Cortactin associates with N-cadherin adhesions and mediates intercellular adhesion strengthening in fibroblasts

Abstract

The regulation of N-cadherin-mediated intercellular adhesion strength in fibroblasts is poorly characterized; this is due, in part, to a lack of available quantitative models. We used a recombinant N-cadherin chimeric protein and a Rat 2 fibroblast, donor-acceptor cell model, to study the importance of cortical actin filaments and cortactin in the strengthening of N-cadherin adhesions. In wash-off assays, cytochalasin D (1 μM) reduced intercellular adhesion by threefold, confirming the importance of cortical actin filaments in strengthening of N-cadherin-mediated adhesions. Cortactin, an actin filament binding protein, spatially colocalized to, and directly associated with, nascent N-cadherin adhesion complexes. Transfection of Rat-2 cells with cortactin-specific, RNAi oligonucleotides reduced cortactin protein by 85% and intercellular adhesion by twofold compared with controls (P<0.005) using the donor-acceptor model. Cells with reduced cortactin exhibited threefold less N-cadherin-mediated intercellular adhesion strength compared with controls in wash-off assays using N-cadherin-coated beads. Immunoprecipitation and immunoblotting showed that N-cadherin-associated cortactin was phosphorylated on tyrosine residue 421 after intercellular adhesion. While tyrosine phosphorylation of cortactin was not required for recruitment to N-cadherin adhesions it was necessary for cadherin-mediated intercellular adhesion strength. Thus cortactin, and phosphorylation of its tyrosine residues, are important for N-cadherin-mediated intercellular adhesion strength.

Recruitment of Phosphoinositide 3-Kinase Defines a Positive Contribution of Tyrosine Kinase Signaling to E-cadherin Function

“…Second, cadherin recruitment of PI 3-kinase was also abolished in SYF-null cells lacking the ubiquitous Src family members, and recruitment was restored by expression of c-Src alone. This positive contribution of Src to cadherin signaling is consistent with the earlier demonstrations that Fyn was necessary for the efficient assembly of cell-cell junctions in keratinocytes (33), and Src inhibitors reduced N-cadherin adhesiveness (37). Furthermore, in other experimental systems, Src is often required for PI 3-kinase to interact with growth factor and cytokine receptors (6,7).…”

“…Several earlier reports also suggested that tyrosine kinases might contribute positively to cadherin function (37). Notably, Calautti et al (33) reported that tyrosine kinase signaling was necessary for differentiating keratinocytes to assemble cadherin-based cell-cell contacts.…”

Cortactin Is Required for N-cadherin Regulation of Kv1.5 Channel Function

“…Functionally, knockdown of cortactin ablated the regulation of Kv1.5 channel activity by N-cadherin in the oocyte expression system, strongly supporting the idea of an N-cadherin-cortical actin-cortactin-Kv1.5 axis. In nonmyocytes, cortactin has been shown to interact with the cadherin-catenin complex (51,52); however, we did not detect N-cadherin-cortactin association by co-immunoprecipitation in heart tissue. This result suggests that N-cadherin-cortactin association is cell type-dependent.…”

A Molecular Clutch between the Actin Flow and N-Cadherin Adhesions Drives Growth Cone Migration

“…Indeed, ␤-catenin is well known to physically link cadherin receptors to actin filaments through binding to ␣-catenin . Conversely, p120 catenin is known to modulate cadherin adhesiveness (Ozawa and Kemler, 1998;Yap et al, 1998;Thoumine et al, 2006) and was reported to bind cortactin (El Sayegh et al, 2004), providing a potential parallel pathway to connect N-cadherin to the actin network. However, the effects of Ncad⌬␤cat and NcadAAA were not completely specific of the Ncad-Fc substrate, as these mutants also reduced neurite extension on laminin-coated glass.…”