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

Abstract

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. 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. Ezrin is an actin-binding protein of the ezrin/radixin/moesin (ERM) 1The abbreviations used are: ERM, ezrin/radixin/moesin; PKA, protein kinase A; GST, glutathione S-transferase; GFP, green fluorescent protein; MEM, minimal essential medium; AP, aminopyrine; IBMX, isobutylmethylxanthine; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; MALDI, matrix-assisted laser desorption ionization; TOF, time-of-flight; MS, mass spectrometry; PKA, cAMP-dependent protein kinase. family of cytoskeleton membrane linker proteins (1Bretscher A. Edwards K. Fehon R.G. Nat. Rev. Mol. Cell. Biol. 2002; 3: 586-599Crossref PubMed Scopus (1137) Google Scholar). 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. 2Urushidani T Hanzel D.K. Forte J.G. Am. J. Physiol. 1989; 256: G1070-G1081Crossref PubMed Google Scholar and 3Yao X Forte J.G. Annu. Rev. Physiol. 2003; 65: 103-131Crossref PubMed Scopus (219) Google Scholar). 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 (4Hunter T. Cooper J.A. Cell. 1981; 24: 741-752Abstract Full Text PDF PubMed Scopus (393) Google Scholar, 5Bretscher A. J. Cell Biol. 1989; 108: 921-930Crossref PubMed Scopus (336) Google Scholar). Our previous studies showed that gastric ezrin is co-distributed with the β-actin isoform in vivo (6Yao X. Chaponnier C. Gabbiani G. Forte J.G. Mol. Biol. Cell. 1995; 6: 541-557Crossref PubMed Scopus (105) Google Scholar) and preferentially bound to the β-actin isoform in vitro (7Yao X. Cheng L. Forte J.G. J. Biol. Chem. 1996; 271: 7224-7229Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). 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 (8Legg J.W. Lewis C.A. Parsons M. Ng T. Isacke C.M. Nat. Cell Biol. 2002; 4: 399-407Crossref PubMed Scopus (206) Google Scholar), 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 Ser291, 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. 9Chen J. Cohn J.A. Mandel L.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7495-7499Crossref PubMed Scopus (104) Google Scholar and 10Urushidani T. Hanzel D.K. Forte J.G. Biochim. Biophys. Acta. 1987; 930: 209-219Crossref PubMed Scopus (46) Google Scholar). Ezrin is phosphorylated on tyrosine residues upon growth factor stimulation (11Gould K.L. Bretscher A. Esch F.S. Hunter T. EMBO J. 1989; 8: 4133-4142Crossref PubMed Scopus (208) Google Scholar, 12Fazioli F. Wong W.T. Ullrich S.J. Sakaguchi K. Appella E. Di Fiore P.P. Oncogene. 1993; 8: 1335-1345PubMed Google Scholar, 13Crepaldi T. Gautreau A. Comoglio P.M. Louvard D. Arpin M. J. Cell Biol. 1997; 138: 423-434Crossref PubMed Scopus (285) Google Scholar). 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 (14Krieg J. Hunter T. J. Biol. Chem. 1992; 267: 19258-19265Abstract Full Text PDF PubMed Google Scholar, 15Berryman M Gary R. Bretscher A. J. Cell Biol. 1995; 131: 1231-1242Crossref PubMed Scopus (180) Google Scholar). 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 (13Crepaldi T. Gautreau A. Comoglio P.M. Louvard D. Arpin M. J. Cell Biol. 1997; 138: 423-434Crossref PubMed Scopus (285) Google Scholar). Thus, it has been proposed that tyrosine phosphorylation of ezrin in mediating its cytoskeletal was by the in phosphorylation of tyrosine 353 was to cell A. Louvard D. Arpin M. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). A identified in F. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar), is localized in a of ERM proteins in ezrin, in and in Using this phosphorylated was in ezrin, and from a of cells and and phosphorylated ERM proteins were shown to be in membrane F. C. Biophys. 1996; PubMed Scopus Google Scholar, T. M. S. M. K. S. S. J. Cell Biol. PubMed Scopus Google Scholar, K. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, A. M. S. F. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). protein kinase and and two and were in to regulate the phosphorylation of the in ERM proteins T. M. S. M. K. S. S. J. Cell Biol. PubMed Scopus Google Scholar, A. M. S. F. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, K. K. J. Cell Biol. PubMed Scopus Google Scholar, A. L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus (180) Google Scholar). of the is to regulate ezrin Using an phosphorylation of in the its with the T. M. S. M. K. S. S. J. Cell Biol. PubMed Scopus Google Scholar). a of mimics the phosphorylated was shown to alter the and of ezrin L. Wong J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the it that the phosphorylation of the interaction to both and D. Bretscher A. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). phosphorylation of an of ERM proteins does not affect its with T. M. S. M. K. S. S. J. Cell Biol. PubMed Scopus Google Scholar, L. Wong J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). However, expression of mutant of ezrin the of by growth K. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, S. S. S. J. Cell Biol. PubMed Scopus (180) Google Scholar), whereas of the of these K. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, M. F. T. Mol. Biol. Cell. PubMed Scopus Google Scholar). To explore the nature of ezrin phosphorylation associated with stimulation of acid mass spectrometric of ezrin phosphorylated in vivo and in vitro to a phosphorylation on Our studies that Ser66 is a substrate of cAMP-dependent protein kinase and suggest that phosphorylation of Ser66 by PKA is critical for parietal cell activation. the that ezrin parietal cell activation, proposed that PKA-mediated phosphorylation of ezrin provides a between the activation of PKA to apical membrane cytoskeletal remodeling associated with acid secretion in parietal cells. and were from was from whereas ezrin was and by Hanzel D.K. T. A. Forte J.G. Am. J. Physiol. 1989; 256: PubMed Google Scholar). the of was by Forte J.G. Am. J. Physiol. 1993; PubMed Google Scholar). and were from was from was from expression ezrin to glutathione were from was by an ezrin into ezrin mutations S66A and S66D were by and a to the the S66D S66D S66A S66A S66D mutant was to phosphorylated ezrin, whereas S66A was as were in of Gastric and glands were from as by X A. Forte J.G. Am. J. Physiol. 1993; PubMed Google Scholar). the was with and and gastric was from the and with minimal essential with was for in a minimal of of and of gastric glands were from the for and in In and two-dimensional glands were in the for of gastric glands was the as by X A. Forte J.G. Am. J. Physiol. 1993; PubMed Google Scholar). glands were with by with of glands was with and were incubated for with by a to the glands from were and and of both the and were in a These data were used to the of to as X A. Forte J.G. Am. J. Physiol. 1993; PubMed Google Scholar). To the stimulation of the data as a of the resting from and glands were in resting stimulated for as (6Yao X. Chaponnier C. Gabbiani G. Forte J.G. Mol. Biol. Cell. 1995; 6: 541-557Crossref PubMed Scopus (105) Google Scholar). the of the glands were and with CHAPS, and cell proteins were for by for were with of for two-dimensional and to for A of was to the by with to the were incubated in with and with were by were by between of To for the nature of ezrin phosphorylation, of gastric glands were with and histamine for and for two-dimensional SDS-PAGE. proteins by two-dimensional were a membrane with an were and the were a of for Mass two-dimensional protein spots were into with a and subjected to by to R. C. E. R. X. Mol. Biol. Cell. 2003; PubMed Scopus Google Scholar). were by of the with acid and and in were and to in a and to in was Mass of of of were with acid and a with a were in a were a on were mass and data was both were in the of and on the the for protein data and data were the protein and a mass of In Phosphorylation of Ezrin by the type ezrin and ezrin proteins were in and the of the proteins was as R. Forte J.G. X. Am. J. Physiol. 2002; PubMed Scopus Google Scholar). proteins bound to were in phosphorylation to To whether Ser66 is a substrate for PKA, of both wild type and S66A mutant, were incubated with of the catalytic subunit of PKA in phosphorylation and of in a of was for and by the of and by SDS-PAGE. was with and by a Cell and of gastric parietal cells from were and as R. C. E. R. X. Mol. Biol. Cell. 2003; PubMed Scopus Google Scholar). of parietal cells were with wild type ezrin and two mutant of ezrin and to the of was incubated in of of was and for cultured parietal cells were with was to the and incubated for by of of cells were in for protein the protein parietal cells were and in To the of ezrin associated with cells were with and by of a and for for to proteins to be into X. K.L. J. Cell Biol. 1997; PubMed Scopus Google Scholar). cells were from a and for as were in whereas the the were with acid by in as X A. Forte J.G. Am. J. Physiol. 1993; PubMed Google Scholar). of parietal cells were stimulated with histamine for as R. C. E. R. X. Mol. Biol. Cell. 2003; PubMed Scopus Google Scholar). cells were and in of a and for and acid cell were by an for were incubated with of for by the of of protein for an were and with in were by and proteins were membrane for was with ezrin to the of was with for by of phosphorylation on a cytolocalization of ezrin, cultured parietal cells were with GFP, wild type and and and in for were with to a resting were with the histamine in the of a proton pump J.G. Forte J.G. J. Cell Sci. PubMed Google Scholar). cells were with for and with by in for to of the and cells were with in by of ezrin and ezrin proteins were by a and with to were on by and in were on a fluorescence a were parietal cells were a to an with a were by an of a of series were with a of in the the of the cultured parietal cells. from were a with data were into for of the of the of apical was and a fluorescent with a were the was to of a the by was as the of the and of the vacuolar were subjected to on and In were by by on and were by and was a of with Cell of ezrin by two-dimensional is shown in for resting and stimulated gastric that stimulation with histamine in a of ezrin spots to acidic with the stimulation-dependent phosphorylation of ezrin (e.g. 10Urushidani T. Hanzel D.K. Forte J.G. Biochim. Biophys. Acta. 1987; 930: 209-219Crossref PubMed Scopus (46) Google Scholar). the induced a in the of the of and a a series of acidic isoforms and an from to with a of PKA, in ezrin on two-dimensional that the phosphorylation of ezrin is from the activation of protein kinase A. Histamine stimulation induced acidic ezrin spots with a to phosphorylation of and the of on ezrin proteins on the of the (e.g. and been as to phosphoprotein and the nature of the phosphorylated To of these acidic spots to induced by histamine two-dimensional of ezrin shown in with acidic of ezrin from the stimulated although was also with an is of protein phosphorylation, the acidic spots suggest that phosphorylation on a of To the ezrin phosphorylation in to activation of acid used gastric glands to the of ezrin phosphorylation by a and in a parallel of glands acid secretion by the A two-dimensional used to the of ezrin phosphorylation is shown as that the with acidic ezrin spots and stimulation with histamine, the of the ezrin spots was with the resting by ezrin and in acid stimulation as is also on the acid response a of stimulation and was for the of the the of protein phosphorylation and to resting by of and ezrin protein phosphorylation with in the (e.g. 10Urushidani T. Hanzel D.K. Forte J.G. Biochim. Biophys. Acta. 1987; 930: 209-219Crossref PubMed Scopus (46) Google Scholar) and suggest that the phosphorylation be for parietal cell activation. To the associated with histamine the acidic ezrin spots (e.g. from the histamine-stimulated were from two-dimensional and subjected to with of two-dimensional from resting were used as were identified by mass mass These spots the ezrin protein but with from mass spectrometric analysis that and for ezrin phosphorylation in parietal cell activation stimulated by Ser66 a of analysis of the acid of ezrin suggested PKA phosphorylation as Ser66 (11Gould K.L. Bretscher A. Esch F.S. Hunter T. EMBO J. 1989; 8: 4133-4142Crossref PubMed Scopus (208) Google Scholar). Our mass spectrometric analysis also to the of Ser66 phosphorylation in response to histamine stimulation. To whether Ser66 is a substrate of PKA, in vitro phosphorylation on recombinant both wild type ezrin and a mutant ezrin in was by wild type and S66A mutant ezrin, the as shown in of the proteins with and the catalytic subunit of PKA in the of 32P into wild type but not S66A mutant ezrin PKA-mediated phosphorylation is since of ezrin with in the of the kinase in of into the wild type Thus, Ser66 of ezrin is a substrate for PKA. To whether Ser66 of ezrin is phosphorylated in response to histamine both wild type and S66A mutant, into cultured parietal cells by stimulation and of proteins from the stimulated cells. a of but not ezrin, from the parietal cell as by ezrin ezrin However, wild type and not mutant S66A ezrin from stimulated parietal indicating that Ser66 is for histamine-stimulated phosphorylation on Thus, that Ser66 of ezrin is involved in histamine stimulation of parietal cells. with the of ezrin cultured parietal cells were with a wild type ezrin analysis cells showed that ezrin protein was the of ezrin in cultured parietal cells a of the expression of in cells is that of To determine whether were in the of the of ezrin and into the with the on the In the case of was in of ezrin in the with previous (e.g. Refs. R. C. E. R. X. Mol. Biol. Cell. 2003; PubMed Scopus Google Scholar and D. Bretscher A. Forte J.G. EMBO J. PubMed Scopus Google Scholar). of wild type is to of wild type is associated with the To for the role of Ser66 phosphorylation in the of ezrin with the two mutant ezrin that mutant proteins Ser66 and phosphorylated ezrin shown in both mutant ezrin proteins a distribution to the wild These that to of to the of localization of the was with that of ezrin by fluorescence microscopy cultured parietal cells were for ezrin an ezrin and for cells were for a and for from and in the to has been in ezrin in cells is localized to the to the apical membrane that been into the cell and to the membrane that the cells. ezrin is with and distribution of the for and was to that of ezrin primarily associated with apical membrane and to a with and distribution of was also not by the and These data that is to the as ezrin and that phosphorylation of Ser66 is not for of ezrin to actin-based cytoskeleton the apical in the morphology of parietal cells with the Ser66 mutant a mutant protein phosphorylated ezrin, apical vacuole of secreting parietal although was shown in the of apical in cells is as as of cells wild type S66A mutant ezrin These data suggest that phosphorylation of Ser66 is not for apical localization of ezrin protein but be involved in apical membrane cytoskeletal of Ser66 is for the apical membrane of cells of apical were as an for apical membrane associated with acid secretion. were from resting and stimulated parietal cells in apical were in the These were from in cells from were In resting were on whereas were stimulated as of type the of S66D was with that of wild type ezrin-expressing resting type the of S66A was with that of wild type ezrin-expressing secreting of apical were as an for apical membrane associated with acid secretion. were from resting and stimulated parietal cells in apical were in the These were from in cells from were In resting were on whereas were stimulated as the of S66D was with that of wild type ezrin-expressing resting the of S66A was with that of wild type ezrin-expressing secreting cells. in a Phosphorylation of Ser66 for of parietal cells by histamine in of the apical canalicular membrane to of and proton into the canalicular In cultured parietal the membrane the apical canalicular membrane has been into the vacuolar stimulation in dilation of apical membrane as and J.G. Forte J.G. J. Cell Sci. PubMed Google Scholar). Because of this stimulated parietal cells in their resting J.G. Forte J.G. J. Cell Sci. PubMed Google Scholar). the expression of S66D mutant ezrin apical membrane whether phosphorylation of Ser66 is critical for apical cytoskeletal remodeling. To this the of stimulation on parietal cells with wild and S66D ezrin with from parietal cells with the histamine and for and for ezrin to the used in for resting parietal and primarily to the in secreting cells. the apical membrane in to surface In the case of secreting cells with wild type S66D mutant the apical canalicular of the (e.g. a and Thus, associated with the cytoskeleton the apical membrane of secreting parietal with the S66D mutant in However, as shown in and mutations in Ser66 a in the and to stimulation. Whereas stimulation of parietal cells wild type S66D mutant ezrin a vacuolar in response to cells S66A ezrin a morphology in dilation of apical is studies revealed that of be used as a for parietal cell J.G. Forte J.G. J. Cell Sci. PubMed Google Scholar). Thus, cells from resting and stimulated with wild type mutant ezrin in that stimulation vacuole to and in wild type and However, the vacuole of stimulated cells was Thus, expression of S66A mutant ezrin attenuates dilation of the suggesting that phosphorylation of ezrin Ser66 is essential for remodeling of the apical cytoskeleton of parietal cells associated with the stimulation. Phosphorylation of Ser66 for Cell of parietal cell activation and of into the apical membrane for proton into canalicular (e.g. Refs. 3Yao X Forte J.G. Annu. Rev. Physiol. 2003; 65: 103-131Crossref PubMed Scopus (219) Google Scholar and J.G. X. Cell Biol. 1996; 6: Full Text PDF PubMed Scopus Google Scholar). studies the of the actin-based cytoskeleton (e.g. J.A. Forte Forte J.G. Full Text PDF PubMed Scopus Google Scholar) and the of ezrin X A. Forte J.G. Am. J. Physiol. 1993; PubMed Google Scholar) in parietal cell activation. the observed to the mutant it was of to the of the phosphorylation of ezrin Ser66 on the of H,K-ATPase from to the apical the distribution of H,K-ATPase in cultured parietal cells wild and S66D mutant ezrin from secreting parietal cells with H,K-ATPase and and distribution is to that shown in the apical membrane and the membrane In resting H,K-ATPase is the as although also on the apical membrane (e.g. J.G. Forte J.G. J. Cell Sci. PubMed Google of cells with wild type ezrin revealed a of H,K-ATPase in the concomitant with an in the apical membrane of indicating that stimulation-induced of H,K-ATPase in cells a of the and H,K-ATPase in the apical membrane of parietal the apical of H,K-ATPase However, for cells S66A mutant ezrin, stimulation to H,K-ATPase to the apical since the in suggesting that phosphorylation of Ser66 be involved in the of examination of parietal cells S66D ezrin that the mutant does the stimulation-induced of that PKA-mediated phosphorylation of ezrin Ser66 is important for proton pump essential for parietal cell activation. a of the linker family of ERM has been in a of as of cell cell motility, and (e.g. A. Edwards K. Fehon R.G. Nat. Rev. Mol. Cell. Biol. 2002; 3: 586-599Crossref PubMed Scopus (1137) Google Scholar). Gastric ezrin was identified by of its phosphorylation with histamine-stimulated gastric acid secretion in parietal cells T. Hanzel D.K. Forte J.G. Biochim. Biophys. Acta. 1987; 930: 209-219Crossref PubMed Scopus (46) Google Scholar). on the the PKA-mediated phosphorylation on ezrin has not been characterized. the that ezrin is substrate of PKA in vivo and in mapped an important PKA-mediated phosphorylation on ezrin to studies that ezrin to is to the apical membrane of cultured parietal to However, mutations of Ser66 on ezrin alter apical membrane dynamics associated with histamine suggesting that phosphorylation of Ser66 on ezrin is for proton pump mobilization and secretion in gastric parietal cells. A role for the cytoskeleton in the of parietal cells has been from studies that and to acid secretion J.A. Forte Forte J.G. Full Text PDF PubMed Scopus Google Scholar). of the apical membrane the parietal cell In from to the secreting the apical canalicular of as the parietal cell to the resting of as a of with of the apical canalicular surface J.G. X. Cell Biol. 1996; 6: Full Text PDF PubMed Scopus Google Scholar, Forte J.G. Full Text PDF PubMed Google Scholar). These studies that actin-based cytoskeletal dynamics to the in parietal cells. Phosphorylation of ezrin (e.g. has been shown to regulate its with the cytoskeleton A. Louvard D. Arpin M. J. Cell Biol. PubMed Scopus Google Scholar). was by the that phosphorylation of the in is for in vitro A. M. S. F. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, F. L. K. Mol. Biol. Cell. PubMed Scopus Google Scholar). Using mutant ezrin the phosphorylated showed that ezrin phosphorylation ezrin is a that triggers the of membrane and indicating that ezrin cytoskeletal Our studies revealed that is phosphorylated and involved in parietal cell activation. is that an kinase for phosphorylation is not to the parietal cell activation Our that phosphorylation of ezrin is and with histamine-stimulated parietal cell secretion. of Ser66 phosphorylation not ezrin with the cytoskeleton but the of apical membrane dilation in response to with the that the of ezrin is for whereas its is for the of ezrin with proteins to the To for ERM involved in membrane association, D. M. Bretscher A. J. Cell Biol. 1997; PubMed Scopus Google Scholar) used as an to a phosphoprotein from cell These showed that the ezrin to the of D. Bretscher A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). However, an for in gastric parietal cells was E. and X. Thus, it is that ezrin to a of the of ezrin with the apical membrane of parietal cells. parietal cell activation of H,K-ATPase from to the apical it be of to how mutant ezrin S66A the apical membrane dynamics and H,K-ATPase the of H,K-ATPase the apical membrane the to of the pump into the apical and of the pump in apical membrane it be important to the phosphorylation of Ser66 Our studies that histamine-stimulated of ezrin in and acid secretion its the suggesting that phosphorylation of is an of the activation In fact, expression of ezrin mutant mimics phosphorylated in of stimulated parietal suggesting that phosphorylation of Ser66 is not for activation. is that a parallel from the Ser66 phosphorylation is for activation. phosphorylation of ezrin in residues be for the activation In fact, phosphorylation and in of In of ezrin phosphorylation associated with histamine stimulation of the role of ezrin in parietal cell secretion. the that ezrin is phosphorylated by PKA on Ser66 and that this phosphorylation is essential for parietal cell activation. that ezrin of H,K-ATPase to the apical that phosphorylation of ezrin proton pump to apical dynamics for secretion in cells. Arpin for ezrin also of for the of this mass data were in the of Mass L.