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At least 19 recordsLinked to original sources

PubMed2004

Src phosphorylates ezrin at tyrosine 477 and induces a phosphospecific association between ezrin and a kelch-repeat protein family member.

Ezrin, a linker between plasma membrane and actin cytoskeleton possesses morphogenic properties and can promote dissemination of tumor cells. Ezrin is phosphorylated on tyrosine, but a detailed picture of the signaling pathways involved in this modification is lacking. The transforming tyrosine kinase Src has various cytoskeletal substrates and is involved in regulation of cellular adhesion. We studied the role of Src in tyrosine phosphorylation of ezrin in adherent cells. We show that ezrin is phosphorylated in human embryonic kidney 293 cells in a Src family-dependent way. In SYF cells lacking Src, Yes, and Fyn, ezrin was not tyrosine-phosphorylated but reintroduction of wild-type Src followed by Src activation or introduction of active Src restored phosphorylation. Mapping of the Src-catalyzed tyrosine in vitro and in vivo by site-directed mutagenesis demonstrated Tyr(477) as the primary target residue. We generated a pTyr(477)-phosphospecific antibody, which confirmed that Tyr(477) becomes phosphorylated in cells in a Src-dependent manner. Tyr(477) phosphorylation did not affect ezrin head-to-tail association or phosphorylation of ezrin on threonine 566, indicating that the function of Tyr(477) phosphorylation is not related to the intramolecular regulation of ezrin. A modified yeast two-hybrid screen in which ezrin bait was phosphorylated by Src identified a novel interaction with a kelch-repeat protein family member, KBTBD2 (Kelch-repeat and BTB/POZ domain containing 2). The Src dependence of the interaction was further verified by affinity precipitation assays. Identification of a functional interplay with Src opens novel avenues for further characterization of the biological activities of ezrin.

Actins↗

PubMed1994

Ezrin has a COOH-terminal actin-binding site that is conserved in the ezrin protein family.

Ezrin, previously also known as cytovillin, p81, and 80K, is a cytoplasmic protein enriched in microvilli and other cell surface structures. Ezrin is postulated to have a membrane-cytoskeleton linker role. Recent findings have also revealed that the NH2-terminal domain of ezrin is associated with the plasma membrane and the COOH-terminal domain with the cytoskeleton (Algrain, M., O. Turunen, A. Vaheri, D. Louvard, and M. Arpin. 1993. J. Cell Biol. 120: 129-139). Using bacterially expressed fragments of ezrin we now demonstrate that ezrin has an actin-binding capability. We used glutathione-S-transferase fusion proteins of truncated ezrin in affinity chromatography to bind actin from the cell extract or purified rabbit muscle actin. We detected a binding site for filamentous actin that was localized to the COOH-terminal 34 amino acids of ezrin. No binding of monomeric actin was detected in the assay. The region corresponding to the COOH-terminal actin-binding site in ezrin is highly conserved in moesin, actin-capping protein radixin and EM10 protein of E. multilocularis, but not in merlin/schwannomin. Consequently, this site is a potential actin-binding site also in the other members of the protein family. Furthermore, the actin-binding site in ezrin shows sequence homology to the actin-binding site in the COOH terminus of the beta subunit of the actin-capping protein CapZ and one of the potential actin-binding sites in myosin heavy chain. The actin-binding capability of ezrin supports its proposed role as a membrane-cytoskeleton linker.

Actins↗

PubMed1996

Membrane-bound ezrin is involved in B-cell receptor-mediated signaling: potential role of an ITAM-like ezrin motif.

Ezrin is a cytoskeleton-plasma membrane linker molecule which is implicated in the T-cell antigen receptor signaling as one of the major tyrosine phosphorylated components. Its function in B-lymphocyte activation has not yet been clarified. Here we studied the potential involvement of ezrin in the B-cell receptor (BCR) signaling in BL41 Burkitt lymphoma cells. Our data demonstrate that ezrin, which shows predominantly cytosolic distribution in unstimulated cells, undergoes only a moderate tyrosine phosphorylation in response to BCR triggering, with no concomitant translocation of the protein from the cytosol to the plasma membrane. Instead, BCR-independent stimulants like oxidant stress induced by phenylarsine oxide, resulted in rapid redistribution of ezrin to the plasma membrane. When BCR triggering was preceded by membrane recruitment of ezrin, it became one of the main and earliest substrates of tyrosine kinases activated by BCR. No detectable influence on distribution or phosphorylation of ezrin was triggered by the tyrosine phosphatase inhibition by orthovanadate, suggesting that these effects of phenylarsine oxide are not attributable to its tyrosine phosphatase inhibitory capacity. The notion that BCR-mediated phosphorylation of ezrin negatively correlates with activation events such as phosphorylation of tyrosine kinase, syk and induction of calcium mobilization response, suggests that ezrin might be implicated in the regulation of transmembrane signaling and cellular responsiveness. As will be discussed, the regulatory function of ezrin may be due to an immunoreceptor tyrosine-based activation motif (ITAM)-like sequence.

Actins↗

PubMed1995

Soluble ezrin purified from placenta exists as stable monomers and elongated dimers with masked C-terminal ezrin-radixin-moesin association domains.

Previous work has indicated that ezrin, a membrane-microfilament linking protein, exists largely as a monomeric protein in solution. Here we purify from human placenta two cytosolic ezrin species that chromatography differently on gel filtration, anion, and cation exchange resins. Both species contain only the ezrin polypeptide, yet they do not readily interconvert in vitro as determined by gel filtration analysis. Determination of the physical properties of the two species indicates that one represents the conventional monomer, whereas the other represents highly asymmetric dimers. Chemical cross-linking data support this conclusion. Purified ezrin monomers normally have a masked C-terminal domain (termed a C-ERMAD) that, upon exposure, can associate with an N-terminal domain (termed N-ERMAD) of another ezrin molecule. Here we show that purified ezrin dimers also have masked C-ERMADs. On the basis of these results, we suggest a working model for the molecular organization of ezrin monomers and dimers and propose a hypothesis that explains the stable coexistence of ezrin monomers and dimers in placenta. Since radixin and moesin, the two other members of the closely related ERM protein family, both contain N- and C-ERMADs, the results we have documented and models proposed for ezrin are likely to apply to radixin and moesin as well.

Binding Sites↗

PubMed2001

The cytoplasmic tail of L-selectin interacts with members of the Ezrin-Radixin-Moesin (ERM) family of proteins: cell activation-dependent binding of Moesin but not Ezrin.

L-selectin regulates the recruitment of naive lymphocytes from the bloodstream to secondary lymphoid organs, mediating their initial capture and subsequent rolling along high endothelial cell surface-expressed ligands in peripheral lymph nodes. In vivo, distribution of L-selectin and cell surface levels determine the tethering efficiency and rolling velocity of leukocytes, respectively. Treatment of naive lymphocytes with phorbol myristate acetate (PMA) induces rapid ectodomain proteolytic down-regulation (shedding) of surface L-selectin via a protein kinase C (PKC)-dependent pathway. In an attempt to isolate proteins that are involved in regulating L-selectin expression, an affinity column was constructed using the 17-amino acid cytoplasmic tail of L-selectin. Affinity purification of extracts from lymphocytes, pre-treated with or without PMA, allowed identification of proteins that interact with the affinity column under one condition but not the other. By using this approach, members of the Ezrin-Radixin-Moesin family of proteins were found to interact specifically with the cytoplasmic tail of L-selectin. Moesin from PMA-stimulated lymphocytes, but not from unstimulated lymphocytes, bound to L-selectin tail. In contrast, ezrin from unstimulated or PMA-stimulated lymphocytes associated with L-selectin tail with equal affinity. Furthermore, the PKC inhibitor Ro 31-8220 significantly reduced the interaction of moesin, but not ezrin, with L-selectin. Alanine mutations of membrane-proximal basic amino acid residues in the cytoplasmic domain of L-selectin identified arginine 357 as a critical residue for both ezrin and moesin interaction. Finally, BIAcore affinity analysis confirmed that N-terminal moesin interacts specifically with L-selectin cytoplasmic tail, with relatively high affinity (K(d) approximately 40 nm). Based on these findings, although moesin and ezrin bind to a similar region of the cytoplasmic tail of L-selectin, moesin binding is dependent on PKC activation, which suggests that ezrin and moesin are regulated differently in lymphocytes.

Amino Acid Sequence↗

PubMed2005

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 NH(2)- 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 NH(2) 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 NH(2)- 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.

Animals↗

PubMed2005

Ezrin in primary cutaneous melanoma.

Ezrin is a member of the ezrin-radixin-moesin family of proteins that link the actin-containing cytoskeleton to the plasma membrane. Ezrin is also connected to signaling molecules involved in the regulation of cell survival, proliferation and migration. Here, we examined the expression of ezrin in 95 primary cutaneous melanomas and correlated ezrin expression with conventional prognostic factors and biomarkers. From 12 patients metastatic tissue samples were also examined. In addition to ezrin staining, Mib-1 proliferation antigen, p53 and Bcl-2 were evaluated. Ezrin immunoreactivity was seen in most tumors; only 19 (20%) melanomas were negative. A total of 48 (51%) tumors had weak immunoreactivity and 28 (29%) strong immunoreactivity. The intensity of ezrin immunoreactivity was associated with tumor thickness (Breslow, P=0.0008) and with tumor invasion level (Clark, P=0.004), thicker tumors having stronger immunoreactivity. Also, there was a correlation between higher Mib-1 index in tumors and strong ezrin expression. All metastatic samples (n=12) showed positive ezrin immunoreactivity. In univariate analysis of survival, patients (n=76) with positive ezrin immunoreactivity had worse clinical disease behavior than those (n=19) without ezrin immunoreactivity, but the difference was not significant (P=0.19). In multivariate analysis of survival, the ezrin immunoreactivity was not a significant marker. The results indicate that ezrin is expressed in most primary melanomas of the skin and in all metastatic tumors. Ezrin expression correlates with tumor thickness and level of invasion suggesting an association between ezrin expression and tumor progression.

Adult↗

PubMed2005

The membrane cytoskeletal crosslinker ezrin is required for metastasis of breast carcinoma cells.

INTRODUCTION: The membrane cytoskeletal crosslinker ezrin participates in several functions including cell adhesion, motility and cell survival, and there is increasing evidence that it regulates tumour progression. However, the role played by ezrin in breast cancer metastasis has not been clearly delineated. METHODS: We examined the role of ezrin in metastasis using a highly metastatic murine mammary carcinoma cell line, namely AC2M2. Stable cell clones that overexpress wild-type ezrin or a dominant-negative amino-terminal domain of ezrin were selected. They were then tested for cell motility and invasion in vitro, and metastasis in a mouse in vivo tumour transplantation model. RESULTS: Parental AC2M2 cells and cells overexpressing wild-type ezrin were transplanted into the mammary fat pad of syngeneic recipient mice; these animals subsequently developed lung metastases. In contrast, expression of the dominant-negative amino-terminal ezrin domain markedly inhibited lung metastasis. Consistent with this effect, we observed that the expression of amino-terminal ezrin caused strong membrane localization of cadherin, with increased cell-cell contact and a decrease in cell motility and invasion, whereas cells expressing wild-type ezrin exhibited strong cytoplasmic expression of cadherins and pseudopodia extensions. In addition, inhibitors of phosphatidylinositol 3-kinase and c-Src significantly blocked cell motility and invasion of AC2M2 cells expressing wild-type ezrin. We further found that overexpression of amino-terminal ezrin reduced levels of Akt pS473 and cytoskeletal-associated c-Src pY418 in AC2M2 cells, which contrasts with the high levels of phosphorylation of these proteins in cells expressing wild-type ezrin. Phosphorylated Erk1/2 was also reduced in amino-terminal ezrin expressing cells, although a mitogen-activated protein kinase kinase (MEK) inhibitor had no detectable effect on cell motility or invasion in this system. CONCLUSION: Our findings indicate that ezrin is required for breast cancer metastasis, and that c-Src and phosphatidylinositol 3-kinase/Akt are effectors of ezrin in the cell motility and invasion stages of the metastatic process. Together, these results suggest that blocking ezrin function may represent a novel and effective strategy for preventing breast cancer metastasis.

Animals↗

PubMed1994

Ezrin has properties to self-associate at the plasma membrane.

Ezrin, a member of a family of proteins involved in the interaction of the microfilament cytoskeleton with the plasma membrane, plays a role in membrane translocation in gastric parietal cells (Hanzel, D., Reggio, H., Bretscher, A., Forte, J. G. and Mangeat, P. (1991). EMBO J. 10, 2363-2373). Human ezrin was expressed in and purified from Escherichia coli. It possesses all the major biophysical, immunological and physiological properties of natural ezrin. Upon microinjection in live gastric HGT-1 cells, ezrin was incorporated into the dorsal microvilli, a site where the endogeneous protein is localized. By coimmunoprecipitation and ezrin-affinity assays, two HGT-1 cell proteins of 77 and 72 kDa behaved as ezrin-binding proteins. In enriched gastric apical membranes, 125I-ezrin labelled proteins of 80, 77 and 72 kDa by overlay assay. The 80 kDa protein was identified as ezrin and the 77 and 72 kDa proteins as gastric forms of proteins structurally related to ezrin, such as radixin and moesin. In insect cells infected with a recombinant baculovirus, one-third of over-expressed ezrin accumulated at the plasma membrane. Ezrin bound a 77 kDa endogenous peripheral membrane protein, behaving as an insect counterpart of the mammalian ezrin family. In addition to the respective role of the amino- and carboxyl-terminal domains of ezrin in linking the membrane and the cytoskeleton (Algrain, M., Turunen, O., Vaheri, A., Louvard, D. and Arpin, M. (1993). J. Cell Biol. 120, 129-139), both domains interacted synergistically in a salt-dependent manner to trigger self-association of ezrin. Ezrin's self-association properties could represent another way of regulating the number of ezrin molecules bound at specific membrane sites.

Animals↗

PubMed2001

Ovarian epithelial carcinoma tyrosine phosphorylation, cell proliferation, and ezrin translocation are stimulated by interleukin 1alpha and epidermal growth factor.

BACKGROUND: Ezrin is a member of the ezrin, radixin, and moesin family. These proteins are membrane-actin cross-linking proteins. Furthermore, ezrin is an important signal transduction protein that undergoes phosphorylation and translocation on stimulation by growth factors. Ezrin phosphorylation and translocation are thought to be correlated with cell motility, invasion, and carcinoma metastasis. Recently, the authors reported that an ezrin antisense phosphorothionate could significantly inhibit endometrial carcinoma cells' penetration in the Matrigel membrane cancer invasion assay. In the current study, the authors measured ezrin content in clinical ovarian epithelial carcinoma (OVCA) specimens and cell lines and investigated whether interleukin (IL)-1alpha and epidermal growth factor (EGF) induce an invasive phenotype in OVCA cells via ezrin phosphorylation and translocation. METHODS: Twenty-five normal ovary, 25 primary OVCA, 21 metastatic OVCA tissue (7 in omentum, 16 in ascites), and 3 OVCA cell lines were collected for Western blot detection of ezrin content. The OVCA cell line SKOV3 was treated with IL-1alpha or EGF. Indirect immunofluorescence staining followed by confocal laser scanning and double-staining electron microscopic immunohistochemistry were used to investigate changes in the intracellular distribution of ezrin and cell morphology after IL-1alpha or EGF treatment. The content of ezrin was measured by Western blotting and analyzed by the National Institutes of Health Image computer program. Immunoprecipitation and Western blot techniques were used for ezrin phosphorylation studies. Genistein was used to block tyrosine phosphorylation. RESULTS: (1) Ezrin was overexpressed in OVCA, with the highest values in metastases. (2) Interleukin-1alpha and EGF significantly increased OVCA tyrosine phosphorylation, ezrin translocation, and cell growth. (3) These effects were abolished by treatment with the tyrosine kinase inhibitor, genistein. (4) Treatment with IL-1alpha or EGF induced an invasive phenotype, i.e., membrane ruffling, and process formation. CONCLUSIONS: High expression and activation of ezrin appear to be related to OVCA metastatic behavior. Interleukin-1alpha and EGF may regulate OVCA invasive behavior by activating ezrin tyrosine phosphorylation, translocation, and cancer cell proliferation. The authors' results may partially explain why OVCA patients with positive macrophage colony stimulating factor (a chemoattractant of IL-1alpha secreting monocytes) or EGF receptors (c-erb B-2) have a poor prognosis.

Blotting, Western↗

PubMed2004

Ezrin regulates NHE3 translocation and activation after Na+-glucose cotransport.

Initiation of Na(+)-glucose cotransport in intestinal epithelial cells leads to activation of the apical Na(+)-H(+) exchanger NHE3 and subsequent increases in cytoplasmic pH (pH(i)). This process requires activation of p38 mitogen-activated protein (MAP) kinase, but additional signaling intermediates have not been identified. One candidate is the cytoskeletal linker protein ezrin, which interacts with NHE3 via specific regulatory proteins. The data show that initiation of Na(+)-glucose cotransport resulted in rapid increases in both apical membrane-associated NHE3 and cytoskeletal-associated ezrin and occurred in parallel with ezrin phosphorylation at threonine 567. Phosphorylation at this site is known to activate ezrin and increase its association with actin. Consistent with a central role for ezrin activation in this NHE3 regulation, an N-terminal dominant negative ezrin construct inhibited both NHE3 recruitment and pH(i) increases after Na(+)-glucose cotransport. Ezrin phosphorylation occurred in parallel with p38 MAP kinase activation, and the latter proceeded normally in cells expressing dominant negative ezrin. In contrast, inhibition of p38 MAP kinase prevented increases in ezrin phosphorylation after initiation of Na(+)-glucose cotransport. Thus, ezrin phosphorylation after Na(+)-glucose cotransport requires p38 MAP kinase activity, but p38 MAP kinase activation does not require ezrin function. These data describe a specific role for ezrin in the coordinate regulation of Na(+)-glucose cotransport and Na(+)-H(+) exchange. Intact ezrin function is necessary for NHE3 recruitment to the apical membrane and NHE3-dependent pH(i) increases triggered by Na(+)-glucose cotransport. The data also define a pathway of p38 MAP kinase-dependent ezrin activation.

Cell Fractionation↗

PubMed1995

Ezrin NH2-terminal domain inhibits the cell extension activity of the COOH-terminal domain.

Overexpression in insect cells of the full coding sequence of the human membrane cytoskeletal linker ezrin (1-586) was compared with that of a NH2-terminal domain (ezrin 1-233) and that of a COOH-terminal domain (ezrin 310-586). Ezrin (1-586), as well as ezrin (1-233) enhanced cell adhesion of infected Sf9 cells without inducing gross morphological changes in the cell structure. Ezrin (310-586) enhanced cell adhesion and elicited membrane spreading followed by microspike and lamellipodia extensions by mobilization of Sf9 cell actin. Moreover some microspikes elongated into thin processes, up to 200 microns in length, resembling neurite outgrowths by a mechanism requiring microtubule assembly. Kinetics of videomicroscopic and drug-interference studies demonstrated that mobilization of actin was required for tubulin assembly to proceed. A similar phenotype was observed in CHO cells when a comparable ezrin domain was transiently overexpressed. The shortest domain promoting cell extension was localized between residues 373-586. Removal of residues 566-586, involved in in vitro actin binding (Turunen, O., T. Wahlström, and A. Vaheri. 1994. J. Cell Biol. 126:1445-1453), suppressed the extension activity. Coexpression of ezrin (1-233) with ezrin (310-586) in the same insect cells blocked the constitutive activity of ezrin COOH-terminal domain. The inhibitory activity was mapped within ezrin 115 first NH2-terminal residues. We conclude that ezrin has properties to promote cell adhesion, and that ezrin NH2-terminal domain negatively regulates membrane spreading and elongation properties of ezrin COOH-terminal domain.

Actins↗

PubMed2005

Significance of cellular distribution of ezrin in pancreatic cystic neoplasms and ductal adenocarcinoma.

HYPOTHESIS: Pancreatic cystic neoplasms and ductal adenocarcinoma manifest diverse clinical features and prognoses, which might be related to cellular distribution of ezrin modulated through various trophic molecules. DESIGN: Laboratory investigation and retrospective analysis. SETTING: Medical school-affiliated university hospital. PATIENTS AND METHODS: Patients with solid pseudopapillary tumor (SPT) (n = 12), mucinous cystic neoplasm (MCN) (n = 18), intraductal papillary mucinous tumor (IPMT) (n = 18), and ductal adenocarcinoma (PA) (n = 73) of the pancreas were studied. Expression of epidermal growth factor (EGF) and its receptor (EGFR) and ezrin were determined using immunohistochemistry. Epidermal growth factor receptor and ezrin expression in sodium butyrate (SB)-treated PA cell line PANC-1 was determined using immunocytochemistry. Messenger RNA expression of ezrin in the PANC-1 cell line treated with SB was determined using reverse transcriptase-polymerase chain reaction. Multivariate analysis of survival of patients with PA was performed. RESULTS: None of 12 SPTs displayed synchronous expression of EGF and EGFR, while all 3 malignant SPTs displayed membranous ezrin expression. One of 18 MCNs displayed synchronous expression of EGF and EGFR, while 4 of 6 borderline malignant and 8 of 8 malignant MCNs displayed membranous ezrin expression. Two of 4 borderline malignant and 11 of 11 malignant IPMTs displayed synchronous expression of EGF and EGFR, and all borderline malignant and malignant IPMTs displayed membranous ezrin expression. Less differentiated PA displayed EGF, EGFR, and membranous ezrin expression more frequently compared with more differentiated PA. Epidermal growth factor receptor expression of PANC-1 cells decreased in an SB dose dependent manner, in which PANC-1 cells became more differentiated and membranous ezrin expression of PANC-1 cells decreased correspondingly. Messenger RNA expression of ezrin in PANC-1 cells also decreased in an SB dose dependent manner. Patients with PA with membranous ezrin expression had a poorer prognosis compared with those without (P = .02). CONCLUSIONS: Membranous translocation of ezrin might play a role during malignant transformation of SPT, MCN, IPMT, and PA, which are either dependent on (IPMT and PA) or independent of (SPT and MCN) the EGF-EGFR pathway. Membranous ezrin expression represents a prognostic factor for PA.

Adenocarcinoma, Mucinous↗

PubMed2006

Abnormal ezrin localization is associated with clinicopathological features in invasive breast carcinomas.

The membrane-cytoskeleton crosslinker ezrin is associated with malignant progression and metastasis in human neoplasias. To study the role of ezrin in breast cancer, we first assessed ezrin expression in a panel of breast cancer cell lines by western blot and confocal microscopy. Western blot revealed no differences in total ezrin levels among these breast cell lines. However, immunofluorescence staining revealed that Estrogen receptor (ER)-positive, noninvasive and nontumorigenic cell lines concentrated ezrin at the apical surface, whereas invasive cell lines localized ezrin in motile structures (membrane ruffles and filopodia) but also had more diffuse cytoplasmic staining. We next studied ezrin expression in 509 breast carcinomas using tissue microarrays. Immunohistochemical staining for ezrin, p53, Ki-67, phospho-Akt, HER2, and hormonal receptors was performed. Ezrin staining in normal breast epithelium localized at the apical, but not lateral, cell surface, whereas, in most breast tumor cases (331, 70.3%), it localized in the cytoplasm. Complete membranous staining occurred in 89 (18.9%) samples, and apical staining was seen in 51 (10.8%) cases. There were significant positive associations between cytoplasmic ezrin localization and adverse tumor characteristics such as high grade, high level of Ki-67 expression, hormonal-receptor negativity, and lymph-node metastases. Apical ezrin staining was associated with favorable clinicopathological features and node-negative tumors. Membranous ezrin staining was associated with high grade, strong HER2 and p-Akt expression. In conclusion, the switch of ezrin localization from the apical membrane to either the complete membrane or to the cytoplasm is correlated with dedifferentiation and adverse features in invasive breast tumors and cancer cell lines.

Breast Neoplasms↗

PubMed2006

Ezrin in osteosarcoma: comparison between conventional high-grade and central low-grade osteosarcoma.

Ezrin is a cytoskeleton linker protein that is actively involved in the regulation of growth and metastatic capacity of cancer cells. Recently, it has been demonstrated that a significant correlation exists between high ezrin expression levels and the poor outcome of pediatric osteosarcoma patients. The expression of ezrin was compared in conventional high-grade and central low-grade osteosarcoma lesions to investigate the role of ezrin overexpression in the metastasis of osteosarcoma. We compared the expression levels of the ezrin protein in 32 cases of high-grade osteosarcomas and 21 cases of low-grade osteosarcomas using immunohistochemistry. Ezrin protein expression levels were examined in three different human osteosarcoma cell lines by Western blotting. In addition, the mRNA expression levels of ezrin in these osteosarcoma cell lines and control fibroblasts were evaluated by real-time quantitative PCR. Ezrin immunoreactivity was present in 43.7% of high-grade osteosarcoma specimens. All low-grade osteosarcomas were negative for ezrin. The expression of ezrin was detected by Western blotting in all three osteosarcoma cell lines. The tested osteosarcoma cell lines showed marked amplification of ezrin mRNA compared to control cells. Taken together, ezrin appears to play a role in the progression of tumors, such as the metastasis of osteosarcoma. However, further data are needed before ezrin can be considered in clinical decision-making about osteosarcoma patients.

Biomarkers, Tumor↗

PubMed1995

Dephosphorylation of ezrin as an early event in renal microvillar breakdown and anoxic injury.

Disruption of the renal proximal tubule (PT) brush border is a prominent early event during ischemic injury to the kidney. The molecular basis for this event is unknown. Within the brush border, ezrin may normally link the cytoskeleton to the cell plasma membrane. Anoxia causes ezrin to dissociate from the cytoskeleton and also causes many cell proteins to become dephosphorylated in renal PTs. This study examines the hypothesis that ezrin dephosphorylation accompanies and may mediate the anoxic disruption of the rabbit renal PT. During normoxia, 73 +/- 3% of the cytoskeleton-associated (Triton-insoluble) ezrin was phosphorylated, but 88 +/- 6% of dissociated (Triton-soluble) ezrin was dephosphorylated. Phosphorylation was on serine/threonine resides, since ezrin was not detectable by an antibody against phosphotyrosine. After 60 min of anoxia, phosphorylation of total intracellular ezrin significantly decreased from 72 +/- 2% to 21 +/- 9%, and ezrin associated with the cytoskeleton decreased from 91 +/- 2% to 58 +/- 2%. Calyculin A (1 microM), the serine/threonine phosphatase inhibitor, inhibited the dephosphorylation of ezrin during anoxia by 57% and also blocked the dissociation of ezrin from the cytoskeleton by 53%. Our results demonstrate that (i) the association of ezrin with the renal microvillar cytoskeleton is correlated with phosphorylation of ezrin serine/threonine residues and (ii) anoxia may cause disruption of the renal brush border by dephosphorylating ezrin and thereby dissociating the brush border membrane from the cytoskeleton.

Animals↗

PubMed2005

Phosphorylation of ezrin on threonine 567 produces a change in secretory phenotype and repolarizes the gastric parietal cell.

Phosphorylation of the membrane-cytoskeleton linker protein ezrin has been functionally linked to acid secretion and vesicle recruitment to the apical secretory membrane in gastric parietal cells. Phosphorylation of the conserved T567 residue of ezrin has been shown to alter the N/C oligomerization of ezrin and promote the formation of actin-rich surface projections in other cells. To test the importance of T567 as a regulatory site for ezrin in parietal cell activation, we incorporated wild-type (WT) and mutant forms of ezrin, including the nonphosphorylatable T567A mutation and a mutant mimicking permanent phosphorylation, T567D. All ezrin constructs included C-terminal cyan-fluorescent protein (CFP) and were incorporated into adenoviral constructs for efficient introduction into cultured parietal cells from rabbit stomach. Fluorescence microscopy was used to localize CFP-ezrin and monitor morphological responses. Accumulation of a weak base (aminopyrine) was used to monitor receptor-mediated acid secretory response of the cultured cells. Similar to endogenous ezrin, WT and T567A CFP-ezrin localized heavily to apical membrane vacuoles with considerably lower levels associated with the surrounding basolateral membrane. Interestingly, H,K-ATPase within cytoplasmic tubulovesicles was incorporated into the apical vacuoles along with WT and T567A mutant ezrin. In these parietal cells secretagogue stimulation produced a striking vacuolar expansion associated with HCl secretion and the secretory phenotype. Expression of T567D CFP-ezrin was quite different, being rarely associated with apical vacuoles. T567D was more typically localized to the basolateral membrane, often associated with long spikes and fingerlike projections. Moreover, the cells did not display secretagogue-dependent morphological changes and, to our surprise, H,K-ATPase was recruited to the T567D CFP-ezrin-enriched basolateral projections. We conclude that T567 phosphorylation, which is probably regulated through Rho signaling pathway, may direct ezrin to membrane-cytoskeletal activity at the basolateral membrane and away from apical secretory activity. The large basolateral expansion is predicted to recruit membranes from sources not normally targeted to that surface.

Adenoviridae↗

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↗