Phosphoinositide binding and phosphorylation act sequentially in the activation mechanism of ezrin

Fiévet, Bruno; Gautreau, Alexis; Roy, Christian; Maestro, Laurence Del; Mangeat, Paul; Louvard, Daniel; Arpin, Monique

doi:10.1083/jcb.200307032

Cited by 337 publications

(425 citation statements)

References 26 publications

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“…As endocytosis and recycling of integrins at the cell rear plays an important role in neutrophil rear release (Lawson and Maxfield, 1995), it is intriguing to speculate that PIPKI␥661 may also promote rear detachment via endocytosis of surface receptors at the cell rear. Alternatively, PIPKI␥661 may promote rear detachment via the synthesis of PtdIns(4,5)P 2 , which regulates the activity of cytoskeleton-associated ERM proteins (Yoshinaga-Ohara et al, 2002;Fievet et al, 2004). A challenge for future investigation will be to define the mechanisms by which PIPKI␥661 and the localized production of PtdIns(4,5)P 2 within the uropod regulates rear retraction.…”

Section: Discussionmentioning

confidence: 99%

Type Iγ PIP Kinase Is a Novel Uropod Component that Regulates Rear Retraction during Neutrophil Chemotaxis

Lokuta

Senetar²,

Bennin

et al. 2007

MBoC

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Cell polarization is necessary for directed migration and leukocyte recruitment to inflamed tissues. Recent progress has been made in defining the molecular mechanisms that regulate chemoattractant-induced cell polarity during chemotaxis, including the contribution of phosphoinositide 3-kinase (PI3K)-dependent phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P 3 ] synthesis at the leading edge. However, less is known about the molecular composition of the cell rear and how the uropod functions during cell motility. Here, we demonstrate that phosphatidylinositol phosphate kinase type I␥ (PIPKI␥661), which generates PtdIns(4,5)P 2 , is enriched in the uropod during chemotaxis of primary neutrophils and differentiated HL-60 cells (dHL-60). Using time-lapse microscopy, we show that enrichment of PIPKI␥661 at the cell rear occurs early upon chemoattractant stimulation and is persistent during chemotaxis. Accordingly, we were able to detect enrichment of PtdIns(4,5)P 2 at the uropod during chemotaxis. Overexpression of kinase-dead PIPKI␥661 compromised uropod formation and rear retraction similar to inhibition of ROCK signaling, suggesting that PtdIns(4,5)P 2 synthesis is important to elicit the backness response during chemotaxis. Together, our findings identify a previously unknown function for PIPKI␥661 as a novel component of the backness signal that regulates rear retraction during chemotaxis.

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Section: Discussionmentioning

confidence: 99%

Type Iγ PIP Kinase Is a Novel Uropod Component that Regulates Rear Retraction during Neutrophil Chemotaxis

Lokuta

Senetar²,

Bennin

et al. 2007

MBoC

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“…This intramolecular association masks the binding sites for plasma membrane proteins and F-actin. An activation step is required to disrupt this association that occurs through conformational changes induced by sequential binding to PIP 2 and phosphorylation of a conserved C-terminal threonine residue (T567 in ezrin; Matsui et al, 1998;Fiévet et al, 2004).A link between the signaling pathways triggered by the small GTPases of the Rho family and activation of ERM proteins has been suggested. The ability of ERM proteins to bind the cytoplasmic domain of CD44 is increased by activation of Rho (Hirao et al, 1996).…”

mentioning

confidence: 99%

Interaction of Ezrin with the Novel Guanine Nucleotide Exchange Factor PLEKHG6 Promotes RhoG-dependent Apical Cytoskeleton Rearrangements in Epithelial Cells

D'Angelo

Aresta

Blangy

et al. 2007

MBoC

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The mechanisms underlying functional interactions between ERM (ezrin, radixin, moesin) proteins and Rho GTPases are not well understood. Here we characterized the interaction between ezrin and a novel Rho guanine nucleotide exchange factor, PLEKHG6. We show that ezrin recruits PLEKHG6 to the apical pole of epithelial cells where PLEKHG6 induces the formation of microvilli and membrane ruffles. These morphological changes are inhibited by dominant negative forms of RhoG. Indeed, we found that PLEKHG6 activates RhoG and to a much lesser extent Rac1. In addition we show that ezrin forms a complex with PLEKHG6 and RhoG. Furthermore, we detected a ternary complex between ezrin, PLEKHG6, and the RhoG effector ELMO. We demonstrate that PLEKHG6 and ezrin are both required in macropinocytosis. After down-regulation of either PLEKHG6 or ezrin expression, we observed an inhibition of dextran uptake in EGF-stimulated A431 cells. Altogether, our data indicate that ezrin allows the local activation of RhoG at the apical pole of epithelial cells by recruiting upstream and downstream regulators of RhoG and that both PLEKHG6 and ezrin are required for efficient macropinocytosis. INTRODUCTIONThe membrane-cytoskeleton linker ezrin is mainly expressed in epithelial cells where it associates to the apical actin-rich structures such as microvilli (Berryman et al., 1993(Berryman et al., , 1995. Recent genetic analyses revealed that ezrin is essential for the morphogenesis of epithelial cells (Fiévet et al., 2007). In ezrin Ϫ/Ϫ mice, morphological defects in the apical domain of intestinal and retinal pigment epithelial cells have been observed (Saotome et al., 2004;Bonilha et al., 2006). In parietal cells, ezrin knockdown impairs the formation of canalicular apical membrane, resulting in severe achlorhydria (Tamura et al., 2005).How ezrin participates in the assembly of the apical actinrich structures such as microvilli is still poorly understood. The association of ezrin and the highly related proteins radixin and moesin with the cortical actin cytoskeleton is strictly regulated. ERM (ezrin, radixin, moesin) proteins contain a conserved globular N-terminal domain, called the FERM domain (Four point one ezrin, radixin, moesin), involved in the binding to both phosphatidylinositol 4,5 bisphosphate (PIP 2 ; Barret et al., 2000) and plasma membrane proteins and a C-terminal F-actin-binding domain that resides in the last 34 amino acids (Turunen et al., 1994;Bretscher et al., 2002). In the cytoplasm, ERM proteins exist in a closed conformation because of an intramolecular interaction between the N-terminal domain and the last 100 amino acids called N-and C-ERMAD (ERM association domain), respectively . This intramolecular association masks the binding sites for plasma membrane proteins and F-actin. An activation step is required to disrupt this association that occurs through conformational changes induced by sequential binding to PIP 2 and phosphorylation of a conserved C-terminal threonine residue (T567 in ezrin; Matsui et al., 1998;Fié...

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“…The subsequent question is whether conformational changes arise upon PIP 2 binding and are sufficient to fully open the molecule leaving the phosphorylation stabilizing the open state. In cellulo, several studies have reported that mutants lacking the PIP 2 binding site show aberrant ERM distribution, with diminished membrane binding and translocation in the cytoplasm [50,65]. If the binding of PIP 2 to ERM contributes also to the activation then a spatially regulated process appears at the membrane: ERM are localized at the plasma membrane with PIP 2 weakening the intra-molecular interaction and the phosphorylation inducing full activation.…”

Section: Conformational Changes Of Erm Upon Pip 2 Bindingmentioning

confidence: 99%

“…On the other hand, it was shown [65] that i) ezrin activation process is dependent on binding to PIP 2 at the plasma membrane and that ii) phosphorylation in the C-terminal domain, occurred only in a second step, resulting in a conformational change of the protein and in its subsequent binding to F-actin. Thus, it is not clear whether phosphorylation is an absolute requirement for membrane binding or if this interaction with the plasma membrane is a primary step, which induces structural changes leading to full activation after phosphorylation.…”

Section: Biochemical Consideration Of Erm Activation At the Plasma Mementioning

confidence: 99%

Model membranes to shed light on the biochemical and physical properties of ezrin/radixin/moesin

2013

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Ezrin, radixin and moesin (ERM) proteins are now more and more recognized to play a key role in a large number of important physiological processes such as morphogenesis, cancer metastasis and virus infection. Several recent reviews extensively discuss their biological functions [1][2][3][4]. In this review, we will first remind the main features of this family of proteins, which are now known as linkers and regulators of the plasma membrane/cytoskeleton linkage. We will then briefly review their implication in pathological processes such as cancer and viral infection. In a second part, we will focus on biochemical and biophysical approaches to study ERM interaction with lipid membranes and conformational change in well-defined environments. In vitro studies using biomimetic lipid membranes, especially large unilamellar vesicles (LUVs), giant unilamellar vesicles (GUVs) and supported lipid bilayers (SLBs) and recombinant proteins help to understand the molecular mechanism of conformational activation of ERM proteins. These tools are aimed to decorticate the different steps of the interaction, to simplify the experiments performed in vivo in much more complex biological environments. KeywordsEzrin; radixin and moesin; biomimetic membranes; phosphoinositol (4,5)-bisphosphate (PIP 2 ); large unilamellar vesicles; supported lipid bilayers; giant unilamelar vesicles Corresponding author: Catherine Picart, CNRS UMR 5628 (LMGP), Grenoble Institute of Technology and CNRS, 3 parvis Louis Néel, F-38016 Grenoble Cedex, France. Europe PMC Funders GroupAuthor Manuscript Biochimie. Author manuscript; available in PMC 2014 July 28. Published in final edited form as:Biochimie. 2013 January ; 95(1): 3-11. doi:10.1016/j.biochi.2012.09.033. Europe PMC Funders Author ManuscriptsEurope PMC Funders Author Manuscripts Biological importance of Ezrin, Radixin and Moesin General overview of ERM proteins (Ezrin, Radixin, Moesin)ERM proteins are localized at the plasma membrane in actin-rich surface structures (Fig 1) [5], such as microvilli, membrane ruffles, and lamellipodia in gut cells, lymphocytes, hepatocytes, spermatozoids, fibroblasts and in a variety of other cell types [5][6][7][8][9]. They are involved in various physiological processes including establishment of cell polarity, cell motility and cell signaling [10]. They act as linkers between the plasma membrane and the cortical actin cytoskeleton. From a structural point of view, ERMs are constituted of 3 domains : a membrane binding domain, also known as FERM (for band 4.1 protein, ezrin, radixin and moesin), and intermediary region and a actin binding domain called C-ERMAD (for C-terminal ERM-association domain) (Fig 2A).The FERM domain, constituted of ~300 amino acids, is characteristic of the proteins of the band 4.1 superfamily. These proteins also present other types of domains, including PDZ, tyrosine phosphatase, SH2-like, tyrosine kinase, kinase-like, myosin head, and PH domains [11]. In ERM proteins, the FERM domain is followed by a long α-helical region, whi...

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Phosphoinositide binding and phosphorylation act sequentially in the activation mechanism of ezrin

Cited by 337 publications

References 26 publications

Type Iγ PIP Kinase Is a Novel Uropod Component that Regulates Rear Retraction during Neutrophil Chemotaxis

Type Iγ PIP Kinase Is a Novel Uropod Component that Regulates Rear Retraction during Neutrophil Chemotaxis

Interaction of Ezrin with the Novel Guanine Nucleotide Exchange Factor PLEKHG6 Promotes RhoG-dependent Apical Cytoskeleton Rearrangements in Epithelial Cells

Model membranes to shed light on the biochemical and physical properties of ezrin/radixin/moesin

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