N-cadherin/p120 Catenin Association at Cell-Cell Contacts Occurs in Cholesterol-rich Membrane Domains and Is Required for RhoA Activation and Myogenesis

Abstract

p120 catenin is a major regulator of cadherin stability at cellcell contacts and a modulator of Rho GTPase activities. In C2C12 myoblasts, N-cadherin is stabilized at cell contacts through its association with cholesterol-rich membrane domains or lipid rafts (LR) and acts as an adhesion-activated receptor that activates RhoA, an event required for myogenesis induction. Here, we report that association of p120 catenin with N-cadherin at cell contacts occurs specifically in LR. We demonstrate that interaction of p120 catenin with N-cadherin is required for N-cadherin association with LR and for its stabilization at cell contacts. LR disruption inhibits myogenesis induction and N-cadherin-dependent RhoA activation as does the perturbation of the N-cadherin-p120 catenin complex after p120 catenin knockdown. Finally, we observe an N-cadherindependent accumulation of RhoA at phosphatidylinositol 4,5-bisphosphate-enriched cell contacts which is lost after LR disruption. Thus, a functional N-cadherin-catenin complex occurs in cholesterol-rich membrane microdomains which allows the recruitment of RhoA and the regulation of its activity during myogenesis induction.Skeletal myogenesis is a multistep process regulated by diffusible molecules and the interaction of muscle cell precursors with their neighbors and the extracellular matrix (1, 2). Particularly, N-cadherin-dependent intercellular adhesion has a major role in cell cycle exit and induction of skeletal muscle differentiation through activation of the Rho family GTPases. RhoA positively regulates MyoD expression and skeletal muscle differentiation because it is required for serum response factor-mediated activation of several muscle-specific gene promoters (3, 4).Dynamic association of cadherin complexes at the plasma membrane (PM) 4 is crucial for cadherin-mediated signaling.Their extracellular domain mediates homophilic cell-cell adhesion, whereas the intracellular domain associates with catenins that produce attachment sites for the F-actin cytoskeleton (5-7). The juxtamembrane domain of the cadherin cytoplasmic tail binds to p120 catenin, which regulates cadherin stability at cell contacts and modulates Rho GTPase activities (8 -11). Cadherin stability is directly dependent on p120 catenin, and in its absence most cadherins are internalized and often degraded, suggesting that p120 catenin controls cadherin turnover at the cell surface (11,12). Moreover, mutations in the E-cadherin region that bind to p120 catenin dissociate the E-cadherin-p120 catenin complex and disrupt strong cell adhesion, although interaction with other catenins remains intact (13). Cadherin stability at cell-cell contacts is also regulated by homophilic binding between extracellular domains and association with the F-actin cytoskeleton (14, 15). Association of N-cadherin with cholesterol-enriched microdomains, called lipid rafts (LR), at cell contacts, also stabilizes N-cadherin (16). Because p120 catenin interaction with cadherins and N-cadherin association with LR at cell contact sites ...

Flotillin micro-domains stabilize Cadherins at cell-cell junctions

“…7B), thus showing that flotillin 1 is associated with GM1-containing membrane microdomains. As previously shown (Causeret et al, 2005;Taulet et al, 2009), we also observed that GM1 patching induced the co-patching of Ncadherin-GFP with GM1 in luciferase shRNA C2C12 myoblasts. In contrast, N-cadherin-GFP co-patching with GM1 was impaired in cells in which flotillin 1 was knocked down (Fig.…”

“…We previously demonstrated that N-cadherin association with cholesterol-rich membrane microdomains results in its association with p120 catenin and its stabilization at CCJs (Causeret et al, 2005;Taulet et al, 2009). We thus assessed whether flotillin 1 knockdown affected p120 catenin interaction with N-and E-cadherin and found that this was the case in both mesenchymal C2C12 myoblasts and epithelial MCF-7 and HCT-116 cells ( Fig.…”

Function of Flotillins in Receptor Tyrosine Kinase Signaling and Endocytosis: Role of Tyrosine Phosphorylation and Oligomerization

“…The complexing of the junctional proteins p120-catenin (p120ctn) and N-Cadherin at cell-cell contact sites was shown to occur in cholesterol rich membrane domains in mouse C2C12 cells (87). A similar observation was made in differentiated human colon adenocarcinoma HT-29 cells, in which the interaction of E-Cadherin with p120ctn preferentially takes place in lipid rafts.…”

Cadherin Point Mutations Alter Cell Sorting and Modulate GTPase Signaling

“…Because we compared proteins with the same overall backbone and cytoplasmic domain, this correlation might not apply for general comparisons across cadherin subtypes due to possible differences in their interactions with GTPases (Anastasiadis et al, 2000;Boulter et al, 2010). Ccadherin and E-cadherin activate Rac1 (Noren et al, 2003;Yap and Kovacs, 2003), but N-cadherin ligation triggers RhoA activation (Charrasse et al, 2002;Comunale et al, 2007;Marrs et al, 2009;Taulet et al, 2009). Whether the latter is due to low N-cadherin affinity and Rac1/RhoA antagonism (Boulter et al, 2010;Burridge and Doughman, 2006;Comunale et al, 2007;Wildenberg et al, 2006), or to differences in GTPase interactions with N-cadherin complexes (Anastasiadis et al, 2000) remains to be determined.…”