INTRODUCTION

Dynamic rearrangement of cell adhesion contacts enables cellsto perform a variety of functions, for example: induction ofmorphological changes during early development, cellmigration, establishment of epithelial-cell polarity and woundhealing. It also allows tumors to metastasise (Takeichi, 1995;Gumbiner, 1996; Adams and Nelson, 1998). Cell adhesion canbe classified into two types: (1) cell-substratum adhesionmediated by molecules such as integrins and (2) cell-celladhesion mediated by molecules such as cadherins.

Cell-substratum adhesionCell-substratum adhesion is mediated by adhesion moleculessuch as integrins, which are receptors for the extracellularmatrix (ECM) components (e.g. fibronectin). Integrins areheterodimers composed of two transmembrane glycoproteinsubunits, α and β. Frequently, they are clustered to formspecialized adhesive structures, focal adhesions and focalcomplexes, in which many signaling molecules areconcentrated (Clark and Brugge, 1995). Focal adhesions arelarge adhesion contacts found at the ends of stress fibers. Focalcomplexes are smaller adhesion contacts found at the tips offilopodia or lamellipodia. The clustering of integrins into focaladhesions and focal complexes enables cells to adhere to ECMcomponents, such as fibronectin. The cytoplasmic domains ofintegrins directly or indirectly bind to talin, α-actinin, vinculin,paxillin and p125 focal-adhesion kinase (FAK), which link

integrins to the actin cytoskeleton. This link between integrinsand the actin cytoskeleton is essential for strong adhesion andintegrin clustering (Clark and Brugge, 1995; Hughes and Pfaff,1998). Cell-substratum adhesion contacts are dynamicallyrearranged during various cellular processes, such aschemoattractant-activated leukocyte adhesion (Laudanna et al.,1996), and cell migration (Gumbiner, 1996). However, littlewas known about the regulatory mechanism underlying theinitiation, maturation and turnover of focal adhesions and focalcomplexes until recently.

Cell-cell adhesion Adhesion molecules such as cadherins mediate cell-celladhesion through homophilic interactions (Takeichi, 1995;Gumbiner, 1996; Adams and Nelson, 1998). The cytoplasmicdomain of E-cadherin directly interacts with β-catenin orplakoglobin (also known as γ-catenin) (Ozawa et al., 1989;Tsukita et al., 1992), which in turn associates with α-catenin.The latter appears to link the cadherin-β-catenin-α-catenincomplex (cadherin-catenins complex) to the actin cytoskeletondirectly, or indirectly through other cytoskeletal proteins suchas α-actinin and vinculin. This link between cadherin and theactin cytoskeleton (via catenins) is essential for strong andrigid adhesion (Tsukita et al., 1992). Indeed, α-catenin-deficient mouse teratocarcinoma F9 cells display a scatteredphenotype under the conditions in which parental or α-catenin-reexpressing cells form compact colonies (Maeno et al., 1999).Loss of α-catenin expression has also been observed in lung

4491Journal of Cell Science 112, 4491-4500 (1999)Printed in Great Britain © The Company of Biologists Limited 1999 JCS0728

The Rho small GTPases, Cdc42, Rac1 and Rho, areimplicated in regulation of integrin-mediated cell-substratum adhesion and cadherin-mediated cell-celladhesion. Identification and characterization of effectors ofthese GTPases have provided insights into their modes ofaction. Rho-kinase, an effector of Rho, regulates integrin-mediated cell-substratum adhesion (focal adhesion) byregulating the phosphorylation state of myosin lightchain (MLC): it directly phosphorylates MLC and alsoinactivates myosin phosphatase. IQGAP1, an effector of

Cdc42 and Rac1, regulates cadherin-mediated cell-celladhesion by interacting with β-catenin and dissociatingα-catenin from the cadherin-catenins complex. ActivatedCdc42 and Rac1 inhibit IQGAP1, thereby stabilizing thecadherin-catenins complex. Cdc42/Rac1 and IQGAP1 thusappear to constitute a switch that regulates cadherin-mediated cell-cell adhesion.

Key words: Cell adhesion, Rho, GTPase, Catenin, Cadherin, Integrin,Rho-kinase, IQGAP1

SUMMARY

COMMENTARY

Cell adhesion and Rho small GTPases

Masaki Fukata1,2, Masato Nakagawa1, Shinya Kuroda1,3 and Kozo Kaibuchi1,*1Division of Signal Transduction, Nara Institute of Science and Technology, Ikoma 630-0101, Japan2Department of Biochemistry, Hiroshima University School of Medicine, Hiroshima 734-8551, Japan3Inheritance and Variation Group, PRESTO, Japan Science and Technology, Kyoto 619-0237, Japan *Author for correspondence (e-mail: kaibuchi@bs.aist-nara.ac.jp)

Accepted 7 October; published on WWW 30 November 1999

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carcinomas (Watabe et al., 1994) and gastric carcinomas(Ochiai et al., 1994) that show scattered cell growth.

Cell-cell adhesion seems to be a static process, but adynamic rearrangement of cell-cell adhesion contactsaccompanies various cellular processes, such as epithelial-cellscattering, dispersal of cancer cells and early embryonic cellmigration (Adams and Nelson, 1998). Indeed, certain epithelialcell lines rapidly migrate even when confluent. This indicatesthat cell-cell adhesion is transiently perturbed in migratingareas (M. Fukata, M. Nakagawa, S. Kuroda and K. Kaibuchi,unpublished data). Recently, studies using optical tweezers andsingle-particle tracking have indicated that approximately 50%of the E-cadherin in the plasma membrane in epithelial cells isconnected to the actin cytoskeleton, probably by α-catenin, butthat the rest appears to be unattached (Sako et al., 1998). Theseobservations suggest that the cell-cell adhesion contacts areconstantly rearranged through remodeling of cadherin-catenincomplexes. However, the mechanism underlying this dynamicrearrangement remains to be clarified.

Rho family small GTPases Rho family GTPases cycle between GDP-bound inactive andGTP-bound active forms, formation of the latter beingstimulated by extracellular signals, such as growth factors andhormones (Bourne et al., 1991; Takai et al., 1995). Members ofthis family, which includes Cdc42, Rac1 and Rho, are involvedin regulation of the cytoskeleton and cell adhesion (Van Aelstand D’Souza-Schorey, 1997; Hall, 1998; Kaibuchi et al., 1999).Cdc42 participates in the formation of filopodia and focalcomplexes (Kozma et al., 1995; Nobes and Hall, 1995) and cell-cell adhesion (Kuroda et al., 1997; Kodama et al., 1999). Rac1is involved in the formation of membrane ruffling and focalcomplexes (Ridley et al., 1992), cell motility (Ridley et al.,1995) and cell-cell adhesion (Braga et al., 1997; Takaishi et al.,1997; Kuroda et al., 1997). Rho has been implicated in theformation of actin stress fibers and focal adhesions (Ridley andHall, 1992), cell-cell adhesion (Braga et al., 1997; Takaishi etal., 1997), cell motility (Takaishi et al., 1994), membraneruffling (Nishiyama et al., 1994), smooth muscle contraction(Hirata et al., 1992; Gong et al., 1996), neurite retraction inneuronal cells (Nishiki et al., 1990; Jalink et al., 1994) andcytokinesis (Kishi et al., 1993; Mabuchi et al., 1993).

The molecular mechanisms underlying the above processesare largely unknown. However, some of the pathways thatconnect Rho family GTPases to control of the cytoskeleton andcell adhesion have been established through identification andcharacterization of specific effectors of these GTPases.Researchers have identified many effectors of the Rho familyGTPases, using affinity chromatography, ligand-overlay assaysor the yeast two-hybrid system (Van Aelst and D’Souza-Schorey,1997; Kaibuchi et al., 1999). Rho effectors include Rho-kinase(also known as ROK or ROCK), the myosin-binding subunit(MBS) of myosin phosphatase, protein kinase N (PKN)/PRK1,rhophilin, rhotekin, citron and mDia1. Rac1 effectors includep21-activated kinase (PAK), IQGAP1, Por1, p140Sra-1 andPOSH, and effectors of Cdc42 include PAK, WASP/N-WASP,IQGAP1 and myotonic dystrophy kinase-related Cdc42-bindingkinase (MRCK). Some of these proteins, such as PAK andIQGAP1, interact with both Rac1 and Cdc42 (Van Aelst andD’Souza-Schorey, 1997; Kaibuchi et al., 1999). Here, we focuson how the Rho family GTPases regulate cell-substratum and

cell-cell adhesion through their effectors. Regulation of thecytoskeleton by these GTPases has been reviewed elsewhere(Van Aelst and D’Souza-Schorey, 1997; Hall, 1998; Kaibuchi etal., 1999).

REGULATION OF CELL-SUBSTRATUM ADHESIONBY RHO GTPASES

Several observations suggest that Rho regulates the formation offocal adhesions associated with actin stress fibers. In serum-starved Swiss 3T3 cells, for example, few focal adhesions andactin stress fibers are observed. However, when these cells arestimulated by lysophosphatidic acid (LPA), new stress fibers andnew focal adhesions at the ends of stress fibers appear. Priortreatment of the cells with C. botulinum C3 toxin, an inhibitor ofRho, inhibits the LPA-induced appearance of focal adhesions andstress fibers. Microinjection of dominant active RhoA (RhoAV14)into the cells induces the formation of focal adhesions and stressfibers (Paterson et al., 1990; Ridley and Hall, 1992).

Cdc42 and Rac1 regulate the formation of focal complexes.In serum-starved Swiss 3T3 cells, bradykinin and platelet-derived growth factor (PDGF) promote the formation of focalcomplexes associated with filopodia and lamellipodia,respectively. Dominant negative Cdc42 (Cdc42N17), whichpreferentially binds GDP rather than GTP, and thereby inhibitsthe activation of endogenous Cdc42 by titrating out its GEF,and dominant negative Rac1 (Rac1N17), inhibit bradykinin-induced formation of focal complexes and PDGF-inducedformation of focal complexes, respectively. Dominant activeCdc42 (Cdc42V12) and dominant active Rac1 (Rac1V12) induceformation of focal complexes associated with filopodia(Kozma et al., 1995; Nobes and Hall, 1995) and lamellipodia(Ridley et al., 1992), respectively.

Analyses using dominant active and dominant negativemutants of small GTPases suggest that activation of Cdc42leads to activation of Rac1, which in turn activates Rho (Ridleyet al., 1992; Nobes and Hall, 1995). Recent experiments,however, show that Rac1/Cdc42 and Rho can be antagonistic insome situations, such as during neurite extension in neuronalcells (Kozma et al., 1997; Leeuwen et al., 1997; Hirose et al.,1998; Amano et al., 1998). In N1E-115 neuroblastoma cells, forexample, Rho is implicated in serum- and LPA-induced neuriteretraction and cell rounding, whereas Cdc42 and Rac1 areimplicated in neurite extension. Downregulation of Rho or Rho-kinase induces neurite extension (Kozma et al., 1997; Hirose etal., 1998; Amano et al., 1998), whereas upregulation of Rac1inhibits LPA-induced neurite retraction (Leeuwen et al., 1997).In addition, a more recent study suggests that the antagonisticrelationship between Rac1/Cdc42 and Rho applies to theregulation of focal complexes and focal adhesions. Theexpression of Cdc42V12 or Rac1V12 causes loss of focaladhesions and stress fibers (Manser et al., 1997). The expressionof Rac1N17 leads to a prominent increase in the size of focaladhesions. The expression of dominant active Rho (RhoL63)causes the transformation of pre-existing focal complexes,which have been induced by dominant active Rac1 (Rac1L61),into focal adhesions. Treatment of the cells with Y27632, aninhibitor of Rho-kinase, causes a shift in the contact patternfrom focal adhesions to peripheral focal complexes, which isaccompanied by the induction of membrane ruffling (Rottner et

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4493Cell adhesion and Rho small GTPases

al., 1999). These observations suggest that Rac1/Cdc42 andRho exert mutually antagonistic effects at some point in theprocess of contact initiation, maturation and turnover, possiblythrough their effectors, PAK and Rho-kinase (see below).

Rnd proteins (Rnd1, Rnd2 and Rnd3/RhoE), a newsubfamily of Rho-related proteins, also appear to regulateintegrin-mediated cell adhesion (Nobes et al., 1998; Guasch etal., 1998). Rnd1 has a much higher affinity for GTP than forGDP and lacks intrinsic GTPase activity, which suggests thatit exists as the GTP-bound form in vivo. Prior injection of wild-type Rnd1 inhibits LPA-induced formation of stress fibers andfocal adhesions, leading to cell rounding. Note that Rnd1 mightalso regulate cadherin-mediated cell adhesion: like Cdc42 andRac1 (Kuroda et al., 1997), it colocalizes with E-cadherin atsites of cell-cell contact through its unique N-terminal region(Nobes et al., 1998). Further studies must address how Rndproteins are regulated and how they exert their functions.

The mechanism of regulation of cell-substratumadhesion by Rho GTPasesThe molecular mechanisms underlying Rho-induced formationof focal adhesions have been elucidated through theidentification and characterization of two of its effectors: Rho-kinase and MBS. The expression of dominant active Rho-kinase induces the formation of stress fibers and focaladhesions in Swiss 3T3 and Madin-Darby canine kidney(MDCK) cells, whereas the expression of dominant negativeRho-kinase inhibits LPA- or RhoAV14-induced formation ofstress fibers and focal adhesions (Leung et al., 1996; Amanoet al., 1997; Ishizaki et al., 1997).

Rho-kinase regulates the phosphorylation of the myosinlight chain (MLC) of myosin II by directly phosphorylatingMLC at Ser19 (Amano et al., 1996) and by inactivating myosinphosphatase through phosphorylation of MBS (Kimura et al.,1996). MLC phosphorylation plays a pivotal role in the actin-myosin interaction for stress fibers in non-muscle cells(Huttenlocher et al., 1995) and in smooth-muscle contraction(Kamm et al., 1985). The expression of the MLC mutantMLCT18D,S19D, which mimics the phosphorylated form ofMLC and causes activation of myosin ATPase and aconformational change in myosin II when reconstituted withmyosin heavy chain in vitro (Kamisoyama et al., 1994;Sweeney et al., 1994; Bresnick et al., 1995), also enhances theformation of stress fibers and focal adhesions (Amano et al.,1998, 1999). Taken together, these observations indicate thatRho, through Rho-kinase, stimulates actomyosin-basedcontractility and that this contractility plays a critical role inthe assembly of stress fibers and focal adhesions. However, thestress fibers induced by activated Rho and those induced bydominant active Rho-kinase are not identical. Dominant activeRho-kinase induced stress fibers are thicker and more sparsethan those induced by activated Rho. A more recent study hasshown that cooperation between mDia1, another effector ofRho, and Rho-kinase is necessary for the formation of fullyrefined stress fibers (Watanabe et al., 1999).

The best-characterized physiological situation in whichregulation of focal adhesions by Rho is implicated is leukocyteadhesion, which is critical to immunity and inflammation(Laudanna et al., 1996). These authors report that inactivationof Rho by C3 blocks agonist-induced lymphocyte α4β1-integrin-mediated adhesion to vascular cell adhesion molecule

1 (VCAM-1) and neutrophil β2-integrin-mediated adhesion tofibrinogen (Laudanna et al., 1996). It remains to be determinedwhether the Rho/Rho-kinase system is involved in theregulation of cell-substratum adhesion in leukocyte adhesion.

The molecular mechanisms by which Cdc42 and Rac1regulate the formation of focal complexes are less wellcharacterized. Several effectors of Rac1 and Cdc42, such asMRCKα and PAK, have been implicated in this process.MRCKα, like Rho-kinase, preferentially phosphorylates MLCat Ser19 in vitro (Leung et al., 1998). Coexpression of MRCKαwith Cdc42V12 enhances the Cdc42V12-induced formation offilopodia. The expression of dominant negative MRCKαblocks Cdc42V12-dependent formation of focal complexes andfilopodia, which suggests that MRCKα regulates the formationof focal complexes and filopodia, presumably by regulating thephosphorylation of MLC.

PAK is also implicated in the development of focalcomplexes and lamellipodia (Manser et al., 1997; Sells et al.,1997). PAK localizes to sites of focal complexes throughinteraction with PIX, a GEF for Rac1 (Manser et al., 1998).This interaction is necessary for PAK-induced lamellipodiaformation (Obermeier et al., 1998). Moreover, PAK-inducedlamellipodia formation is inhibited by Rac1N17. Hence, theCdc42-dependent activation of PAK leads to a PIX-mediatedactivation of Rac1. However, whether and, if so how, PAKgenerates focal complexes downstream of Cdc42 or Rac1 hasyet to be elucidated.

PAK might however be involved in the dissolution of focaladhesions induced by Rho. PAK phosphorylates andsuppresses myosin light chain kinase, which leads to reducedlevels of phosphorylation of MLC (Sanders et al., 1999). Theexpression of a constitutively active PAK, similar to expressionof Cdc42V12 or Rac1V12, causes a loss of focal adhesions andstress fibers (Manser et al., 1997; Zhao et al., 1998). Thus, PAKmight regulate the turnover of focal adhesions downstream ofCdc42 and Rac1 (Fig. 1). Two groups, however, reported thatPAK phosphorylates MLC at Ser19 in vitro (Chew et al., 1998;Van Eyk et al., 1998) and that the expression of dominant activePAK, but not kinase-dead PAK, increases the level ofphosphorylation of MLC in vivo (Sells et al., 1999). Thus, thephysiological role of PAK in the regulation of phosphorylationof MLC remains unresolved.

Upstream regulation of Rho GTPases in cell-substratum adhesionRecently, two groups reported that integrin-mediated cell-substratum adhesion, like stimulation by growth factors,induces the activation of the Rho family GTPases (Price et al.,1998; Ren et al., 1999). The engagement of integrins withfibronectin activates Cdc42, which subsequently activates Rac1during cell spreading 5 minutes after the attachment of cells tothe substratum (Price et al., 1998). Moreover, activation of Rhocan occur later independently of the activation of Cdc42 (Priceet al., 1998).

How does integrin-mediated cell adhesion activate Rhofamily GTPases? Regarding Rac1, one potential candidate isVav, which is a GEF for Rac1. The GEF activity of Vav towardRac1 is enhanced by tyrosine phosphorylation (Crespo et al.,1997; Han et al., 1997). Yron et al. (1999) have presentedevidence to suggest that integrin-mediated cell adhesioninduces tyrosine phosphorylation of Vav. Thus, integrin-

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mediated cell adhesion probably activates Rac1 viaphosphorylation of Vav. Further work will be required if weare to elucidate how integrin-mediated cell adhesion activatesthe Rho family GTPases.

REGULATION OF CELL-CELL ADHESION BY RHOGTPASES

Recently, several investigations have suggested that the Rhofamily GTPases are required for cadherin-mediated cell-celladhesion. Microinjection of Rac1N17 or C3 inhibits theaccumulation of cadherin at sites of cell-cell contact whenkeratinocytes are transferred from low-calcium medium tostandard medium (which restores calcium-dependent cell-celladhesion) (Braga et al., 1997). The effects of Rac1 and Rho onthe localization of cadherin probably depend on the maturationstatus of the junction (i.e. on the time after induction ofcontacts and on confluence) and the cell types (Braga et al.,1999); for example, Rac1 and Rho are required for localizationof E-cadherin to sites of cell-cell contact in keratinocytes,whereas they are not required for the VE-cadherin localizationin human umbilical cord endothelial cells.

Takaishi et al. (1997) have shown that, in MDCK cells stablyexpressing Rac1V12, the immunofluorescent intensities of E-cadherin, β-catenin and actin filaments at sites of cell-cellcontact are increased relative to those in the parental cells. Bycontrast, in cells expressing Rac1N17, the immunofluorescentintensities of E-cadherin, β-catenin and actin filaments arereduced (Takaishi et al., 1997). Microinjection of C3 inhibitscadherin-mediated cell-cell adhesion in wild-type MDCKcells, but not in MDCK cells expressing Rac1V12. Thissuggests that Rho activity is required for cadherin-mediatedcell-cell adhesion but not for a Rac1-regulated cadherin-mediated cell-cell adhesion pathway. (Takaishi et al., 1997).

In addition, we have shown that Cdc42, Rac1 and Rho arerequired for E-cadherin-mediated cell-cell adhesion in MDCK

cells (Kuroda et al., 1997). Microinjection of Rho GDI, anegative regulator of Cdc42, Rac1 and Rho, results inperturbation of cell-cell adhesion in MDCK cells. Co-microinjection of either Cdc42V12 or Rac1V12, but notRhoAV14, with Rho GDI reverses the inhibitory effect (Kurodaet al., 1997). In addition, Tiam1, which colocalizes with E-cadherin at sites of cell-cell contact and has GEF activity forRac1, is implicated in the regulation of cell-cell adhesion inMDCK cells (Hordijk et al., 1997). Expression of Tiam1 orRac1V12 inhibits scatter factor/hepatocyte growth factor(HGF)-induced cell scattering of MDCK cells, probably byincreasing E-cadherin activity (Hordijk et al., 1997). Thus,Cdc42, Rac1 and Rho, as well as Tiam1, appear to regulatecadherin-mediated cell-cell adhesion.

The above experiments did not indicate whether the Rhofamily GTPases regulate E-cadherin-mediated cell-celladhesion by acting directly on the cadherin-catenins complexor by acting indirectly on the actin cytoskeleton or othercytoskeleton components. This issue has been addressed by useof the cell dissociation assay (a quantitative assay for E-cadherin activity) in three stable cell lines derived from mouseL fibroblasts (which do not express endogenous cadherins): (1)EL cells that express E-cadherin and adhere to each other inan E-cadherin-dependent manner (Nagafuchi et al., 1994); (2)nEαCL cells expressing an E-cadherin mutant in which thecytoplasmic domain is deleted and replaced by the C-terminaldomain of α-catenin, and the cadherin-catenins complex is notremodeled (Nagafuchi et al., 1994); (3) LAP86 cells thatexpress neuropilin-1, which is a calcium-independent cell-adhesion molecule (Kawakami et al., 1996), and require neitheran actin-based cytoskeleton (which EL and nEαCL cellsrequire) nor catenins for adhesion (Fukata, M. et al., 1999).

Expression of Cdc42N17 or Rac1N17 in EL cells markedlyreduces E-cadherin activity, whereas expression of dominantnegative RhoA (RhoAN19) slightly reduces it (Fukata, M. et al.,1999). In contrast, expression of Cdc42N17, Rac1N17 orRhoAN19 in nEαCL cells slightly reduces the mutant E-

M. Fukata and others

IntegrinPlasma membrane

ECM

F actin

Myosin

Vinculin

Talin

Vinculin

Talin

Focal adhesionassembly

Vinculin

Talin

β

Vinculin

Talin

Stress fiber(Contraction)

p

Rho

GTP

Rho-kinase MBS

Rac1Cdc42

GTP

PAK

?

Focal adhesiondisassembly

p

pp

β β βαααα

Fig. 1. Antagonistic effect of Rho andRac1/Cdc42 on the formation of focaladhesions. Rho, through Rho-kinase, playsa critical role in the formation of focaladhesions and stress fibers by activatingmyosin II, whereas Rac1/Cdc42, throughPAK, disassembles focal adhesions andstress fibers. α, integrin α; β, integrin β.

4495Cell adhesion and Rho small GTPases

cadherin activity. Furthermore, expression of Cdc42N17,Rac1N17 or RhoAN19 in LAP86 cells does not affect adhesion.Since β-catenin, the E-cadherin cytoplasmic domain and theN-terminal region of α-catenin are not required for theadhesion in nEαCL cells, and the effect of Cdc42N17 orRac1N17 in nEαCL cells is less than that in EL cells, Cdc42and Rac1 appear to regulate E-cadherin-mediated celladhesion, acting directly on the cadherin-catenins complex.Since RhoAN19 has similar effects on E-cadherin activity in ELcells and nEαCL cells (whose adhesion requires the actincytoskeleton) and no effect in LAP86 cells (whose adhesiondoes not require the actin cytoskeleton), Rho presumably

regulates E-cadherin activity indirectly by acting on the actincytoskeleton (Fukata, M. et al., 1999).

The mechanism of regulation of cell-cell adhesionby Rho GTPasesWe have recently proposed a mechanism by which Cdc42 andRac1 might regulate E-cadherin activity (Kuroda et al., 1998,1999). We and others have shown that an effector of Cdc42 andRac1, IQGAP1, colocalizes with cadherin-catenins at sites ofcell-cell contact (Hart et al., 1996; Kuroda et al., 1996, 1998).IQGAP1 might therefore regulate E-cadherin-mediated cell-cell adhesion. IQGAP1 accumulates at sites of cell-cell contact

Strong adhesion

F actin

EE

GTPIQGAP1

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Rho GDIGDP

E

Weak adhesion

E

αβ

α

αβ

αβ

E

EE E E

RhoGTP

Rho GDIGDP

E E

β

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Rho

Cdc42/Rac1

Fig. 2. Role of IQGAP1 in the regulation ofE-cadherin-mediated cell-cell adhesion.Cdc42 (and Rac1) and IQGAP1 can serve aspositive and negative regulators of cadherinactivity, respectively. E, E-cadherin; α, α-catenin; β, β-catenin.

RhoAN19 ++Rac1N17 +++

5~15 min

Cdc42V12 +++Rac1 V12 ++

2~ 6 h

Scattering( Cell adhesion ↓)

Membrane ruffling ( Motility ↑ )

HGF, phorbol ester

Cdc42N17+ RhoAV14 +

Fig. 3. Role of the Rho family GTPasesin the regulation of cell scattering.Hepatocyte growth factor (HGF) orphorbol ester induces membrane rufflingfollowed by cell scattering in MDCKcells. Activation of Rac1, Rho and Cdc42is probably necessary for membraneruffling, and inactivation of Cdc42, Rac1and Rho is probably essential fordisruption of cell-cell adhesion duringcell scattering. + represents the extent ofinhibitory activity of each GTPase mutanttoward membrane ruffling and cellscattering.

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in EL cells but not in nEαCL cells, which indicates that thisaccumulation depends on the presence of the cytoplasmicdomain of E-cadherin and β-catenin (Kuroda et al., 1998).IQGAP1 directly interacts with β-catenin and the cytoplasmicdomain of E-cadherin both in vitro and in vivo, but not withα-catenin (Kuroda et al., 1998). Since the binding affinity ofIQGAP1 for β-catenin is higher than that of E-cadherin in vitro(half-maximal binding is at 20 nM and 400 nM, respectively)and in vivo (Kuroda et al., 1998), it is likely that IQGAP1functions mainly through β-catenin. IQGAP1 interacts with theN-terminal region of β-catenin (residues 1-183), whichcontains the α-catenin-binding domain (residues 120-151)(Fukata, M. et al., 1999).

IQGAP1 and α-catenin compete for binding to β-catenin,and IQGAP1 dissociates α-catenin from the β-catenin-α-catenin complex in vitro (Fukata, M. et al., 1999). In addition,overexpression of IQGAP1 in EL cells, but not in nEαCL cells,results in the dissociation of α-catenin from the cadherin-catenins complex and in a decrease in the level of E-cadherin-mediated cell-cell adhesion (Kuroda et al., 1998). Thus,IQGAP1 appears to regulate E-cadherin-mediated adhesion invivo by causing α-catenin to dissociate from the cadherin-catenins complex (Kuroda et al., 1998).

How do Cdc42 and Rac1 regulate the E-cadherin-mediatedcell-cell adhesion through IQGAP1? Cdc42 and Rac1 boundto guanosine 5′-(3-O-thio)triphosphate (GTPγS), a non-hydrolyzable analog of GTP, inhibit the interaction betweenIQGAP1 and β-catenin in vitro, whereas their GDP-boundforms and GTPγS-RhoA do not (Fukata, M. et al., 1999).Coexpression of Cdc42V12 with IQGAP1 in EL cells inhibitsthe dissociation of α-catenin from the cadherin-cateninscomplex induced by IQGAP1 (Fukata, M. et al., 1999) andcounteracts the inhibitory effect of IQGAP1 on the E-cadherin-mediated cell-cell adhesion (Kuroda et al., 1998). ActivatedCdc42 must therefore suppress the inhibitory action ofIQGAP1 by preventing IQGAP1 from interacting with β-catenin and thereby stabilize the cadherin-catenins complex.

These observations suggest a mechanism by which Cdc42and Rac1, together with IQGAP1, regulate cadherin-mediatedcell-cell adhesion (Fig. 2). In their GTP-bound, active forms,Cdc42 and Rac1 interact with IQGAP1, preventing it frominteracting with β-catenin and thereby stabilizing the cadherin-catenins complex. This results in strong cell-cell adhesion. Incontrast, in their GDP-bound, inactive forms, Cdc42 and Rac1cannot interact with IQGAP1, and IQGAP1 interacts with β-catenin, dissociating α-catenin from the cadherin-cateninscomplex. This results in weak cell-cell adhesion. Such a modelcan explain the dynamic rearrangement of cell-cell adhesion.A mixture of E-cadherin-β-catenin-α-catenin and E-cadherin-β-catenin-IQGAP1 complexes should exist at sites of cell-cellcontact. The ratio between the two complexes could determinethe adhesive activity. The fact that approximately 50% of E-cadherin appears to be connected to the actin cytoskeleton,probably via α-catenin in EL cells, but that the rest appears tobe free, is consistent with this (Sako et al., 1998).

Most GTPases known thus far exert their functions bybinding to effectors and changing the activities of effectors. Forexample, GTP-bound Gsα binds to adenylyl cyclase, causingits activation, and GTP-bound Ras binds to Raf, causing itsactivation. The interaction between GTP-bound Cdc42/Rac1and IQGAP1, which inhibits IQGAP1 function by preventing it

from interacting with β-catenin, represents a different case. Thissequestration of IQGAP1 by Cdc42 and Rac1 might be a novelmechanism for GTPases action. Giα acts similarly on adenylylcyclase (type V), which is inhibited by GTP-bound Giα but notby GDP-bound Giα, through direct interaction (Taussig et al.,1994; Dessauer et al., 1998; Wittpoth et al., 1999).

Analyses by electron microscope have recently revealed thatthe morphology of the cell-cell adhesion site in MDCK cellsstably expressing Rac1V12 is different from that of thoseexpressing Cdc42V12. The lateral membranes in the formerintermingle more markedly than those in cells expressingCdc42V12 (Takaishi et al., 1997; Kodama et al., 1999), whichsuggests that IQGAP1, downstream of Cdc42 and Rac1,regulates cadherin activity cooperatively with Cdc42- or Rac1-specific effectors, such as MRCK.

MRCK is another effector of Cdc42 and localizes to sites ofcell-cell contact when coexpressed with Cdc42V12 in HeLacells (Leung et al., 1998). It can phosphorylate MLC (Leunget al., 1998), which suggests that MRCK regulates actomyosin-based contractility at sites of cell-cell contact by such amechanism. Cadherin activity could therefore be duallyregulated through direct regulation of the cadherin-cateninscomplex by IQGAP1 and indirect regulation of actin filamentsby MRCK downstream of Cdc42 and Rac1.

Upstream regulation of Rho GTPases in cell-celladhesionThe above model and analogies with integrin-mediated celladhesion lead us to speculate that E-cadherin-mediated cell-cell adhesion induces activation of the Rho family GTPases.Very recent observations support this idea. The engagement ofE-cadherins in homophilic calcium-dependent cell-cellinteractions results in a rapid phosphoinositide 3-kinase (PI 3-kinase)-dependent activation of Akt/PKB in MDCK cells (Peceet al., 1999). Activated PI 3-kinase activates Rac1 (Kotani etal., 1994, 1995; Hawkins et al., 1995; Reif et al., 1996).Therefore, it may prove worthwhile to examine whether cell-cell contact induces the activation of the Rho family GTPases.Indeed, by measuring the level of GTP-bound Rac1, we havealready found that, during cadherin-mediated cell adhesion, theamount of activated Rac1 increases (M. Nakagawa, M. Fukata,S. Kuroda and K. Kaibuchi, unpublished data).

The role of tyrosine phosphorylationOver the past ten years, a considerable number of studies onthe regulation of cadherin function have been performed. Themechanisms underlying regulation, however, are not fullyunderstood. Among them, tyrosine phosphorylation of β-catenin is the best-studied mechanism. Increased levels oftyrosine phosphorylation of β-catenin frequently characterizedysfunctional cadherin-mediated cell-cell adhesion induced byv-Src (Matsuyoshi et al., 1992; Behrens et al., 1993) or bytreatment with growth factors such as epidermal growth factor(EGF) or HGF (Shibamoto et al., 1994). Treatment of cellswith pervanadate, a potent inhibitor of tyrosine phosphatases,also induces perturbation of cadherin-mediated cell-celladhesion, which is accompanied by tyrosine phosphorylationof β-catenin and dissociation of α-catenin from the cadherin-catenins complex (Ozawa and Kemler, 1998a). Therefore, thetyrosine phosphorylation of β-catenin probably plays a crucialrole in regulation of cadherin function.

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Given these findings, together with our model for IQGAP1-mediated regulation of cadherin, it is tempting to speculate thattyrosine-phosphorylated β-catenin recruits IQGAP1 or GTPase-activating proteins (GAPs) for Cdc42 and/or Rac1 to sites of cell-cell contact. This would cause a reduction in cadherin activitythrough inhibition of Cdc42 and/or Rac1 activity. Takeda et al.(1995), however, have reported that tyrosine phosphorylation ofβ-catenin is not required for the reduction in cadherin activityinduced by v-Src, given that expression of v-Src reduces cadherinactivity in nEαCL cells as well as in EL cells. Thus, thephysiological role of the tyrosine phosphorylation of β-catenin incadherin function remains unresolved.

Cadherin dimerizationAnalyses by NMR spectroscopy and X-ray crystallographysuggest that the extracellular domain of cadherin forms adimer. They show that the two monomers interact withmonomers from different dimers on the opposite membrane(see Fig. 2), which leads to zipper-like adhesion (Overduin etal., 1995; Shapiro et al., 1995). Therefore, dimerization ofcadherin is another crucial contributor to strong adhesion. Themechanism, however, underlying dimer formation remains tobe elucidated. Recent studies suggest that the membrane-proximal region of the cadherin cytoplasmic domain, which isseparate from the distal β-catenin-binding domain, regulatescadherin dimerization and activity. In the case of E-cadherin,the membrane-proximal region inhibits dimerization andadhesive activity (Ozawa and Kemler, 1998b), whereas in thecase of C-cadherin, this region supports lateral clustering of C-cadherin (Yap et al., 1998). Thus, the role of the membrane-proximal region of cadherin remains to be determined.

Given that p120ctn (Ozawa and Kemler, 1998b; Yap et al.,1998) and δ-catenin (Lu et al., 1999), members of theArmadillo/β-catenin family, bind to the membrane-proximalregion of the cadherin cytoplasmic domain, they mightmodulate E-cadherin-mediated cell-cell adhesion by regulatingdimerization of cadherin. Recently, p120ctn was shown toinhibit cadherin activity in some carcinoma cell lines (Aono etal., 1999; Ohkubo and Ozawa, 1999), and the expression of δ-catenin increases the cell scattering induced by HGF in MDCKcells (Lu et al., 1999). Further studies are necessary if we areto elucidate how p120ctn and δ-catenin regulate cadherinactivity and which signalling pathway (e.g. tyrosine kinase orRho family GTPases) is involved. Given that IQGAP1 binds toE-cadherin as well as β-catenin in vitro and in vivo (Kuroda etal., 1998), it might be also involved in the regulation of E-cadherin dimerization.

Physiological processes in which the Rho familyGTPases regulate cadherin activityCadherin-mediated cell-cell adhesion is dynamicallyrearranged in various situations, such as during cell scattering,compaction of early embryogenesis, wound-induced cellmigration and tumorigenesis (Takeichi, 1995; Gumbiner, 1996;Adams and Nelson, 1998). The physiological processes inwhich the Cdc42/Rac1/IQGAP1 system operates remain to beclarified, however.

Cell scattering provides one of the most prominent examplesof dynamic rearrangement of cell-cell adhesion (Takeichi, 1995;Gumbiner, 1996). HGF or phorbol ester induces membraneruffling and centrifugal spreading of MDCK cells in colonies and

subsequently stimulates cell-cell dissociation followed by cellscattering (Hartmann et al., 1994). Dysfunction of E-cadherin-mediated cell-cell adhesion is believed to be essential for cellscattering. Rho family GTPases are implicated in the regulationof cell scattering induced by HGF or phorbol ester (Takaishi etal., 1994, 1995; Ridley et al., 1995). Microinjection of RhoAN19

or Rac1N17 inhibits HGF-induced membrane ruffling andsubsequent cell scattering of MDCK cells (Fukata, Y. et al.,1999). Moreover, microinjection of Cdc42V12 or Rac1V12 intoMDCK cells blocks the disruption of cell-cell adhesion duringthe HGF- and phorbol ester-induced cell scattering (Kodama etal., 1999; M. Fukata, M. Nakagawa, S. Kuroda and K. Kaibuchi,unpublished data). Thus, activation of Rho and Rac1 is probablynecessary for membrane ruffling, and the inactivation of Cdc42and Rac1 appears to be essential for disruption of cell-celladhesion during the cell scattering (Fig. 3). In addition, α-catenin-deficient mouse teratocarcinoma F9 cells display thescattered phenotype, under the conditions in which parental or α-catenin-reexpressing cells form compact colonies (Maeno et al.,1999). Taken together, these results suggest that we shouldexamine whether IQGAP1 plays a pivotal role in the suppressionof cadherin activity during the cell scattering.

Dynamic rearrangement in E-cadherin-mediated cell-celladhesion underlies the compaction of the eight-cell embryo,in which the embryo develops from a collection of looselyadherent blastomeres into a tightly packed epithelium calleda blastocyst (Fleming and Johnson, 1988). Gastrulationprovides another example of dynamic rearrangement of thecadherin-catenins complex. In sea urchin embryos, cadherinis localized to sites of cell-cell contact throughoutgastrulation, whereas α-catenin staining at the sites decreasesmarkedly (Miller and McClay, 1997a,b). Further studies mustdetermine whether the Cdc42/Rac1/IQGAP1 system isinvolved in the regulation of cell-cell adhesion duringcompaction and gastrulation.

CONCLUSION

Over the past several years, researchers have madeconsiderable effort to elucidate the mechanisms by which Rhofamily GTPases regulate the cytoskeleton and cell adhesions.Many effectors have been identified and characterized. As aresult, some of the pathways that connect these GTPases tocontrol of the cytoskeleton and cell adhesion have beenestablished. For example, we now know that Rho-kinase isessential for integrin-mediated cell adhesion and is involved inMLC phosphorylation, which leads to the formation of focaladhesions. In addition, Cdc42 and Rac1, together withIQGAP1, are involved in regulation of cadherin-mediated cell-cell adhesion. The next step is to elucidate the physiologicalphenomena taking advantage of these mechanisms.

We thank Drs M. Takeichi, A. Nagafuchi, S. Tsukita (KyotoUniversity, Japan) and Dr W. J. Nelson (Stanford University, USA)for helpful discussion during the course of our work, and N. Itoh, A.Kawajiri and M. Yamaga for preparing the manuscript. The originalwork by the authors has been supported by the Japan Society of thePromotion of Science Research for the Future, by the Ministry ofEducation, Science and Culture, Japan, by the Human FrontierScience Program, and by a grant from Kirin Brewery CompanyLimited.

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