and chief cells, but not in mucous neck cells. In resting parietal cells, KCNJ15 was mainly found in puncta throughout the cytoplasm but was distinct from H(+)-K(+)-ATPase. Upon stimulation, KCNJ15 and H(+)-K(+)-ATPase become colocalized on the apical membranes, as suggested by immunofluorescence staining. Western blot analysis of the resting and the stimulated membrane fractions confirmed this observation. From nonsecreting preparations, KCNJ15-containing vesicles sedimented after a 4-h centrifugation at 100,000 g, but not after a 30-min spin, which did sediment most of the H(+)-K(+)-ATPase-containing tubulovesicles. Most of the KCNJ15 containing small vesicle population was depleted upon stimulation of parietal cells, as indicated by the fact that the KCNJ15 signal was shifted to a large membrane fraction that sedimented at 4,000 g. Our results demonstrate that, in nonsecreting parietal cells, KCNJ15 is stored in vesicles distinct from the H(+)-K(+)-ATPase-enriched tubulovesicles. Furthermore, upon stimulation, KCNJ15 and H(+)-K(+)-ATPase both translocate to the apical membrane for active acid secretion. Thus KCNJ15 can be added to the family of apical K(+) channels in gastric parietal cells.