Here, we identified signaling downstream of Src that is responsible for induction of the “adhesion switch” phenotype we described previously in KM12C colon cancer cells (Avizienyte et al., 2002
). MEK/ERK and MLCK/myosin activities, which others have shown to be directly linked in a biochemical pathway needed for cell migration (Klemke et al., 1997
), are necessary for the changes that lead to enhanced integrin adhesion assembly and reduced cell-cell contact formation associated with loss of proper E-cadherin regulation (previously reported in Avizienyte et al., 2002
). We show that phospho-ERK and phospho-MLC, downstream readouts of MEK and MLCK activities, respectively, accumulate at newly forming integrin-dependent adhesions, whereas inhibitors of MEK or MLCK activities block the Src- or serum-induced formation of integrin-associated protrusive adhesion structures (Figures and ). Recent data showed strong and persistent activation of ERK and MLCK in extending cell pseudopodia, whereas inhibition of MEK or MLCK activities prevented pseudopodia extension, although not retraction (Brahmbhatt and Klemke, 2003
). More recently, it has been reported that Src and FAK signal through ERK and MLCK to induce focal adhesion turnover during cell migration, although how exactly increased contractility leads to dissociation of these structures is still unclear (Webb et al., 2004
). Our data suggest that generation of contractility at newly forming adhesion structures in epithelial cancer cells is required for extension of cellular protrusions. At the same time, E-cadherin localization to the cell membrane, is restored by preventing active Src from causing peripheral accumulation of phospho-myosin, and presumably from generating contractility there, as judged by loss of membrane-associated phospho-myosin staining when MEK/ERK, ROCK, or MLCK activities are inhibited (Figures , , ). Our results indicate that the Src SH3 and SH2 domains are required for the peripheral accumulation of phospho-myosin. This may reflect the fact that these Src domains are required to target Src to the periphery () and to localize Src kinase activity for the local activation of ERK, ROCK, and MLCK. Alternatively, or in addition, the Src SH2 and SH3 domains may recruit, either directly or indirectly, protein complexes that may cause localized contractile force generation at integrin adhesion sites, resulting in suppression of E-cadherin-mediated cell-cell contact formation. Although we have been able to detect a biochemical complex between endogenous Src and MLC in KM12C cells (our unpublished results), we have not established whether Src directly interacts with myosin and where this complex is localized in the cell. Nevertheless, the Src-induced epithelial to mesenchymal switch, which is associated with increased vimentin expression, is tightly linked to peripheral phosphomyosin activity.
The normal balance of integrin- and cadherin-mediated adhesion requires precise and dynamic regulation of the peripheral actin cytoskeleton. Therefore, it is likely that this is perturbed by Src-induced peripheral accumulation of phospho-MLC and enhanced acto-myosin contractility. Relieving contractility by inhibitors of MEK or MLCK may restore normal cytoskeletal remodeling, enabling E-cadherin–mediated adhesions to form in the presence of calcium.
Previous studies have reported that RhoA-stimulated contractility is required for the Ras-induced mesenchymal phenotype of MCF10A breast cancer cells (Zhong et al., 1997
). However, in these cells, blocking RhoA-induced contractility with C3 exotransferase or a dominant-inhibitory RhoA protein, suppresses focal adhesion organization but fails to restore normal cell-cell contacts (Zhong et al., 1997
). As RhoA activity is reported to be necessary for cadherin-dependent contacts to form between epithelial cells (Braga et al., 1997
; Takaishi et al., 1997
; Jou and Nelson, 1998
; reviewed in Fukata and Kaibuchi, 2001
), the findings that increased contractility is associated with oncogene-induced E-cadherin deregulation, in some cases mediated by RhoA activation, indicates that the role of RhoA in epithelial cell-cell contact dynamics is complex. In keeping with this, different RhoA effectors can contribute in opposing ways to cell-cell junctions. Specifically, the RhoA effector kinase ROCK and acto-myosin contractility disrupts junctions between a variety of tumorigenic and nontumorigenic epithelial cells, whereas signaling through Dia1 is linked to stabilization of adherens junction complexes (Sahai and Marshall, 2002
). Thus, the balance of signaling through these two RhoA effector pathways in a particular cellular context is likely to determine the net balance of cell-cell contact assembly and disassembly and may explain the apparently paradoxical positive and negative effects of RhoA. Our results show that ROCK activity is involved in accumulation of phosphorylated MLC at the cell periphery in active Src-expressing KM12C cells and that interfering with ROCK activity in these cells can restore E-cadherin–mediated cell-cell contacts.
Recent work examined the status of cellular RhoA activity upon induction of contact between epithelial cells either at high density or after plating Chinese hamster ovary cells expressing C-cadherin onto the extracellular domain of C-cadherin (Noren et al., 2001
). In both cases, substantial reduction of GTP-loading on RhoA was observed, leading Burridge and colleagues to propose that it may be necessary to keep cellular contractility low to avoid tension being applied to the newly formed cell-cell junctions (Noren et al., 2001
). Indeed, during the process of chicken embryo fibroblast spreading, the rate of spreading is inversely related to myosin activity (Wakatsuki et al., 2003
). It therefore appears that relaxation of contractile forces may be a common feature of the early stages of actin remodeling events that accompany formation of a number of adhesion types. We show here that suppressing peripheral accumulation of phospho-myosin, which normally occurs as a result of integrin engagement, promotes E-cadherin–associated contacts to form between KM12C colon cancer cells.
In conclusion, the Src SH3 and SH2 domains cooperate with MEK/ERK, MLCK and ROCK signaling to promote peripheral accumulation of phospho-myosin and to maintain a mesenchymal phenotype. When peripheral accumulation of phospho-myosin is blocked, E-cadherin can relocalize to membrane contact sites between cells and KM12C cells adopt an epithelial-like phenotype. Taken together, our data indicate that there is reciprocal, and interdependent, regulation of integrin- and cadherin-associated adhesions and that signals which regulate both adhesion types converge on a peripherally targeted pool of cellular phosphomyosin, presumably controlling localized contractility. This implicates spatially regulated contractile force as a critical determinant of epithelial cell plasticity, particularly in cells that can switch between epithelial and mesenchymal-like states.