The relationship between the cadherin-stabilization and Rho-regulatory functions of p120 remains unclear, as do the mechanisms regulating actomyosin contractility at the AJ. In this study, we demonstrate for the first time that ROCK1, a major RhoA effector, physically associates with the cadherin complex through p120. Our data favor a model whereby p120 localizes RhoA signaling to the cadherin complex, in part by recruiting upstream regulators (p190A; Wildenberg et al., 2006
) and downstream effectors (ROCK1).
Importantly, the association of ROCK1 with the cadherin complex is dependent on p120. ROCK1 specifically coimmunoprecipitates with wild-type E-cadherin, but not with p120-uncoupled 764AAA E-cadherin, from cross-linked A431D cell lysates. The failure to recruit ROCK1 to the cadherin complex likely contributes to the altered cytoskeletal organization previously reported in A431D cells expressing 764AAA E-cadherin (Thoreson et al., 2000
). The time course of ROCK1 recruitment to the AJ is consistent with establishment of a junctional actin network (Zhang et al., 2005
). Junctional actin is not necessary for the establishment of cell–cell contacts but is instead involved in stabilizing clustered cadherins. Interestingly, ROCK1 depletion does not eliminate cell–cell adhesion or affect cadherin stability but instead disrupts the normal organization of the cadherin complex and F-actin at cell–cell junctions. In the case of the 764AAA E-cadherin, p120 is not bound to the complex, and ROCK1 therefore does not associate with the cadherins and presumably is not properly localized to modulate junctional actin. While AJs are weakened following ROCK1 depletion, pharmaceutical inhibition of ROCK or myosin II dramatically disrupts cell–cell contacts (), which is consistent with several lines of evidence indicating a requirement for ROCK and myosin II activity in cadherin function (Shewan et al., 2005
). The more dramatic effect of pharmaceutical inhibition of ROCK or myosin II on cell–cell junctions, compared with ROCK1 depletion, suggests that either 1) ROCK2 contributes to cadherin function or 2) incomplete ROCK1 knockdown permits low levels of cell–cell adhesion. Depletion of ROCK2, however, has little or no detectable effect on cell–cell adhesion, indicating that, at least in A431 cells, ROCK1 is the primary isoform active at cell junctions. This conclusion is supported by the identification of ROCK1, but not ROCK2, in p120 ReCLIP experiments.
Recent studies utilizing human embryonic stem cells (hESCs) have implicated ROCK and myosin II activity in the regulation of AJs. In hESCs, inhibition or depletion of myosin IIA led to dramatic loss of E-cadherin due to down-regulation of p120 (Li et al., 2010
). Similarly, inhibition of either ROCK or myosin II activity disrupts cell–cell adhesion in both human and murine embryonic stem cells (Harb et al., 2008
). However, ROCK1 depletion of pharmacological inhibition of ROCK and myosin II had no effect on cadherin levels in our experiments. Thus it appears that inhibition of actomyosin contractility in A431 epidermoid carcinoma cells affects the distribution, rather than the stability, of the cadherin complex ().
More importantly, however, studies in hESC systems indicate a link between contractility, cadherins, and the capacity for self-renewal. Dissociation of hESCs causes ROCK-dependent anoikis (Watanabe et al., 2007
; Smutny et al., 2010
) due to excess actomyosin contractility (Chen et al., 2010
). Disruption of the AJs and subsequent dissociation of hESCs leads to an increase in RhoA activation and apoptosis (Ohgushi et al., 2010
), whereas inhibition of ROCK activity allows hESCs to maintain self-renewal capacity in the absence of other factors, including feeder cells, Matrigel, and even tissue culture–treated dishes (Harb et al., 2008
). In hESCs, E-cadherin promotes the expression of stem cell markers, suggesting that p120 functionally contributes to pluripotency through its obligatory role in stabilizing E-cadherin (Li et al., 2010
). Given our data, we suggest that p120 also contributes to these processes through regulation of cadherin-associated RhoA/ROCK1 signaling.
The relationship between cadherins, ROCK, and self-renewal is likely relevant to the tumor stem cell hypothesis, which suggests that tumors are maintained by subpopulations of “stem cells” with the capacity to self-renew and support tumor formation (Chaffer and Weinberg, 2011
). A role for p120 in tumor stem cell survival is supported by our previous studies in Madin-Darby canine kidney cells transformed by oncogenic Src or Rac1. While expression of these oncogenes permits anchorage-independent growth, a classical assay for tumorigenicity, p120 loss reverses this effect in a manner dependent on ROCK activity (Dohn et al., 2009
). In this case, the absence of p120 elevates ROCK activity, which apparently promotes anoikis of unanchored tumorigenic cells, a scenario analogous to that reported in hESCs (Ohgushi et al., 2010
A functional relationship between p120 and RhoA, upstream of ROCK1, has been identified in several earlier studies (Anastasiadis et al., 2000
; Wildenberg et al., 2006
; Castaño et al., 2007
; Yanagisawa et al., 2008
). In this study, we report that ROCK1 itself associates with p120, although it remains unclear whether the interaction is direct. Interestingly, we have also detected p190A in ReCLIP experiments from MCF-7 cells (Table S2), consistent with previous observations in NIH 3T3 cells (Wildenberg et al., 2006
). Thus, in addition to suppressing RhoA by various means, p120 recruits a major RhoA activator to modulate downstream signaling at the cadherin complex. Taken together, these data point to a dynamic RhoA complex with Rho effectors (ROCK1) and Rho suppressors (p190A and p120) forming a functional nexus of RhoA signaling. This allows rapid, localized cycling between activation and suppression of RhoA/ROCK signaling in response to specific cues, such as Rac1 activation (). Notably, a similar model of localized Rho regulation, involving a DDR1-Par3/Par6-p190A complex that associates with E-cadherin, has been proposed to regulate collective cell migration, a process that requires precisely localized regulation of contractility to enable motility of the group while maintaining individual cell–cell adhesions (Hidalgo-Carcedo et al., 2011
FIGURE 9: Dynamic regulation of RhoA activity at the cadherin complex. A schematic illustrating a possible mechanism of Rho regulation at the cadherin complex. p120 appears to recruit both ROCK1 and p190A RhoGAP to the cadherin complex and facilitate cycling between (more ...)
In conclusion, we have identified a novel p120-dependent interaction between ROCK1 and the cadherin complex. Our data further support a model in which Rho activity is dynamically regulated at the cadherin complex, which could facilitate a variety of processes (depending on cellular context), including cadherin clustering, collective cell migration, and self-renewal. Our findings suggest a more complex role for p120 in the control of RhoA signaling than previously appreciated, with p120 recruiting upstream regulators and downstream effectors of RhoA to the cadherin complex in order to focus the effects of RhoA signaling at cell–cell junctions.