The pathway linking small GTP-binding proteins of the Rhofamily to cytoskeletal components and novel signaling kinasecascadesJ. Silvio Gutkind and Lynn Vitale-Cross

The Ras superfamily of GTPases comprises more than 50members. Whereas Ras is known to play a central role in cellproliferation, the Rho-family of Ras-related smallGTP-binding proteins have been shown to regulate severalaspects of cytoskeleton functioning. Recently, we and othershave shown that members of the Rho-family of GTPases alsocontrol signaling cascades communicating the membrane tothe nucleus, and that these GTP-binding proteins areintegral components of growth-regulatory pathways. In thisarticle, we will focus on the recent identification of moleculeslinking the Rho-family of GTPases to the cytoskeleton and tosignaling kinase cascades.

Key words: actin / cytoskeleton / GTP-binding proteins /kinases / oncogenes / Ras / Rho

©1996 Academic Press Ltd

THE RAS SUPERFAMILY of GTPases comprises more than50 members, which have been divided into six familiesbased upon sequence similarity: Ras, Rho, Arf, Sar,Ran and Rab.1 These proteins function as molecularswitches, cycling between an active GTP-bound stateand an inactive GDP-bound state. Ras is known to playa central role in proliferative signaling by a variety ofcell surface growth factor receptors (see later). TheRho-family of GTP-binding proteins consists of theRac, Rho and Cdc42 subfamilies, and have beenshown to regulate several aspects of cytoskeletonfunctioning.2 The Rac subfamily includes Rac1 andRac2, the former involved in the regulation oflamellipodia (pleat-shaped protrusions at the cellperiphery) and membrane ruffling,3 and the latter inNADPH oxidase-catalysed superoxide formation inneutrophils.4 The Rho subfamily has at least threemembers, RhoA, RhoB and RhoC. RhoA has been

shown to participate in the formation of actin-stressfibers, as well as in mediating redistribution ofcytoskeletal components.1,2 The Cdc42 group consistsof Cdc42Hs (referred here as Cdc42), G25K andRhoG,2 and participates in the formation of filipodia,that are thin finger-like cytoplasmic extensions.Recently, we and others have shown that Rho-relatedGTPases also control signaling cascades linking themembrane to the nucleus.5 Thus, the emergingpicture is that members of the Rho family of GTP-binding proteins control the organization of the actincytoskeleton, and are also integral components ofgrowth-regulatory pathways. In this article, we willfocus on the recent identification of molecules linkingthe Rho-family of GTPases to the cytoskeleton and tosignaling kinase cascades.

Rho as a regulator of the cytoskeleton

Inactivation of cellular Rho proteins by ADP-ribosyla-tion upon treatment of intact cells with the C3 toxinfrom Clostridium botulinum, or microinjection of GTP-loaded Rho into fibroblasts, led to the establishmentof a critical role for Rho in the formation of stressfibers, focal adhesions, and cell motility.2,6 However,molecules linking Rho to the cytoskeleton have justbegun to be identified. Rho has been shown toregulate the activity of a number of enzymes, such asphosphatidylinositol 3-kinase, phosphatidylinositol4-phosphate 5-kinase and phospholipase D, but adirect link between these second-messenger generat-ing enzymes and the cytoskeleton has not been firmlyestablished. Very recently, it has been reported thatRho can physically associate to a number of proteins,including protein kinase N, a kinase closely relatedbut distinct from protein kinase C,7,8 and to a novelserine-threonine kinase, termed Rho-kinase.9 Bothkinases were shown to be activated upon binding toGTP-bound Rho, and these findings prompted exten-sive investigation regarding the role of these newly

From the Molecular Signaling Unit, Laboratory of CellularDevelopment and Oncology, National Institute of Dental Research,National Institutes of Health, 9000 Rockville Pike, Building 30,Room 212, Bethesda, MA 20892-4330, USA

seminars in CELL & DEVELOPMENTAL BIOLOGY, Vol 7, 1996: pp 683–690

©1996 Academic Press Ltd1084-9521/96/050683 + 08 $25.00/0

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discovered kinases as candidates for connecting Rhoto cytoskeletal components.

Increasing evidence has accumulated to suggestthat continuous turnover of intermediate filaments isinvolved in maintaining the intermediate filamentstructure and function in living cells. Interestingly,using the yeast two-hybrid system as an experimentalapproach to identify downstream targets for PKN,Ono’s laboratory recently reported the physical asso-ciation of PKN to the neurofilament L protein,10 asubunit of neuron-specific intermediate filament.Furthermore, they found that PKN associates to thehead-rod domains of neurofilament proteins M andH, as well as to that of vimentin. Additional workwould be necessary to explore the biological conse-quences of the interaction between the regulatorydomain of PKN and intermediate filaments, as well asto test whether neurofilaments and vimentin act asadaptors promoting the activity of PKN towardsadjacent cytoskeletal targets. Interestingly, it was alsoshown that these neurofilament proteins are in-vitrosubstrates for PKN, and that PKN phosphorylationinhibits their polymerization in vitro.10 Takentogether, these findings provide compelling evidenceto suggest that Rho might regulate the assembly–disassembly of intermediate filaments, including neu-rofilaments and vimentin, through the activation ofPKN.

The search for proteins that associate to GTP-loaded Rho led to the purification of a protein ofapproximately 138 kD,11 whose sequence was almostidentical to that of the 110-kD regulatory subunit ofrat smooth muscle protein phosphatase 1M, which is ahomolog of the myosin-binding subunit (MBS) ofmyosin phosphatase from chicken. Furthermore,myosin light chain phosphatase activity was specifi-cally detected in the eluate from GTP-RhoA affinitycolumns. Myosin phosphatase is composed of threesubunits, MBS, a 37 kD type 1 phosphatase catalyticsubunit, and a 20 kD regulatory subunit. RhoA inter-acts specifically with myosin phosphatase composedonly of MBS and the catalytic subunit, thus suggestingthat the interaction of RhoA with myosin phosphatasemay result in dissociation of the 20 kD regulatorysubunit from the holoenzyme. RhoA was also shownto promote the association of MBS with membranes,presumably by forming a complex with MBS at thelevel of the plasma membrane. Interestingly, asdiscussed above, activated Rho interacts with, andstimulates the enzymatic activity of PKN and Rho-kinase, and the latter was shown to phosphorylateMBS and to inhibit myosin phosphatase. This Rho-

dependent inhibition of myosin phosphatase, eitherby membrane localization and phosphorylation and/or by promoting the dissociation of the 20 kD reg-ulatory subunit, results in a net increase in myosinlight chain phosphorylation by other protein kinases.Such phosphorylated myosin then polarizes actin-myosin bundles leading to the contraction of smoothmuscle, or to stress fiber formation in other, non-muscle cells. These recent findings can finally explainhow RhoA can activate myosin and control stress fiberformation. Furthermore, it might also help explainwhy the effect of Rho on the cytoskeleton is cell typespecific,6 as inhibition of myosin phosphatase wouldresult in a net increase in phosphorylation only onthose cell types exhibiting constitutive myosin lightchain kinase activity. We can conclude that the recentidentification of downstream targets for Rho, includ-ing PKN and Rho-kinase and MBS represent the firststep towards the elucidation of the molecular basis ofcytoskeletal control by Rho-like proteins (See Figure1). In addition, these recent studies might haveimportant implications regarding Rho-binding pro-teins. Specifically, the COOH-terminal domain ofMBS contains a polybasic region followed by a leucinezipper-like motif, and PKN contains a similar poly-basic region followed by a leucine zipper-like motif inthe protein region that interacts with Rho, thussuggesting that this shared structure mediates inter-action with Rho.

Other cytoskeletal functions of Rho-related GTPa-ses are much less understood. For example, inpolarized epithelia Rho is involved in the regulation

Figure 1. Regulation of cytoskeletal structures by Rho. Seetext for details.

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of tight junctions and the organization of perijunc-tional actin.12 In Drosophila, Rac1 is required foractin assembly at adherens junctions of the wing discepithelium and for axonal outgrowth.13,14 Cdc42regulates polarization in helper T cells toward anti-gen-presenting cells,15 and is also involved in theregulation of polarized cell growth in yeast.16 TheseGTPases are also involved in the establishment of avariety of adhesive structures, which include focaladhesion complexes, adherens junctions and tightjunctions. Furthermore, recent work suggests thatRho and Rac are integral components of a multi-protein complex recruited upon integrin activation.17

It is possible that some of the recent advances in themolecular dissection of structural proteins and signal-ing pathways controlled by Rho related GTP-bindingproteins will shed more light on the role of theseGTPases in the formation of specialized adhesioncomplexes, as well as in their morphological andmotility functions.

Proline-rich proteins link Rho-related GTPasesto actin

Another area of active investigation involves the studyof the mechanisms by which members of the Rhofamily of GTPases regulate the polymerization anddepolymerization of actin filaments in nonmusclecells. Two classes of actin-binding proteins control theturnover between the monomeric (G-actin) andpolymeric (F-actin) forms: (1) sequestering proteins,which inhibit actin polymerization by binding toG-actin; and (2) capping proteins, which inhibit actinpolymerization by binding to the barbed (fast-polym-erizing) ends of filaments. How Rho-related GTPasesregulate the activity of actin-binding proteins has justbegun to be elucidated.

Using permeabilized platelets as an experimentalmodel, it has been recently shown that activated Raccan cause the uncapping of barbed ends.18 Fur-thermore, Rac was shown to stimulate phosphatidyli-nositol(4,5)-biphosphate (PIP2) synthesis, the Rac-induced-uncapping of filaments was inhibited byPIP2-binding peptides and thus, it has been suggestedthat Rac controls the activity of capping proteins byregulating PIP2 synthesis. Similar mechanisms can beproposed for Rho and Cdc42, however, the identity ofthe capping protein controlled by any of these smallGTP-binding proteins remains to be established.

One such candidate is profilin, a ubiquitouslyexpressed actin-monomer binding protein.19 Profilin

is unique in having both positive and negative effectson actin polymerization. It promotes depolymeriza-tion by binding monomeric actin, and promotespolymerization by increasing the rate of nucleotideexchange.20 Profilin has been associated with micro-filaments in highly dynamic areas of the cell, such asthe leading lamella and the tips of nascent stress fibersof fibroblasts.21 It binds to actin, PIP2 and poly-L-proline. Binding of PIP2 and G-actin is mutuallyexclusive, which led to the view that profilin mightlink signal transducing pathways activated by tyrosinekinase and G protein-coupled receptors with themembrane-attached microfilament system21,22 (seeFigure 2).

Another possible mechanism for the control ofactin polymerization by profilin might depend on itsability to bind poly-L-proline-containing proteins.That observation prompted the search for naturalligands containing proline clusters, leading to theidentification of vasodilator-stimulated phosphopro-tein (VASP) as such a partner. VASP was originallydescribed as a substrate for cGMP and cAMP-depend-ent protein kinases in platelets,23 but was later foundto be widely expressed. Of interest, VASP has beenidentified as a component of focal adhesion sites, andcolocalizes with profilin in nascent focal contacts(Figure 2). VASP binds profilin through a proline richregion, consisting in a G(P)5 motif as a single copy oras a three-fold tandem repeat in human and canineVASP, respectively.24 Taken together, these findingssuggest that actin filament assembly is controlled by asignaling pathway affecting the availability of PIP2,and by cAMP and cGMP-dependent pathways actingon VASP (see Figure 2).

Recent findings revealed that small GTP-bindingproteins of the Rho family can also regulate cytoskele-tal organization through proline-rich proteins. Partic-ularly, two groups provided evidence that WASP, theprotein implicated in the Wiskott-Aldrich immunode-ficiency syndrome, acts as a downstream effector ofCdc42.25,26 In turn, ectopic expression of WASP in avariety of different cell lines causes actin polymeriza-tion at sites that are enriched in WASP, in a Cdc42-dependent manner.25 WASP contains several polypro-line regions, and shares extensive sequence homologywith VASP. WASP expressions appear to be restrictedto lymphocytes, platelets, neutrophils and macro-phages, and have been shown to bind a number ofSH3 containing proteins, including NCK.27 Further-more, two novel putative protein domains wereidentified in WASP, and termed WH1 and WH2,25

which are also present in other proteins believed to be

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involved in cytoskeletal organization, such as VASP,verprolin and the protein product of the Drosophilagene enabled.25 Thus, it is tempting to speculate thatWH1 and WH2 domains might be involved in linkingsmall GTP-binding proteins and signaling pathways tostructural components of the cytoskeleton. The eluci-dation of the mechanism by which Cdc42 and WASPregulate the acting cytoskeleton is still unknown, andthe subject of intensive investigation.

Proliferative signaling through Rho-relatedproteins

Although the function of the Rho-family of GTPbinding proteins is still not fully understood, therecent observation that several oncogenes, includingdbl and ost,28,29 induce the exchange of GDP for GTPbound to Rho-like proteins have suggested that these

small G proteins might participate in growth control-ling pathways. Supporting this notion, a large numberof exchange factors for Rho family members hasrecently been identified by virtue of their oncogenicpotential in NIH3T3 fibroblasts. Furthermore, severalreports have demonstrated that activated mutants ofRho and Rac can cause malignant transformation ofNIH3T3 cells,30,31 and Rac and Rho were also shownto be critical for transformation by activatedRas.30,32,33

Ras has been shown to play a central role in cellproliferation and differentiation. One of Ras1 func-tions is to regulate the activity of mitogen-activatedprotein kinases (MAPKs), also known as extracellularsignal-regulated kinases (ERKs). These protein ser-ine/threonine kinases are rapidly activated uponstimulation of a variety of cell-surface receptors, andthey play a key role converting extracellular stimuli tointracellular signals that, in turn, control the expres-

Figure 2. Regulation of actin polymerization by signaling pathways and small GTP-binding proteinsacting on proline-rich proteins. See text for details.

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sion of genes essential for many cellular processes.34

At the molecular level, Ras exchanges GDP for GTPupon activation of Ras-specific guanine–nucleotideexchange factors. In the GTP-bound state, Ras thenphysically associates with the N-terminal region ofRaf,35 thereby recruiting this serine-threonine kinaseto the plasma membrane and allowing its activation bya still unknown mechanism. In turn, Raf activates alinear cascade of protein kinases, defined sequentiallyas MAPK kinase, such as MEK1 and MEK2, whichultimately phosphorylate MAPKs on both threonineand tyrosine residues, thereby increasing their enzy-matic activity.34

Recently, it has been shown that a novel family ofkinases structurally related to MAPK, stress activatedprotein kinases (SAPKs) or jun N-terminal kinases(JNKs), phosphorylate in vivo the N-terminal trans-activating domain of the c-Jun protein. Although anumber of studies have demonstrated that activity ofc-Jun and JNK is elevated in cells transformed byoncogenic ras genes,36 many growth factors known toactivate the Ras-MAPK pathway fail to elevate theenzymatic activity of JNK in several cellular systems.5,37

In addition, using the expression of an epitope-taggedJNK1 (HA-JNK) in COS-7 cells as a model system toexplore the mechanism of activation of JNK, we foundthat the small GTP-binding protein Ras could weaklyactivate JNK, utilizing a signaling pathway distinctfrom that regulating MAPK.5 Interestingly, it hasrecently been reported that GTP-bound forms of theRho-related proteins Rac1 and Cdc42 can specificallyassociate and activate a novel serine-threonine kinase,which was designated Pak.38 This situation is highlyanalogous to that of the Ras–Raf interaction, thussuggesting that the Rho family of GTP bindingproteins might also initiate activity of a kinasecascade.38 This observation and the failure of Ras tofully activate JNK prompted us to ask whether the Rhofamily of G proteins participates in signaling to theJNK pathway.

We have recently shown that expression of activatedforms of Rac1 and Cdc42 can efficiently stimulate JNKwithout affecting MAPK activity.5 Particularly, acti-vated Rac1 and Cdc42 were the most potent JNKinducers among those we have tested, and althoughactivated RhoA is known to induce the most dramaticchanges in the cytoskeletal structure it stimulated JNKonly to a very limited extent. Furthermore, we havefound that treatment of cells with cytoskeletal disrupt-ing agents such as cytochalasin D fails to block JNKactivation induced by Rho-like proteins (unpublishedobservation). We concluded that members of the Rho

family of low molecular weight GTP binding proteinscan effectively stimulate signaling pathways leading toJNK activation, probably through a mechanism dis-tinct from that involved in cytoskeletalreorganization.

The functioning of Rho-like GTP binding proteinsis tightly regulated in vivo by proteins that controltheir GDP/GTP state. For example, GTPase activatingproteins (GAPs)1 as well as nucleotide dissociationinhibitors (GDI) are negative modulators, whereasguanine nucleotide exchange factors (GEFs)39 pro-mote the exchange of GDP for GTP thus activatingRho-like proteins. Several GEFs for this family havebeen described, including the protein product of thedbl and ost oncogenes. Recent work demonstrated thatthese exchange factors for Rho-like proteins canpotently induce JNK activity.5 Because a growing list ofproteins exhibits a domain of homology to Dbl (DHdomain)39 we have explored whether additional DHcontaining proteins could also activate JNK. Ofinterest, we found that the protein product of the vavoncogene potently induces JNK in a Rac-dependentmanner (unpublished observation). However, neithermammalian SOS nor the product of the ect2 onco-gene40 were capable of raising JNK activity (unpub-lished observation) in spite of each possessing a DHdomain. Thus, these findings support the emergingnotion that functional activity as a Rho-like GEFrather than the mere presence of a DH domain isrequired for coupling to the JNK pathway.

The signaling elements functioning downstream ofRac and Rho in the control of cell proliferation arestill unknown. As described above, Rho has beenshown to regulate several enzymes involved in phos-pholipid metabolism, and Rho-kinase and PKN, andRac and Cdc42 activate JNK. Nevertheless, the role ofthese enzymes in growth control is yet to be estab-lished. Furthermore, Rho activates the serum respon-sive factor, SRF, utilizing a pathway that is independ-ent from JNK and MAPK, suggesting that Rho mightactivate an as yet unidentified signaling pathwaycontrolling transcription.41

Whether the growth regulatory and cytoskeletaleffects elicited by GTP-binding proteins of the Rhofamily utilize separate or identical pathways is stillunclear. However, several lines of evidence suggestthat the cytoskeletal and growth control pathwaysdownstream of Rac, Cdc42 and Rho diverge.6 Indeed,the recent finding that WASP can act as a Cdc42effector, and that WASP blocks JNK activation byCdc42 (JSG, unpublished) suggests that signalingpathways downstream from CDC42 bifurcate. While

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WASP controls actin cytoskeleton, other kinase(s),such as Pak and MLK, might regulate transcriptionalactivation.

Conclusions

We can conclude that a number of remarkablediscoveries over the past year has revealed the

existence of multiple distinct pathways regulated bysmall GTP-binding proteins of the Rho family. Highlyconserved from yeast to mammals, these pathwaysinclude modules of sequential kinase cascades (seeFigure 3), some of which might control geneticprograms involved in cell proliferation, transforma-tion and/or programmed cell death, as well as distinctpathways controlling the organization of the actincytoskeleton. The identification of molecules involvedin each of these pathways represents one of the mainchallenges in this rapidly growing field.

Figure 3. Signaling from the membrane to the nucleus through small GTP-binding proteins of theRas and Rho family acting on distinct kinase cascades. The pathway connecting cell surfacereceptors to low molecular weight GTP-binding proteins, as well as the identity of biologicallyrelevant substrates for these kinase cascades are yet to be fully elucidated.

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