2006;25:8C19. complex through transient interaction with several of its up- and downstream effectors, including ROCK1. INTRODUCTION CellCcell adhesion is mediated, in part, by homophilic interactions between cadherins (e.g., E-cadherin) Anastrozole at the adherens junction (AJ) of adjacent cells. Cadherins are regulated by the catenins p120-catenin (hereafter p120), -catenin, and -catenin, which interact with the cytoplasmic domain of cadherins. p120 binds to the juxta-membrane domain of classical cadherins Anastrozole and stabilizes the cadherin complex at the plasma membrane (Thoreson 2011 ). ROCK1 recovery from this study is summarized in Table 1. Fifteen distinct peptides, covering multiple regions of ROCK1 were recovered, covering 12.8% of the Anastrozole total amino acid sequence (Figure 1A). No ROCK1 peptides were detected in the control pulldowns with an irrelevant immunoglobulin G (IgG). Sequence alignment analysis revealed that all but two peptides (mapped to the highly conserved kinase domain) were specific to ROCK1 (Table 1). Additionally, ROCK1 was detected in p120 ReCLIP samples from Caco-2 colorectal adenocarcinoma, MCF-7 mammary adenocarcinoma, and MCF-10A mammary epithelial cells, suggesting this interaction is relevant in several epithelial cell types (Supplemental Table S1). Open in a separate window FIGURE 1: Identification of ROCK1 as a p120 binding partner. (A) Distribution of ROCK1 peptides recovered from p120 ReCLIP eluates described previously (Smith test. p120 regulates ROCK1 localization A p120 knockdown-reconstitution system (Davis test and a cutoff of p 0.05; NS indicates no significant difference. (D to G) Effects of ROCK1 depletion. Immunofluorescence analysis of ROCK1 (red) and p120 (D), E-cadherin (E), ezrin (F), and F-actin (G) in wild-type A431 cells and A431 cells expressing ROCK1 shRNA. Where applicable, ROCK1 knockdown cells are outlined in white (F, G). Scale bars: 10 m. Strikingly, distribution of p120 and E-cadherin was dramatically altered in the ROCK1i cells, particularly at cellCcell junctions (Figure 7, D and E, respectively). As assessed by p120 and E-cadherin immunofluorescence, AJs were discontinuous, with multiple gaps between cells, compared with the tight, continuous AJs in wild-type cells. -catenin, -catenin, plakoglobin, and P-cadherin were similarly affected in ROCK1i cells (Figure S2), suggesting that ROCK1 depletion affects the entire cadherin complex. Membrane localization of ezrin was also disrupted (Figure 7F), indicating that ROCK activity was deficient in ROCK1i cells. Analysis of LAMA5 the actin cytoskeleton using phalloidin revealed that the junctional actin network of ROCK1i cells was disrupted (Figure 7G), which could account for the disruption of the cadherin complex. Similar disruption of p120 distribution was observed in ROCK1-depleted MCF-7 cells (Figure S3). These effects appear to be specific to ROCK1, as ROCK2 depletion does not affect p120, E-cadherin, ezrin, or actin staining (Figure S4). To determine whether the disorganization of the cadherin complex in ROCK1i cells was the result of disruption of the actomyosin pathway, we used chemical inhibitors to ablate ROCK and myosin II activity in wild-type A431. In parallel, we examined the effects of ROCK activation on the cadherin complex by treating cells with a RhoA activator. Cells were serum-starved and treated with dimethyl sulfoxide (DMSO) Anastrozole vehicle alone (Figure 8A), 5 M lysophosphatidic acid (LPA) (Figure 8C), 10 M Y-27632 (Figure 8D), or 10 M blebbistatin (Figure 8E) for 24 h, then processed for immunofluorescence analysis to detect p120 and ROCK1. For comparison, ROCK1i cells were also serum-starved and analyzed (Figure 8B). Activation of RhoA with LPA did not affect p120 or ROCK1 localization, but Y-27632 and blebbistatin resulted in large cells with disorganized AJs, as assessed by p120 staining. The effects of Y-27632 and blebbistatin treatments were similar to ROCK1 depletion, but both inhibitors caused a more dramatic disruption of overall cell morphology than ROCK1 depletion. Thus the effects of ROCK1 depletion are comparable to the effects of global ROCK inhibition. Furthermore, inhibition of myosin II produces the same phenotype as ROCK inhibition. These results suggest that Anastrozole disruption of ROCK-dependent actomyosin contractility is responsible for the cellCcell adhesion defects observed in ROCK1i cells. Open in a separate window FIGURE 8: Inhibition, but not activation, of ROCK activity mimics the effects of ROCK1 depletion. Immunofluorescence analysis p120 (green) and ROCK1 (red) of vehicle-treated wild-type A431 cells (A), A431 cells expressing ROCK1 shRNA, outlined in white (B), and wild-type A431 cells treated with 5 M LPA (C), 10 M Y-27632 (D), or 10 M blebbistatin (E) for 24 h. Scale bar: 10 m. DISCUSSION The relationship between the cadherin-stabilization and Rho-regulatory.