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

Chen, Jing; Cohn, Jonathan; Mandel, Lazaro J.

doi:10.1073/pnas.92.16.7495

Cited by 102 publications

(70 citation statements)

References 26 publications

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“…Indeed, it has been argued that phosphorylation may serve as the physiological activator to release the intramolecular association of the inactive ERM proteins, allowing them to bind to both their membrane target and actin . Furthermore, there is a strong correlation between ezrin serine/threonine phosphorylation and its association with the cytoskeleton in the kidney epithelium (Chen et al, 1995). We have demonstrated that phosphorylation of moesin correlates with its association with the Triton X-100 insoluble fraction in vivo, and consistent with this, activated RhoA increases the percentage of total moesin that is detergent insoluble.…”

Section: Discussionsupporting

confidence: 57%

RhoA-Dependent Phosphorylation and Relocalization of ERM Proteins into Apical Membrane/Actin Protrusions in Fibroblasts

Shaw

Henry

Solomon

et al. 1998

MBoC

172

122

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The ERM proteins (ezrin, radixin, and moesin) are a group of band 4.1-related proteins that are proposed to function as membrane/cytoskeletal linkers. Previous biochemical studies have implicated RhoA in regulating the association of ERM proteins with their membrane targets. However, the specific effect and mechanism of action of this regulation is unclear. We show that lysophosphatidic acid stimulation of serum-starved NIH3T3 cells resulted in relocalization of radixin into apical membrane/actin protrusions, which was blocked by inactivation of Rho by C3 transferase. An activated allele of RhoA, but not Rac or CDC42Hs, was sufficient to induce apical membrane/actin protrusions and localize radixin or moesin into these structures in both Rat1 and NIH3T3 cells. Lysophosphatidic acid treatment led to phosphorylation of radixin preceding its redistribution into apical protrusions. Significantly, cotransfection of RhoAV14 or C3 transferase with radixin and moesin revealed that RhoA activity is necessary and sufficient for their phosphorylation. These findings reveal a novel function of RhoA in reorganizing the apical actin cytoskeleton and suggest that this function may be mediated through phosphorylation of ERM proteins. INTRODUCTIONEzrin, radixin, and moesin (the ERM proteins) are three closely related proteins in the band 4.1 superfamily, members of which are known to serve as membrane-cytoskeletal linkers . The ERM proteins are found in dynamic plasma membrane/actin interfaces such as ruffling membranes, cleavage furrows, and microvilli; ezrin is a component of microvilli in a number of polarized epithelia (Bretscher, 1983;Hanzel et al., 1991;Berryman et al., 1993;Franck et al., 1993;Winckler et al., 1994;Amieva and Furthmayr, 1995). Furthermore, antisense experiments have demonstrated that these proteins are critical for the formation of surface microvilli in a lymphoma cell line as well as maintenance of cell-cell and cell-substratum adhesion in certain epithelial cells (Takeuchi et al., 1994). Expression of a dominant negative allele of ezrin has also been shown to cause loss of microvilli in polarized epithelial cells (Crepaldi et al., 1997). It was recently reported that ezrin is involved in the reorganization of ICAM-2 into uropods in lymphoblastoma cells, sensitizing them to natural killer cells (Helander et al., 1996). This again suggests that the ERM proteins may help to reorganize both the cytoskeleton and membrane proteins to promote cell-cell adhesion. These proteins are also closely related to the product of the neurofibromatosis type 2 (NF2) tumor suppressor gene merlin, which is mutated in certain cancer tumor types (Rouleau et al., 1993;Trofatter et al., 1993).Several studies suggest that the ERM proteins have a bipartite structure analogous to that proposed for band 4.1, composed of an amino-terminal domain re- † Present address: University of Iowa College of Medicine, Department of Physiology and Biophysics, 400 EMRB, Iowa City, IA 52245. sponsible for binding to integral membrane targets [incl...

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Section: Discussionsupporting

confidence: 57%

RhoA-Dependent Phosphorylation and Relocalization of ERM Proteins into Apical Membrane/Actin Protrusions in Fibroblasts

Shaw

Henry

Solomon

et al. 1998

MBoC

172

122

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“…Some signals may open these closed forms of ERM proteins to activate their cross-linking activity. ERM proteins were reported to be phosphorylated on tyrosine-as well as serine/threonine-residues in vivo (Gould et al, 1986;Hanzel et al, 1991;Egerton et al, 1992;Thuillier et al, 1994;Chen et al, 1995;Nakamura et al, 1995;Crepaldi et al, 1997) as well as to bind to PIP 2 (Niggli et al, 1995;Hirao et al, 1996). Furthermore, a close relationship between ERM proteins and Rho-dependent signaling has been identi®ed; the C-terminal ends of ERM proteins are phosphorylated in the downstream of Rho (Matsui et al, 1998), and the Nterminal halves of ERM proteins bind directly to Rho-GDI, Rho GDP dissociation inhibitor (Hirao et al, 1996;Takahashi et al, 1997).…”

Section: Introductionmentioning

confidence: 99%

Expression level, subcellular distribution and Rho-GDI binding affinity of merlin in comparison with ezrin/radixin/moesin proteins

et al. 1999

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Merlin, a neuro®bromatosis type-2 tumor suppressor, shows signi®cant sequence similarity to ERM (Ezrin/ Radixin/Moesin) proteins, general actin ®lament/plasma membrane cross-linkers, which are regulated in a Rhodependent manner. To understand its physiological functions, we compared merlin with ERM proteins in vivo and in vitro. Quantitative immunoblotting revealed that the molar ratio of merlin/ERM in cultured epithelial or non-epithelial cells was *0.14 or *0.05, respectively. After centrifugation of cell homogenate, merlin was mostly recovered in the insoluble fraction, whereas almost half of ERM proteins were found in the soluble fraction. Merlin and ERM proteins were concentrated at microvilli when introduced into ®broblasts. In contrast, in epithelial cells, introduced merlin was co-distributed with E-cadherin in lateral membranes, whereas ERM proteins were concentrated in apical microvilli. Finally, we examined the binding anity of merlin to Rho GDP dissociation inhibitor (Rho-GDI), to which N-terminal halves of ERM proteins but not the full-length molecules speci®cally bind. In vitro binding assays revealed that the N-terminal halves of merlin isoform-I and -II as well as full-length merlin isoform-II bound to Rho-GDI with similar binding anity to ERM proteins. Immunoprecipitation con®rmed these ®ndings in vivo. These ®ndings do not favor the notion that merlin functions simply in a redundant or competitive manner to ERM proteins.

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“…ERM localization to the apical membrane and association with CD44 is dictated by phosphorylation mediated by Rho activation (Chen et al, 1995;Hirao et al, 1996;Matsui et al, 1998;Shaw et al, 1998a;Matsui et al, 1999). Rho activation induces the rapid phosphorylation of ERM proteins on a conserved threonine residue (T558, moesin; T564, radixin; T567, ezrin; T576, merlin) to promote the formation of microvillar structures (Oshiro et al, 1998;Shaw et al, 1998a).…”

Section: Introductionmentioning

confidence: 99%

Effect of merlin phosphorylation on neurofibromatosis 2 (NF2) gene function

2004

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The neurofibromatosis 2 (NF2) tumor suppressor gene product, merlin, belongs to the ezrin-radixin-moesin (ERM) subgroup of the Protein 4.1 family, which links cell surface glycoproteins to the actin cytoskeleton. Previous studies have suggested that phosphorylation of merlin, similar to other ERM proteins, may regulate its function. To determine whether merlin phosphorylation has functional consequences for merlin suppression of cell growth and motility, we generated doxycycline-regulatable RT4 schwannoma cell lines that inducibly express full-length merlin with mutations at two potential phosphorylation sites (amino-acid residues S518 and T576). Whereas a mutation at S518 that mimics constitutive phosphorylation (S518D) abrogates the ability of merlin to suppress cell growth and motility, the S518A merlin mutant, which mimics nonphosphorylated merlin, functions equivalently to wild-type merlin. Similar mutations involving T576, the analogous phosphorylation site in ERM proteins important for regulating their function, had no effect. In contrast to other functionally inactive missense merlin mutants, the regulated overexpression of S518D merlin resulted in dramatic changes in cell shape and the elaboration of filopodial extensions. These results provide the first direct demonstration that the S518D merlin mutation, which mimics merlin phosphorylation, impairs not only merlin growth and motility suppression but also leads to an acquisition of a novel phenotype previously ascribed to ERM proteins.

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

Cited by 102 publications

References 26 publications

RhoA-Dependent Phosphorylation and Relocalization of ERM Proteins into Apical Membrane/Actin Protrusions in Fibroblasts

RhoA-Dependent Phosphorylation and Relocalization of ERM Proteins into Apical Membrane/Actin Protrusions in Fibroblasts

Expression level, subcellular distribution and Rho-GDI binding affinity of merlin in comparison with ezrin/radixin/moesin proteins

Effect of merlin phosphorylation on neurofibromatosis 2 (NF2) gene function

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