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PubMed2002

Ezrin, a membrane-cytoskeletal linking protein, is highly expressed in atypical endometrial hyperplasia and uterine endometrioid adenocarcinoma.

We previously demonstrated that ezrin transcription was required for in vitro invasion and was involved in the acquisition of metastatic potential in endometrial cancer cells. In order to determine the functional role of ezrin in endometrial cancer, we examined ezrin protein expression in 20 cancerous and 33 non-cancerous tissues by immunohistochemistry and Western blot analysis. The specimens included 20 uterine endometrioid adenocarcinomas (UEC), seven simple endometrial hyperplasias (sH), seven complex endometrial hyperplasias (cH), seven atypical endometrial hyperplasias (aH), and 12 samples of normal endometrium (NE). Tissues of primary (P) and metastatic (M) lesions of endometrial cancers were obtained from five patients. Ezrin was specifically expressed in UEC and its precursor lesions. Ezrin expression was significantly higher in aH (P<0.05) and UEC (P<0.001) compared with NE, sH, and cH. In addition, ezrin was significantly highly expressed in M compared with P (P<0.05). Ezrin expression was associated with neither clinical stage nor histopathologic grade of UEC. In immunohistochemistry, ezrin was localized in the membrane of metastasized cancer cells, although ezrin was mainly distributed in the cytoplasm of most cancer cells and some endometrial hyperplastic cells. On Western blot analysis, ezrin was also detected in both cytosolic and membrane fractions in aH and UEC, whereas ezrin was detected in only cytosolic fraction in sH and cH. These data suggest that expression and subcellular distribution of ezrin protein play an important role in development and progression of UEC.

Adult↗
PubMed1997

Essential functions of ezrin in maintenance of cell shape and lamellipodial extension in normal and transformed fibroblasts.

BACKGROUND: Changes in cell shape and motility are important manifestations of oncogenic transformation, but the mechanisms underlying these changes and key effector molecules in the cytoskeleton remain unknown. The Fos oncogene induces expression of ezrin, the founder member of the ezrin/radixin/moesin (ERM) protein family, but not expression of the related ERM proteins, suggesting that ezrin has a distinct role in cell transformation. ERM proteins have been suggested to link the plasma membrane to the actin-based cytoskeleton and are substrates and anchoring sites for a variety of protein kinases. Here, we examined the role of ezrin in cellular transformation. RESULTS: Fos-mediated transformation of Rat-1 fibroblasts resulted in an increased expression and hyperphosphorylation of ezrin, and a concomitant increased association of ezrin with the cortical cytoskeleton. We tagged ezrin with green fluorescent protein and examined its distribution in normal and Fos-transformed fibroblasts: ezrin was concentrated at the leading edge of extending pseudopodia of Fos-transformed Rat-1 cells, and was mainly cytosolic in normal Rat-1 cells. Functional ablation of ezrin by micro-CALI (chromophore-assisted laser inactivation) blocked plasma-membrane ruffling and motility of Fos-transformed fibroblasts. Ablation of ezrin in normal Rat-1 cells caused a marked collapse of the leading edge of the cell. CONCLUSIONS: Ezrin plays an important role in pseudopodial extension in Fos-transformed Rat-1 fibroblasts, and maintains cell shape in normal Rat-1 cells. The increased expression, hyperphosphorylation and subcellular redistribution of ezrin upon fibroblast transformation coupled with its roles in cell shape and motility suggest a critical role for ezrin in oncogenic transformation.

Animals↗
PubMed1995

Indirect association of ezrin with F-actin: isoform specificity and calcium sensitivity.

Whereas it has been demonstrated that muscle and nonmuscle isoactins are segregated into distinct cytoplasmic domains, the mechanism regulating subcellular sorting is unknown (Herman, 1993a). To reveal whether isoform-specific actin-binding proteins function to coordinate these events, cell extracts derived from motile (Em) versus stationary (Es) cytoplasm were selectively and sequentially fractionated over filamentous isoactin affinity columns prior to elution with a KCl step gradient. A polypeptide of interest, which binds specifically to beta-actin filament columns, but not to muscle actin columns has been conclusively identified as the ERM family member, ezrin. We studied ezrin-beta interactions in vitro by passing extracts (Em) over isoactin affinity matrices in the presence of Ca(2+)-containing versus Ca(2+)-free buffers, with or without cytochalasin D. Ezrin binds and can be released from beta-actin Sepharose-4B in the presence of Mg2+/EGTA and 100 mM NaCl (at 4 degrees C and room temperature), but not when affinity fractionation of Em is carried out in the presence of 0.2 mM CaCl2 or 2 microM cytochalasin D. N-acetyl-(leucyl)2-norleucinal and E64, two specific inhibitors of the calcium-activated protease, calpain I, protect ezrin binding to beta actin in the presence of calcium. Moreover, biochemical analysis of endothelial lysates reveals that a calpain I cleavage product of ezrin emerges when cell locomotion is stimulated in response to monolayer injury. Immunofluorescence analysis of leading lamellae reveals that anti-ezrin and anti-beta-actin IgGs can be simultaneously co-localized, extending the results of isoactin affinity fractionation of Em-derived extracts and suggesting that ezrin and beta-actin interact in vivo. To test the hypothesis that ezrin binds directly to beta-actin, we performed three sets of studies under a wide range of physiological conditions (pH 7.0-8.5) using purified pericyte ezrin and either alpha- or beta-actin. These included co-sedimentation, isoactin affinity fractionation, and co-immunoprecipitation. Results of these experiments reveal that purified ezrin does not directly bind to beta-actin filaments, either in solution or while isoactins are covalently cross-linked to Sepharose-4B. This is in contrast to our finding that ezrin and beta-actin could be co-immunoprecipitated or co-sedimented from Em-derived cell lysates. To explore whether calcium transients occur in cellular domains enriched in ezrin and beta-actin, we mapped cellular free calcium in endothelial monolayers crawling in response to injury.(ABSTRACT TRUNCATED AT 400 WORDS)

Actin Cytoskeleton↗
PubMed2005

Intermediate filaments interact with dormant ezrin in intestinal epithelial cells.

Ezrin connects the apical F-actin scaffold to membrane proteins in the apical brush border of intestinal epithelial cells. Yet, the mechanisms that recruit ezrin to the apical domain remain obscure. Using stable CACO-2 transfectants expressing keratin 8 (K8) antisense RNA under a tetracycline-responsive element, we showed that the actin-ezrin scaffold cannot assemble in the absence of intermediate filaments (IFs). Overexpression of ezrin partially rescued this phenotype. Overexpression of K8 in mice also disrupted the assembly of the brush border, but ezrin distributed away from the apical membrane in spots along supernumerary IFs. In cytochalasin D-treated cells ezrin localized to a subapical compartment and coimmunoprecipitated with IFs. Overexpression of ezrin in undifferentiated cells showed a Triton-insoluble ezrin compartment negative for phospho-T567 (dormant) ezrin visualized as spots along IFs. Pulse-chase analysis showed that Triton-insoluble, newly synthesized ezrin transiently coimmunoprecipitates with IFs during the first 30 min of the chase. Dormant, but not active (p-T567), ezrin bound in vitro to isolated denatured keratins in Far-Western analysis and to native IFs in pull-down assays. We conclude that a transient association to IFs is an early step in the polarized assembly of apical ezrin in intestinal epithelial cells.

Animals↗
PubMed2000

Interaction between two isoforms of the NF2 tumor suppressor protein, merlin, and between merlin and ezrin, suggests modulation of ERM proteins by merlin.

The product of the neurofibromatosis type II (NF2) tumor suppressor gene, merlin, is closely related to the ezrin-radixin-moesin (ERM) family, a group of proteins believed to link the cytoskeleton to the plasma membrane. Mutation in the NF2 locus is associated with Schwann cell tumors (schwannomas). The two predominant merlin isoforms, I and II, differ only in the carboxy-terminal 16 residues and only isoform I is anti-proliferative. Merlin lacks an actin-binding domain conserved among ezrin, radixin and moesin. Because merlin, ezrin and moesin are co-expressed in Schwann cells, and all homodimerize, we have examined whether merlin and ezrin dimerize with one another. We found by immunoprecipitation and yeast two-hybrid assays that both merlin isoforms interact with ezrin. The interaction occurs in a head-to-tail orientation, with the amino-terminal half of one protein interacting with the carboxy-terminal half of the other. The two merlin isoforms behave differently in their interaction with ezrin. Isoform I binds only ezrin whose carboxy-terminus is exposed, whereas isoform II binds ezrin regardless of whether ezrin is in the open or closed conformation. The heterodimerization of merlin is a much stronger interaction than the interaction between either merlin isoform and ezrin, and can inhibit merlin-ezrin binding. This suggests that, in vivo, merlin dimerization could regulate merlin-ERM protein interaction, and could thus indirectly regulate other interactions involving ERM proteins.

Animals↗
PubMed2000

Tyrosine phosphorylation and cellular redistribution of ezrin in MDCK cells treated with pervanadate.

Ezrin is a key protein in membrane-cytoskeleton interaction and is expressed primarily in actin-rich surface projections. Activation in protein tyrosine phosphorylation apparently regulates the structure and function of ezrin. In this study, we found that pervanadate (PV, the complexes of vanadate with hydrogen peroxide) caused an increase in tyrosine phosphorylation of ezrin and affected its cellular redistribution. Treatment of Madin-Darby canine kidney (MDCK) cells with pervanadate resulted in a dramatically increased tyrosine phosphorylation of ezrin within two to five min and the level reached the maximum after 60 min. This was accompanied by an alteration in the subcellular distribution of ezrin. Immunofluorescence and scanning laser confocal microscopy analysis revealed that, after PV stimulation, ezrin was redistributed from cytosol to the apical and lateral membrane domains. This occurred within five min, and more obvious redistribution to the lateral membrane domain was observed after 30 min. Furthermore, immunoblotting of ezrin in cell fractionation experiments showed that, in PV-treated MDCK cells, cytosolic ezrin was translocated to the membrane fraction, while there was no change in the level of ezrin associated with the actin-cytoskeleton. Therefore, cytoplasmic signaling may result in activation of ezrin in tyrosine phosphorylation, which is induced by PV stimulation. These results suggest that ezrin has qualities that might play a role in modulation of cell shape and adhesion.

Amino Acid Sequence↗
PubMed2006

Expression of ezrin in glial tubes in the adult subventricular zone and rostral migratory stream.

Ezrin is a member of the ERM (ezrin-radixin-moesin) family of membrane-cytoskeletal linking proteins. ERM proteins are involved in a wide variety of cellular functions including cell motility, signal transduction, cell-cell interaction and cell-matrix recognition. A recent in situ hybridization study showed that the mRNA encoding ezrin is expressed in neurogenic regions of the mature brain including the subventricular zone (SVZ) and rostral migratory stream (RMS); however, the specific cell types expressing ezrin and their relationship to migrating and proliferating cells in these regions have not been characterized previously. In this study, we used immunocytochemistry to perform double labeling with a variety of cell-type specific markers to characterize the expression of ezrin in the SVZ and RMS of adult mice. Ezrin was expressed at high levels in both the SVZ and RMS where ezrin-immunopositive processes formed a trabecular network surrounding the proliferating and migrating cells. Ezrin-positive cells co-labeled with the glial makers S100beta and GFAP (glial fibrillary acidic protein), but only minimally with the early neuronal markers beta III tubulin and polysialylated form of neural cell adhesion molecule 1 (PSA-NCAM), indicating that ezrin was expressed primarily in the glial tube cells. Ezrin positive cells also expressed beta-catenin, a membrane-complex protein previously implicated in the regulation of stem-cell proliferation and neuronal migration. Glial tube cells act as both precursors of, and a physical channel for, migrating neuroblasts. Bi-directional signals between glial tube cells and migrating neuroblasts have been shown to regulate the rates of both proliferation of the precursor cells and migration of the newly generated neuroblasts. Our finding that ezrin and beta-catenin are both present at the cell membrane of the glial tube cells suggests that these proteins may be involved in those signaling processes.

Animals↗
PubMed1997

A dual involvement of the amino-terminal domain of ezrin in F- and G-actin binding.

Human recombinant ezrin, or truncated forms, were coated in microtiter plate and their capacity to bind actin determined. F-actin bound ezrin with a Kd of 504 +/- 230 nM and a molecular stoichiometry of 10.6 actin per ezrin. Ezrin bound both alpha- and beta/gamma-actin essentially as F-form. F-actin binding was totally prevented or drastically reduced when residues 534-586 or 13-30 were deleted, respectively. An actin binding activity was detected in amino-terminal constructs (ezrin 1-310 and 1-333) provided the glutathione S-transferase moiety of the fusion protein was removed. Series of carboxyl-terminal truncations confirmed the presence of this actin-binding site which bound both F- and G-actin. The F- and G-actin-binding sites were differently sensitive to various chemical effectors and distinct specific ezrin antibodies. The internal actin-binding site was mapped between residues 281 and 333. The association of ezrin amino-terminal fragment to full-length ezrin blocked F-actin binding to ezrin. It is proposed that, in full-length ezrin, the F-actin-binding site required the juxtaposition of the distal-most amino- and carboxyl-terminal residues of the ezrin molecule.

Actins↗
PubMed1998

The carboxyl-terminal region of EBP50 binds to a site in the amino-terminal domain of ezrin that is masked in the dormant molecule.

EBP50 (ezrin-radixin-moesin-binding phosphoprotein 50) was recently identified by affinity chromatography on the immobilized NH2-terminal domain of ezrin. Here we map and characterize the regions in EBP50 and ezrin necessary for this association. Using blot overlays and in solution binding assays, the COOH-terminal 30 residues of EBP50 were found to be sufficient for an association with residues 1-286 of ezrin. EBP50 did not bind to full-length (1-585) ezrin, indicating that the EBP50 binding site is masked in the full-length molecule. Ezrin contains two complementary self-association domains known as N- and C-ERMADs (ezrin-radixin-moesin-association domains), encompassing residues 1-296 and 479-585, respectively. An ezrin 1-583 construct lacking the two terminal residues necessary for this association was found to have an unmasked EBP50 binding site. Moreover, binding of EBP50 and the C-ERMAD to ezrin residues 1-296 was found to be mutually exclusive, with the C-ERMAD having a higher affinity. These results suggest that in full-length ezrin, the binding site for EBP50 is masked through an intramolecular N/C-ERMAD association. Based on these and additional results, we propose a model whereby dormant ezrin can be activated to bind EBP50 on its NH2-terminal end and F-actin on its COOH-terminal end. Since EBP50 is proposed to bind membrane proteins through its PDZ domains, this provides a molecular description of the regulated linkage of microfilaments to membranes in cell surface microvilli.

Amino Acid Sequence↗
PubMed2003

The amino-terminal domains of the ezrin, radixin, and moesin (ERM) proteins bind advanced glycation end products, an interaction that may play a role in the development of diabetic complications.

The presence of advanced glycation end products (AGEs) formed because of hyperglycemia in diabetic patients has been strongly linked to the development of diabetic complications and disturbances in cellular function. In this report, we describe the isolation and identification of novel AGE-binding proteins from diabetic rat kidneys. The proteins were purified by cation exchange and AGE-modified bovine serum albumin (AGE-BSA) affinity chromatography. NH2-terminal and internal sequencing identified the proteins as the NH2-terminal domains of ezrin, radixin, and moesin (ERM proteins). Using BIAcore biosensor analysis, human N-ezrin-(1-324) bound to immobilized AGE-BSA with a KD of 5.3 +/- 2.1 x 10 -7 m, whereas full-length ezrin-(1-586) and C-ezrin-(323-586) did not bind. Other glycated proteins such as AGE-RNase, N in -carboxymethyllysine (CML)-BSA, and glycated human serum albumin isolated from hyperglycemic diabetic sera competed with the immobilized AGE-BSA for binding to N-ezrin, but non-glycated BSA and RNase did not. Thus N-ezrin binds to AGEs in a glycation- and concentration-dependent manner. Phosphorylated ezrin plays a crucial role in cell shape changes, cell attachment, and cell adhesion. The effect of AGE-BSA on ezrin function was studied in a tubulogenesis model in which LLC-PK1 cell tubule formation is dependent on phosphorylated ezrin. Addition of AGE-BSA completely inhibited the ability of the cells to produce tubules. Furthermore, in vitro tyrosine phosphorylation of N-ezrin and ezrin was also inhibited by AGE-BSA. These proteins represent a novel family of intracellular binding molecules for glycated proteins and provide a potential new target for therapeutic intervention in the prevention or treatment of diabetic complications.

Animals↗
PubMed2006

Androgen induction of prostate cancer cell invasion is mediated by ezrin.

Ezrin is a key signaling molecule that regulates cell survival, adhesion migration, and invasion. We have previously shown that ezrin is regulated by androgen in rat prostate and that its expression is increased in prostate cancer and in prostate intraepithelial neoplasia. We have used the androgen-sensitive cell line LNCaP-FGC to investigate the role of ezrin in androgen-induced cell invasion. We found that androgen treatment of LNCaP-FGC cells induces ezrin expression, an effect that is inhibited by the androgen receptor antagonist, bicalutamide. In addition, androgen treatment induces the phosphorylation of ezrin in Thr-567 and Tyr-353 in a sequential manner. This is mediated through protein kinase C alpha and Src tyrosine kinase, respectively. Androgen treatment induces the translocation of both protein kinase C alpha and ezrin to the cell membrane and their association. Inhibition of ezrin function using short interference RNA or the overexpression of T567A and Y353F-ezrin mutants significantly reduces androgen-induced Matrigel invasion but does not affect cell proliferation or cell adhesion. Matrigel invasion of the androgen-insensitive prostate cancer cell lines PC-3 and LNCaP-R is also dependent on ezrin. In summary, we have shown that androgens regulate ezrin at transcriptional and posttranscriptional levels. Hormonal regulation of ezrin phosphorylation is required for androgen-induced cell invasion.

Amino Acid Substitution↗
PubMed2006

Altered expression of ezrin in esophageal squamous cell carcinoma.

Ezrin is a membrane-cytoskeletal linker belonging to the ezrin-radixin-moesin (ERM) family and has been suggested to be involved in tumorigenesis. In this study we investigated ezrin expression pattern in normal esophageal mucosa and esophageal squamous cell carcinoma (ESCC) and the correlation with clinical characteristics. Immunohistochemical staining showed a tendency for ezrin to translocate from membrane to cytoplasm in the progression from normal epithelium to invasive carcinoma of the esophagus. By Western blot, we found that ezrin expression was downregulated in 13 ESCC specimens and upregulated in 36 others. Moreover, quantitative real-time RT-PCR demonstrated that ezrin mRNA level in normal esophageal mucosa was 3.60 +/- 3.60 times that in ESCC (p<0.001). Proliferating cell nuclear antigen (PCNA) expression level was higher in ezrin downregulated group compared with that in ezrin upregulated group (p<0.05). However, there was no significant association between ezrin expression and clinical characteristics. The results suggested that the localization of ezrin by immunohistochemistry may be useful in the diagnosis of ESCC, and ezrin may play a suppressive role in the tumorgenesis of ESCC.

Biomarkers, Tumor↗
PubMed1996

Ezrin expression in stromal cells of capillary hemangioblastoma. An immunohistochemical survey of brain tumors.

Ezrin is a cytoskeleton-associated protein that appears to link actin filaments to the plasma membrane. Immunocytochemical studies suggest that ezrin is expressed in epithelial cells but not in mesenchymal cells. In addition, ezrin is expressed by certain epithelial tumors, such as renal cell adenocarcinomas. Ezrin serves as a tyrosine kinase substrate, and is phosphorylated in epidermal growth factor-stimulated cells. Ezrin may thus mediate regulatory signals in different cell functions. We studied the distribution of ezrin in 104 cases of primary tumors of the central nervous system (CNS) by immunocytochemistry. Special interest was focused on capillary hemangioblastoma, owing to its resemblance to renal cell adenocarcinoma, and on malignant gliomas, owing to their frequent epidermal growth factor receptor amplification. The stromal cells of hemangioblastomas were found to be strongly positive for ezrin. No expression was detected in gliomas and, except for hemangioblastomas, ezrin expression was restricted to those few CNS tumors that show epithelial differentiation, ie, choroid plexus papillomas, craniopharyngiomas, ependymomas, and cysts. The diffuse cytoplasmic expression of ezrin in the stromal cells of capillary hemangioblastoma may indicate that stromal cells overexpress ezrin or express ezrin with deficient binding properties.

Antibodies, Monoclonal↗
PubMed2006

PKA-mediated protein phosphorylation regulates ezrin-WWOX interaction.

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 cAMP-dependent protein kinase activation cascade to the regulated HCl secretion in gastric parietal cells. Our recent studies have mapped the PKA-mediated phosphorylation site to Ser(66) and established its functional role in parietal cell activation [R. Zhou et al., Characterization of protein kinase A-mediated phosphorylation of ezrin in gastric parietal cell activation, J. Biol. Chem. 278 (2003) 35651-35659], but the underlying basis for this regulation is not known. Here, we provide the first evidence that PKA-mediated phosphorylation of Ser(66)regulates the interaction of ezrin with WWOX, a WW domain-containing protein. Our biochemical study reveals that ezrin directly binds to the first WW domain of WWOX via its C-terminal tyrosine-containing polyproline sequence (470)PPPPPPVY(477). Mutational analyses further demonstrate that tyrosine(477) is essential for the ezrin-WWOX interaction. In addition, our study shows that PKA-mediated phosphorylation of ezrin is essential and sufficient for the apical localization of WWOX protein as disruption of ezrin-WWOX interaction eliminated the apical localization of WWOX. Finally, our study demonstrates the essential role of ezrin-WWOX interaction in the apical membrane remodeling associated with H,K-ATPase recruitment. Taken together, these results define a novel molecular mechanism underlying phospho-regulation of ezrin function by PKA in parietal cell activation.

Amino Acid Sequence↗
PubMed2003

Ezrin immunoreactivity in relation to survival in serous ovarian carcinoma patients.

OBJECTIVE: Ezrin is a membrane-cytoskeleton linker protein, which regulates cell polarity and signaling. Increased ezrin expression in astrocytomas and uveal melanomas is correlated with unfavorable prognostic factors and reduced patient survival. We investigated ezrin IR in normal ovarian surface epithelium and serous ovarian carcinomas, and its relation with clinical parameters and patient outcome. METHODS: Tissue microarray blocks were constructed of all serous ovarian carcinoma tissue samples removed at a primary operation in Helsinki University Central Hospital between 1964 and 1999 and of healthy ovarian tissue samples. Ezrin expression was assessed by indirect immunohistochemistry using a monoclonal 3C12 ezrin antibody. Tissue samples (n = 440) were scored for the intensity of ezrin immunoreactivity, and the scores were compared with patient age, the stage and grade of disease, and disease outcome. RESULTS: Healthy ovarian epithelium showed strong polarized ezrin immunoreactivity. In serous ovarian carcinoma, the reactivity varied from strong (15.0% of samples) to moderate (57.3%) or weak/negative (27.7%) and the subcellular distribution was typically diffuse. Weak or negative expression of ezrin was associated with shorter survival (P = 0.027) but also with an advanced age of the patients (P = 0.0001), and a higher histological grade of the disease (P = 0.032). In Cox multivariate survival analysis, ezrin immunoreactivity had no independent effect on survival, when controlling for the stage and grade of the disease, and patient age. CONCLUSIONS: In contrast with astrocytomas and uveal melanomas, negative or weak ezrin immunoreactivity in serous ovarian carcinoma correlates with poor patient outcome.

Adolescent↗
PubMed2001

Ezrin interacts with focal adhesion kinase and induces its activation independently of cell-matrix adhesion.

Ezrin, a membrane-cytoskeleton linker, is required for cell morphogenesis, motility, and survival through molecular mechanisms that remain to be elucidated. Using the N-terminal domain of ezrin as a bait, we found that p125 focal adhesion kinase (FAK) interacts with ezrin. We show that the two proteins coimmunoprecipitate from cultured cell lysates. However, FAK does not interact with full-length ezrin in vitro, indicating that the FAK binding site on ezrin is cryptic. Mapping experiments showed that the entire N-terminal domain of FAK (amino acids 1-376) is required for optimal ezrin binding. While investigating the role of the ezrin-FAK interaction, we observed that, in suspended kidney-derived epithelial LLC-PK1 cells, overproduction of ezrin promoted phosphorylation of FAK Tyr-397, the major autophosphorylation site, creating a docking site for FAK signaling partners. Treatment of the cells with a Src family kinase inhibitor reduced the phosphorylation of Tyr-577 but not that of Tyr-397, indicating that ezrin-mediated FAK activation does not require the activity of Src kinases. Altogether, these observations indicate that ezrin is able to trigger FAK activation in signaling events that are not elicited by cell-matrix adhesion.

Animals↗
PubMed2003

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.

Adenosine Triphosphate↗
PubMed2003

Identification and relevance of the CD95-binding domain in the N-terminal region of ezrin.

The CD95 (Fas/APO-1) linkage to the actin cytoskeleton through ezrin is an essential requirement for susceptibility to the CD95-mediated apoptosis in CD4+ T cells. We have previously shown that moesin was not involved in the binding to CD95. Here we further support the specificity of the ezrin/CD95 binding, showing that radixin did not bind CD95. The ezrin region specifically and directly involved in the binding to CD95 was located in the middle lobe of the ezrin FERM domain, between amino acids 149 and 168. In this region, ezrin, radixin, and moesin show 60-65% identity, as compared with the 86% identity in the whole FERM domain. Transfection of two different human cell lines with a green fluorescent protein-tagged ezrin mutated in the CD95-binding epitope, induced a marked inhibition of CD95-mediated apoptosis. In these cells, the mutated ezrin did not co-localize or co-immunoprecipitate with CD95. Further analysis showed that the mutated ezrin, while unable to bind CD95, was fully able to bind actin, thus preventing the actin linkage to CD95. Altogether, our results support the specificity of ezrin in the association to CD95 and the importance of the ezrin-to-CD95 linkage in CD95-mediated apoptosis. Moreover, this study suggests that a major role of ezrin is to connect CD95 to actin, thus allowing the CD95 polarization on the cells and the occurrence of the following multiple cascades of the CD95 pathway.

Actins↗