Ezrin regulates NHE3 translocation and activation after Na
            <sup>+</sup>
            -glucose cotransport

Zhao, Huiren; Shiue, Harn; Palkon, Sara; Wang, Yingmin; Cullinan, Patrick; Burkhardt, Janis K.; Musch, Mark W.; Chang, Eugene B.; Turner, Jerrold R.

doi:10.1073/pnas.0308400101

Cited by 89 publications

(101 citation statements)

References 34 publications

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“…11 Ezrin is also preferentially degraded and resynthesized after oxidative stress 16 and the phosphorylation at threonine 567 (Thr-567) depends on the p38 MAP-kinase activity. 17 However, the detailed signal transduction pathways of phosphorylated ezrin remain unclear.…”

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Phosphorylated ezrin is located in the nucleus of the osteosarcoma cell

et al. 2010

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The survival of osteosarcoma patients is connected to metastasis. The ezrin expression is associated with the development of metastasis and poor outcome in osteosarcoma. Ezrin is present in the cytoplasm and after phosphorylation assumes an active form and links F-actin to the cell membrane. This study evaluated ezrin and phosphorylated ezrin at site Tyr354 and Thr567 expression and its subcellular localization in osteosarcoma. We studied 50 osteosarcoma patients (mean follow-up 9.8 years). Ezrin expression was assessed using immunohistochemical and immunofluorescence analysis on tissue microarray and cultured cells of human osteosarcoma 143B. The western blot analysis was carried out on cultured cells. The majority of osteosarcomas, showing cytoplasmic positivity for ezrin, phosphorylated and unphosphorylated, were associated with membranous and nuclear positivity for phosphorylated ezrin Thr567 and phosphorylated ezrin Tyr354, respectively. Ezrin expression was associated with high-grade osteosarcoma (P ¼ 0.04), with metastasis (P ¼ 0.04) and with tumors that developed metastasis (P ¼ 0.04); phosphorylated ezrin Thr567 expression was present mostly in tumors with metastasis (P ¼ 0.01) and in osteosarcomas that did not develop metastasis (P ¼ 0.002). The osteosarcoma patients with ezrin expression have a short survival. The cytoplasmic ezrin expression in osteosarcoma matches its role of membrane-cytoskeleton linker protein. The subcellular trafficking of ezrin is not blocked and it is linked to ezrin phosphorylation, also in cancer. The phosphorylated ezrin Tyr354 nuclear localization suggests its possible role as a nuclear factor in osteosarcoma. The phosphorylated ezrin Thr567 phosphorylation may not be necessary in osteosarcoma metastatic progression but it was modulated. The ezrin expression is associated with more aggressive osteosarcomas and with metastasis.

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Phosphorylated ezrin is located in the nucleus of the osteosarcoma cell

et al. 2010

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“…ϩ -Glucose Cotransport-SGLT1-expressing Caco-2 cells were maintained as described previously (25,28). For experimental use, cells were grown as confluent monolayers (25).…”

Section: Cell Culture and Stimulation Of Sglt1-mediated Namentioning

confidence: 99%

“…NHE3 activity can be regulated by the actin cytoskeleton (21)(22)(23). Although this may be caused in part by regulation of NHE3 at the plasma membrane, it is also clear that translocation of NHE3 to the plasma membrane contributes significantly to NHE3 up-regulation (24,25). Although the mechanisms that regulate NHE3 translocation are incompletely understood, recent studies suggest that Akt may be involved in this process.…”

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confidence: 99%

“…This increased NHE3 activity requires ezrin phosphorylation at threonine 567 and is at least partly the result of NHE3 exocytosis (25). Although both ezrin phosphorylation and NHE3 activation require p38 MAP kinase activation (25,27), we considered it unlikely that p38 MAP kinase could phosphorylate ezrin directly because the residues surrounding threonine 567 lack the proline residues typical of p38 MAP kinase substrates. The goal of these studies was to identify the kinase that phosphorylates ezrin, thereby triggering NHE3 exocytosis, after the initiation of Na ϩ -glucose cotransport.…”

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Akt2 Phosphorylates Ezrin to Trigger NHE3 Translocation and Activation

Shiue

Musch

Wang

et al. 2005

Journal of Biological Chemistry

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Initiation of Na؉ -glucose cotransport in intestinal absorptive epithelia causes NHE3 to be translocated to the apical plasma membrane, leading to cytoplasmic alkalinization. We reported recently that this NHE3 translocation requires ezrin phosphorylation. However, the kinase that phosphorylates ezrin in this process has not been identified. Because Akt has also been implicated in NHE3 translocation, we investigated the hypothesis that Akt phosphorylates ezrin. After initiation of Na ؉ -glucose cotransport, Akt is activated with kinetics that parallel those of ezrin phosphorylation. Inhibition of p38 MAP kinase, which blocks ezrin phosphorylation, also prevents Akt activation. Purified Akt directly phosphorylates recombinant ezrin at threonine 567 in vitro in an ATP-dependent manner. This in vitro phosphorylation can be prevented by Akt inhibitors. In intact cells, inhibition of either phosphoinositide 3-kinase, an upstream regulator of Akt, or inhibition of Akt itself using inhibitors validated in vitro prevents ezrin phosphorylation after initiation of Na ؉ -glucose cotransport. Specific small interfering RNA knockdown of Akt2 prevented ezrin phosphorylation in intact cells. Pharmacological Akt inhibition or Akt2 knockdown also prevented NHE3 translocation and activation after initiation of Na ؉ -glucose cotransport, confirming the functional role of Akt2. These studies therefore identify Akt2 as a critical kinase that regulates ezrin phosphorylation and activation. This Akt2-dependent ezrin phosphorylation leads to NHE3 translocation and activation.

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“…These findings indicate reciprocal activation of NHE3 and CFTR. CFTR and NHE3 interact via a shared regulatory complex consisting of NHE regulatory factors, ezrin, and PKA (11,41). It is unclear, however, whether CFTR and NHE3 are coexpressed in the same cells.…”

Section: Discussionmentioning

confidence: 99%

CFTR inhibition augments NHE3 activity during luminal high CO₂exposure in rat duodenal mucosa

Mizumori

Choi²,

Guth³

et al. 2008

American Journal of Physiology-Gastrointestinal and Liver Physi

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We hypothesized that the function of duodenocyte apical membrane acid-base transporters are essential for H ϩ absorption from the lumen. We thus examined the effect of inhibition of Na ϩ /H ϩ exchanger-3 (NHE3), cystic fibrosis transmembrane regulator (CFTR), or apical anion exchangers on transmucosal CO 2 diffusion and HCO 3 Ϫ secretion in rat duodenum. Duodena were perfused with a pH 6.4 high CO 2 solution or pH 2.2 low CO2 solution with the NHE3 inhibitor, S3226, the anion transport inhibitor, DIDS, or pretreatment with the potent CFTR inhibitor, CFTRinh-172, with simultaneous measurements of luminal and portal venous (PV) pH and carbon dioxide concentration ([CO2] Ϫ loss by NHE3 inhibition and reduced intracellular acidification during CFTR inhibition is consistent with activation or unmasking of NHE3 activity by CFTR inhibition, increasing cell surface H ϩ available to neutralize luminal HCO 3 Ϫ with consequent CO2 absorption. NHE3, by secreting H ϩ into the luminal microclimate, facilitates net transmucosal HCO 3 Ϫ absorption with a mechanism similar to proximal tubular HCO 3 Ϫ absorption.CO 2 absorption; bicarbonate secretion; portal venous PCO2 EPITHELIAL HCO 3 Ϫ SECRETION is considered to be the primary defense against duodenal injury from gastric acid. One of the unexplained mysteries of duodenal HCO 3 Ϫ secretion is why it is needed, given that secretion into the lumen from pancreas and liver is more than adequate to fully neutralize the bulk luminal content (13). Epithelial HCO 3 Ϫ secretion has thus been hypothesized to neutralize a distinct "preepithelial layer" adjacent to the apical plasma membrane of the mucosa (10, 24). Moreover, the upper intestine absorbs HCO 3 Ϫ by an electroneutral Na ϩ -dependent mechanism, hypothesized to be Na ϩ /H ϩ exchange (37). Since the epithelial Na ϩ /H ϩ exchanger (NHE) 3, which helps facilitate intestinal neutral NaCl absorption, is highly expressed in duodenum (31), we wondered whether duodenal NHE3 additionally functions as an H ϩ secretor, as it does in the proximal tubule (33, 38), facilitating HCO 3 Ϫ absorption. The duodenum must also absorb ϳ450 mmol of gastric H ϩ /24 h to decrease luminal hydrogen concentration ([H ϩ ]) by 6 log orders over its 15-cm length (12). HCl in the duodenal lumen is neutralized by HCO 3 Ϫ secreted by the pancreas and duodenal epithelium. This generates extremely high luminal CO 2 pressures (PCO 2 Ͼ 400 Torr), which are dissipated by the proximal jejunum (32). Gastric mucosal CO 2 and H ϩ permeability is low, since pyloric obstruction leads to severe metabolic alkalosis due to the inability of the normal stomach to absorb substantial quantities of H ϩ or CO 2 (16). Thus the duodenum is the major site for intestinal H ϩ and CO 2 absorption.We have demonstrated sequential transmucosal CO 2 and H ϩ movement from duodenal lumen into the portal vein (PV), facilitated by epithelial cytosolic and membrane-bound carbonic anhydrases (CAs) in the rat (27). Cellular acidification by luminal CO 2 /H ϩ rapidly induces protective responses su...

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

Cited by 89 publications

References 34 publications

Phosphorylated ezrin is located in the nucleus of the osteosarcoma cell

Phosphorylated ezrin is located in the nucleus of the osteosarcoma cell

Akt2 Phosphorylates Ezrin to Trigger NHE3 Translocation and Activation

CFTR inhibition augments NHE3 activity during luminal high CO₂exposure in rat duodenal mucosa

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

Cited by 89 publications

References 34 publications

Phosphorylated ezrin is located in the nucleus of the osteosarcoma cell

Phosphorylated ezrin is located in the nucleus of the osteosarcoma cell

Akt2 Phosphorylates Ezrin to Trigger NHE3 Translocation and Activation

CFTR inhibition augments NHE3 activity during luminal high CO2exposure in rat duodenal mucosa

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Product

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

CFTR inhibition augments NHE3 activity during luminal high CO₂exposure in rat duodenal mucosa