Rho activation at aglanceRachel J. BuchsbaumDivision of Hematology/Oncology and MolecularOncology Research Institute, Tufts-New EnglandMedical Center, 750 Washington Street, Boston,MA 02111, USAe-mail: rbuchsbaum@tufts-nemc.org

Journal of Cell Science 120, 1149-1152Published by The Company of Biologists 2007doi:10.1242/jcs.03428

Cells receive a multitude of stimuli –chemical (such as cytokines and growthfactors) and physical (such asmechanical stresses or adhesion toextracellular matrix or other cells) – thatinfluence cell function by affectingintracellular signaling pathways. Veryoften these stimuli involve cell surfacereceptors or other molecules thatfunction through activation of the Rho

family of small GTPases. Rho GTPasescontrol multiple cellular processes,including actin and microtubuledynamics, gene expression, the cellcycle, cell polarity and membranetransport, through their ability to bind tonumerous downstream effectors, whichlead to diverse parallel downstreamsignaling pathways (Schwartz, 2004).

Over 20 members of the Rho family havebeen identified in mammalian cells(Wennerberg and Der, 2005). These arerepresented here by the canonical proteinsRho, Rac and Cdc42 and have been thesubject of numerous excellent reviews(Bishop and Hall, 2000; Ridley, 2001;Boettner and Van Aelst, 2002; Ridley etal., 2003; Burridge and Wennerberg,2004; Jaffe and Hall, 2005). Like theclassic monomeric Ras GTPase, mostRho proteins act as switches by cyclingbetween active (GTP-bound) and inactive

(GDP-bound) conformations. (Four Rhofamily members are GTPase deficient andbind GTP constitutively; little is knownabout their regulation.) There are threeclasses of regulatory proteins that affectthe activation state of these cycling Rhomolecules: guanine nucleotide exchangefactors (GEFs), which promote exchangeof GTP for GDP; GTPase-activatingproteins (GAPs), which enhance theintrinsic GTP-hydrolysis activity, leadingto GTPase inactivation; and guanine-nucleotide-dissociation inhibitors (GDIs),which bind to prenylated GDP-boundRho proteins and allow translocationbetween membranes and the cytosol.Currently, the best understood regulatorsof Rho activation in response to upstreamstimuli are the GEFs.

GEFs for Rho family proteins have beenidentified in bacteria, plants, yeast,worms, fruit flies and humans. Most

1149Cell Science at a Glance

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RSK and MSK at a GlanceRho Activation at a Glanceand MSK at a Glanceand MSK at a GlanceRachel J. Buchsbaum

Rho Activation at a Glance

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TKs

APC

RasGTP

Gβ Gγ

Cbl-bhSiah1

GF LPA EphThrombin

PKA

14-3-3

HIV-1gp41 P

Ubi-Ubi

p130CAS

nm23HI

PIP2

PKA

PIP3

Vav

P-Rex1SOS

PAK

βγ

Net1

Net1

Ect2

ROSp190RhoGAP

p115RhoGEF

Eps8

Abi1

Asef

Tiam1

aPKC

Par6

WASP

MLK2

R-Ras

MKK7

RhoG

Rho

Plasma membrane

Key

Nucleus

Rac

Cbl

LMWPTPASE

Cdc42

COOL-1

COOL-2

COOL-2

COOL-2

GTPases

Non-classical GEFs

GEF activators

GEF inhibitors

GTPase effectors

Scaffolds andprotein complexes

Classical GEFs

RasGRF

DOCK180

DOCK180

SopE, SopE2

S. typhimurium

IRSp53

WAVE2

S6KF MLK3

Effectors

EphexinEphexin

Integrins

Plexins

RTK

LPA, Thrombin, etc.

Eph receptorsGPCRs

Ephexin

ITSN

ELMO

CrkII

Fgd1

Actin

Fgd1

Golgi

Lbc

GEF-H1

CNK1

p115RhoGEF LARG PDZ-RhoGEF

KalirinTrio

Dbs

Src

Dbl

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ECM

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2Par

3

Spin

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Fak

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RhoGEF activity is mediated by catalyticDH (Dbl homology) domains, whichstabilize GTP-free Rho intermediates,effectively leading to GTP loadingowing to high intracellular GTP levels.DH domains contain three conservedregions and form related structures ofelongated bundles of �-helices, in whichamino acid variations confer specificitytowards individual GTPases. Almost allDH-containing Rho family GEFscontain a PH (pleckstrin homology)domain immediately C-terminal to theDH domain. DH-associated PH domainshave several regulatory roles with regardto DH domain and GEF function,including modulation of exchangeactivity, interactions with phospholipidsand proteins, and membrane targeting.

The identification of over 60 mammalianRho family GEFs to date has revealed acomplex array of regulatory mechanismsfor these proteins. The poster depicts thebest-studied GEFs and major themes intheir regulation. A comprehensivedescription is beyond the scope of thisoverview and I apologize to thoseauthors whose work is not included.However, several excellent reviews havebeen published (Schmidt and Hall, 2002;Erickson and Cerione, 2004; Rossman etal., 2005).

A common theme in the regulation ofRho family GEFs is relief ofintramolecular inhibitory interactions.This is illustrated by the constitutiveactivation of N-terminally truncatedDbl, Vav, Asef, Tiam1, Ect2 and Net1mutants and C-terminally truncatedp115RhoGEF and Lbc mutants. Oftenthe associated PH domain is involved inthis intramolecular inhibition. In theRhoGEF Dbl, for example, the N-terminus binds to the PH domain,blocking access of the GTPase to the DHdomain. Phosphorylation by Ack1 orinteraction with heterotrimeric G protein�� subunits may relieve the inhibition. Inthe RacGEF Vav, an interaction betweenDH and PH domains that masks the Rac-binding site is induced by binding ofphosphatidylinositol 4,5-bisphosphate(PIP2) to the PH domain and relieved bybinding of phosphatidylinositol 3,4,5-trisphosphate (PIP3). Complex inhibitoryintramolecular interactions have beendescribed for the dual Ras-Rac exchangefactor SOS, which possesses both aCDC25-like exchange domain for Ras as

well as a DH domain to promote GTP-GDP exchange on Rac. PIP3 bindingrelieves a similar DH-PH interaction thatblocks GTP-GDP exchange on Rac,while the DH-PH region itself blocks anallosteric Ras-binding site on SOS thatregulates Ras activity. Recentlypublished work indicates that EGF-receptor-dependent phosphorylation of atyrosine residue near a downstreamregulatory region is required to unmaskthe exchange activity of the Cdc42 GEFCOOL-1/�PIX (Feng et al., 2006).

GEFs have a number of other functionaldomains, many of which couple toupstream receptors or other signalingmolecules. It is thus not surprising thatprotein-protein interactions also regulateGEF activity. Activated G�13, releasedfrom its �� subunits by LPA- orthrombin-mediated stimulation of G-protein-coupled receptors (GPCRs), canbind to and stimulate several RhoGEFs,including Dbl, p115RhoGEF, PDZ-RhoGEF and LARG. The G�� units bindand activate Dbl, as mentioned above. Thehematopoietic RacGEF P-Rex1 isactivated by binding to both PIP3 andspecific G�� subunits. In the RacGEFAsef, interaction between the N-terminalABR region and the armadillo repeatdomain of the adenomatous polyposis coliprotein (APC) relieves autoinhibition andpromotes Rac exchange activity. SomeGEFs, such as Dbl, Dbs, and RasGRF1and RasGRF2, form homo- or hetero-oligomers through DH domaininteractions that are required for fullfunction of the protein, whereasdimerization of others (PDZ-RhoGEF,LARG, p115RhoGEF) is inhibitory. Inthe case of the multidomain GEFs Kalirinand Trio that contain separate DHdomains for Rac and Rho, alternativesplicing leads to multiple isoforms thathave different functional activities duringdevelopment.

GEF activity can also be downregulatedby interaction with inhibitory proteins– for example, the inhibition of Vav bybinding of the C-terminus to Cbl-b orhSiah1, the inhibition of p115RhoGEFby binding of the C-terminus to the HIV-1gp41 protein, and the inhibition ofTiam1 by binding of the N-terminus tothe tumor suppressor nm23H1.Association of microtubules with theRhoGEF GEF-H1 inhibits its exchangeactivity toward Rho. Oligomerization of

the A-kinase-anchoring protein (AKAP)Lbc through C-terminal leucine zippersequences is required for inhibition of itsexchange activity by PKA and 14-3-3. Inaddition, Rho family GEFs may also bedownregulated by being targeted fordegradation. Examples include SOCS1-stimulated Vav polyubiquitylation, Ras-stimulated polyubiquitylation of thedual Ras-RacGEF RasGRF2, theubiquitylation of murine SOS2, and theCbl-directed ubiquitylation of COOL-1/�PIX.

Multiple levels of regulation have beenidentified in the case of some Rho familyGEFs. Interaction of the PH domain ofRacGEF Vav with PIP3, for example,allows tyrosine phosphorylation ofresidues interacting with the GTPase-binding pocket by Src/Syk tyrosinekinases (TKs) in response to T cellreceptor signaling, further opening upaccess to the Rac-binding site. Theactivation of P-Rex1 by PIP3 and G��subunits is blunted by PKA-mediatedphosphorylation, and the exchangeactivity of the RacGEF Tiam1 ismodulated by both phosphoinositidebinding and by phosphorylation onthreonine and tyrosine.

Change in intracellular location andlocalized activation of Rho GTPases isanother mechanism for regulation of Rhofamily GEFs. Sometimes the PH domainmediates translocation – for example, thelocalization of Dbl and Lbc to actin stressfibers. Some GEFs, such as Tiam1 andFgd1 (a Cdc42GEF), contain a secondPH domain. In the case of Tiam1, thesecond PH-domain is required formembrane translocation; in Fgd1, aproline-rich N-terminal region, ratherthan the PH domains, localizes theprotein to subcortical actin and Golgistructures. Other Rho family GEFs arerecruited to membranes by adaptorproteins, direct binding, or other proteininteractions. Adaptor proteins such asGrb2 and SLP-76 are required forlocalization of Vav to activated B- and T-cell receptors. The DH-PH domains ofephexin directly interact with thetransmembrane receptor tyrosine kinaseEphA4. Binding of G�13 after LPA orthrombin stimulation inducesredistribution of p115RhoGEF from thecytosol to the plasma membrane. Andupon nuclear envelope breakdown priorto mitosis, the RhoGEFs Ect2 and Net1

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are released from the nucleus, where theyare otherwise sequestered, to activatethe Rho-mediated contraction of theactomyosin ring that drives cytokinesis.

GTPases themselves often regulate Rhofamily GEF activity. For example,several RhoGEFs activated by G�-GTP,including p115RhoGEF, LARG andPDZ-RhoGEF, also contain an RGSdomain. This domain functions as a GAPfor G� subunits, thus allowing the GEFsto fine-tune signals coming fromGPCRs. The RacGEF Tiam1 is activatedby Ras-GTP through its Ras-bindingdomain. Similarly, binding of Rac-GTPto the PH domain of the RhoGEF Dbspromotes Rho activation. During theprocess of actin remodeling and cellspreading, Rac activation generatesreactive oxygen species that inhibit thelow-molecular-weight protein tyrosinephosphatase (LMW-PTPase), leading toincreased tyrosine phosphorylation andactivation of p190RhoGAP andsubsequent downregulation of Rhoactivity. In a complex process probablyinvolving both allosteric and directeffects, Cdc42-GTP activates and Rac-GTP inhibits the RacGEF activity ofdimeric COOL-2/�PIX (Baird et al.,2005).

Another emerging theme in Rho familyGEF function is a role specifyingsignaling downstream of Rho GTPases,often through participation inmultimolecular complexes. This can bethrough direct binding of the GEF to theimmediate GTPase effector proteindownstream – for example, binding ofCOOL-2/�PIX to Pak and binding ofintersectin (ITSN) to Wiskott-Aldrichsyndrome protein (WASP). Alternativelythis can be through binding of the GEF toscaffold proteins that also form a complexwith GTPase effectors – for example,binding of Tiam1 and RasGRF1 to thep38 scaffold JIP2, and binding of Tiam1to the F-actin–protein-phosphatase-1–S6kinase scaffold spinophilin and thePar3 component of the Par polaritycomplex (Mertens et al., 2006). Acombination of both mechanisms isdisplayed by Tiam1, which can alsointeract with IRSp53, a small adaptormolecule that binds activated Rac as wellas the Arp2/3–G-actin scaffold WAVE2.Our understanding of the complexcontributions of scaffold proteins to GEF-directed GTPase signaling is just

beginning. For example, the humanortholog of Connector Enhancer of Ksr1(hCNK1), a multi-domain adaptor proteinfirst implicated in Ras signalingpathways, may also function as a scaffoldprotein for Rho-dependent Jnk activationthrough binding RhoGEFs Net1 andp115RhoGEF, activated Rho, MLK2 andMKK7 (Jaffe et al., 2005).

In some cases where GEFs activate morethan one GTPase, formation of a complexcan also direct GEFs towards specificGTPases. The dual Ras-Rac-GEF SOS,for example, contains a proline-rich C-terminus that can interact with the SH3domains of either Grb2 or Abi1/E3b1.Interaction of SOS with Grb2 enablesrecruitment of the complex to tyrosinekinase receptors and activation of Ras. Bycontrast, interaction of SOS with Abi1leads to formation of a complex with Eps8and activation of Rac. Similarly, theGTPase activation profile of ephexin ismodulated depending on the activationstate of the EphA4 receptor that it binds.When unstimulated, ephexin can activateCdc42 and possibly Rac in addition toRho, but EphA4 clustering leads toenhanced Rho activation and suppressesCdc42/Rac activation. The GEFspecificity of COOL-2/�PIX depends onits monomer-dimer equilibrium. As notedabove, the dimer promotes Rac activation.However, interaction with G��-PAKcomplexes promotes dissociation tomonomers, which can activate either Racor Cdc42. Finally, although Ras-GRF1and Ras-GRF2 can each potentiallyactivate both Ras and Rac GTPases,RasGRF1 preferentially activates Rac topromote LTD (long-term depression) andRas-GRF2 preferentially activates Ras topromote LTP (long-term potentiation) inthe hippocampus, at least in part becausethey are associated with different subsetsof NMDA glutamate receptors in thebrain (Li et al., 2006). The fact that GEFsboth turn on GTPase signaling andspecifically direct the downstream effectsof that signaling may explain why higherorganisms have evolved so many moreGEFs than GTPases.

There are proteins that lack the classicaltandem DH-PH motif but exhibitnucleotide exchange activity toward Rhofamily members. SWAP-70, part of theB cell DNA recombination complex, andrelated family members, possess PHdomains N-terminal to DH-like domains

and function upstream of Rho GTPases.In humans the CZH/DOCK180superfamily members lack DH-PHdomains altogether but possess DOCKhomology domains required fornucleotide exchange through an as-yet-unidentified mechanism. Canonicalmembers act as RacGEFs, others areCdc42GEFs, and some remain to becharacterized. Bacteria, which lack theirown Rho GTPases, have multiplemechanisms for modulating the activityof mammalian Rho family proteins. Thebacteria Salmonella typhimurium injectsthe non-DH-containing SopE (Rac andCdc42) and SopE2 (Rac) proteins intomammalian cells, where they function asGEFs in order to facilitate bacterialinternalization. And in plants, whichcontain numerous Rop (Rho of plants)proteins, a new family of Rho GEFs hasrecently been identified. These contain anovel PRONE (plant-specific Ropnucleotide exchanger) domain (Berkenet al., 2005).

GEF activation of Rho GTPases, like allsignaling pathways, does not take placein an isolated linear fashion but occursthrough the serial formation ofmacromolecular complexes at precisepoints in time and space that arecurrently incompletely understood.Further insight into how Rho proteins areactivated by upstream signals willrequire more knowledge of how theeffects of GEFs are coordinated withthose of GAPs and GDIs, along withother signaling molecules. An exampleof this is integrin signaling, itself acomplex system involving multipleligands and multiple receptors. There aremany examples of integrin activationleading to signaling through Rhoproteins, but the details of the regulatoryproteins involved are often unclear.Integrin ligation is known to triggersignaling through activation of Vav andformation of DOCK180 complexescontaining either p130CAS and CrkII orthe Rac-like RhoG and the DOCK180-binding protein Elmo (Hsia et al., 2003;Katoh and Negishi, 2003; Gakidis et al.,2004). However, integrin signaling alsoleads to Rho inactivation through Src-mediated activation of RhoGAP and Racactivation through release of RhoGDI(Dib et al., 2001; Del Pozo et al., 2002).

Furthermore, pathways involvingmultiple GTPases are often coordinately

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triggered during complex biologicalprocesses. An example of this is seenduring axon guidance in the developingnervous system. Binding of thetransmembrane glycoproteinsemaphorin 4D to its transmembranereceptor plexin-B1 leads to Rhoactivation through stimulation of theRhoGEFs LARG and PRG,sequestration of active Rac by a GTPase-binding domain on plexin B1, andinactivation of the Ras family memberR-Ras through a plexin GAP domain,which leads to integrin detachment,repulsion of the axonal growth cone, andturning of the developing axon (Krugeret al., 2005). Understanding thecoordinated regulation of RhoGEFactivity with that of GAPs, GDIs andother Ras family proteins will constitutethe next frontier in the study of RhoGTPases.

ReferencesBaird, D., Feng, Q. and Cerione, R. A. (2005). The Cool-2/�-Pix protein mediates a Cdc42-Rac signaling cascade.Curr. Biol. 15, 1-10.Berken, A., Thomas, C. and Wittinghofer, A. (2005). Anew family of RhoGEFs activates the Rop molecularswitch in plants. Nature 436, 1176-1180.

Bishop, A. L. and Hall, A. (2000). Rho GTPases and theireffector proteins. Biochem. J. 348, 241-255.Boettner, B. and Van Aelst, L. (2002). The role of RhoGTPases in disease development. Gene 286, 155-174.Burridge, K. and Wennerberg, K. (2004). Rho and Ractake center stage. Cell 116, 167-179.Del Pozo, M. A., Kiosses, W. B., Alderson, N. B., Meller,N., Hahn, K. M. and Schwartz, M. A. (2002). Integrinsregulate GTP-Rac localized effector interactions throughdissociation of Rho-GDI. Nat. Cell Biol. 4, 232-239.Dib, K., Melander, F. and Andersson, T. (2001). Role ofp190RhoGAP in beta 2 integrin regulation of RhoA inhuman neutrophils. J. Immunol. 166, 6311-6322.Erickson, J. W. and Cerione, R. A. (2004). Structuralelements, mechanism, and evolutionary convergence ofRho protein-guanine nucleotide exchange factorcomplexes. Biochemistry 43, 837-842.Feng, Q., Baird, D., Peng, X., Wang, J., Ly, T., Guan,J.-L. and Cerione, R. A. (2006). Cool-1 functions as anessential regulatory node for EGF receptor- and Src-mediated cell growth. Nat. Cell Biol. 8, 945-956.Gakidis, M. A., Cullere, X., Olson, T., Wilsbacher, J.L., Zhang, B., Moores, S. L., Ley, K., Swat, W.,Mayadas, T. and Brugge, J. S. (2004). Vav GEFs arerequired for beta2 integrin-dependent functions ofneutrophils. J. Cell Biol. 166, 273-282.Hsia, D. A., Mitra, S. K., Hauck, C. R., Streblow, D. N.,Nelson, J. A., Ilic, D., Huang, S., Li, E., Nemerow, G.R. and Leng, J. (2003). Differential regulation of cellmotility and invasion by FAK. J. Cell Biol. 160, 753-767.Jaffe, A. B. and Hall, A. (2005). Rho GTPases:biochemistry and biology. Annu. Rev. Cell Dev. Biol. 21,247-269.Jaffe, A. B., Hall, A. and Schmidt, A. (2005).Association of CNK1 with Rho guanine nucleotideexchange factors controls signaling specificitydownstream of Rho. Curr. Biol. 15, 405-412.Katoh, H. and Negishi, M. (2003). RhoG activates Rac1by direct interaction with the Dock180-binding proteinElmo. Nature 424, 461-464.Kruger, R. P., Aurandt, J. and Guan, K. L. (2005).

Semaphorins command cells to move. Nat. Rev. Mol. CellBiol. 6, 789-800.Li, S., Tian, X. and Feig, L. A. (2006). Distinct roles forRas-guanine nucleotide-releasing factor 1 (Ras-GRF1)and Ras-GRF2 in the induction of long-term potentiationand long-term depression. J. Neurosci. 26, 1721-1729.Mertens, A. E. E., Pegtel, D. M. and Collard, J. G.(2006). Tiam1 takes PARt in cell polarity. Trends Cell Biol.16, 308-316.Ridley, A. (2001). Rho family proteins: coordinating cellresponses. Trends Cell Biol. 11, 471-477.Ridley, A., Schwartz, M. A., Burridge, K., Firtel, R. A.,Ginsberg, M. H., Borisy, G., Parsons, J. T. and Horwitz,A. R. (2003). Cell migration: integrating signals from frontto back. Science 302, 1704-1709.Rossman, K. L., Der, C. J. and Sondek, J. (2005). GEFmeans go: turning on Rho GTPases with guaninenucleotide-exchange factors. Nat. Rev. Mol. Cell Biol. 6,167-180.Schmidt, A. and Hall, A. (2002). Guanine nucleotideexchange factors for Rho GTPases: turning on the switch.Genes Dev. 16, 1587-1609.Schwartz, M. (2004). Rho signalling at a glance. J. CellSci. 117, 5457-5458.Wennerberg, K. and Der, C. J. (2005). Rho-familyGTPases: it’s not only Rac and Rho (and I like it). J. CellSci. 117, 1301-1312.

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Cell Science at a Glance on the WebElectronic copies of the poster insert areavailable in the online version of this articleat jcs.biologists.org. The JPEG images canbe downloaded for printing or used asslides.

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