Regulation of actin cytoskeleton by Rho-family GTPases and their associated proteins

seminars in

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

Regulation of actin cytoskeleton by Rho-family GTPases and their associated proteins Louis Lim*,†, Christine Hall* and Clinton Monfries*,†

cells. In the interest of brevity, we have not considered Rho-associated proteins involved in phospholipid metabolism and actin polymerization/depolymerization events, including PI-3 kinase and phosphatidylinositol 4-phosphate 5-kinase.

Cdc42, Rac and Rho GTPase each regulates distinct morphological changes in response to specific growth factors. These changes which involve actin-containing cytoskeletal structures may underlie aspects of growth and development. Proteins binding to the active GTP-bound form of the GTPase including the Cdc42/Rac activated PAK, and the Rho associated kinase ROK can act as morphological effectors as can the RacGAP chimaerin. In fibroblasts and neuronal-type cells, the growth factors evoke morphological changes by activating individual GTPase pathways or Rac-Rho and Cdc42-Rac hierarchical pathways. There is also evidence for Cdc42-Rho antagonism. The morphological outcome will depend on the level of activation of the different GTPases by their stimulatory growth factor.

Rho-family GTPases and their associated proteins The Rho-family GTPases with approximately 30% sequence similarity to Ras include RhoA, RhoB, RhoC, Rac1, Rac2 and Cdc42Hs/G25K. They are highly conserved, functioning in all eukaryotic cells. As with all Ras-like GTPases they function as molecular switches, cycling between active GTP-bound and inactive GDP-bound states, in pathways integrating signals from diverse receptors in response to extracellular stimuli.4 The GTP/GDP cycle of the Rho GTPases is modulated by guanine nucleotide exchange factors (GEFs) which up-regulate by stimulating GTP-loading, GDP-dissociation inhibitors which inhibit exchange activity and maintain the GDP-bound p21 in a soluble complex and downregulating GTPase activating proteins (GAPs) which stimulate their intrinsic GTPase. There is a large family of sequence-related GAPs for the Rho-family proteins with bcr-homology domain, including abr, chimaerin, p190 Rho GAP.5 A family of GEFs exists with homology to the oncogene dbl, e.g. dbs, ost, tiam.6 Some of these GEFs have cell transforming potential, which led to their initial isolation. Proteins interacting only with the GTP-bound form of the Rho-family and which represent effector targets have recently been isolated. They include the Cdc42/ Rac activated serine/threonine kinase PAK,7 the tyrosine kinase ACK,8 which contain related GTPasebinding domains and Rho-interacting serine/threonine kinases.9-12 Other targets are the Wiskott Aldrich Syndrome protein, WASP,13,14 PI-3 kinase15 and phosphatidylinositol 4-phosphate 5-kinase.16,17

Key words: actin morphology / chimaerin / PAK / Rho GTPases / ROK ©1996 Academic Press Ltd

MANY OF THE CELL’S responses to its environment involve dynamic changes in shape. Cell growth and cytokinesis, cell motility, substrate adhesion and the formation of cellular contacts, phagocytosis and local movements of the plasma membrane all require a series of rearrangements of the actin cytoskeleton, regulated by the Ras-related family of Rho GTPases. Cell proliferation, secretion, intracellular trafficking of components, and nuclear import, centrally involve other Ras superfamily subgroups, Ras, Rab, Arf and Ran. The Rho family also plays a critical role in cell growth and transformation, co-operating with Ras in cell transformation,1 in cytokinesis2 and cell cycle progression.3 This short essay deals with the relationships of the Rho GTPases and associated protein kinases and GAPs in promoting changes in actin-based morphology, particularly in fibroblasts and neuroblastoma From the *Institute of Neurology, 1 Wakefield Street, London, WC1N 1PJ, UK and †Glaxo-IMCB Group, Institute of Molecular and Cell Biology, National University of Singapore, Singapore 119260 ©1996 Academic Press Ltd 1084-9521/96/050699 + 08 $25.00/0

699

L. Lim et al All these GTPase-interacting proteins have a multidomain structure, some with several domains/motifs such as SH2, SH3, PH (plekstrin homology), proline rich sequences, phorbol ester binding domains, in addition to one or more catalytic domains.

mutants demonstrated their specificities in effecting actin reorganization.23,24 The dominant negative mutants have a higher affinity for GDP and block endogenous protein signalling, possibly by titrating GEFs.25 Rho is selectively inactivated by Clostridium botulinum C3 transferase, which ADP-ribosylates the critical Asn41.26,27 Microinjection of constitutively-activated RhoAG14V into Swiss 3T3 fibroblasts generated stress fibre formation and the assembly of focal adhesions.23 These structures were also rapidly generated by LPA whose action was inhibited by C3 transferase, LPA effects, but not those of microinjected Rho, can be inhibited by tyrphostin, suggesting that there is tyrosine kinase activity both upstream28 and downstream29 of Rho in this pathway. Rho-dependent stress fibres were also observed following the formation of lamellipodia or membrane ruffling and pinocytosis as a later response (20–30 min) to microinjected constitutively activated Rac1G12V, and to PDGF, EGF or insulin (although they did not accumulate to such high density as those elicited as an immediate response to LPA or bombesin). These morphological effects were mediated by Rac1 acting downstream of PDGF, EGF and insulin, being inhibited by dominant negative Rac1G12VT17N.24 This led to the concept of a hierarchical relationship between Rac and Rho, with Rho being activated by certain factors through Rac. Rac signalling to Rho may involve Rac-mediated arachidonic acid production and subsequent leukotriene-dependent Rho activation.30 Bombesin activates Rac and Rho independently in fibroblasts.24 Ras induces membrane ruffling31 and acts upstream of Rac in fibroblasts.24 Evidence that Rho as well as Rac mediate morphological events associated with Ras-dependent transformation can be found in a recent review.1

Actin cytoskeleton and cell morphology The simplest protrusive actin-containing structures which function in shape changes are the needle-like filopodia, thought to have a sensory role at the leading edge of motile cells18 and in neuronal growth cones.19,20 Lamellipodia, sheet-like protrusions, may form a web between two filopodia or generate ruffles as the membrane moves forward and lifts. Actin stress fibres are long bundles, traversing the cells which abut the plasma membrane at focal adhesions, which form attachment points to the substratum. The focal adhesion complexes contain various components such as viculin, paxillin, p125FAK and talin, linking to integrins at the inner membrane surface. At the leading edge of cells these are transient attachments. The plasma membrane closely interconnects with the underlying actin network and in the formation of peripheral protrusions, actin polymerization at the leading edge can theoretically produce the force to push the membrane forward. Forces generating movement in the actin cytoskeleton arise from actin filament polymerization, with associated ATP hydrolysis, and from forces generated by motor proteins, such as myosin.21,22 Other cytoskeletal components such as microtubules, spindle assembly components and intermediate filaments functioning independently in processes affecting cell morphology, also intercommunicate with the actin cytoskeleton.

Rho and Rac relationships in fibroblast morphology Cdc42 relationships with Rac and Rho Much of our understanding of the role of Rac and Rho has come from studies on fibroblasts which show distinct morphological responses to certain growth factors present in serum. These factors act through heterotrimeric G-protein coupled serpentine receptors (e.g. lysophosphatidic acid [LPA], bombesin) or receptor tyrosine kinases (e.g. PDGF, EGF) which feed into signalling pathways activating Rho and Rac. Microinjection of recombinant wild type Rac and Rho or constitutively-activated, GTPase defective RhoAG14V, Rac1G12V and dominant negative Rac1T17N

Microinjection of Cdc42Hs into Swiss 3T3 fibroblasts was found to promote formation of peripheral actin microspikes (PAMs) which include filopodia.32 Subsequently lamellipodia formation and membrane ruffling occurred. This Cdc42-induced ruffling was inhibited by co-injection of dominant negative Rac1T17N. Treatment with bradykinin also resulted in similar morphological effects, with formation of PAMs or filopodia being followed by ruffling. Bradykinin is thought to act through serpentine receptor-linked 700

Rho GTPases, associated proteins and actin morphology formation was also induced by microinjection of C3 transferase, but not when co-injected with dominant negative Cdc42HsT17N. Correspondingly, co-injection with RhoA abolished the effects of Cdc42Hs. Thus, as with fibroblasts, these two GTPases appear to compete; in this case Cdc42 promoting outgrowth and Rho retraction of neurites. The neurotransmitter acetylcholine (ACh) can also exhibit growth factor properties.35 ACh treatment of NE-115 cells, as with Cdc42 injection, promoted sequential formation of filopodia and lamellipodia along neurites and in their growth cones. This involves the muscarinic G-protein linked receptor, but ACh was only effective when applied in a concentration gradient. However while filopodia formation was inhibited by injection of dominant negative Cdc42HsT17N prior to ACh treatment, lamellipodia formation was unaffected. The latter was inhibited by pre-injection with dominant negative Rac1T17N. Thus ACh can activate Rac independently or through Cdc42. In keeping with a Cdc42-RhoA competition, ACh inhibited the neurite retraction effects of LPA. Furthermore, cells which exhibited neurite outgrowth when starved of serum (presumably containing LPA) no longer did so when injected with Cdc42HsT17N.

G-proteins. These effects of bradykinin were inhibited by prior microinjection of dominant negative Cdc42HsT17N while Rac1T17N only inhibited ruffling/ lamellipodia formation. These results indicate that bradykinin acts by activating Cdc42 which subsequently activates Rac. Cdc42Hs microinjection while promoting PAMs also elicited a reduction in stress fibres.32 It is possible that this reflects the utilization by the two types of structures of a common pool of actin, or of similar actin-binding proteins. Whatever the mechanism, this finding indicates Cdc42 to oppose Rho-mediated effects. The relationships of Cdc42, Rac and Rho were also examined in another study.33 Microinjection into Swiss 3T3 fibroblasts of constitutively activated RacG12V triggered assembly of fine focal complexes around lamellipodia, which were distinct from Rhotype focal adhesions. Small focal complexes along filopodia and at the cell periphery were also detected in response to constitutively activated Cdc42G12V when both Rac and Rho were simultaneously inactivated (by co-injection of inhibitory RacT17N and C3 transferase). A combination of Cdc42G12V, RacT17N and C3 transferase generated numerous filopodia within 5 min; Cdc42G12V and C3 transferase generated both lamellipodia and filopodia over the same period. However in the absence of C3 transferase, Cdc42G12V alone or with RacT17N were reported to generate filopodia only after a longer period (20 min). Although RacT17N can apparently block Cdc42induced formation of Rho- and Rac-type focal complexes in confluent cells, whether Cdc42 or Cdc42activating extrinsic factors feed via Rac through to Rho seems uncertain. The above findings are consistent with Cdc42-Rho antagonism, with inhibition of Rho facilitating Cdc42-type effects.

PAK as an effector In the rat, there are at least three PAK isoforms, α-PAK7 and β-PAK36 highly expressed in brain, and the ubiquitous γ-PAK.37 Homologues from other species include mouse β-PAK (mPAK-3)38 and human γ-PAK (hPAK65).39 The GTPase-binding and kinase domains are related7 to those of S. cerevisiae Ste20p, a central component of the pheromone MAPK pathway.40,41 The pheromone receptors act through heterotrimeric G-protein β and γ subunits in a signalling cascade now known to link Cdc24 (the GEF for Cdc42) and Cdc42 with the PAK-like Ste20p.42,43 In mammalian cells, Cdc42 and Rac are involved not in the MAPK pathway but in the JNK/SAPK cascade,44,45 with p38 MAPK being activated by Rho.46 There is some evidence that PAK may act in this pathway.47,48 PAK may thus participate in both nuclear signalling and morphological events. The morphological activities of α-PAK have been studied in epithelial HeLa cells, lacking this PAK isoform.49 On co-transfection with either Cdc42G12V or RacG12V, α-PAK translocated from the cytosol to

Rho-family GTPases and neuronal morphology Recent studies have documented the importance of the Rho GTPases in cytoskeletal dynamics of neuronal-type cells during development/differentiation. In NIE-115 neuroblastomas and PC12 cells, LPA causes growth cone collapse, neurite retraction and cell rounding. This effect is blocked by treatment with C3 transferase which by itself promotes neurite outgrowth.34 In NIE-115 cells, microinjection of Cdc42Hs directly promoted formation of filopodia and lamellipodia in growth cones and along neurites.35 This 701

L. Lim et al The Rho-binding domain was not essential for the morphological activities of transfected ROKα. Possibly over-expression of an active ROKα overcomes the need for RhoA-binding to effect its translocation to peripheral sites9 where ROKα presumably acts.

Cdc42- or Rac-like focal complexes containing paxillin, vinculin and talin. No gross morphological changes occurred on injection of DNA encoding α-PAK, suggesting that its intracellular activation was tightly regulated. This difficulty was circumvented by injecting DNA encoding constitutively active α-PAK mutants. Although formation of PAMs or ruffles did not occur, there was considerable loss of stress fibres and focal adhesions. This loss was similar to the effects of introducing Cdc42G12V or Rac1G12V. These data support a role for PAK downstream of both Cdc42 and Rac in the dissolution of stress fibres/focal adhesions. WASP with a related Cdc42-binding domain is expressed in haemopoietic cells14,50 and has sequence similarity to other proline-rich proteins, including VASP, which is present in focal adhesions, actin filaments and dynamic membrane structures.51 Actin clustering or aggregation occurred in transfected cells overexpressing WASP, but not when these cells are injected with dominant negative Cdc42T17N.14 The significance of this is still unclear.

ROK and Rho, focal adhesions and stress fibres In fibroblasts, LPA- and Rho-mediated cellular contraction preceded formation of stress fibres.56 It was proposed that contraction was initiated by Rhomediated phosphorylation of myosin light chains57 and subsequent assembly of myosin filaments. This was followed by alignment of actin filaments (stress fibres) on the myosin filaments and consequential peripheral aggregation of integrins connected to actin filaments by actin-binding proteins. The Rhoeffector ROK can phosphorylate and inactivate the myosin-binding subunit of myosin phosphatase58 which results in activation of other kinases responsible for myosin mobilization. This proposal provides a plausible role for stress fibres in the formation of integrin-containing focal adhesion complexes. Certainly at mitosis, concomitant loss of stress fibres accompanies the rounding up of cells; with an actinbased contractile ring then forming at the cleavage furrow during cytokinesis, for which Rho is also required.2

ROK as an effector There are several kinases binding to Rho-GTP including ROKα,9-11, ROKβ,10 p160ROCK 12 and PKC-related PKN.52,53 The ROKs and ROCKs belong to a kinase family including the myotonic dystrophy kinase54 and the Drosophila ‘warts’ gene controlling cell growth and morphology.55 Whereas Cdc42 stimulates PAK activity 100-fold or more, ROKα activity is increased only 2–5 fold by RhoA. ROKα is ubiquitously expressed. In HeLa cells on transfection with RhoA or RhoAG14V, cytosolic ROKα is translocated to membranes co-localizing with actin microfilaments at the cell periphery, and at the cleavage furrow in mitotic cells.9 In Hela cells transfection with ROKα DNA resulted in expression of active ROKα whose activity was not substantially increased on injection of RhoAG14V. ROKα expression generated formation of stress fibres and focal adhesions.10 This ROKα-mediated formation occurred even in the presence of dominant negative RhoT19N or C3 transferase, showing ROKα to be downstream of Rho. A kinase dead mutant ROKα was ineffective. Both N-terminal and C-terminal regions were important. C-terminal truncation caused extensive actin condensation while N-terminal deletion resulted in disassembly of stress fibres and focal adhesions. These represent dominant-positive and -negative mutants.

GAPS as effectors The multidomain structure of these GAPs e.g. Abr, Bcr, chimaerin (α- and β-) and p190 Rho GAP indicates that targeting to specific locations and coupling to different effector processes may be important for their function. The different GAP domains, taken in isolation, can selectively downregulate different GTPases and inhibit specific morphological activities. For example, p190 shows RhoGAP activity in vivo, inhibiting stress fibre formation but not PDGF-induced ruffling.59 Microinjection of the GAP domains of Bcr,59 3BP160 and chimaerin32 prevents membrane ruffling in 3T3 fibroblasts, in line with their Rac-GAP activities. Nevertheless, these GAPs may have other than a down-regulating role. Thus microinjection of full length n(α1)-chimaerin into fibroblasts induces the simultaneous formation of lamellipodia and filopodia.61 These underwent cycles of dissolution and 702

Rho GTPases, associated proteins and actin morphology ple, PDGF activation of Rho is blocked by dominant negative RacT17N; this mutant does not affect Rho effector functions directly since it does not affect LPAinduction (Rho-mediated) of stress fibres. Similarly, Bradykinin activation of Rac is blocked by dominant negative Cdc42T17N; this mutant does not affect phorbol ester-induction (Rac-mediated) of ruffles and thus does not act on Rac effectors. Although separate Rac-Rho and Cdc42-Rac hierarchies exist, it is by no means certain that these extend linearly, i.e. Cdc42-Rac-Rho (see Figure 1). We cannot exclude that perhaps the hierarchy exists and it is the presence of activated Cdc42 which prevents Rho-mediated events from occurring, possibly through competition for common morphological components. There is certainly evidence of a Cdc42Rho antagonism. Thus in fibroblasts, Cdc42-induced formation of PAMs occurs at the expense of stress fibres and focal adhesions, which are promoted by Rho. In keeping with this the Cdc42-GTP activated PAK can induce disassembly of the Rho-induced structures (Figure 2). Whether this involves inhibition of ROK merits further investigation. However since

formation, mimicking morphological events at the leading edge of fibroblasts and neuronal growth cones. The formation of lamellipodia and filopodia was inhibited by dominant negative Rac1T17N and Cdc42HsT17N, respectively. Chimaerin’s GTPase-binding- but not GAP- activity was required. There was an associated loss of Rho-type focal adhesions and formation of Cdc42- and Rac-type focal complexes. (In fibroblasts, transfection with α1-chimaerin cDNA resulted in a reduced adhesion and decreased formation of focal adhesions62.) As expected from Cdc42/ Rac1 effects on neuroblastoma cells,35 chimaerin also promoted the simultaneous formation of lamellipodia and filopodia in their neurite growth cones.61

GTPase hierarchy, antagonism and morphological differentiation The use of appropriate mutants has provided good evidence that morphological effects of trophic and growth factors are mediated by Rho GTPases in specific and sometimes hierarchical ways. For exam-

Bradykinin

PDGF Insulin EGF

Acetylcholine

LPA

Bombesin

Ras Cdc42-GDP dbl

Rac-GDP tiam

Cdc42-GTP

Filopodia PAM Focal complexes

Rho-GDP chimaerin Bcr 3BP1

Rac-GTP

Lamellipodia Pinocytosis Focal complexes

lbc

p190RhoGAP Rho-GTP

Contractility Stress fibres Focal adhesions

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 and subsequently Rho24 in Swiss 3T3 fibroblasts. Separate Cdc42-Rac and Rac-Rho hierarchies exist but whether these extend into a Cdc42-Rac-Rho hierarchy is uncertain, particularly since Cdc42dependent formation of peripheral actin microspikes (PAMs) is inversely correlated with Rhodependent formation of stress fibres.32 See text for more discussion of Cdc42-Rho antagonism. Ras acts upstream of Rac.24 Rho is rapidly activated by LPA and other factors, some of which may be cell-specific.23,34 Bombesin can separately activate Rac and Rho.24 In NIE-115 neuroblastoma cells acetylcholine can activate Cdc42 and Rac independently (although microinjected Cdc42 will also activate 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. The Cdc42- and Rac-focal complexes and Rho-focal adhesions are all different.33,49

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L. Lim et al Ack WASP Cdc42-GTP n-(α1-)chimaerin Rac-GTP

Rho-GTP

actin polymerization

Filopodia PAK Ruffles PKN ROK Myosin phosphatase-P Myosin light chain-P

Contractility

Stress fibres Focal adhesions

Figure 2. Effector actions of proteins associated with Rho-family GTPases. A number of potential effectors bind to Rho-family GTPases in their GTP-bound ‘active’ state. These include, for Cdc42GTP, ACK,8 WASP13,14 and PAK7 which also binds Rac-GTP. Rho-GTP binds ROK/ROCK.9-12 PKClike PKN phosphorylates neurofilament subunits.64 ROK acts downstream of Rho-GTP in stimulating formation of stress fibres and focal adhesions.10 The myosin-binding sub-unit of myosin phosphatase binds Rho-GTP and is a substrate of ROK. It is down-regulated by phosphorylation, resulting in an increased phosphorylation status of myosin light chain kinase58 leading to enhanced myosin contractility, driving the formation of stress fibres and focal adhesions.56 There may be other mechanisms underlying ROK stimulation of stress fibres and focal adhesions. Activated PAK causes dissolution of stress fibres;49 thus an effector of Cdc42 (or Rac) can downregulate Rho-dependent morphology. This is consistent with Cdc42 and Rho having antagonistic roles. The Rac-GAP n(α1)-chimaerin acting through both Cdc42-GTP and Rac-GTP promotes cyclical formation of filopodia and ruffles, for which GAP activity is not required.61

References

Rac-GTP can also stimulate PAK as well as activate Rho-induced pathways, this implies a difference in the activity of PAK in Cdc42- and Rac- pathways. In neuroblastoma cells, this competition between Cdc42 and Rho, involving their differential activation, perhaps shapes at any one time the appropriate morphological response to opposing trophic signals such as ACh and LPA. The dynamics of retraction and extension of neuritic structures, particularly at the growth cone, may be an essential aspect of the neuronal response to changes in the metabolic milieu which determine proper differentiation (or death). When the balance of active and inactive GTPases is upset, as in transgenic mouse cerebellar Purkinje neurones expressing constitutionally active RacG12V the morphological consequences can be severe — axon terminals are reduced and there are supernumerary small dendritic spines, indicative of abnormal development of different neuronal processes.63

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