Association Of Ezrin Research Articles

Immunohistochemical assessment of ezrin and moesin in colorectal carcinoma

ABSTRACTEzrin and moesin are important molecules of the ERM family of proteins, which regulate cell adhesion and migration. The aim of this study was to evaluate the intensity and pattern of ezrin and moesin expression in colorectal carcinoma (CRC) together with correlating their expression with the clinico-pathologic features of this neoplasm. This study was carried out on 48 CRC and 10 adenoma specimens. All adenoma and 95% of CRC cases showed both ezrin and moesin expression. Ezrin was predominantly cytoplasmic in adenoma cases in comparison to membranous localization in carcinoma cases. Moesin was predominantly expressed in stroma (inflammatory cells and fibroblasts) in carcinoma (89.1%) compared with adenoma (50%). High H-score of ezrin expression was associated with adenocarcinoma type (P = .024) and was inversely correlated with mitotic count (P = .005). High H-score of moesin expression was associated with early Dukes staging of CRC (P = .016), absence of lymph node involvement (P = .022), and low number of involved lymph nodes (P = .04). The association of ezrin with favorable prognostic parameters may be due to its prominent membranous localization. The stroma of CRC could stand against invasion by expression of moesin. Ezrin and moesin are independently expressed from each other.

Phosphorylation of ezrin on Thr567 is required for the synergistic activation of cell spreading by EPAC1 and protein kinase A in HEK293T cells

Recent studies have demonstrated that the actin binding protein, ezrin, and the cAMP-sensor, EPAC1, cooperate to induce cell spreading in response to elevations in intracellular cAMP. To investigate the mechanisms underlying these effects we generated a model of EPAC1-dependent cell spreading based on the stable transfection of EPAC1 into HEK293T (HEK293T–EPAC1) cells. We found that direct activation of EPAC1 with the EPAC-selective analogue, 8-pCPT-2′-O-Me-cAMP (007), promoted cell spreading in these cells. In addition, co-activation of EPAC1 and PKA, with a combination of the adenylate cyclase activator, forskolin, and the cAMP phosphodiesterase inhibitor, rolipram, was found to synergistically enhance cell spreading, in association with cortical actin bundling and mobilisation of ezrin to the plasma membrane. PKA activation was also associated with phosphorylation of ezrin on Thr567, as detected by an electrophoretic band mobility shift during SDS-PAGE. Inhibition of PKA activity blocked ezrin phosphorylation and reduced the cell spreading response to cAMP elevation to levels induced by EPAC1-activation alone. Transfection of HEK293T–EPAC1 cells with inhibitory ezrin mutants lacking the key PKA phosphorylation site, ezrin-Thr567Ala, or the ability to associate with actin, ezrin-Arg579Ala, promoted cell arborisation and blocked the ability of EPAC1 and PKA to further promote cell spreading. The PKA phospho-mimetic mutants of ezrin, ezrin-Thr567Asp had no effect on EPAC1-driven cell spreading. Our results indicate that association of ezrin with the actin cytoskeleton and phosphorylation on Thr567 are required, but not sufficient, for PKA and EPAC1 to synergistically promote cell spreading following elevations in intracellular cAMP.

718 Enhanced Cytoskeletal Association of Ezrin By ESPF Is Involved in EPEC-Mediated Suppression of NHE3 Activity

Sodiumhydrogen exchanger 3 (NHE3) plays an important role in sodium and fluid absorption throughout the intestine. Our previous work showed that enteropathogenic Escherichia coli (EPEC) significantly decreases NHE3 activity in human intestinal epithelial Caco-2 cells and PS120 fibroblasts transfected with NHE3. Mutation of the translocated effector protein EspF (∆espF) prevented the change in NHE3 activity. The aim of this study was to determine the mechanism by which EspF reduces NHE3 activity. NHE3 activity was assayed as 22Na+ uptake utilizing NHE inhibitors EIPA and HOE-694. EspF has several effects on intestinal epithelial cells including induction of cell death, alteration of vesicle trafficking via interactions with sorting nexin 9 (SNX9) and enhancing the cytoskeletal association of ezrin. An EspF point mutant that prevents cell death, L16E was used to determine if this phenotype was related to the decrease in NHE3. Complementation of ∆espF with the L16E mutation restored the decrease in NHE3 activity suggesting that cell death is not the cause of reduced NHE3 activity (WT EPEC -35%, ∆espF +3%, L16E -29%, p<0.05, n=15, NHE3 activity as percent change from uninfected). EspF interacts with SNX9 via proline rich repeats (PRR) and alters vesicle trafficking. As NHE3 is regulated by apical recycling, we tested the effect of EspFD3, a strain harboring point mutations in each PRR that is impaired for SNX9 binding. EspF-D3 restored the decrease in NHE3 activity in a ∆espF mutant (WT EPEC -52%, ∆espF 0%, +espF -38%, +espF-D3 -45%, n=15). Thus, interference with vesicle trafficking is not involved in EspF-mediated modulation of NHE3 activity. We published previously that EspF enhances the cytoskeletal association of ezrin. Ezrin regulates NHE3 activity by binding directly to NHE3 or to NHERF1/2. Expression of the scaffolding proteins NHERF1 and NHERF2 in PS120 fibroblasts enhances the NHE3 response to EPEC (NHE3 only -36%, +NHERF1 -56%, +NHERF2 -65%, n=9 p<0.05). To determine if ezrin was involved in EPEC-induced decrease in NHE3 activity, the effect of expressing full-length versus dominant negative ezrin was studied. While Caco-2 cells expressing full-length ezrin exhibited the expected 2-fold decrease in NHE3 activity in response to EPEC infection, expression of dominant negative ezrin prevented this response causing a minor but not significant, increase in NHE3 activity (p<0.01, n=9). Our data suggest that EspF reduces NHE3 activity, at least in part, via its effects on ezrin. These events likely contribute to the diarrhea associated with EPEC infection.

Association of Ezrin with Intercellular Adhesion Molecule-1 and -2 (ICAM-1 and ICAM-2): REGULATION BY PHOSPHATIDYLINOSITOL 4,5-BISPHOSPHATE

Ezrin is a cytoplasmic linker molecule between plasma membrane components and the actin-containing cytoskeleton. We studied whether ezrin is associated with intercellular adhesion molecule (ICAM)-1, -2, and -3. In transfected cells, ICAM-1 and ICAM-2 colocalized with ezrin in microvillar projections, whereas an ICAM-1 construct attached to cell membrane via a glycophosphatidylinositol anchor was uniformly distributed on the cell surface. An interaction of ICAM-2 and ezrin was seen by affinity precipitation, microtiter binding assay, coimmunoprecipitation, and surface plasmon resonance methods. The calculated KD value was 3.3 x 10(-7) M. Phosphatidylinositol 4, 5-bisphosphate (PtdIns(4,5)P2) induced an interaction of ezrin and ICAM-1 and enhanced the interaction of ezrin and ICAM-2, but ICAM-3 did not bind ezrin even in the presence of PtdIns(4,5)P2. PtdIns(4, 5)P2 was shown to bind to cytoplasmic tails of ICAM-1 and ICAM-2, which are the first adhesion proteins demonstrated to interact with PtdIns(4,5)P2. The results indicate an interaction of ezrin with ICAM-1 and ICAM-2 and suggest a regulatory role of phosphoinositide signaling pathways in regulation of ICAM-ezrin interaction.

Ezrin: a protein requiring conformational activation to link microfilaments to the plasma membrane in the assembly of cell surface structures.

The cortical cytoskeleton of eucaryotic cells provides structural support to the plasma membrane and also contributes to dynamic processes such as endocytosis, exocytosis, and transmembrane signaling pathways. The ERM (ezrin-radixin-moesin) family of proteins, of which ezrin is the best studied member, play structural and regulatory roles in the assembly and stabilization of specialized plasma membrane domains. Ezrin and related molecules are concentrated in surface projections such as microvilli and membrane ruffles where they link the microfilaments to the membrane. The present knowledge about ezrin is discussed from an historical perspective. Both biochemical and cell biological studies have revealed that ezrin can exist in a dormant conformation that requires activation to expose otherwise masked association sites. Current results indicate that activated ezrin monomers or head-to-tail oligomers associate directly with F-actin through a domain in its C terminus, and with the membrane through its N-terminal domain. The association of ezrin with transmembrane proteins can be direct, as in the case of CD44, or indirect through EBP50. Other binding partners, including the regulatory subunit of protein kinase A and rho-GDI, suggest that ezrin is an integral component of these signaling pathways. Although the membrane-cytoskeletal linking function is clear, further studies are necessary to reveal how the activation of ezrin and its association with different binding partners is regulated.

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.