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

Abstract

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...

Specific Phosphorylation of p120-Catenin Regulatory Domain Differently Modulates Its Binding to RhoA

“…4A). Assays using different mutants of RhoA confirmed our conclusion: p120-catenin interacted better with the RhoA Thr19Asn mutant, an inactive RhoA mutant (4,32), than with the RhoA Gln63Leu mutant, a RhoA mutant with impaired GTPase activity and, therefore, constitutively bound to GTP molecules (15) (Fig. 4B, lanes 3 and 4).…”

Intermediate Filaments Interact with Dormant Ezrin in Intestinal Epithelial Cells

“…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.…”

Role of Rho family GTPases in epithelial morphogenesis

“…Interestingly, inhibition of Rho causes removal of cadherins from junctions before significant changes in cell morphology (i.e., cell rounding and retraction) can be observed, suggesting that Rho may play a role in adherens junction formation by stimulating cadherin clustering (Braga et al 1997(Braga et al , 1999. Rho also is thought to function in the intracellular targeting of proteins, such as c-Src and ERM (Fincham et al 1996;Kotani et al 1997;Shaw et al 1998;Timpson et al 2001). An alternative scenario for the role of Rho in adherens junction formation therefore could be that Rho recruits accessory proteins to nascent junctions.…”