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

Abstract

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...

Intramolecular Interactions between the Dbl Homology (DH) Domain and the Carboxyl-terminal region of Myosin II-interacting Guanine Nucleotide Exchange Factor (MyoGEF) Act as an Autoinhibitory Mechanism for the Regulation of MyoGEF Functions

“…Our findings demonstrate that exogenous expression of the MyoGEF carboxylterminal region interferes with MyoGEF functions, leading to cytokinesis defects and decreasing breast cancer cell invasion. It has been shown that the carboxyl-terminal region of MyoGEF can interact with centrosome/spindle-associated protein (CSPP), ezrin, and GIPC1 (46,47,58). Aurora B and Plk1 can phosphorylate the carboxyl-terminal region of MyoGEF (31,32).…”

“…The ezrin-radixinmoesin proteins can link the plasma membrane to actin filaments and play a critical role in organizing the cell cortex and promoting breast tumor metastasis (73,74). It has been shown that ezrin can bind to the carboxyl-terminal region (residues 579 -790) of MyoGEF/PLEKHG6 (58). In addition, ezrin is localized to membrane ruffles and required for membrane ruffling (75)(76)(77).…”

GIPC1 Interacts with MyoGEF and Promotes MDA-MB-231 Breast Cancer Cell Invasion

“…A line of evidence indicates that GIPC1 is implicated in endocytosis (15,16,38,39). Furthermore, depletion of PLEKHG6/MyoGEF represses dextran uptake in EGF-stimulated A431 cells (40), suggesting that MyoGEF may also play a role in regulating endocytosis. However, it is not clear at present whether the GIPC1-MyoGEF interaction is implicated in regulating membrane vesicle trafficking.…”

Ezrin mediates c-Myc actions in prostate cancer cell invasion

“…The effects of c-Myc on invasion were blocked by the expression of dominant-negative ezrin variants. Therefore, it is likely that pathways downstream from ezrin, such as RhoA/ Cdc42 or Akt activation (D'Angelo et al, 2007;Prag et al, 2007), are required for c-Myc-induced invasion. On the other hand, our data also support a more general role for ezrin as a positive regulator of c-Myc protein levels.…”