The Apical Na+/H+ Exchanger Isoform NHE3 Is Regulated by the Actin Cytoskeleton

Kurashima, Kazuyoshi; D’Souza, Sudhir J.A.; Szászi, Katalin; Ramjeesingh, Ravi; Orlowski, John; Grinstein, Sergio

doi:10.1074/jbc.274.42.29843

Cited by 110 publications

(123 citation statements)

References 40 publications

(43 reference statements)

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“…It connects NHERF/EBP-50 (which in turn binds important membrane proteins such as CFTR and NHE-3) to F-actin and also enables protein kinase A-mediated regulation of apical channels (Kurashima et al, 1999;Louvet-Vallée, 2000;Weinman et al, 2000Weinman et al, , 2001. Because ezrin is the earliest protein of this complex scaffold to be recruited, it has long been considered as an organizer of the brush border (Bretscher et al, 1997).…”

Section: Introductionmentioning

confidence: 99%

Intermediate Filaments Interact with Dormant Ezrin in Intestinal Epithelial Cells

Wald

Oriolo

Casanova

et al. 2005

MBoC

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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. INTRODUCTIONEzrin, a member of the ezrin-radixin-moesin (ERM) family, is a conspicuous component of the apical F-actin-based scaffold in simple epithelial cells. It connects NHERF/EBP-50 (which in turn binds important membrane proteins such as CFTR and NHE-3) to F-actin and also enables protein kinase A-mediated regulation of apical channels (Kurashima et al., 1999;Louvet-Vallée, 2000;Weinman et al., 2000Weinman et al., , 2001. Because ezrin is the earliest protein of this complex scaffold to be recruited, it has long been considered as an organizer of the brush border (Bretscher et al., 1997). In support of that view, ezrin knockout mice show a distinctive brush-border phenotype with much shorter microvilli that resemble undifferentiated stages (Saotome et al., 2004). Furthermore, expression of ezrin in fibroblasts is sufficient to organize microvilli (Shaw et al., 1998). After translation, ezrin initially adopts a "dormant" configuration, in which the C-and N-terminal domains bind to each other. On phosphorylation in T567, it changes to an open "active" configuration, freeing the C-terminal domain (C-ERMAND) to bind F-actin and the N-terminal domain (N-ERMAND) to bind NHERF or transmembrane proteins (Bretscher et al., 2000). Although the mechanism of activation and assembly of ezrin has been studied in great detail by several groups, the mechanisms that ensure that ezrin assembly occur mostly under the apical membrane of simple epithelial cells remain obscure. Fievet et al. (2004) have shown that binding to phosphatidylinositol 4,5-diphosphate (PIP 2 ) is a prerequisite for activation. However, PIP 2 is not known to be apically polarized in epithelial cells, rather it is basol...

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Section: Introductionmentioning

confidence: 99%

Intermediate Filaments Interact with Dormant Ezrin in Intestinal Epithelial Cells

Wald

Oriolo

Casanova

et al. 2005

MBoC

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“…In addition, activation of small GTPases of the Rho and Ras families is associated with stimulation of NHE1 (17,18). However, disruption of the actin cytoskeleton with cytochalasin B did not significantly alter basal NHE1 activity (14), unlike the detrimental effects observed for NHE3. Thus, the NHE isoforms are differentially responsive to the state of actin polymerization.…”

Section: Members Of the Namentioning

confidence: 73%

“…Specifically, depolymerization of actin filaments by scavenging monomeric actin with latrunculin B, or by capping their barbed ends with cytochalasins, led to a profound inhibition of NHE3 activity. Unlike the effects of wortmannin, however, the inhibition induced by cytoskeletal disassembly was not associated with a decrease in the amount of NHE3 at the plasma membrane (14). Therefore, the activity of NHE3 is likely to be modulated by multiple regulatory mechanisms.…”

Section: Members Of the Namentioning

confidence: 85%

“…3A), resembling the effects of latrunculin B and cytochalasin (14). The depression of NHE3 activity is not likely the result of a generalized detrimental effect of RhoA(T19N) myc , since the transfectants retained BCECF and were effectively acid-loaded by the ammonium pre-pulse.…”

Section: Transfection Of Ap-1 Cells With Rac1 Cdc42mentioning

confidence: 91%

“…The activity of NHE3 is also acutely sensitive to the state of assembly of the actin cytoskeleton (14). Specifically, depolymerization of actin filaments by scavenging monomeric actin with latrunculin B, or by capping their barbed ends with cytochalasins, led to a profound inhibition of NHE3 activity.…”

Section: Members Of the Namentioning

confidence: 99%

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RhoA and Rho Kinase Regulate the Epithelial Na+/H+ Exchanger NHE3

Szászi

Kurashima

Kapùs

et al. 2000

Journal of Biological Chemistry

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The activity of the Na ؉ /H ؉ exchanger NHE3 isoform, which is found primarily in epithelial cells, is sensitive to the state of actin polymerization. Actin assembly, in turn, is controlled by members of the small GTPase Rho family, namely Rac1, Cdc42, and RhoA. We therefore investigated the possible role of these GTPases in modulating NHE3 activity. Cells stably expressing NHE3 were transiently transfected with inhibitory forms of Rac1, Cdc42, or RhoA and transport activity was assessed using microfluorimetry. NHE3 activity was not adversely affected by either dominant-negative Rac1 or Cdc42. By contrast, the inhibitory form of RhoA greatly depressed NHE3 activity, without noticeably altering its subcellular distribution. NHE3 activity was equally reduced by inhibiting p160 Rho-associated kinase I (ROK), a downstream effector of RhoA, with the selective antagonist Y-27632 and a dominant-negative form of ROK. Furthermore, inhibition of ROK reduced the phosphorylation of myosin light chain. A comparable net dephosphorylation was achieved by the myosin light chain kinase inhibitor ML9, which similarly inhibited NHE3. These data suggest that optimal NHE3 activity requires a functional RhoA-ROK signaling pathway which acts, at least partly, by controlling the phosphorylation of myosin light chain and, ultimately, the organization of the actin cytoskeleton.

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The future of interventional cardiology

White

2012

Cathet Cardio Intervent

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The Apical Na+/H+ Exchanger Isoform NHE3 Is Regulated by the Actin Cytoskeleton

Cited by 110 publications

References 40 publications

Intermediate Filaments Interact with Dormant Ezrin in Intestinal Epithelial Cells

Intermediate Filaments Interact with Dormant Ezrin in Intestinal Epithelial Cells

RhoA and Rho Kinase Regulate the Epithelial Na+/H+ Exchanger NHE3

The future of interventional cardiology

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