Regulation of actin cytoskeleton by Rho-family GTPases andtheir associated proteinsLouis Lim*,†, Christine Hall* and Clinton Monfries*,†

Cdc42, Rac and Rho GTPase each regulates distinctmorphological changes in response to specific growth factors.These changes which involve actin-containing cytoskeletalstructures may underlie aspects of growth and development.Proteins binding to the active GTP-bound form of theGTPase including the Cdc42/Rac activated PAK, and theRho associated kinase ROK can act as morphologicaleffectors as can the RacGAP chimaerin. In fibroblasts andneuronal-type cells, the growth factors evoke morphologicalchanges by activating individual GTPase pathways orRac-Rho and Cdc42-Rac hierarchical pathways. There is alsoevidence for Cdc42-Rho antagonism. The morphologicaloutcome will depend on the level of activation of the differentGTPases by their stimulatory growth factor.

Key words: actin morphology / chimaerin / PAK / RhoGTPases / ROK

©1996 Academic Press Ltd

MANY OF THE CELL’S responses to its environmentinvolve dynamic changes in shape. Cell growth andcytokinesis, cell motility, substrate adhesion and theformation of cellular contacts, phagocytosis and localmovements of the plasma membrane all require aseries of rearrangements of the actin cytoskeleton,regulated by the Ras-related family of Rho GTPases.Cell proliferation, secretion, intracellular traffickingof components, and nuclear import, centrally involveother Ras superfamily subgroups, Ras, Rab, Arf andRan. The Rho family also plays a critical role in cellgrowth and transformation, co-operating with Ras incell transformation,1 in cytokinesis2 and cell cycleprogression.3

This short essay deals with the relationships of theRho GTPases and associated protein kinases andGAPs in promoting changes in actin-based morphol-ogy, particularly in fibroblasts and neuroblastoma

cells. In the interest of brevity, we have not consideredRho-associated proteins involved in phospholipidmetabolism and actin polymerization/depolymeriza-tion events, including PI-3 kinase and phosphatidyli-nositol 4-phosphate 5-kinase.

Rho-family GTPases and their associatedproteins

The Rho-family GTPases with approximately 30%sequence similarity to Ras include RhoA, RhoB,RhoC, Rac1, Rac2 and Cdc42Hs/G25K. They arehighly conserved, functioning in all eukaryotic cells.As with all Ras-like GTPases they function as molec-ular switches, cycling between active GTP-bound andinactive GDP-bound states, in pathways integratingsignals from diverse receptors in response to extrac-ellular stimuli.4 The GTP/GDP cycle of the RhoGTPases is modulated by guanine nucleotideexchange factors (GEFs) which up-regulate by stim-ulating GTP-loading, GDP-dissociation inhibitorswhich inhibit exchange activity and maintain theGDP-bound p21 in a soluble complex and down-regulating GTPase activating proteins (GAPs) whichstimulate their intrinsic GTPase.

There is a large family of sequence-related GAPs forthe Rho-family proteins with bcr-homology domain,including abr, chimaerin, p190 Rho GAP.5 A family ofGEFs exists with homology to the oncogene dbl, e.g.dbs, ost, tiam.6 Some of these GEFs have celltransforming potential, which led to their initialisolation.

Proteins interacting only with the GTP-bound formof the Rho-family and which represent effector targetshave recently been isolated. They include the Cdc42/Rac activated serine/threonine kinase PAK,7 thetyrosine kinase ACK,8 which contain related GTPase-binding domains and Rho-interacting serine/threo-nine kinases.9-12 Other targets are the Wiskott AldrichSyndrome protein, WASP,13,14 PI-3 kinase15 and phos-phatidylinositol 4-phosphate 5-kinase.16,17

From the *Institute of Neurology, 1 Wakefield Street, London,WC1N 1PJ, UK and †Glaxo-IMCB Group, Institute of Molecularand Cell Biology, National University of Singapore, Singapore119260

seminars in CELL & DEVELOPMENTAL BIOLOGY, Vol 7, 1996: pp 699–706

©1996 Academic Press Ltd1084-9521/96/050699 + 08 $25.00/0

699

All these GTPase-interacting proteins have a multi-domain structure, some with several domains/motifssuch as SH2, SH3, PH (plekstrin homology), prolinerich sequences, phorbol ester binding domains, inaddition to one or more catalytic domains.

Actin cytoskeleton and cell morphology

The simplest protrusive actin-containing structureswhich function in shape changes are the needle-likefilopodia, thought to have a sensory role at theleading edge of motile cells18 and in neuronal growthcones.19,20 Lamellipodia, sheet-like protrusions, mayform a web between two filopodia or generate rufflesas the membrane moves forward and lifts. Actin stressfibres are long bundles, traversing the cells which abutthe plasma membrane at focal adhesions, which formattachment points to the substratum. The focaladhesion complexes contain various componentssuch as viculin, paxillin, p125FAK and talin, linking tointegrins at the inner membrane surface. At theleading edge of cells these are transient attachments.The plasma membrane closely interconnects with theunderlying actin network and in the formation ofperipheral protrusions, actin polymerization at theleading edge can theoretically produce the force topush the membrane forward. Forces generatingmovement in the actin cytoskeleton arise from actinfilament polymerization, with associated ATP hydroly-sis, and from forces generated by motor proteins, suchas myosin.21,22 Other cytoskeletal components such asmicrotubules, spindle assembly components andintermediate filaments functioning independently inprocesses affecting cell morphology, also intercommu-nicate with the actin cytoskeleton.

Rho and Rac relationships in fibroblastmorphology

Much of our understanding of the role of Rac andRho has come from studies on fibroblasts which showdistinct morphological responses to certain growthfactors present in serum. These factors act throughheterotrimeric G-protein coupled serpentine recep-tors (e.g. lysophosphatidic acid [LPA], bombesin) orreceptor tyrosine kinases (e.g. PDGF, EGF) which feedinto signalling pathways activating Rho and Rac.Microinjection of recombinant wild type Rac and Rhoor constitutively-activated, GTPase defectiveRhoAG14V, Rac1G12V and dominant negative Rac1T17N

mutants demonstrated their specificities in effectingactin reorganization.23,24 The dominant negativemutants have a higher affinity for GDP and blockendogenous protein signalling, possibly by titratingGEFs.25 Rho is selectively inactivated by Clostridiumbotulinum C3 transferase, which ADP-ribosylates thecritical Asn41.26,27

Microinjection of constitutively-activated RhoAG14V

into Swiss 3T3 fibroblasts generated stress fibreformation and the assembly of focal adhesions.23

These structures were also rapidly generated by LPAwhose action was inhibited by C3 transferase, LPAeffects, but not those of microinjected Rho, can beinhibited by tyrphostin, suggesting that there istyrosine kinase activity both upstream28 and down-stream29 of Rho in this pathway.

Rho-dependent stress fibres were also observedfollowing the formation of lamellipodia or membraneruffling and pinocytosis as a later response(20–30 min) to microinjected constitutively activatedRac1G12V, and to PDGF, EGF or insulin (although theydid not accumulate to such high density as thoseelicited as an immediate response to LPA or bombe-sin). These morphological effects were mediated byRac1 acting downstream of PDGF, EGF and insulin,being inhibited by dominant negative Rac1G12VT17N.24

This led to the concept of a hierarchical relationshipbetween Rac and Rho, with Rho being activated bycertain factors through Rac. Rac signalling to Rhomay involve Rac-mediated arachidonic acid produc-tion and subsequent leukotriene-dependent Rho acti-vation.30 Bombesin activates Rac and Rho independ-ently in fibroblasts.24

Ras induces membrane ruffling31 and actsupstream of Rac in fibroblasts.24 Evidence that Rho aswell as Rac mediate morphological events associatedwith Ras-dependent transformation can be found in arecent review.1

Cdc42 relationships with Rac and Rho

Microinjection of Cdc42Hs into Swiss 3T3 fibroblastswas found to promote formation of peripheral actinmicrospikes (PAMs) which include filopodia.32 Subse-quently lamellipodia formation and membrane ruf-fling occurred. This Cdc42-induced ruffling was inhib-ited by co-injection of dominant negative Rac1T17N.Treatment with bradykinin also resulted in similarmorphological effects, with formation of PAMs orfilopodia being followed by ruffling. Bradykinin isthought to act through serpentine receptor-linked

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G-proteins. These effects of bradykinin were inhibitedby prior microinjection of dominant negativeCdc42HsT17N while Rac1T17N only inhibited ruffling/lamellipodia formation. These results indicate thatbradykinin acts by activating Cdc42 which subse-quently activates Rac.

Cdc42Hs microinjection while promoting PAMsalso elicited a reduction in stress fibres.32 It is possiblethat this reflects the utilization by the two types ofstructures of a common pool of actin, or of similaractin-binding proteins. Whatever the mechanism, thisfinding indicates Cdc42 to oppose Rho-mediatedeffects.

The relationships of Cdc42, Rac and Rho were alsoexamined in another study.33 Microinjection intoSwiss 3T3 fibroblasts of constitutively activatedRacG12V triggered assembly of fine focal complexesaround lamellipodia, which were distinct from Rho-type focal adhesions. Small focal complexes alongfilopodia and at the cell periphery were also detectedin response to constitutively activated Cdc42G12V

when both Rac and Rho were simultaneously inacti-vated (by co-injection of inhibitory RacT17N and C3transferase). A combination of Cdc42G12V, RacT17N

and C3 transferase generated numerous filopodiawithin 5 min; Cdc42G12V and C3 transferase generatedboth lamellipodia and filopodia over the same period.However in the absence of C3 transferase, Cdc42G12V

alone or with RacT17N were reported to generatefilopodia only after a longer period (20 min).Although RacT17N can apparently block Cdc42-induced formation of Rho- and Rac-type focal com-plexes in confluent cells, whether Cdc42 or Cdc42-activating extrinsic factors feed via Rac through toRho seems uncertain. The above findings are con-sistent with Cdc42-Rho antagonism, with inhibition ofRho facilitating Cdc42-type effects.

Rho-family GTPases and neuronal morphology

Recent studies have documented the importance ofthe Rho GTPases in cytoskeletal dynamics of neu-ronal-type cells during development/differentiation.In NIE-115 neuroblastomas and PC12 cells, LPAcauses growth cone collapse, neurite retraction andcell rounding. This effect is blocked by treatment withC3 transferase which by itself promotes neuriteoutgrowth.34

In NIE-115 cells, microinjection of Cdc42Hsdirectly promoted formation of filopodia and lamelli-podia in growth cones and along neurites.35 This

formation was also induced by microinjection of C3transferase, but not when co-injected with dominantnegative Cdc42HsT17N. Correspondingly, co-injectionwith RhoA abolished the effects of Cdc42Hs. Thus, aswith fibroblasts, these two GTPases appear to com-pete; in this case Cdc42 promoting outgrowth andRho retraction of neurites.

The neurotransmitter acetylcholine (ACh) can alsoexhibit growth factor properties.35 ACh treatment ofNE-115 cells, as with Cdc42 injection, promotedsequential formation of filopodia and lamellipodiaalong neurites and in their growth cones. Thisinvolves the muscarinic G-protein linked receptor, butACh was only effective when applied in a concentra-tion gradient. However while filopodia formation wasinhibited by injection of dominant negativeCdc42HsT17N prior to ACh treatment, lamellipodiaformation was unaffected. The latter was inhibited bypre-injection with dominant negative Rac1T17N. ThusACh can activate Rac independently or throughCdc42.

In keeping with a Cdc42-RhoA competition, AChinhibited the neurite retraction effects of LPA. Fur-thermore, cells which exhibited neurite outgrowthwhen starved of serum (presumably containing LPA)no longer did so when injected with Cdc42HsT17N.

PAK as an effector

In the rat, there are at least three PAK isoforms,α-PAK7 and â-PAK36 highly expressed in brain, andthe ubiquitous γ-PAK.37 Homologues from otherspecies include mouse â-PAK (mPAK-3)38 and humanγ-PAK (hPAK65).39 The GTPase-binding and kinasedomains are related7 to those of S. cerevisiae Ste20p, acentral component of the pheromone MAPK path-way.40,41 The pheromone receptors act through het-erotrimeric G-protein â and γ subunits in a signallingcascade now known to link Cdc24 (the GEF forCdc42) and Cdc42 with the PAK-like Ste20p.42,43 Inmammalian cells, Cdc42 and Rac are involved not inthe MAPK pathway but in the JNK/SAPK cascade,44,45

with p38 MAPK being activated by Rho.46 There issome evidence that PAK may act in this pathway.47,48

PAK may thus participate in both nuclear signallingand morphological events.

The morphological activities of α-PAK have beenstudied in epithelial HeLa cells, lacking this PAKisoform.49 On co-transfection with either Cdc42G12V

or RacG12V, α-PAK translocated from the cytosol to

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701

Cdc42- or Rac-like focal complexes containing pax-illin, vinculin and talin. No gross morphologicalchanges occurred on injection of DNA encodingα-PAK, suggesting that its intracellular activation wastightly regulated. This difficulty was circumvented byinjecting DNA encoding constitutively active α-PAKmutants. Although formation of PAMs or ruffles didnot occur, there was considerable loss of stress fibresand focal adhesions. This loss was similar to the effectsof introducing Cdc42G12V or Rac1G12V. These datasupport a role for PAK downstream of both Cdc42 andRac in the dissolution of stress fibres/focaladhesions.

WASP with a related Cdc42-binding domain isexpressed in haemopoietic cells14,50 and has sequencesimilarity to other proline-rich proteins, includingVASP, which is present in focal adhesions, actinfilaments and dynamic membrane structures.51 Actinclustering or aggregation occurred in transfected cellsoverexpressing WASP, but not when these cells areinjected with dominant negative Cdc42T17N.14 Thesignificance of this is still unclear.

ROK as an effector

There are several kinases binding to Rho-GTP includ-ing ROKα,9-11, ROKâ,10 p160ROCK 12 and PKC-relatedPKN.52,53 The ROKs and ROCKs belong to a kinasefamily including the myotonic dystrophy kinase54 andthe Drosophila ‘warts’ gene controlling cell growth andmorphology.55 Whereas Cdc42 stimulates PAK activity100-fold or more, ROKα activity is increased only 2–5fold by RhoA.

ROKα is ubiquitously expressed. In HeLa cells ontransfection with RhoA or RhoAG14V, cytosolic ROKαis translocated to membranes co-localizing with actinmicrofilaments at the cell periphery, and at thecleavage furrow in mitotic cells.9 In Hela cells trans-fection with ROKα DNA resulted in expression ofactive ROKα whose activity was not substantiallyincreased on injection of RhoAG14V. ROKα expressiongenerated formation of stress fibres and focal adhe-sions.10 This ROKα-mediated formation occurredeven in the presence of dominant negative RhoT19N

or C3 transferase, showing ROKα to be downstream ofRho. A kinase dead mutant ROKα was ineffective.Both N-terminal and C-terminal regions were impor-tant. C-terminal truncation caused extensive actincondensation while N-terminal deletion resulted indisassembly of stress fibres and focal adhesions. Theserepresent dominant-positive and -negative mutants.

The Rho-binding domain was not essential for themorphological activities of transfected ROKα. Possiblyover-expression of an active ROKα overcomes theneed for RhoA-binding to effect its translocation toperipheral sites9 where ROKα presumably acts.

ROK and Rho, focal adhesions and stressfibres

In fibroblasts, LPA- and Rho-mediated cellular con-traction preceded formation of stress fibres.56 It wasproposed that contraction was initiated by Rho-mediated phosphorylation of myosin light chains57

and subsequent assembly of myosin filaments. Thiswas followed by alignment of actin filaments (stressfibres) on the myosin filaments and consequentialperipheral aggregation of integrins connected toactin filaments by actin-binding proteins. The Rho-effector ROK can phosphorylate and inactivate themyosin-binding subunit of myosin phosphatase58

which results in activation of other kinases responsiblefor myosin mobilization. This proposal provides aplausible role for stress fibres in the formation ofintegrin-containing focal adhesion complexes. Cer-tainly at mitosis, concomitant loss of stress fibresaccompanies the rounding up of cells; with an actin-based contractile ring then forming at the cleavagefurrow during cytokinesis, for which Rho is alsorequired.2

GAPS as effectors

The multidomain structure of these GAPs e.g. Abr,Bcr, chimaerin (α- and â-) and p190 Rho GAPindicates that targeting to specific locations andcoupling to different effector processes may beimportant for their function. The different GAPdomains, taken in isolation, can selectively down-regulate different GTPases and inhibit specific mor-phological activities. For example, p190 shows Rho-GAP activity in vivo, inhibiting stress fibre formationbut not PDGF-induced ruffling.59 Microinjection ofthe GAP domains of Bcr,59 3BP160 and chimaerin32

prevents membrane ruffling in 3T3 fibroblasts, in linewith their Rac-GAP activities.

Nevertheless, these GAPs may have other than adown-regulating role. Thus microinjection of fulllength n(α1)-chimaerin into fibroblasts induces thesimultaneous formation of lamellipodia and filopo-dia.61 These underwent cycles of dissolution and

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Cdc42-GDP

Cdc42-GTP

dbl

Bradykinin

FilopodiaPAMFocal complexes

Acetylcholine

Rac-GDP

Rac-GTP

tiam

PDGFInsulin EGF

LamellipodiaPinocytosisFocal complexes

Bombesin

Rho-GDP

Rho-GTP

lbc

LPA

ContractilityStress fibresFocal adhesions

Ras

chimaerinBcr3BP1

p190RhoGAP

formation, mimicking morphological events at theleading edge of fibroblasts and neuronal growthcones. The formation of lamellipodia and filopodiawas inhibited by dominant negative Rac1T17N andCdc42HsT17N, respectively. Chimaerin’s GTPase-bind-ing- but not GAP- activity was required. There was anassociated loss of Rho-type focal adhesions andformation of Cdc42- and Rac-type focal complexes.(In fibroblasts, transfection with α1-chimaerin cDNAresulted in a reduced adhesion and decreased forma-tion of focal adhesions62.) As expected from Cdc42/Rac1 effects on neuroblastoma cells,35 chimaerin alsopromoted the simultaneous formation of lamellipodiaand filopodia in their neurite growth cones.61

GTPase hierarchy, antagonism andmorphological differentiation

The use of appropriate mutants has provided goodevidence that morphological effects of trophic andgrowth factors are mediated by Rho GTPases inspecific and sometimes hierarchical ways. For exam-

ple, PDGF activation of Rho is blocked by dominantnegative RacT17N; this mutant does not affect Rhoeffector functions directly since it does not affect LPA-induction (Rho-mediated) of stress fibres. Similarly,Bradykinin activation of Rac is blocked by dominantnegative Cdc42T17N; this mutant does not affectphorbol ester-induction (Rac-mediated) of ruffles andthus does not act on Rac effectors.

Although separate Rac-Rho and Cdc42-Rac hier-archies exist, it is by no means certain that theseextend linearly, i.e. Cdc42-Rac-Rho (see Figure 1). Wecannot exclude that perhaps the hierarchy exists andit is the presence of activated Cdc42 which preventsRho-mediated events from occurring, possiblythrough competition for common morphologicalcomponents. There is certainly evidence of a Cdc42-Rho antagonism. Thus in fibroblasts, Cdc42-inducedformation of PAMs occurs at the expense of stressfibres and focal adhesions, which are promoted byRho. In keeping with this the Cdc42-GTP activatedPAK can induce disassembly of the Rho-inducedstructures (Figure 2). Whether this involves inhibitionof ROK merits further investigation. However since

Figure 1. Activation of Rho-family GTPases associated with specific changes in morphology.Bradykinin activates Cdc42 and subsequently Rac;32 PDGF, insulin and EGF activate Rac andsubsequently Rho24 in Swiss 3T3 fibroblasts. Separate Cdc42-Rac and Rac-Rho hierarchies exist butwhether these extend into a Cdc42-Rac-Rho hierarchy is uncertain, particularly since Cdc42-dependent formation of peripheral actin microspikes (PAMs) is inversely correlated with Rho-dependent formation of stress fibres.32 See text for more discussion of Cdc42-Rho antagonism. Rasacts upstream of Rac.24 Rho is rapidly activated by LPA and other factors, some of which may becell-specific.23,34 Bombesin can separately activate Rac and Rho.24 In NIE-115 neuroblastoma cellsacetylcholine can activate Cdc42 and Rac independently (although microinjected Cdc42 will alsoactivate Rac.35) GTPase activation is up-regulated by GEFs and down-regulated by GAPs. However,the exact in-vivo specificities of the family of GEFs (and some GAPs) are not fully established. TheCdc42- and Rac-focal complexes and Rho-focal adhesions are all different.33,49

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AckWASP actin polymerization

FilopodiaCdc42-GTP

n-(α1-)chimaerin

Rac-GTP

PAK

Ruffles

Rho-GTPPKN

ROK

Myosin phosphatase-P

Myosin light chain-P

ContractilityStress fibresFocal adhesions

Rac-GTP can also stimulate PAK as well as activateRho-induced pathways, this implies a difference in theactivity of PAK in Cdc42- and Rac- pathways.

In neuroblastoma cells, this competition betweenCdc42 and Rho, involving their differential activation,perhaps shapes at any one time the appropriatemorphological response to opposing trophic signalssuch as ACh and LPA. The dynamics of retraction andextension of neuritic structures, particularly at thegrowth cone, may be an essential aspect of theneuronal response to changes in the metabolic milieuwhich determine proper differentiation (or death).When the balance of active and inactive GTPases isupset, as in transgenic mouse cerebellar Purkinjeneurones expressing constitutionally active RacG12V

the morphological consequences can be severe —axon terminals are reduced and there are super-numerary small dendritic spines, indicative of abnor-mal development of different neuronal processes.63

Acknowledgements

We thank the Glaxo-Singapore Research Fund forsupport.

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