- Email: [email protected]
Regulation of cadherin-mediated cell–cell adhesion by the Rho family GTPases
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Regulation of cadherin-mediated cell–cell adhesion by the Rho family GTPases Kozo Kaibuchi*, Shinya Kuroda, Masaki Fukata and Masato Nakagawa Reports in the past two years have shown that Cdc42, Rac1, and Rho — belonging to the Rho small GTPase family — participate in the regulation of cadherin-mediated cell–cell adhesion. IQGAP1, an effector of Cdc42 and Rac1, interacts with cadherin and β-catenin and induces the dissociation of α-catenin from the cadherin–catenins complex leading to disruption of cell–cell adhesion: activated Cdc42 and Rac1 counteract the effect of IQGAP1. Thus, Cdc42 and Rac1 appear to regulate cadherinmediated cell–cell adhesion acting through IQGAP1. Addresses Division of Signal Transduction, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma 630-0101, Japan *e-mail: [email protected] Current Opinion in Cell Biology 1999, 11:591–596 0955-0674/99/$ — see front matter © 1999 Elsevier Science Ltd. All rights reserved. Abbreviations EL cells GAP GDI GEF HGF MDCK cells PAK
E-cadherin expressing L cells GTPase-activating protein GDP-dissociation inhibitor GDP/GTP exchange factor hepatocyte growth factor Madin-Darby canine kidney cells p21-activated kinases
dynamically remodelled; however, the mechanism underlying the dynamic rearrangement of cell–cell adhesion remains to be clarified. The Rho small GTPase family (including Rho, Rac, and Cdc42) participates in the regulation of the actin cytoskeleton and various cell adhesion events [7,8•]. Rho has been implicated in the formation of actin stress fibres and focal adhesions [9], cell morphology [10], cell aggregation [11], cell–cell adhesion [12,13], cell motility [14], membrane ruffling [15], smooth muscle contraction [16,17], neurite retraction in neuronal cells [18,19], and cytokinesis [20,21]. Rac is involved in membrane ruffling [22], cell motility [23], actin polymerisation [24], and cell–cell adhesion [12,13,25]. Cdc42 participates in filopodia formation [26,27] and cell–cell adhesions [25]. The molecular mechanisms underlying the above processes were largely unknown, until recently. Some of the molecular pathways that connect the Rho family GTPases to control of the cytoskeleton and cell adhesion have been established. The major emphasis in this review is on how the Rho family GTPases regulate cell–cell adhesion. The above functions regulated by the Rho family GTPases are not described here in detail: for such information, the reader is referred to other reviews [7,8•].
The Rho family GTPases Introduction Cell–cell adhesions are mediated by adhesion molecules, such as cadherins and their associated cytoplasmic proteins, α-catenin and β-catenin [1–3]. E-cadherin, a classic cadherin, is a Ca2+-dependent homophilic cell–cell adhesion molecule [1–3]. The cytoplasmic domain of E-cadherin interacts with β-catenin or plakoglobin (also called γ-catenin) [4,5]. Plakoglobin and β-catenin also interact with α-catenin, which has been thought to directly or indirectly link the complex composed of cadherin, β-catenin and α-catenin (cadherin–catenins complex) to the actin cytoskeleton. Strong adhesion requires linkage of cadherin to the actin cytoskeleton via catenins [5,6]. Cell–cell adhesion seems to be a static process but dynamic rearrangements of cell–cell adhesion should occur during various cellular processes, such as epithelial cell scattering, dispersal of cancer cells, and early embryonic cell migration [6]. Indeed, certain cell lines including Madin-Darby canine kidney (MDCK) cells rapidly migrate even in confluent conditions, indicating that the cell–cell adhesion process is transiently perturbed in migrating areas, enabling dynamic migration (M Fukata, S Kuroda, M Nagawa, K Kaibuchi, unpublished data). The cell–cell adhesion appears to be constantly rearranged in real time, suggesting that the cadherin–catenins complex is
The Rho family GTPases exhibit both GDP/GTP-binding and GTPase activities. They have two interconvertible forms; GDP-bound inactive and GTP-bound active forms [28,29]. The conversion from the GDP-bound to GTPbound form is stimulated by GDP/GTP exchange factor (GEF) and inhibited by GDP-dissociation inhibitor (GDI) [29,30]. The conversion from the GTP-bound to GDPbound form is catalysed by GTPase-activating protein (GAP) [29,30]. On the basis of recent studies, the activity of the Rho family GTPases appears to be regulated cyclically, as follows (Figure 1). In the cytosol of resting cells, the Rho family GTPases are present in the GDP-bound form complexed with Rho GDI. When cells are stimulated by certain extracellular signals, Rho GDI dissociates, specific GEFs for the Rho family GTPases are activated and the GTPases are targeted to cell membranes by their carboxy-terminal prenyl group [29,31,32]. The GDP-bound form of the Rho family GTPases is then converted to the GTP-bound form. The GTP-bound form, attached to membranes, interacts with specific effectors to exert specific functions. GAPs stimulate the GTPase activity of the Rho family GTPases and reconvert them to the inactive GDP-bound form. Rho GDI can then form a complex with the GDP-bound form and extract it from the membrane back into the cytosol.
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Figure 1
Plasma membrane GTP•
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Mode of activation of Rho family GTPases. When Rho GDI is dissociated from Rho family GTPases in a certain manner, Rho GEF, which is thought to be activated by Gα13 or tyrosine phosphorylation, appears to stimulate the dissociation of GDP from the Rho family GTPases and thereby stimulate the association of GTP with the Rho family GTPases. The GTP-bound form attached to membranes through its carboxy-terminal prenyl group, interacts with its specific targets. Rho GAP acts as a negative regulator by enhancing the GTPase activity of the Rho family GTPases and reconverting them to the inactive GDP-bound form. Rho GDI can then form a complex with the GDP-bound form and extract it from the membrane back into the cytosol. Rho, Rho family GTPases; GEF, GDP/GTP exchange factor; GDI, GDP dissociation inhibitor; GAP, GTPase activating protein.
The mode of action of the Rho family GTPases has been well studied, and many effector molecules for these small GTPases have been identified [7,33•]. A number of proteins have been identified as effectors for Rho. These include Rho-kinase (ROK/ROCK), the myosin-binding subunit (MBS) of myosin phosphatase, protein kinase N (PKN)/PRK1, rhophilin, rhotekin, citron, and p140 mDia. A number of proteins have been identified as effectors for Rac including p21-activated kinases (PAKs), IQGAP1, Por1, p140Sra-1 and POSH, and as effectors for Cdc42 including PAKs, WASP/N-WASP, IQGAP1 and MRCK. Several of these proteins such as PAKs and IQGAP1 interact with both Rac and Cdc42.
Roles of the Rho family GTPases in the regulation of cadherin activity Recent work has suggested that the Rho family GTPases including Cdc42, Rac1, and Rho are required for cadherinmediated cell–cell adhesion [12,13,25,34••,35•,36•]. It has been shown that microinjection of dominant-negative Rac1Asn17, which preferentially binds GDP rather than GTP and thereby inhibits the activation of endogenous Rac1 by titrating out its GEF [22,30], or of botulinum toxin C3, which inactivates Rho by ADP-ribosylation [37], reduces the accumulation of cadherin at cell–cell contact sites when keratinocytes are transferred from low calcium media to standard media to induce calcium-dependent cell–cell adhesion [12].
The effects of Rac1 and Rho on localisation of cadherin are probably dependent on the cell types [35•]; Rac1 and Rho are required for the localisation of E-cadherin at cell–cell contact sites in keratinocytes, whereas they are not required for the VE-cadherin localisation in human umbilical cord endothelial cells. It has also been reported that in MDCK cells stably expressing dominant-active Rac1Val12 — which is thought to be the constitutively GTP-bound active form in the cells [22] — immunofluorescent intensities of E-cadherin, β-catenin, and actin filaments at cell–cell contact sites increased more than those in the parental cells [13]. In the MDCK cells expressing Rac1Asn17, those of Ecadherin, β-catenin and actin filament decreased. We have found that Cdc42 as well as Rac1 is required for the regulation of cell–cell adhesion in MDCK cells [25]. Microinjection of Rho GDI, a negative regulator for Cdc42, Rac1, and Rho, results in the perturbation of cell–cell adhesion in MDCK cells. Co-injection of either Cdc42Val12 or Rac1Val12 with Rho GDI reverses the inhibitory action of Rho GDI, whereas that of RhoVal14 with Rho GDI does not. In addition, it has also been shown that Tiam1, one of the GEFs for Rac1, is involved in the regulation of cell–cell adhesion in MDCK cells [38]. Expression of Tiam1 or Rac1Val12 inhibits hepatocyte growth factor (HGF)-induced scattering of MDCK cells probably by increasing E-cadherin-mediated cell–cell adhesion. These observations have led to the suggestion that Cdc42, Rac1, and Rho as well as Tiam1 regulate cadherin-mediated cell–cell adhesion either directly or indirectly. Direct evidence has not yet been presented, however, that the Rho family GTPases regulate cadherin activity. To provide such evidence, we used L cells expressing E-cadherin (EL cells) and L cells expressing an E-cadherin mutant in which the cytoplasmic domain was deleted and replaced by the carboxy-terminal domain of α-catenin (nEαCL cells) [39]. Both cell lines display cell–cell adhesive activity in an E-cadherin-dependent manner [39]. We have recently shown that, using the dissociation assay, a quantitative assay for E-cadherin activity [39], expression of Cdc42Asn17 or Rac1Asn17 in EL cells markedly reduces the E-cadherin activity, whereas expression of RhoAsn19 slightly reduces it [36•]. In contrast, expression of Cdc42Asn17, Rac1Asn17, or RhoAAsn19, in nEαCL cells slightly reduces the mutant Ecadherin activity. This result strongly suggests that Cdc42 and Rac1 directly regulate the E-cadherin activity, whereas RhoA indirectly regulates it possibly through the rearrangement of the actin cytoskeleton. Consistent with this, treatment with cytochalasin D, which prevents actin polymerisation and thereby disrupts the linkage of cadherin to actin cytoskeleton, reduces the E-cadherin activity in both EL and nEαCL cells [40,41].
Mode of action of the Rho family GTPases in the regulation of cadherin activity The next question is how Cdc42 and Rac1 regulate E-cadherin activity. Among the effectors of Cdc42 and Rac1,
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interacting with β-catenin, and thereby stabilises the cadherin–catenins complex.
IQGAP1 has been shown to accumulate at the cell–cell contact sites [34••,42,43]. This raises the possibility that IQGAP1 regulates the E-cadherin activity downstream of Cdc42 and Rac1. IQGAP1 accumulates at cell–cell contact sites in EL cells, but not in nEαCL cells, indicating that this accumulation is dependent on E-cadherin and β-catenin [34••]. A recent study has shown that IQGAP1 directly interacts with β-catenin and the cytoplasmic domain of E-cadherin both in vitro and in vivo [34••]. IQGAP1 interacts with the amino-terminal end of β-catenin (residues 1–183) [36•], which overlaps with the α-catenin-binding domain (residues 120–151) [44]. IQGAP1 inhibits the binding of α-catenin to β-catenin, and causes α-catenin to dissociate from its complex with β-catenin in vitro [36•]. In addition, overexpression of IQGAP1 in EL cells results in the dissociation of αcatenin from the cadherin–catenins complex and in concomitant reduction of E-cadherin activity [34••]. The overexpression of IQGAP1 in nEαCL cells does not affect the mutant E-cadherin activity. Thus, IQGAP1 appears to regulate E-cadherin activity in vivo at least through the dissociation of α-catenin from the cadherin–catenins complex [34••].
On the basis of these observations, we propose a mechanism by which Cdc42 and Rac1 together with IQGAP1 regulate the cadherin-mediated cell–cell adhesion (Figure 2). When Cdc42 and Rac1 are in the GTP-bound active forms at cell–cell contact sites, Cdc42 and Rac1 interact with IQGAP1 and thereby prohibit IQGAP1 from interacting with β-catenin. This results in the stabilisation of the cadherin–catenins complex. When Cdc42 and Rac1 are in the GDP-bound inactive forms, Cdc42 and Rac1 cannot interact with IQGAP1, and IQGAP1 interacts with β-catenin, thereby dissociating α-catenin from the cadherin–catenins complex. This state confers relatively weak adhesive activity, which is mediated by E-cadherin unlinked to the actin cytoskeleton. Thus, Cdc42 and Rac1 positively regulate cadherin-mediated cell–cell adhesion by the suppression of the activity of IQGAP1 to perturb the cadherin–catenins complex. This state confers relatively strong adhesive activity, which is mediated by E-cadherin linked to the actin cytoskeleton. Thus, Cdc42 and Rac1, and IQGAP1 can serve as positive and negative molecular switches of the cadherin activity, respectively. Following this proposal, it can be speculated that cell–cell contact induces the activation of the Rho family GTPases. In this regard, it has been shown that cell to substratum contact, which is mediated via integrins, induces the activation of the Rho family GTPases including Rho, Cdc42, and Rac1 [45•,46•], and that E-cadherin interacts with certain types of integrins at cell–cell contact sites [47]. Like that of growth factor receptors, engagement of E-cadherin results in the tyrosine phosphorylation of certain proteins [48] and recruitment of adapter proteins such as Shc to Ecadherin [49]. Hence, it may prove worthwhile to examine
Do Cdc42 and Rac1 regulate the IQGAP1 activity on E-cadherin–catenins complex? Cdc42 and Rac1 bound to the nonhydrolysable GTP analogue GTPγS inhibit the interaction of IQGAP1 with β-catenin, whereas their GDPbound forms or GTPγS·RhoA do not inhibit in vitro [36•]. Co-expression of Cdc42Val12 with IQGAP1 in EL cells inhibits the dissociation of α-catenin from the cadherin–catenins complex induced by IQGAP1 [36•] and concomitantly restores the E-cadherin activity [34••]. Thus, this result indicates that activated Cdc42 suppresses the inhibitory action of IQGAP1 by preventing IQGAP1 from Figure 2 Role of IQGAP1 in the regulation of E-cadherin-mediated cell–cell adhesion. When Cdc42 and Rac1 are in the GDP-bound inactive form, Cdc42 and Rac1 cannot interact with IQGAP1, and IQGAP1 interacts with β-caterin thereby dissociating α-catenin from the cadherin–catenins complex. This state confers the weak adhesive activity. In contrast, when Cdc42 and Rac1 are in the GTP-bound active form at sites of cell–cell contact, Cdc42 and Rac1 interact with IQGAP1, and thereby, prohibit IQGAP1 from interacting with β-catenin. This results in the stabilization of the cadherin–catenins complex. Thus, Cdc42 and Rac1 positively regulate cadherin-mediated cell–cell adhesion by the suppression of the activity of IQGAP1 to perturb the cadherin–catenins complex. This state confers the strong adhesive activity. Thus, Cdc42 and Rac1, and IQGAP1 can serve as positive and negative molecular switches of the cadherin activity, respectively. E, E-cadherin; α, α-catenin; β, β-catenin.
Weak adhesion
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GDP• Cdc42/Rac1 IQGAP1
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Actin Current Opinion in Cell Biology
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whether cell–cell contact induces the activation of the Rho family GTPases. One may ask why IQGAP1 is colocalised with E-cadherin and β-catenin at cell–cell contact sites, even though it inhibits cell–cell adhesion. This can be explained by the notion that E-cadherin stays as a complex with either α-catenin and βcatenin or β-catenin and IQGAP1 at cell–cell contact sites. There should be a mixture of E-cadherin/α-catenin/β-catenin complex and E-cadherin/β-catenin/IQGAP1 complex. The ratio between the two may determine the adhesive activity. In this regard, it should be noted that about one half of E-cadherin appears to be connected to the actin cytoskeleton probably via α-catenin in EL cells, whereas the rest appears to be free from the actin cytoskeleton [50•].
Other mechanisms that account for the regulation of cadherin activity Numerous attempts have been made to clarify the regulation of the cadherin function. One of the most intriguing phenomena proposed is tyrosine phosphorylation of β-catenin. Increased tyrosine phosphorylation of β-catenin appears to correlate with dysfunction of cadherin-mediated cell–cell adhesion, induced by the expression of v-Src [51,52] or treatment with growth factors, such as epidermal growth factor or hepatocyte growth factor (HGF) [53]. It has been shown that treatment of cells with pervanadate, a potent inhibitor of tyrosine phosphatases, induces perturbation of the cadherin-mediated cell–cell adhesion [54•]. In accordance with dysfunction of cadherin-mediated cell–cell adhesion, tyrosine phosphorylation of β-catenin and dissociation of α-catenin from the cadherin–catenins complex occur [54•]. Therefore, the tyrosine phosphorylation of β-catenin was thought to be important for the regulation of the cadherin function. On the basis of these findings together with our model, it is tempting to speculate that tyrosine-phosphorylated β-catenin recruits IQGAP1 or GAPs for Cdc42 and/or Rac1 to the cell–cell contact sites, resulting in the reduction of cadherin activity through the inhibition of Cdc42 and/or Rac1 activity. It has been reported, however, that the tyrosine phosphorylation of β-catenin is not required for the reduction of cadherin activity induced by v-Src, as the expression of v-Src reduces the cadherin function of nEαCL cells as well as EL cells [55]. Thus, the physiological role of the tyrosine phosphorylation of β-catenin in cadherin function remains to be clarified.
Physiological processes in which the Rho family GTPases regulate cadherin activity It remains to be determined in which physiological processes the above Cdc42/Rac1/IQGAP1 system operates. Dynamic rearrangement of cell–cell adhesion should occur in various situations [2,56]. Cell scattering provides one of the most prominent examples of dynamic rearrangement of cell–cell adhesion [2,56]. Scatter factor/HGF and phorbol ester stimulate the motility of epithelial cells such as MDCK cells, initially inducing membrane ruffling and
centrifugal spreading of cell colonies followed by a disruption of cell–cell adhesions and then cell scattering [57]. The expression of v-Src in MDCK cells suppresses the cadherinmediated cell–cell adhesion with concomitant disruption of adherens junctions where the cadherin–catenins complex acts as an adhesion molecule without affecting other junctions such as tight junctions [58]. Therefore, cell scattering accompanies the dynamic rearrangement in cadherin-mediated cell–cell adhesion. Accumulating evidence indicates that the Rho family GTPases regulate membrane ruffling and cell scattering induced by HGF or phorbol ester [14,23,59,60]. It has been shown that HGF-induced membrane ruffling and cell scattering of keratinocytes (308R cells) are inhibited by microinjection of either Rho GDI or Clostridium botulinum C3 toxin, but not by microinjection of RacAsn17 [14]. The action of Rho GDI is counteracted by co-microinjection of activated Rho, but not of activated Rac, indicating that Rho is necessary for the HGF-induced cell scattering. Conversely, it has been reported that Rac, rather than Rho, is necessary for the HGF-induced membrane ruffling and cell scattering of MDCK cells [23]. These apparent contradictory results concerning the roles of Rho and Rac might be explained by the differences in cell types and reagents used. A more recent study has shown that the expression of Rho Asn19 or RacAsn17 inhibits the HGF-induced membrane ruffling and cell scattering of MDCK cells [61]. In addition, we have found that microinjection of Cdc42Val12 or Rac1Val12 into MDCK cells blocks the disruption of cell–cell adhesion during the HGF-induced cell scattering of MDCK cells but not membrane ruffling (M Fukata, S Kuroda, M Nakagawa, K Kaibuchi, unpublished data). Thus, it is likely that the inactivation of Cdc42 and Rac1 is necessary for disruption of cell–cell adhesion during the cell scattering but not for cell motility. Although evidence is not available that this process involves IQGAP1, it is intriguing to examine whether IQGAP1 plays a role in the suppression of cadherin activity possibly through the dissociation of α-catenin from the cadherin–catenins complex. It has been shown that the dynamic rearrangement in E-cadherin-mediated cell–cell adhesion underlies the compaction of the eight-cell embryo by which the embryo develops from a collection of loosely adherent blastomeres into a tightly packed epithelium called a blastocyst [62]. This morphogenic process entails the rapid activation of E-cadherin at the cell surface without marked change of its expression [63–65]. Although the mechanism regulating E-cadherin activity in embryos remains unknown, it is possible that remodelling of the cadherin–catenins complex is involved. Gastrulation also provides an example of dynamic rearrangement of the cadherin–catenins complex. In sea urchin embryo, cadherin is localised at the cell–cell contact sites throughout gastrulation, whereas α-catenin staining at the sites decreases markedly [66,67]. Further studies are
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necessary to determine whether the Cdc42/Rac1/IQGAP1 system is involved in the regulation of cell–cell adhesion during gastrulation.
Conclusions Much effort have been made over the past several years to understand the mechanism by which the Rho family GTPases regulate the cytoskeletons and cell adhesion. As a result, some of the molecular pathways that connect the Rho family GTPases to control of the cytoskeleton have been established. In addition, recent evidence indicates that Cdc42 and Rac1 together with IQGAP1 are involved in the regulation of cadherin-mediated cell–cell adhesion. Cdc42 and Rac1, and IQGAP1 can serve as positive and negative molecular switches of the cadherin activity, respectively. Although cadherin activity is dynamically regulated in various processes, the molecular mechanism controlling the activity has yet to be elucidated. A study on Cdc42, Rac1, and IQGAP1 as molecular switches should provide insight into the regulation of cadherin-mediated cell–cell adhesion.
Acknowledgements We thank Naohiro Itoh and Aie Kawajiri for preparing the manuscript. The original work by the authors has been supported by the Japan Society of the Promotion of Science Research for the Future, by the Ministry of Education, Science, and Culture, Japan, by the Human Frontier Science Program, and by a grant from Kirin Brewery Company Limited.
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33. Kaibuchi K, Kuroda S, Amano M: Regulation of the cytoskeleton • and cell adhesion by the Rho family GTPases in mammalian cells. Annu Rev Biochem 1999, 68:459-486. The emphasis of this review is on molecules that interact with the Rho family GTPases and their roles in the regulation of cytoskeleton and cell adhesion. 34. Kuroda S, Fukata M, Nakagawa M, Fujii K, Nakamura T, Ookubo T, •• Izawa I, Nagase T, Nomura N, Tani H et al.: Role of IQGAP1, a target of the small GTPases Cdc42 and Rac1, in regulation of E-cadherinmediated cell–cell adhesion. Science 1998, 281:832-835. This paper reported for the first time the mechanism by which the Rho family GTPases including Cdc42 and Rac1 regulate E-cadherin-mediated cell–cell adhesion. IQGAP1, which is an effector of Cdc42 and Rac1 appears to regulate cell–cell adhesion through the cadherin–catenin pathway acting downstream of Cdc42 and Rac1. 35. Braga VMM, Del Maschio A, Machesky L, Dejana E: Regulation of • cadherin function by Rho and Rac: modulation by junction maturation and cellular context. Mol Biol Cell 1999, 10:9-22. This paper reported that the regulation of cadherin adhesiveness by the Rho family GTPases is influenced by the maturation status of the junction and the cellular context. 36. Fukata M, Kuroda S, Nakagawa M, Kawajiri A, Itoh N, Shoji I, Matsuura Y, • Yonehara S, Fujisawa H, Kikuchi A, Kaibuchi K: Cdc42 and Rac1 regulate the interaction of IQGAP1 with β-catenin. J Biol Chem 1999, 274:26044-26050. This paper described that Cdc42 and Rac1 negatively regulate the IQGAP1 function by inhibiting the interaction of IQGAP1 with β-catenin, leading to stabilisation of the cadherin–catenins complex. 37.
Aktories K: Rho proteins: targets for bacterial toxins. Trends Microbiol 1997, 5:282-288.
38. Hordijk PL, ten Klooster JP, van der Kammen RA, Michiels F, Oomen LC, Collard JG: Inhibition of invasion of epithelial cells by Tiam1-Rac signaling. Science 1997, 278:1464-1466. 39. Nagafuchi A, Ishihara S, Tsukita S: The roles of catenins in the cadherin-mediated cell adhesion: functional analysis of Eα-catenin fusion molecules. J Cell Biol 1994, 127:235-245. cadherin–α 40. Angres B, Barth A, Nelson WJ: Mechanism for transition from initial to stable cell–cell adhesion: kinetic analysis of E-cadherinmediated adhesion using a quantitative adhesion assay. J Cell Biol 1996, 134:549-557. 41. Imamura Y, Itoh M, Maeno Y, Tsukita S, Nagafuchi A: Functional domains of α-catenin required for the strong state of cadherinbased cell adhesion. J Cell Biol 1999, 144:1311-1322. 42. Hart MJ, Callow MG, Souza B, Polakis P: IQGAP1, a calmodulinbinding protein with a rasGAP-related domain, is a potential effector for Cdc42Hs. EMBO J 1996, 15:2997-3005. 43. Kuroda S, Fukata M, Kobayashi K, Nakafuku M, Nomura N, Iwamatsu A, Kaibuchi K: Identification of IQGAP as a putative target for the small GTPases, Cdc42 and Rac1. J Biol Chem 1996, 271:23363-23367. 44. Aberle H, Butz S, Stappert J, Weissig H, Kemler R, Hoschuetzky H: Assembly of the cadherin–catenin complex in vitro with recombinant proteins. J Cell Sci 1994, 107:3655-3663.
50. Sako Y, Nagafuchi A, Tsukita S, Takeichi M, Kusumi A: Cytoplasmic • regulation of the movement of E-cadherin on the free cell surface as studied by optical tweezers and single particle tracking: corralling and tethering by the membrane skeleton. J Cell Biol 1998, 140:1227-1240. The translational movement of E-cadherin in the plasma membrane in epithelial cells, and the mechanism of its regulation were studied using single particle tracking and optical tweezers 51. Matsuyoshi N, Hamaguchi M, Taniguchi S, Nagafuchi A, Tsukita S, Takeichi M: Cadherin-mediated cell-cell adhesion is perturbed by v-Src tyrosine phosphorylation in metastatic fibroblast. J Cell Biol 1992, 118:703-714. 52. Behrens J, Vakaet L, Friis R, Winterhager E, Van Roy F, Mareel MM, Birchmeier W: Loss of epithelial differentiation and gain of invasiveness correlates with tyrosine phophosrylation of the Eβ-catenin complex in cells transformed with a cadherin/β temperature-sensitive v-SRC gene. J Cell Biol 1993, 120:757-766. 53. Shibamoto S, Hayakawa K, Takeuchi K, Hori T, Oku N, Miyazawa K, Kitamura N, Takeichi M, Ito F: Tyrosine phosphorylation of β-catenin and plakoglobin enhanced by hepatocyte growth factor and epidermal growth factor in human carcinoma cells. Cell Adhes Commun 1994, 1:295-305. 54. Ozawa M, Kemler R: Altered cell adhesion activity by pervanadate • due to the dissociation of α-catenin from the E-cadherin–catenin complex. J Biol Chem 1998, 273:6166-6170. Pervanadate treatment of cells caused tyrosine phosphorylation of E-cadherin and β-catenin. In the treated cells, a significant amount of α-catenin was dissociated from the E-cadherin–catenins complex. 55. Takeda H, Nagafuchi A, Yonemura S, Tsukita S, Behrens J, Birchmeier W: v-Src kinase shifts the cadherin-based cell adhesion from the strong to the weak state and β-catenin is not required for the shift. J Cell Biol 1995, 131:1839-1847. 56. Takeichi M: Morphogenetic roles of classic cadherins. Curr Opin Cell Biol 1995, 7:619-627. 57.
Hartmann G, Weidner KM, Schwarz H, Birchmeier W: The motility signal of scatter factor/hepatocyte growth factor mediated through the receptor tyrosine kinase met requires intracellular action of Ras. J Biol Chem 1994, 269:21936-21939.
58. Warren SL, Nelson WJ: Nonmitogenic morphoregulatory action of pp60 v-Src on multicellular epithelial structures. Mol Cell Biol 1987, 7:1326-1337. 59. Takaishi K, Kikuchi A, Kuroda S, Kotani K, Sasaki T, Takai Y: Involvement of rho p21 and its inhibitory GDP/GTP exchange protein (Rho GDI) in cell motility. Mol Cell Biol 1993, 13:72-79. 60. Takaishi K, Sasaki T, Kameyama T, Tsukita S, Tsukita S, Takai Y: Translocation of activated Rho from the cytoplasm to membrane ruffling area, cell–cell adhesion sites and cleavage furrows. Oncogene 1995, 11:39-48. 61. Fukata Y, Oshiro N, Kinoshita N, Kawano Y, Matsuoka Y, Bennett V, Matsuura Y, Kaibuchi K: Phosphorylation of adducin by rho-kinase plays a crucial role in cell motility. J Cell Biol 1999, 145:347-361. 62. Fleming TP, Johnson MH: From egg to epithelium. Annu Rev Cell Biol 1988, 4:459-485.
45. Price LS, Leng J, Schwartz MA, Bokoch GM: Activation of Rac and • Cdc42 by integrins mediates cell spreading. Mol Biol Cell 1998, 9:1863-1871. This paper describes that integrin-dependent adhesion of cell to substratum leads to the rapid activation of Cdc42 and Rac.
63. Kemler R, Babinet C, Eisen H, Jacob F: Surface antigen in early differentiation. Proc Natl Acad Sci USA 1977, 74:4449-4452.
46. Ren XD, Kiosses WB, Schwartz MA: Regulation of the small GTP• binding protein Rho by cell adhesion and the cytoskeleton. EMBO J 1999, 18:578-585. Plating Swiss 3T3 cells of fibronectin-coated dishes elicits a transient inhibition of Rho, followed by a phase of Rho activation. This indicates that integrin-dependent adhesion of cell to substratum induces Rho activation.
65. Sefton M, Johnson MH, Clayton L: Synthesis and phosphorylation of uvomorulin during mouse early development. Development 1992, 115:313-318.
47.
Higgins JM, Mandlebrot DA, Shaw SK, Russell GJ, Murphy EA, Chen YT, Nelson WJ, Parker CM, Brenner MB: Direct and regulated β7 with E-cadherin. J Cell Biol 1998, interaction of integrin αEβ 140:197-210.
64. Vestweber D, Gossler A, Boller K, Kemler R: Expression and distribution of cell adhesion molecule uvomorulin in mouse preimplantation embryos. Dev Biol 1987, 124:451-456.
66. Miller JR, McClay DR: Changes in the pattern of adherens junctionassociated β-catenin accompany morphogenesis in the sea urchin embryo. Dev Biol 1997, 192:310-322. 67.
Miller JR, McClay DR: Characterization of the role of cadherin in regulating cell adhesion during sea urchin development. Dev Biol 1997, 192:323-339.
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Regulation of cadherin-mediated cell–cell adhesion by the Rho family GTPases Kozo Kaibuchi*, Shinya Kuroda, Masaki Fukata and Masato Nakagawa Reports in the past two years have shown that Cdc42, Rac1, and Rho — belonging to the Rho small GTPase family — participate in the regulation of cadherin-mediated cell–cell adhesion. IQGAP1, an effector of Cdc42 and Rac1, interacts with cadherin and β-catenin and induces the dissociation of α-catenin from the cadherin–catenins complex leading to disruption of cell–cell adhesion: activated Cdc42 and Rac1 counteract the effect of IQGAP1. Thus, Cdc42 and Rac1 appear to regulate cadherinmediated cell–cell adhesion acting through IQGAP1. Addresses Division of Signal Transduction, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma 630-0101, Japan *e-mail: [email protected] Current Opinion in Cell Biology 1999, 11:591–596 0955-0674/99/$ — see front matter © 1999 Elsevier Science Ltd. All rights reserved. Abbreviations EL cells GAP GDI GEF HGF MDCK cells PAK
E-cadherin expressing L cells GTPase-activating protein GDP-dissociation inhibitor GDP/GTP exchange factor hepatocyte growth factor Madin-Darby canine kidney cells p21-activated kinases
dynamically remodelled; however, the mechanism underlying the dynamic rearrangement of cell–cell adhesion remains to be clarified. The Rho small GTPase family (including Rho, Rac, and Cdc42) participates in the regulation of the actin cytoskeleton and various cell adhesion events [7,8•]. Rho has been implicated in the formation of actin stress fibres and focal adhesions [9], cell morphology [10], cell aggregation [11], cell–cell adhesion [12,13], cell motility [14], membrane ruffling [15], smooth muscle contraction [16,17], neurite retraction in neuronal cells [18,19], and cytokinesis [20,21]. Rac is involved in membrane ruffling [22], cell motility [23], actin polymerisation [24], and cell–cell adhesion [12,13,25]. Cdc42 participates in filopodia formation [26,27] and cell–cell adhesions [25]. The molecular mechanisms underlying the above processes were largely unknown, until recently. Some of the molecular pathways that connect the Rho family GTPases to control of the cytoskeleton and cell adhesion have been established. The major emphasis in this review is on how the Rho family GTPases regulate cell–cell adhesion. The above functions regulated by the Rho family GTPases are not described here in detail: for such information, the reader is referred to other reviews [7,8•].
The Rho family GTPases Introduction Cell–cell adhesions are mediated by adhesion molecules, such as cadherins and their associated cytoplasmic proteins, α-catenin and β-catenin [1–3]. E-cadherin, a classic cadherin, is a Ca2+-dependent homophilic cell–cell adhesion molecule [1–3]. The cytoplasmic domain of E-cadherin interacts with β-catenin or plakoglobin (also called γ-catenin) [4,5]. Plakoglobin and β-catenin also interact with α-catenin, which has been thought to directly or indirectly link the complex composed of cadherin, β-catenin and α-catenin (cadherin–catenins complex) to the actin cytoskeleton. Strong adhesion requires linkage of cadherin to the actin cytoskeleton via catenins [5,6]. Cell–cell adhesion seems to be a static process but dynamic rearrangements of cell–cell adhesion should occur during various cellular processes, such as epithelial cell scattering, dispersal of cancer cells, and early embryonic cell migration [6]. Indeed, certain cell lines including Madin-Darby canine kidney (MDCK) cells rapidly migrate even in confluent conditions, indicating that the cell–cell adhesion process is transiently perturbed in migrating areas, enabling dynamic migration (M Fukata, S Kuroda, M Nagawa, K Kaibuchi, unpublished data). The cell–cell adhesion appears to be constantly rearranged in real time, suggesting that the cadherin–catenins complex is
The Rho family GTPases exhibit both GDP/GTP-binding and GTPase activities. They have two interconvertible forms; GDP-bound inactive and GTP-bound active forms [28,29]. The conversion from the GDP-bound to GTPbound form is stimulated by GDP/GTP exchange factor (GEF) and inhibited by GDP-dissociation inhibitor (GDI) [29,30]. The conversion from the GTP-bound to GDPbound form is catalysed by GTPase-activating protein (GAP) [29,30]. On the basis of recent studies, the activity of the Rho family GTPases appears to be regulated cyclically, as follows (Figure 1). In the cytosol of resting cells, the Rho family GTPases are present in the GDP-bound form complexed with Rho GDI. When cells are stimulated by certain extracellular signals, Rho GDI dissociates, specific GEFs for the Rho family GTPases are activated and the GTPases are targeted to cell membranes by their carboxy-terminal prenyl group [29,31,32]. The GDP-bound form of the Rho family GTPases is then converted to the GTP-bound form. The GTP-bound form, attached to membranes, interacts with specific effectors to exert specific functions. GAPs stimulate the GTPase activity of the Rho family GTPases and reconvert them to the inactive GDP-bound form. Rho GDI can then form a complex with the GDP-bound form and extract it from the membrane back into the cytosol.
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Figure 1
Plasma membrane GTP•
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Mode of activation of Rho family GTPases. When Rho GDI is dissociated from Rho family GTPases in a certain manner, Rho GEF, which is thought to be activated by Gα13 or tyrosine phosphorylation, appears to stimulate the dissociation of GDP from the Rho family GTPases and thereby stimulate the association of GTP with the Rho family GTPases. The GTP-bound form attached to membranes through its carboxy-terminal prenyl group, interacts with its specific targets. Rho GAP acts as a negative regulator by enhancing the GTPase activity of the Rho family GTPases and reconverting them to the inactive GDP-bound form. Rho GDI can then form a complex with the GDP-bound form and extract it from the membrane back into the cytosol. Rho, Rho family GTPases; GEF, GDP/GTP exchange factor; GDI, GDP dissociation inhibitor; GAP, GTPase activating protein.
The mode of action of the Rho family GTPases has been well studied, and many effector molecules for these small GTPases have been identified [7,33•]. A number of proteins have been identified as effectors for Rho. These include Rho-kinase (ROK/ROCK), the myosin-binding subunit (MBS) of myosin phosphatase, protein kinase N (PKN)/PRK1, rhophilin, rhotekin, citron, and p140 mDia. A number of proteins have been identified as effectors for Rac including p21-activated kinases (PAKs), IQGAP1, Por1, p140Sra-1 and POSH, and as effectors for Cdc42 including PAKs, WASP/N-WASP, IQGAP1 and MRCK. Several of these proteins such as PAKs and IQGAP1 interact with both Rac and Cdc42.
Roles of the Rho family GTPases in the regulation of cadherin activity Recent work has suggested that the Rho family GTPases including Cdc42, Rac1, and Rho are required for cadherinmediated cell–cell adhesion [12,13,25,34••,35•,36•]. It has been shown that microinjection of dominant-negative Rac1Asn17, which preferentially binds GDP rather than GTP and thereby inhibits the activation of endogenous Rac1 by titrating out its GEF [22,30], or of botulinum toxin C3, which inactivates Rho by ADP-ribosylation [37], reduces the accumulation of cadherin at cell–cell contact sites when keratinocytes are transferred from low calcium media to standard media to induce calcium-dependent cell–cell adhesion [12].
The effects of Rac1 and Rho on localisation of cadherin are probably dependent on the cell types [35•]; Rac1 and Rho are required for the localisation of E-cadherin at cell–cell contact sites in keratinocytes, whereas they are not required for the VE-cadherin localisation in human umbilical cord endothelial cells. It has also been reported that in MDCK cells stably expressing dominant-active Rac1Val12 — which is thought to be the constitutively GTP-bound active form in the cells [22] — immunofluorescent intensities of E-cadherin, β-catenin, and actin filaments at cell–cell contact sites increased more than those in the parental cells [13]. In the MDCK cells expressing Rac1Asn17, those of Ecadherin, β-catenin and actin filament decreased. We have found that Cdc42 as well as Rac1 is required for the regulation of cell–cell adhesion in MDCK cells [25]. Microinjection of Rho GDI, a negative regulator for Cdc42, Rac1, and Rho, results in the perturbation of cell–cell adhesion in MDCK cells. Co-injection of either Cdc42Val12 or Rac1Val12 with Rho GDI reverses the inhibitory action of Rho GDI, whereas that of RhoVal14 with Rho GDI does not. In addition, it has also been shown that Tiam1, one of the GEFs for Rac1, is involved in the regulation of cell–cell adhesion in MDCK cells [38]. Expression of Tiam1 or Rac1Val12 inhibits hepatocyte growth factor (HGF)-induced scattering of MDCK cells probably by increasing E-cadherin-mediated cell–cell adhesion. These observations have led to the suggestion that Cdc42, Rac1, and Rho as well as Tiam1 regulate cadherin-mediated cell–cell adhesion either directly or indirectly. Direct evidence has not yet been presented, however, that the Rho family GTPases regulate cadherin activity. To provide such evidence, we used L cells expressing E-cadherin (EL cells) and L cells expressing an E-cadherin mutant in which the cytoplasmic domain was deleted and replaced by the carboxy-terminal domain of α-catenin (nEαCL cells) [39]. Both cell lines display cell–cell adhesive activity in an E-cadherin-dependent manner [39]. We have recently shown that, using the dissociation assay, a quantitative assay for E-cadherin activity [39], expression of Cdc42Asn17 or Rac1Asn17 in EL cells markedly reduces the E-cadherin activity, whereas expression of RhoAsn19 slightly reduces it [36•]. In contrast, expression of Cdc42Asn17, Rac1Asn17, or RhoAAsn19, in nEαCL cells slightly reduces the mutant Ecadherin activity. This result strongly suggests that Cdc42 and Rac1 directly regulate the E-cadherin activity, whereas RhoA indirectly regulates it possibly through the rearrangement of the actin cytoskeleton. Consistent with this, treatment with cytochalasin D, which prevents actin polymerisation and thereby disrupts the linkage of cadherin to actin cytoskeleton, reduces the E-cadherin activity in both EL and nEαCL cells [40,41].
Mode of action of the Rho family GTPases in the regulation of cadherin activity The next question is how Cdc42 and Rac1 regulate E-cadherin activity. Among the effectors of Cdc42 and Rac1,
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interacting with β-catenin, and thereby stabilises the cadherin–catenins complex.
IQGAP1 has been shown to accumulate at the cell–cell contact sites [34••,42,43]. This raises the possibility that IQGAP1 regulates the E-cadherin activity downstream of Cdc42 and Rac1. IQGAP1 accumulates at cell–cell contact sites in EL cells, but not in nEαCL cells, indicating that this accumulation is dependent on E-cadherin and β-catenin [34••]. A recent study has shown that IQGAP1 directly interacts with β-catenin and the cytoplasmic domain of E-cadherin both in vitro and in vivo [34••]. IQGAP1 interacts with the amino-terminal end of β-catenin (residues 1–183) [36•], which overlaps with the α-catenin-binding domain (residues 120–151) [44]. IQGAP1 inhibits the binding of α-catenin to β-catenin, and causes α-catenin to dissociate from its complex with β-catenin in vitro [36•]. In addition, overexpression of IQGAP1 in EL cells results in the dissociation of αcatenin from the cadherin–catenins complex and in concomitant reduction of E-cadherin activity [34••]. The overexpression of IQGAP1 in nEαCL cells does not affect the mutant E-cadherin activity. Thus, IQGAP1 appears to regulate E-cadherin activity in vivo at least through the dissociation of α-catenin from the cadherin–catenins complex [34••].
On the basis of these observations, we propose a mechanism by which Cdc42 and Rac1 together with IQGAP1 regulate the cadherin-mediated cell–cell adhesion (Figure 2). When Cdc42 and Rac1 are in the GTP-bound active forms at cell–cell contact sites, Cdc42 and Rac1 interact with IQGAP1 and thereby prohibit IQGAP1 from interacting with β-catenin. This results in the stabilisation of the cadherin–catenins complex. When Cdc42 and Rac1 are in the GDP-bound inactive forms, Cdc42 and Rac1 cannot interact with IQGAP1, and IQGAP1 interacts with β-catenin, thereby dissociating α-catenin from the cadherin–catenins complex. This state confers relatively weak adhesive activity, which is mediated by E-cadherin unlinked to the actin cytoskeleton. Thus, Cdc42 and Rac1 positively regulate cadherin-mediated cell–cell adhesion by the suppression of the activity of IQGAP1 to perturb the cadherin–catenins complex. This state confers relatively strong adhesive activity, which is mediated by E-cadherin linked to the actin cytoskeleton. Thus, Cdc42 and Rac1, and IQGAP1 can serve as positive and negative molecular switches of the cadherin activity, respectively. Following this proposal, it can be speculated that cell–cell contact induces the activation of the Rho family GTPases. In this regard, it has been shown that cell to substratum contact, which is mediated via integrins, induces the activation of the Rho family GTPases including Rho, Cdc42, and Rac1 [45•,46•], and that E-cadherin interacts with certain types of integrins at cell–cell contact sites [47]. Like that of growth factor receptors, engagement of E-cadherin results in the tyrosine phosphorylation of certain proteins [48] and recruitment of adapter proteins such as Shc to Ecadherin [49]. Hence, it may prove worthwhile to examine
Do Cdc42 and Rac1 regulate the IQGAP1 activity on E-cadherin–catenins complex? Cdc42 and Rac1 bound to the nonhydrolysable GTP analogue GTPγS inhibit the interaction of IQGAP1 with β-catenin, whereas their GDPbound forms or GTPγS·RhoA do not inhibit in vitro [36•]. Co-expression of Cdc42Val12 with IQGAP1 in EL cells inhibits the dissociation of α-catenin from the cadherin–catenins complex induced by IQGAP1 [36•] and concomitantly restores the E-cadherin activity [34••]. Thus, this result indicates that activated Cdc42 suppresses the inhibitory action of IQGAP1 by preventing IQGAP1 from Figure 2 Role of IQGAP1 in the regulation of E-cadherin-mediated cell–cell adhesion. When Cdc42 and Rac1 are in the GDP-bound inactive form, Cdc42 and Rac1 cannot interact with IQGAP1, and IQGAP1 interacts with β-caterin thereby dissociating α-catenin from the cadherin–catenins complex. This state confers the weak adhesive activity. In contrast, when Cdc42 and Rac1 are in the GTP-bound active form at sites of cell–cell contact, Cdc42 and Rac1 interact with IQGAP1, and thereby, prohibit IQGAP1 from interacting with β-catenin. This results in the stabilization of the cadherin–catenins complex. Thus, Cdc42 and Rac1 positively regulate cadherin-mediated cell–cell adhesion by the suppression of the activity of IQGAP1 to perturb the cadherin–catenins complex. This state confers the strong adhesive activity. Thus, Cdc42 and Rac1, and IQGAP1 can serve as positive and negative molecular switches of the cadherin activity, respectively. E, E-cadherin; α, α-catenin; β, β-catenin.
Weak adhesion
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whether cell–cell contact induces the activation of the Rho family GTPases. One may ask why IQGAP1 is colocalised with E-cadherin and β-catenin at cell–cell contact sites, even though it inhibits cell–cell adhesion. This can be explained by the notion that E-cadherin stays as a complex with either α-catenin and βcatenin or β-catenin and IQGAP1 at cell–cell contact sites. There should be a mixture of E-cadherin/α-catenin/β-catenin complex and E-cadherin/β-catenin/IQGAP1 complex. The ratio between the two may determine the adhesive activity. In this regard, it should be noted that about one half of E-cadherin appears to be connected to the actin cytoskeleton probably via α-catenin in EL cells, whereas the rest appears to be free from the actin cytoskeleton [50•].
Other mechanisms that account for the regulation of cadherin activity Numerous attempts have been made to clarify the regulation of the cadherin function. One of the most intriguing phenomena proposed is tyrosine phosphorylation of β-catenin. Increased tyrosine phosphorylation of β-catenin appears to correlate with dysfunction of cadherin-mediated cell–cell adhesion, induced by the expression of v-Src [51,52] or treatment with growth factors, such as epidermal growth factor or hepatocyte growth factor (HGF) [53]. It has been shown that treatment of cells with pervanadate, a potent inhibitor of tyrosine phosphatases, induces perturbation of the cadherin-mediated cell–cell adhesion [54•]. In accordance with dysfunction of cadherin-mediated cell–cell adhesion, tyrosine phosphorylation of β-catenin and dissociation of α-catenin from the cadherin–catenins complex occur [54•]. Therefore, the tyrosine phosphorylation of β-catenin was thought to be important for the regulation of the cadherin function. On the basis of these findings together with our model, it is tempting to speculate that tyrosine-phosphorylated β-catenin recruits IQGAP1 or GAPs for Cdc42 and/or Rac1 to the cell–cell contact sites, resulting in the reduction of cadherin activity through the inhibition of Cdc42 and/or Rac1 activity. It has been reported, however, that the tyrosine phosphorylation of β-catenin is not required for the reduction of cadherin activity induced by v-Src, as the expression of v-Src reduces the cadherin function of nEαCL cells as well as EL cells [55]. Thus, the physiological role of the tyrosine phosphorylation of β-catenin in cadherin function remains to be clarified.
Physiological processes in which the Rho family GTPases regulate cadherin activity It remains to be determined in which physiological processes the above Cdc42/Rac1/IQGAP1 system operates. Dynamic rearrangement of cell–cell adhesion should occur in various situations [2,56]. Cell scattering provides one of the most prominent examples of dynamic rearrangement of cell–cell adhesion [2,56]. Scatter factor/HGF and phorbol ester stimulate the motility of epithelial cells such as MDCK cells, initially inducing membrane ruffling and
centrifugal spreading of cell colonies followed by a disruption of cell–cell adhesions and then cell scattering [57]. The expression of v-Src in MDCK cells suppresses the cadherinmediated cell–cell adhesion with concomitant disruption of adherens junctions where the cadherin–catenins complex acts as an adhesion molecule without affecting other junctions such as tight junctions [58]. Therefore, cell scattering accompanies the dynamic rearrangement in cadherin-mediated cell–cell adhesion. Accumulating evidence indicates that the Rho family GTPases regulate membrane ruffling and cell scattering induced by HGF or phorbol ester [14,23,59,60]. It has been shown that HGF-induced membrane ruffling and cell scattering of keratinocytes (308R cells) are inhibited by microinjection of either Rho GDI or Clostridium botulinum C3 toxin, but not by microinjection of RacAsn17 [14]. The action of Rho GDI is counteracted by co-microinjection of activated Rho, but not of activated Rac, indicating that Rho is necessary for the HGF-induced cell scattering. Conversely, it has been reported that Rac, rather than Rho, is necessary for the HGF-induced membrane ruffling and cell scattering of MDCK cells [23]. These apparent contradictory results concerning the roles of Rho and Rac might be explained by the differences in cell types and reagents used. A more recent study has shown that the expression of Rho Asn19 or RacAsn17 inhibits the HGF-induced membrane ruffling and cell scattering of MDCK cells [61]. In addition, we have found that microinjection of Cdc42Val12 or Rac1Val12 into MDCK cells blocks the disruption of cell–cell adhesion during the HGF-induced cell scattering of MDCK cells but not membrane ruffling (M Fukata, S Kuroda, M Nakagawa, K Kaibuchi, unpublished data). Thus, it is likely that the inactivation of Cdc42 and Rac1 is necessary for disruption of cell–cell adhesion during the cell scattering but not for cell motility. Although evidence is not available that this process involves IQGAP1, it is intriguing to examine whether IQGAP1 plays a role in the suppression of cadherin activity possibly through the dissociation of α-catenin from the cadherin–catenins complex. It has been shown that the dynamic rearrangement in E-cadherin-mediated cell–cell adhesion underlies the compaction of the eight-cell embryo by which the embryo develops from a collection of loosely adherent blastomeres into a tightly packed epithelium called a blastocyst [62]. This morphogenic process entails the rapid activation of E-cadherin at the cell surface without marked change of its expression [63–65]. Although the mechanism regulating E-cadherin activity in embryos remains unknown, it is possible that remodelling of the cadherin–catenins complex is involved. Gastrulation also provides an example of dynamic rearrangement of the cadherin–catenins complex. In sea urchin embryo, cadherin is localised at the cell–cell contact sites throughout gastrulation, whereas α-catenin staining at the sites decreases markedly [66,67]. Further studies are
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necessary to determine whether the Cdc42/Rac1/IQGAP1 system is involved in the regulation of cell–cell adhesion during gastrulation.
Conclusions Much effort have been made over the past several years to understand the mechanism by which the Rho family GTPases regulate the cytoskeletons and cell adhesion. As a result, some of the molecular pathways that connect the Rho family GTPases to control of the cytoskeleton have been established. In addition, recent evidence indicates that Cdc42 and Rac1 together with IQGAP1 are involved in the regulation of cadherin-mediated cell–cell adhesion. Cdc42 and Rac1, and IQGAP1 can serve as positive and negative molecular switches of the cadherin activity, respectively. Although cadherin activity is dynamically regulated in various processes, the molecular mechanism controlling the activity has yet to be elucidated. A study on Cdc42, Rac1, and IQGAP1 as molecular switches should provide insight into the regulation of cadherin-mediated cell–cell adhesion.
Acknowledgements We thank Naohiro Itoh and Aie Kawajiri for preparing the manuscript. The original work by the authors has been supported by the Japan Society of the Promotion of Science Research for the Future, by the Ministry of Education, Science, and Culture, Japan, by the Human Frontier Science Program, and by a grant from Kirin Brewery Company Limited.
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