**3.2 Effectors of the Rho GTPases**

The Rho GTPases have been implicated in a wide varity of cellular processes, including cytoskeletal organization, cell adhesion to the substratum, cell polarity, and transcriptional activation. Several lines of evidence indicate that Rho GTPases link plasma membrane receptors to the assembly and organization of the actin cytoskeleton. Rho GTPases control individual aspects of the actin cytoskeleton through distinct effector proteins. In fact, over 60 targets of the three common Rho GTPases (Rho, Rac, Cdc42) have been found (Fig. 2).

Fig. 2. Regulators and mammalian targets of the Rho family GTPases.

Transmembrane receptors activate Rho GTPases through GEFs such as Tiam-1 or adaptor proteins. Activated Rho GTPases bind to and activate protein kinases, including these of the MRCK, PAK and ROCK families. The effector proteins then interact with several proteins with distinct effects on the actin cytoskeleton and cellular morphology. See text for details.

#### **3.2.1 Rho signaling**

562 Breast Cancer – Focusing Tumor Microenvironment, Stem Cells and Metastasis

GTP/GDP exchange ratio, but are released from the GDI and translocated to the membranes

The Rho GTPases have been implicated in a wide varity of cellular processes, including cytoskeletal organization, cell adhesion to the substratum, cell polarity, and transcriptional activation. Several lines of evidence indicate that Rho GTPases link plasma membrane receptors to the assembly and organization of the actin cytoskeleton. Rho GTPases control individual aspects of the actin cytoskeleton through distinct effector proteins. In fact, over 60 targets of the three common Rho GTPases (Rho, Rac, Cdc42) have

during the course of cell activation (Takai, Sasaki et al. 1995).

Fig. 2. Regulators and mammalian targets of the Rho family GTPases.

Transmembrane receptors activate Rho GTPases through GEFs such as Tiam-1 or adaptor proteins. Activated Rho GTPases bind to and activate protein kinases, including these of the MRCK, PAK and ROCK families. The effector proteins then interact with several proteins with distinct effects on the actin cytoskeleton and cellular morphology. See text for details.

**3.2 Effectors of the Rho GTPases** 

been found (Fig. 2).

Rho was originally studied for its role in regulate the formation of stress fibers and focal adhesion (FA) complexes (Nobes and Hall 1995) which precursors actomyosin assembly and contractile potential, both of which are required for the cellular movement. Rho is also involved in cell-cell adhesion. In particular, inactivation of RhoA by C3 transferase disrupts the organization of actin filaments at cell-cell contact, leading to the inhibition of the proper formation of both adherens junctions (AJs) and tight junctions (TJs) (Braga, Machesky et al. 1997; Takaishi, Sasaki et al. 1997). For example, in normal mammary epithelial cells, MCF10 cells, E-cadherin cytoskeletal links in AJs was disrupted by C3 transferase. In addition, inhibition of Rho blocks the formation of new AJs in MCF10 cells (Zhong, Kinch et al. 1997). It has been suggested that the function of Rho can be either promoted or antagonized by Rac and Cdc42, depending on different variables, such as cellular context, stimulus, and extracellular matrix (ECM) (Zhang, Nie et al. ; Narumiya and Morii 1993; Nobes and Hall 1995). In Swiss 3T3 fibroblasts, the Rho GTPases have been placed in a hierarchical order where Cdc42 activates Rac, and Rac activates Rho (Nobes and Hall 1995); however, in N1E-115 neuroblastoma and Madine-Darby canine kidney (MDCK) cells, constitutively activated Rac down-regulates Rho (Leeuwen, Kain et al. 1997; Michiels and Collard 1999).

Rho is widely studied for its involvement in the acquisition of migratory, invasive, and metastatic phenotypes. Expression of a dominant negative form of RhoA led to the attenuation of membrane ruffling, lamellipodia formation and migration (O'Connor, Nguyen et al. 2000). In addition, RhoA localization to lamellipodia was blocked by inhibiting phosphodiesterase activity while enhanced by inhibiting cAMP-dependent protein kinase activity (O'Connor, Nguyen et al. 2000). Furthermore, activation of Rho either by LPA treatment or by stimulating the actomyosin system has been associated with the migratory ability of tumor cells. For example, in an experimental metastasis model, NIH3T3 fibroblasts expressing a constitutively active form of RhoA were injected into the tail vein of nude mice and formed increased number metastasis nodules in the lung (del Peso, Hernandez-Alcoceba et al. 1997). Moreover, in the absence of serum, activated RhoA is capable of promoting invasion of cultured rat MM1 hepatoma cells through a mesothelial cell monolayer (Yoshioka, Matsumura et al. 1998). Although these are not oncogenes by themselves, RhoA and RhoC are frequently found to be overexpressed in clinical cancers (Sahai and Marshall 2002), and RhoC has been repeatedly associated with metastasis. For example, the expression of RhoA, RhoB and RhoC in 33 pancreatic ductal adnocarcinoma cases were examined in a study (Suwa, Ohshio et al. 1998), it was found that the expression level of RhoC was higher in tumors than in non-malignant tissues, higher in metastatic lesions than in primary tumors, and correlated with perineural invasion and lymph node metastasis as well as poorer prognosis. Although early studies showed that RhoB has a positive role in cell growth, more recent studies suggested that RhoB is down-regulated in human tumors, and its expression inversely correlates with tumor aggressiveness. For example, RhoB protein is found expressed in normal lung tissue and is lost progressively throughout lung cancer progression (Mazieres, Antonia et al. 2004). In line with this, higher expression of RhoB is associated with favorable prognosis in bladder cancer (Kamai, Tsujii et al. 2003). It has been suggested that RhoB can act as a tumor suppressor, since it is activated in response to several stress stimuli, such as DNA damage and hypoxia, inhibits tumor growth, cell migration, and invasion, and has proapoptotic functions in cells (Huang and Prendergast 2006).

Rho GTPases and Breast Cancer 565

In classical Swiss 3T3 fibroblast model, activation of Cdc42 leads to filopodia formation, Rac results in lamellipodia formation and membrane ruffling, and Rho results in stress fibers formation (Nobes and Hall 1995). The cytoskeletal rearrangements caused by Rho GTPases activation play a key role in cell motility. In addition to their effects on the actin cytoskeleton and motility, Rac and Cdc42 also play a role in cell-cell adhesion in epithelial cells. Expression of a constitutively active form of Rac in MDCK cells or keratinocytes leads to an increase in E-cadherin complex members and F-actin at cell-cell contacts, while a dominant negative mutant was found to disrupt cell-cell adhesions (Braga, Machesky et al. 1997; Takaishi, Sasaki et al. 1997; Jou and Nelson 1998). A number of studies have suggested that Cdc42 plays an important role in establishing the initial polarization of epithelial cells, which is required for the proper formation of cell-cell adhesions. For example, transfection of a dominant negative form of Cdc42 in MDCK cells results in the selective depolarization of basolateral membrane proteins due to inhibition of membrane transport (Kroschewski, Hall et al. 1999). Expression of a constitutively active form of Cdc42 in MDCK cells increased AJs and blocked cellular migration induced by HGF/SF (Kodama, Takaishi et al.

Given the importance of Rac and Cdc42 in the regulation of cell cytoskeletal, adhesion and motility, it has been widely considered that they play important roles in cellular processes related to invasion and metastasis. The first evidence of Rac's role in invasion was obtained when Rac-specific GEF T-lymphoma invasion and metastasis (Tiam-1) was identified in a retroviral insertional mutagenesis screen. Virus-infected T-lymphoma cells were repeatedly selected for *in vitro* invasion through a layer of fibroblasts and the proviral insertions in invasive clones were used to identify the Tiam-1 gene (Habets, Scholtes et al. 1994). Subsequently, Rac, and later Cdc42, were shown to also confer an invasive potential to these T-lymphoma cells (Michiels, Habets et al. 1995; Stam, Michiels et al. 1998). More evidence for Rac and Cdc42's involvement in invasion and metastasis has been provided since then. Expression of the laminin-receptor α6β4 integrin in the melanoma cell line MDA-MB-435 promotes invasiveness in a Rac and PI3-kinase-dependent manner (Shaw, Rabinovitz et al. 1997). In addition, constitutively active forms of Rac and Cdc42 in breast carcinoma cell line T47D promote invasion through a collagen matrix. However, this invasion can be blocked by PI3-Kinase inhibitors, indicating that PI3-kinase acts downstream of Rac and Cdc42

A number of Rac and Cdc42 effectors have been identified. Some of these have been found to specifically mediate cell motility, whereas others play a more prominent role in mediating cell adhesion. It is well established that WASP and MRCKs are Cdc42 specific effectors that regulate actin organization and filopodia formation which promotes a more motile phenotype (Aspenstrom, Lindberg et al. 1996; Miki, Miura et al. 1996). In addition, members of the p21-activated kinase family (PAK), downstream of Rac and Cdc42, play important roles in cytoskeletal-mediated changes that affect motility (Manser, Leung et al. 1994). The scalffold proteins IQGAP and Par-6, both of which can be activated by Cdc42 and rac,

The scaffold protein N-WASP binds to Arp2/3 complexes that are crucial for the assembly of within filopodia (Kolluri, Tolias et al. 1996). It has been shown that both N-WASP and Arp2/3 complexes are required for Cdc42 to trigger actin filament assembly (Welch, DePace

**3.2.3 Rac and Cdc42 signaling** 

(Keely, Westwick et al. 1997).

**3.2.4 Effectors of Rac and Cdc42** 

promote cell polarization and contribute to cell-cell adhesion.

1999).

#### **3.2.2 Effectors of Rho**

There are two major effectors that are downstream of Rho: Rho associated coiled-coil forming kinase (ROCK/Rho kinase/ROK) (Leung, Manser et al. 1995; Ishizaki, Maekawa et al. 1996) and mammalian homolog of Drosophila diaphanous (mDia) (Watanabe, Madaule et al. 1997; Wasserman 1998). While mDia is a formin molecule that can catalyze actin nucleation, polymerization,and produce long, straight actin filaments (Goode and Eck 2007), ROCK is a serine/threonine kinase that phosphorylates a number of substrates (Riento and Ridley 2003). The actions of ROCK and mDia on actin and myosin are believed to work together to induce actomyosin bundles in cells. Expression of an active form of mDia induces stress fibers in cultured cells, and treatment of these cells with a specific ROCK inhibitor, Y-27632 (Narumiya, Ishizaki et al. 2000), causes dissolution of the bundles, leaving the cells with diffusely distributed actin filaments (Watanabe, Kato et al. 1999). It has also been reported that ROCK and mDia are required in contractile ring formations (Kosako, Yoshida et al. 2000; Watanabe, Ando et al. 2008).

At least six substrates of ROCK are known to play roles in actin cytoskeletal reorganization, including myosin light chain (MLC), myosin-binding subunit of MLC phosphatase, LIMkinase, adducin, ezrin/radixin/moesin (ERM) family of proteins, and Na+/H+ exchange protein (NHE1). Among the six substrates, MLC-phosphatase, MLC, and LIM-kinase, are the three best studied ROCK effectors and have been found to play important roles in driving ROCK's physiological function on the actin cytoskeleton. ROCK inactivates myosinbinding subunit of MLC-phosphatase by phosphorylation (Kimura, Ito et al. 1996; Uehata, Ishizaki et al. 1997). ROCK is also able to phosphorylate myosin light chain directly (Maekawa, Ishizaki et al. 1999). These two actions of ROCK increase the myosin light chain phosphorylation, stimulate cross-linking of actin by myosin and enhance actomyosin contractility. ROCK also phosphorylates and activates LIM-kinase, which in turn phosphorylates and inactivates actin-depolymerizing and severing factor, cofilin (Amano, Ito et al. 1996). The later action of ROCK results in stabilization of existing actin filaments and increase in their content.

The ROCK effectors adducin and the ERM family of proteins regulate actin cytoskeleton in a more direct way. ROCK has been shown to phosphorylate adducin (Kimura, Fukata et al. 1998; Fukata, Oshiro et al. 1999), which, together with spectrin, is an important component of the cortical actin network underlying the plasma membrane (Gardner and Bennett 1987). ROCK-phosphorylated adducin interacts with filamentous-actin (F-actin), and its localization suggests a role in regulating cellular migration. In HGF/SF-stimulated MDCK cells, phosphoadducin localizes to membrane ruffles, and ROCK-phosphorylated adducin localizes to the leading edge of migrating NRK49F fibroblasts in wound healing assays (Fukata, Oshiro et al. 1999); while the introduction of nonphosphorylatable adducin into MDCK and NRK49F cells inhibited membrane ruffling and migration, as did a dominant negative ROCK mutant (Fukata, Oshiro et al. 1999). ROCK can also phosphorylate the ERM proteins that are important for linking actin filaments to the plasma membrane (Matsui, Maeda et al. 1998). Interestingly, it has been demonstrated that the TSC1 tumor suppressor hamartin regulates cell adhesion to cell substrates through the ERM family of actin-binding proteins and RhoA (Lamb, Roy et al. 2000). Finally, NHE1 is well known as a ubiquitous Na+/H+ exchange protein that enables stress fiber formation (Tominaga, Ishizaki et al. 1998).

There are two major effectors that are downstream of Rho: Rho associated coiled-coil forming kinase (ROCK/Rho kinase/ROK) (Leung, Manser et al. 1995; Ishizaki, Maekawa et al. 1996) and mammalian homolog of Drosophila diaphanous (mDia) (Watanabe, Madaule et al. 1997; Wasserman 1998). While mDia is a formin molecule that can catalyze actin nucleation, polymerization,and produce long, straight actin filaments (Goode and Eck 2007), ROCK is a serine/threonine kinase that phosphorylates a number of substrates (Riento and Ridley 2003). The actions of ROCK and mDia on actin and myosin are believed to work together to induce actomyosin bundles in cells. Expression of an active form of mDia induces stress fibers in cultured cells, and treatment of these cells with a specific ROCK inhibitor, Y-27632 (Narumiya, Ishizaki et al. 2000), causes dissolution of the bundles, leaving the cells with diffusely distributed actin filaments (Watanabe, Kato et al. 1999). It has also been reported that ROCK and mDia are required in contractile ring formations (Kosako,

At least six substrates of ROCK are known to play roles in actin cytoskeletal reorganization, including myosin light chain (MLC), myosin-binding subunit of MLC phosphatase, LIMkinase, adducin, ezrin/radixin/moesin (ERM) family of proteins, and Na+/H+ exchange protein (NHE1). Among the six substrates, MLC-phosphatase, MLC, and LIM-kinase, are the three best studied ROCK effectors and have been found to play important roles in driving ROCK's physiological function on the actin cytoskeleton. ROCK inactivates myosinbinding subunit of MLC-phosphatase by phosphorylation (Kimura, Ito et al. 1996; Uehata, Ishizaki et al. 1997). ROCK is also able to phosphorylate myosin light chain directly (Maekawa, Ishizaki et al. 1999). These two actions of ROCK increase the myosin light chain phosphorylation, stimulate cross-linking of actin by myosin and enhance actomyosin contractility. ROCK also phosphorylates and activates LIM-kinase, which in turn phosphorylates and inactivates actin-depolymerizing and severing factor, cofilin (Amano, Ito et al. 1996). The later action of ROCK results in stabilization of existing actin filaments

The ROCK effectors adducin and the ERM family of proteins regulate actin cytoskeleton in a more direct way. ROCK has been shown to phosphorylate adducin (Kimura, Fukata et al. 1998; Fukata, Oshiro et al. 1999), which, together with spectrin, is an important component of the cortical actin network underlying the plasma membrane (Gardner and Bennett 1987). ROCK-phosphorylated adducin interacts with filamentous-actin (F-actin), and its localization suggests a role in regulating cellular migration. In HGF/SF-stimulated MDCK cells, phosphoadducin localizes to membrane ruffles, and ROCK-phosphorylated adducin localizes to the leading edge of migrating NRK49F fibroblasts in wound healing assays (Fukata, Oshiro et al. 1999); while the introduction of nonphosphorylatable adducin into MDCK and NRK49F cells inhibited membrane ruffling and migration, as did a dominant negative ROCK mutant (Fukata, Oshiro et al. 1999). ROCK can also phosphorylate the ERM proteins that are important for linking actin filaments to the plasma membrane (Matsui, Maeda et al. 1998). Interestingly, it has been demonstrated that the TSC1 tumor suppressor hamartin regulates cell adhesion to cell substrates through the ERM family of actin-binding proteins and RhoA (Lamb, Roy et al. 2000). Finally, NHE1 is well known as a ubiquitous Na+/H+ exchange protein that enables stress

**3.2.2 Effectors of Rho** 

Yoshida et al. 2000; Watanabe, Ando et al. 2008).

fiber formation (Tominaga, Ishizaki et al. 1998).

and increase in their content.

#### **3.2.3 Rac and Cdc42 signaling**

In classical Swiss 3T3 fibroblast model, activation of Cdc42 leads to filopodia formation, Rac results in lamellipodia formation and membrane ruffling, and Rho results in stress fibers formation (Nobes and Hall 1995). The cytoskeletal rearrangements caused by Rho GTPases activation play a key role in cell motility. In addition to their effects on the actin cytoskeleton and motility, Rac and Cdc42 also play a role in cell-cell adhesion in epithelial cells. Expression of a constitutively active form of Rac in MDCK cells or keratinocytes leads to an increase in E-cadherin complex members and F-actin at cell-cell contacts, while a dominant negative mutant was found to disrupt cell-cell adhesions (Braga, Machesky et al. 1997; Takaishi, Sasaki et al. 1997; Jou and Nelson 1998). A number of studies have suggested that Cdc42 plays an important role in establishing the initial polarization of epithelial cells, which is required for the proper formation of cell-cell adhesions. For example, transfection of a dominant negative form of Cdc42 in MDCK cells results in the selective depolarization of basolateral membrane proteins due to inhibition of membrane transport (Kroschewski, Hall et al. 1999). Expression of a constitutively active form of Cdc42 in MDCK cells increased AJs and blocked cellular migration induced by HGF/SF (Kodama, Takaishi et al. 1999).

Given the importance of Rac and Cdc42 in the regulation of cell cytoskeletal, adhesion and motility, it has been widely considered that they play important roles in cellular processes related to invasion and metastasis. The first evidence of Rac's role in invasion was obtained when Rac-specific GEF T-lymphoma invasion and metastasis (Tiam-1) was identified in a retroviral insertional mutagenesis screen. Virus-infected T-lymphoma cells were repeatedly selected for *in vitro* invasion through a layer of fibroblasts and the proviral insertions in invasive clones were used to identify the Tiam-1 gene (Habets, Scholtes et al. 1994). Subsequently, Rac, and later Cdc42, were shown to also confer an invasive potential to these T-lymphoma cells (Michiels, Habets et al. 1995; Stam, Michiels et al. 1998). More evidence for Rac and Cdc42's involvement in invasion and metastasis has been provided since then. Expression of the laminin-receptor α6β4 integrin in the melanoma cell line MDA-MB-435 promotes invasiveness in a Rac and PI3-kinase-dependent manner (Shaw, Rabinovitz et al. 1997). In addition, constitutively active forms of Rac and Cdc42 in breast carcinoma cell line T47D promote invasion through a collagen matrix. However, this invasion can be blocked by PI3-Kinase inhibitors, indicating that PI3-kinase acts downstream of Rac and Cdc42 (Keely, Westwick et al. 1997).

#### **3.2.4 Effectors of Rac and Cdc42**

A number of Rac and Cdc42 effectors have been identified. Some of these have been found to specifically mediate cell motility, whereas others play a more prominent role in mediating cell adhesion. It is well established that WASP and MRCKs are Cdc42 specific effectors that regulate actin organization and filopodia formation which promotes a more motile phenotype (Aspenstrom, Lindberg et al. 1996; Miki, Miura et al. 1996). In addition, members of the p21-activated kinase family (PAK), downstream of Rac and Cdc42, play important roles in cytoskeletal-mediated changes that affect motility (Manser, Leung et al. 1994). The scalffold proteins IQGAP and Par-6, both of which can be activated by Cdc42 and rac, promote cell polarization and contribute to cell-cell adhesion.

The scaffold protein N-WASP binds to Arp2/3 complexes that are crucial for the assembly of within filopodia (Kolluri, Tolias et al. 1996). It has been shown that both N-WASP and Arp2/3 complexes are required for Cdc42 to trigger actin filament assembly (Welch, DePace

Rho GTPases and Breast Cancer 567

analysis has shown that MCF10A breast cells stably transfected with wild type RhoC or a constitutively active mutant of RhoC overexpress genes associated with invasion and metastasis (Wu, Wu et al. 2004). Other RhoGTPases are also involved in breast tumors. RhoA is found overexpressed in breast tumor tissues but not in the normal tissue (Fritz, Brachetti et al. 2002). The expression of dominant negative RhoA in rat mammary adenocarcinoma cells affects tumor cell growth *in vivo* and reduces intravasation into the peripheral blood, resulting in decrease in lung colonization ability (Fritz, Just et al. 1999). Other studies have indirectly shown an important role of RhoA in breast carcinogenesis. For example, highly metastatic MDA-MB-231 cells that were treated with HMG-CoA reductase inhibitor, namely cerivastain, showed reduced proliferation and invasion through Matrigel, in a RhoA- but not Ras-dependent manner (Denoyelle, Vasse et al. 2001). However, poorly metastatic breast cancer cells such as MCF-7 are less sensitive to cerivastain treatment, indicating that RhoA might be more significantly overexpressed in late stages of breast

Rho proteins are also important players in breast tumor progression and metastasis exerted by the CD44 hyaluronan receptor (Bourguignon 2001). CD44 is expressed in human breast tumors and promotes cell growth and metastasis in tumor cells. Studies have found that RhoA and CD44 directly interact with each other *in vivo* in highly metastatic human breast cancer cell lines. Accordingly, inhibition of Rho signaling results in the abrogation of the metastatic phenotype elicited by CD44 (Bourguignon, Zhu et al. 1999). RhoA has also been found to be involved in insulin signaling via Shc in human breast cancer (Finlayson, Chappell et al. 2003). Overexpression of insulin receptors correlates with development, progression and outcome of breast cancer, and insulin signaling involves hyperphosphorylation of Shc. Hence, Shc leads to the activation of geranyl transferases, which results in an increased amount of prenylated RhoA in the tumor tissue compared with normal mammary tissue (Finlayson, Chappell et al. 2003). Furthermore, RhoA has been reported to increase the metastatic potential of tumor cells via its ability to promote tumor angiogenesis through the downregulation of thrombospodin-1 (Tsp-1) (Watnick, Cheng et al. 2003). Rho pathway is part of the downstream signaling cascade that is activated by PI3K

and leads to ROCK stimulation, Myc phosphorylation and Tsp-1 repression.

The involvement of Rac GTPases in breast cancer was first reported in rodents (Bouzahzah, Albanese et al. 2001). Expression of a dominant negative Rac1 mutant indicated that Rac1 affects tumor cell growth and metastasis *in vivo*. Deregulation of Rac3, closely related to Rac1, has also been detected in breast cancer (Mira, Benard et al. 2000). Rac3 maps to chromosome band 17q25.3, a region known to contain candidate tumor suppressor genes both in breast and ovarian cancers (Morris, Haataja et al. 2000). Highly proliferative breast cancer cells, T47D and MCF-7, but not normal breast cell lines, contain constitutively active Rac3 in a Ras-independent manner (Morris, Haataja et al. 2000). It has also been shown that expression of a dominant negative mutant Rac3 (N17) leads to inhibition of S-phase entry and cellular proliferation in breast tumor cells, which indicate that Rac3 may promote cell growth (Leung, Nagy et al. 2003). Further, the Rac-PAK signaling pathway is essential for receptor tyrosine kinase ErbB2-mediated transformation of human breast epithelial cancer cells (Mazieres, Antonia et al. 2004). Activation of Rac-PAK1 pathway by ErbB2 homodimers can induce growth factor-independent proliferation and promote disruptions to the three-dimensional (3D) mammary acinar-like structures, via activation of the Erk and

cancer as with other tumors.

**4.2 Rac GTPases in breast tumors** 

et al. 1997; Miki, Sasaki et al. 1998). Therefore, N-WASP may promote cellular motility through proper filopodia formation. MRCKs α and β are Cdc42 specific effectors that can phosphorylate MLC via a ROCK-like kinase domain (Leung, Chen et al. 1998). It is well accepted that phosphorylation of MLC is required for actomyosin complex assembly and contraction. Overexpression of MRCKα and Cdc42 synergizes to promote filopodia formation, while a MRCKα kinase-deficient mutant inhibits the formation of Cdc42-induced filopodia (Leung, Chen et al. 1998). Therefore, MRCDs are believed to play important roles in cytoskeletal organization and contraction, and contribute to migration. PAK, a protein kinase downstream of Rac and Cdc42, plays a crucial role in actin dynamics and adhesion (Manser, Leung et al. 1994). PAK has been demonstrated to phosphorylate and inactivate MLCK, subsequently causing a decrease in MLC phosphorylation (Sanders, Matsumura et al. 1999). Thus, inactivation of MLCK leads to stress fiber and focal adhesion disassembly. Moreover, PAK controls the actin cytoskeletion through the phosphorylation and subsequent activation of LIM-kinase. Phosphor-LIM-kinsae can further phosphorylate and inactivate the actin-depolymerizing protein cofilin, thus inhibiting actin depolymerization when Rac is activated and causing extreme membrane ruffling (Arber, Barbayannis et al. 1998; Yang, Higuchi et al. 1998). The IQGAP1 and IQGAP2 scaffolding effectors of Cdc42 and Rac regulate cell-cell adhesion through actin polymerization and sequestration of βcatenin (Kuroda, Fukata et al. 1996; Erickson, Cerione et al. 1997). *In vitro*, IQGAP oligomerizes and cross-links F-catin it has also been found to complex with Cdc42 and Factin *in vivo* (Fukata, Kuroda et al. 1997). In addition, one study has shown that the IQGAP protein also competes with α-catenin for binding to β-catenin, thus preventing Ecadherin/α-catenin/β-catenin complex from attaching to the actin cytoskeleton, and thereby disrupting cell-cell contacts (Erickson, Cerione et al. 1997). Another scaffolding protein, Par-6, was identified using activated Cdc42 and TC10 mutants as baits in yeast two-hybrid screens (Joberty, Petersen et al. 2000; Qiu, Abo et al. 2000). It is known that Par-6 binds to a second scaffolding protein, Par-3, and both Par-6 and Par-3 bind independently to atypical protein kinase C (aPKC) isioforms (Lin, Edwards et al. 2000). In addition, endogenous Par-3 localizes to TJs in MDCK cells, overexpression of Par-6 or the N-terminal portion of Par-3 (the Par-6-interaction responsible region) disrupts TJ formation (Joberty, Petersen et al. 2000).
