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European Journal of Cell Biology 85 (2006) 947–959

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Rho GTPase signaling in Dictyostelium discoideum:Insights from the genome

Georgia Vlahou, Francisco Rivero�

Center for Biochemistry of the Medical Faculty and Center for Molecular Medicine Cologne, University of Cologne,

Joseph-Stelzmann-Str. 52, D-50931 Cologne, Germany

Abstract

Rho GTPases are ubiquitously expressed across the eukaryotes where they act as molecular switches participating inthe regulation of many cellular processes. We present an inventory of proteins involved in Rho-regulated signalingpathways in Dictyostelium discoideum that have been identified in the completed genome sequence. In Dictyostelium

the Rho family is encoded by 18 genes and one pseudogene. Some of the Rho GTPases (Rac1a/b/c, RacF1/F2 andRacB) are members of the Rac subfamily, and one, RacA, belongs to the RhoBTB subfamily. The Cdc42 and Rhosubfamilies, characteristic of metazoa and fungi, are absent. The activities of these GTPases are regulated by twomembers of the RhoGDI family, by eight members of the Dock180/zizimin family and by a surprisingly large numberof proteins carrying RhoGEF (42 genes) or RhoGAP (43 genes) domains or both (three genes). Most of these showdomain compositions not found in other organisms, although some have clear homologs in metazoa and/or fungi.Among the (in many cases putative) effectors found in Dictyostelium are the CRIB domain proteins (WASP and tworelated proteins, eight PAK kinases and a novel gelsolin-related protein), components of the Scar/WAVE complex, 10formins, four IQGAPs, two members of the PCH family, numerous lipid kinases and phospholipases, and componentsof the NADPH oxidase and the exocyst complexes. In general, the repertoire of Rho signaling components ofDictyostelium is similar to that of metazoa and fungi.r 2006 Elsevier GmbH. All rights reserved.

Keywords: Dictyostelium; Rho GTPase; Guanine nucleotide exchange factor; Guanine nucleotide dissociation inhibitor; GTPase-

activating protein; Formin; Dock180; IQGAP; WASP; Exocyst

Introduction

Rho GTPases constitute a family of small GTPaseswithin the Ras superfamily. Initially, Rho GTPases wereimplicated in most actin-regulated processes such asmotility, adhesion, morphogenesis and membrane traf-ficking (including phagocytosis, pinocytosis and exocy-tosis). However, the spectrum of processes for which

e front matter r 2006 Elsevier GmbH. All rights reserved.

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ing author. Tel.: +49221 478 6987;

8 6979.

ess: francisco.rivero@uni-koeln.de (F. Rivero).

regulation by Rho GTPases has been documented hasexpanded enormously, and include, to mention a few,microtubule organization, cytokinesis, gene expression,cell cycle progression, apoptosis and tumorigenesis. Infact, it has become evident that few cellular processes arenot directly or indirectly controlled by Rho familyproteins (Wennerberg and Der, 2004; Burridge andWennerberg, 2004; Jaffe and Hall, 2005).

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Like other small GTPases, those of the Rho familyact as molecular switches, cycling between an activeGTP-bound and an inactive GDP-bound state. Threemajor classes of proteins modulate the activation/inactivation cycle. Guanine nucleotide exchange factors(GEFs) catalyze the exchange of GDP for GTP toactivate the switch. GTPase activating proteins (GAPs)stimulate the intrinsic GTPase activity, and thusinactivate the switch. Guanine nucleotide-dissociationinhibitors (GDIs) block spontaneous activation andregulate cycling of the GTPase between membranes andcytosol. Activation enables Rho GTPases to undergoconformation-specific interactions with a multitude ofeffectors that elicit rearrangements of the actin cyto-skeleton and many other cellular responses.

Rho GTPases are ubiquitously expressed across theeukaryotes. In human the Rho family consists of 21genes, most of them still poorly characterized, that canbe grouped into eight distinct subfamilies. Three ofthem, Rac, Rho and Cdc42 have been extensivelystudied, but functional studies on members of the otherfamilies are accumulating (Wennerberg and Der, 2004).Studies on Dictyostelium discoideum are increasinglycontributing to the understanding of basic cellularprocesses regulated by Rho GTPases, like actin organi-zation, chemotaxis and endocytosis (Rivero andSomesh, 2002). These studies are benefiting from theadvantages of D. discoideum as a model organism. Theseinclude the complexity of the actin cytoskeleton and thesignaling networks, which is comparable to that ofleukocytes and other highly motile cells (Parent, 2004;Affolter and Weijer, 2005; Rivero and Eichinger, 2005),and the genetic tractability of the organism. Theavailability of a completely sequenced and assembledgenome constitutes a powerful tool that is facilitatingthe design of functional studies (Eichinger et al., 2005).

In this review, we present an inventory of proteinsinvolved in Rho-regulated signaling pathways inDictyostelium that have been identified in the completedgenome sequence (Table 1) (Eichinger et al., 2005). Weare not discussing here functional studies performedover the years on some of these proteins; for this thereader is referred to recent comprehensive reviews on thematter (Rivero and Somesh, 2002; Weeks et al., 2005).

Rho GTPases

A previous study using the partially complete andunassembled genome sequence identified 16 genesencoding Rho GTPases (Rivero et al., 2001). Theavailability of the complete genome sequence has setthe inventory to 19 genes, one of them, racK, beingapparently a pseudogene (Eichinger et al., 2005; Weekset al., 2005). Details on the phylogenetic relationships ofthe Dictyostelium Rho GTPases will be presented here

only briefly (Fig. 1). Based on phylogenetic analyses,several Rho GTPases can be loosely grouped in the Racsubfamily: Rac1a, Rac1b, Rac1c, RacF1, RacF2, RacBand the GTPase domain of RacA (see below) (Riveroet al., 2001; Weeks et al., 2005). All other Dictyostelium

Rho GTPases were named Rac for historical reasonsbut they do not have a clear affiliation, although someare closer to Rac than to members of other subfamilies.RacI, RacM, RacN and RacO cluster significantlytogether, suggesting that they might also function inthe same processes; together with RacJ, these four countamong the most divergent Rho family members. Thereare no representatives of the Cdc42, Rho or othersubfamilies in Dictyostelium. In fact Cdc42 and Rhoappear to be specializations of fungi and metazoa. Invertebrates the Rho family has undergone considerableexpansion, differentiating into eight subfamilies (Wen-nerberg and Der, 2004; Salas-Vidal et al., 2005) (Fig. 1).In this review we will use the term Rho GTPase as thegeneric family name, and only where needed specificsubfamily names will be used.

The expression pattern and the function of Dictyo-

stelium Rho GTPases have been discussed recently(Rivero and Somesh, 2002; Weeks et al., 2005). Ingeneral, knockout, overexpression and gain-of-functionmutants have been used to investigate the physiologicalroles of a limited number of Dictyostelium RhoGTPases, and clear roles for Rho GTPases in regulationof morphology, chemotaxis, endocytosis and vesicletrafficking, cytokinesis and development have beenestablished.

The Dictyostelium genome harbors a member of theRhoBTB subfamily of atypical Rho GTPases, racA.This subfamily has representatives in metazoa (althoughit is missing in Caenorhabditis elegans), but is absent infungi and plants. In RhoBTB proteins the GTPase isfollowed by a tandem repeat of BTB (broad-complex,tramtrack and bric-a-brac) domains. The BTB domainhas been recently identified as the adaptor domain incullin-3-dependent ubiquitin ligase complexes, and infact mammalian RhoBTB proteins are able to interactwith cullins (Wilkins et al., 2004, and our unpublishedobservations). This suggests that members of thissubfamily could function to target other proteins forproteasomal degradation, but the exact mechanisms andhow they are connected to the proposed role ofmammalian RHOBTB genes as tumor suppressorsremain to be established. Remarkably, the GTPasedomain of RacA is more related to Rac than toRhoBTB, and presumably constitutes a relict of theorigin of this subfamily in an ancient rac gene thatacquired a C-terminal extension at an early stage.

More recently a new subfamily of GTPases, Miro(mitochondrial Rho) has been incorporated to the Rhofamily (Fransson et al., 2003), although the ascription ofMiro to this family is controversial. Miro proteins consist

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Table 1. Proteins involved in Rho signaling in Dictyostelium discoideum and their occurrence in other phyla

Protein class Number of

genes

Relevant

domain or

componenta

Closest relatives in other

organisms

Occurrenceb Interaction

with Rho

GTPasesc

Rho GTPases

Rac-like 6 GTPase Rac (P), F, M

RhoBTB-like 1 GTPase RhoBTB M

Other RhoGTPasesd 12 GTPase Rac (Unique) U

Dissociation inhibitors

RhoGDI1 1 RhoGDI RhoGDI E +

RhoGDI2 1 RhoGDI RhoGDI (Unique) U �

Exchange factors

RhoGEFe 45 RhoGEF (DH) Mostly unique F, M +

CZH 8 CZH2 (DHR-2) DOCK180, MBC,

CED-5

E +

Darlin 1 SmgGDS, Yeb3p F, M +

GTPase-activating proteins

RhoGAPe 46 RhoGAP Mostly unique E +

Effectors and other Rho GTPase-binding proteins

Scar complex 5 PIR121 WAVE complex P, M na

WASP 1 CRIB WASP F, M +

WASP-related 2 CRIB WASP/WAVE (Unique) U +

PAK 9 CRIB PAK kinases F, M +

Gelsolin-related 1 CRIB U +

Formins 10 GBD Formins (P), F, M +

IQGAP-related 4 GRD IQGAP F, M +

PCH family 2 HR1 CIP4, Toca-1, Cdc15p F, M na

Lipid kinasesf X19 p85 Class I PI-3-kinases

(p110), PI-4-P5K, DGK

E na

Phospholipasesg 5 Phospholipase C,

Phospholipase D1/2

E na

NADPH oxidase X5 p67phox NADPH oxidase E na

Exocyst complex 8 Sec3, Exo70 Exocyst complex E na

LIS1 1 LIS1, Pac1 E +

LimE 1 LIM proteins U (+)

The table is based on published information and in additional inspection of the Dictyostelium genome either for domains characteristic of each of the

protein classes or using the BLAST server at DictyBase (http://www.dictybase.org) with additional metazoan or fungal proteins that do not display

defined domains as query. Because Rho signaling is an expanding field, no claim of completeness can be made. Conversely, for many of the protein

families included in the table participation in Rho signaling has been documented in some species but not in others, therefore a role in Dictyostelium

should be taken as putative.aRelevant domains or components refer, apart from the GTPase, to those involved in interactions with the Rho GTPase if they have been

determined. Abbreviations of the relevant domains: CRIB, Cdcd42 and Rac interactive binding (also known as PBD, p21-binding domain); CZH2,

CDM-zizimin homology 2 domain (also known as DHR-2, Dock homology region 2); GAP, GTPase-activating protein; GBD, Rho GTPase-binding

domain of formins; GDI, guanine nucleotide dissociation inhibitor; GEF, guanine nucleotide exchange factor (the RhoGEF domain is also known as

DH, Dbl homology domain); GRD, RasGAP-related domain; HR1, protein kinase C-related kinase homology region 1.bOccurrence refers in general to the presence of a protein with equivalent domain architecture. In the cases of large families it just indicates that the

relevant domain is represented. U, unique (the protein has no relatives in plants, fungi or metazoa, or differs from relatives due to an unusual domain

composition); E, eukaryotes; P, plants; F, fungi; M, metazoa (for E, F, P, and M the protein might be missing from any particular species). When in

parentheses, occurrence indicates that related proteins with a different domain architecture exist in that particular phylum.cIn general, not all members of a family have been assayed for interaction with Rho GTPases, and conversely, in most cases only a reduced number

of Rho GTPases have been used in these assays; (+) indicates that the interaction might be indirect; na, not assayed. Data are gathered from the

literature and from our own unpublished results.dThis group includes RacC–E and RacG–O. RacK is a pseudogene.eThree genes encode proteins with both RhoGAP and RhoGEF domains.fClass I PI 3-kinases are not present in plants and fungi. The p85 regulatory subunit, that in vertebrates interacts with Cdc42 and Rac1 is

apparently missing in Dictyostelium. This does not exclude a potential regulation by Rho GTPases through other mechanisms, therefore PI 3-kinases

have not been excluded from the table.gIncludes a single phospholipase C as well four phospholipase D proteins related to mammalian PLD1 and PLD2.

G. Vlahou, F. Rivero / European Journal of Cell Biology 85 (2006) 947–959 949

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Fig. 1. Phylogenetic tree of the Rho family. Complete sets of Rho proteins from several species were used. This comprises the 18

Dictyostelium discoideum (Dd) Rho proteins (RacK was excluded), 6 from Saccharomyces cerevisiae (Sc), 12 from Arabidopsis

thaliana (At), 5 from Caenorhabditis elegans (Ce), 7 from Drosophila melanogaster (Dm) and 20 from Homo sapiens (Hs). Human

RhoBTB3 was not included in the analysis. Ascription of Miro proteins to the Rho family is controversial, therefore these proteins

have been excluded. The phylogenetic tree was constructed using the neighbor joining method as already described (Rivero et al.,

2001). Dictyostelium Rho GTPases are highlighted. Well-defined subfamilies have been labeled. An asterisk indicates a significant

cluster of Dictyostelium Rho GTPases. Plants exclusively possess Rop proteins. Both animals and yeast have members of the Rho

and the Cdc42 subfamilies, but yeast does not have Rac proteins (although filamentous fungi do). Dictyostelium has representatives

of the Rac subfamily, but lacks Rho and Cdc42 proteins. Note that the DdRacA GTPase domain is more closely related to Rac

proteins than to RhoBTB proteins, although RacA has the domain architecture of a RhoBTB protein. The scale bar indicates 10%

divergence. Only the innermost nodes where bootstrapping values were significant are indicated.

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of two GTPase domains, two EF-hand motifs placedbetween the GTPases and a short C-terminal transmem-brane region that specifically targets the protein to theouter mitochondrial membrane. Only the N-terminalGTPase bears similarity (although very low) to other Rhoproteins, and the Rho-specific insert loop (a short aminoacid insertion that contributes to determine the specificityof functions of Rac against GTPases of other families) isapparently missing. Miro proteins are found in plants,fungi and metazoa and appear to be involved inmitochondrial homeostasis, morphogenesis and axonaltransport (Fransson et al., 2003; Guo et al., 2005). InDictyostelium a single gene, gemA, encodes an unchar-acterized Miro protein with approximately 50% similar-ity to the human counterparts.

RhoGEFs

Three major families of Rho GEFs have beenrecognized in eukaryotes, each characterized by anunrelated short domain that displays nucleotide ex-change activity. These domains are the RhoGEF domain(also known as DH or Dbl-homology domain), theCZH2 (CDM-zizimin homology) domain (also known asDOCKER or DHR2, Dock homology region 2), and therecently described RopGEF. The last is exclusivelyfound in plants (Berken et al., 2005).

The RhoGEF domain consists of about 180 aminoacids that fold into 10–15 a-helices arranged in anoblong helical bundle. This domain is almost invariablyfollowed by a pleckstrin homology (PH) domain that isthought to regulate the exchange activity of theRhoGEF domain, acts as a membrane anchor andparticipates in interactions with other signaling compo-nents. In Dictyostelium 45 genes encode proteins with aRhoGEF domain (Fig. 2; for details see SupplementaryFig. 1), a number that lies between the �23 ofDrosophilamelanogaster and the 69 found in the humangenome (Rossman et al., 2005). Based on a sequencecomparison, there is a high degree of homogeneityacross the RhoGEF domains of Dictyostelium, and ingeneral well-defined clades cannot be recognized, evenamong proteins with a similar domain composition.Two pairs of proteins are an exception: DDB0187910and DDB0187913 on one side and DDB0192204 andDDB0189592 on the other.

The RhoGEF-PH module appears alone (in Dictyo-

stelium in 10 cases) or more frequently in combinationwith a broad diversity of other domains, as it is also thecase in other organisms (Schmidt and Hall, 2002;Rossman et al., 2005). The domain architecture ofmany RhoGEFs appears unique to Dictyostelium. Forexample, armadillo repeats or a myosin motor domainoccur only in Dictyostelium RhoGEFs. A myosin

molecule with a RhoGEF domain as in MyoM hasnot been identified thus far in other organisms, but inmammals a myosin with a RhoGAP domain has beendescribed (Chieregatti et al., 1998). A total of 8 proteinscarry one or more CH (calponin homology) domains atthe N-terminus. The combination CH-RhoGEF iscommon in metazoa and fungi, like in Vav, a-PIX andCdc24, but in Dictyostelium these proteins have a uniquedomain architecture (see Rivero and Eichinger, 2005, fordetails): almost all have one IQ domain, lack SH3 andC1 domains and in some cases bear additional domains,like VHP (villin headpiece), PX (Phox-homologous) orArfGAP. In a few proteins the RhoGEF domainappears in combination with other signaling domains,like RhoGAP, RasGAP, RasGEF, ArfGAP or a proteinkinase, some of them also found in metazoan RhoGEFsalthough in different arrangements. One interestingprotein is DDB0186714, where the RhoGEF-PHmodule is placed at the C-terminus of a region withseveral transmembrane segments, a structure thatresembles that of RasGEF-W (Wilkins et al., 2005).Four proteins carry C-terminal FYVE domains andhave homologs in Ustilago maydis and in metazoan Fgdand frabin, a class of actin-associated proteins (Ikedaet al., 2001).

To date only three RhoGEFs have been characterizedin terms of interaction with Rho GTPases, and mutantsexist that reveal roles in chemotaxis, actin organizationand cell morphology. The C-terminal region of Rac-GAP1, which harbors two RhoGEF domains, displaysexchange activity for Rac1a, but not for RacC or RacE(Knetsch et al., 2001). The RhoGEF domain of MyoMdisplays a similar specificity (including additionallyRac1b) (Geissler et al., 2000). By contrast, RacGEF1is more potent on RacB than on Rac1b, and does notcatalyze exchage on RacC and RacE (Park et al., 2004).

CZH proteins constitute a recently described family ofRhoGEFs present in all eukaryotes. They are unrelatedto the RhoGEF family described above, and theirmechanism for nucleotide exchange is not known. CZHproteins are composed of an N-terminal CZH1 domainand a C-terminal not yet well-defined CZH2 domain.These domains were initially named DHR1 and DHR2,respectively (Cote and Vuori, 2002). The CZH1 domainmay interact with the CZH2 domain and inhibit theinteraction with the GTPase. A recent phylogeneticanalysis has divided CZH2 proteins into two majorsubfamilies, the Dock180- and the zizimin-relatedsubfamily. Presently only few CZH proteins have beencharacterized to some extent: mammalian Dock180 andrelated proteins, C. elegans CED-5 and fruit flymyoblast city, for example, are involved in cell migra-tion, engulfment of apoptotic cells, T-cell activation andneurite outgrowth (see Meller et al. (2005) for a review).The CZH family is represented by 8 members inDictyostelium, 4 in each subfamily. All 8 have the

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Fig. 2. Domain structure of predicted Dictyostelium proteins with RhoGEF and RhoGAP domains. The Dictyostelium genome

encodes 42 proteins with a RhoGEF domain, 43 proteins with a RhoGAP domain and 3 proteins with both. The number of

representatives of each subfamily is indicated on the left. In this simplified cartoon the protein lengths as well as size and position of

the domains are not drawn at scale. In several subfamilies some members carry additional domains that have not been depicted here.

Detailed depictions including sequence identifiers can be seen in Supplementary Figs. 1 and 2. The domains shown are: ArfGap,

GTPase-activating protein for the small GTPase ARF; C2, protein kinase C conserved region 2; CH, calponin-homology domain;

FBOX, a receptor for ubiquitination targets; FCH, Fes/CIP4-homology domain; FYVE, a class of zinc finger domain present in

Fab1, YOTB, Vac, and EEA1; IPPc, inositol polyphosphate 5-phosphatase domain; IQ, calmodulin-binding motif; LRR, leucine-

rich repeat; PH, pleckstrin-homology domain; PX, phox-homologous domain; RasGAP, GTPase-activating protein for Ras-like

small GTPases; RasGEF, guanine nucleotide exchange factor for Ras-like small GTPases; RCC1, regulator of chromosome

condensation 1; SEC14, domain in homologs of an S. cerevisiae phosphatidylinositol transfer protein (Sec14p); SH3, Src-homology

3 domain; TM, transmembrane region(s); VHP, villin headpiece; WD, WD40 repeats; ZnF, zinc finger.

G. Vlahou, F. Rivero / European Journal of Cell Biology 85 (2006) 947–959952

typical domain architecture CZH1–CZH2. Except foran N-terminal SH3 domain, characteristic of theDock180 subfamily, in two of the proteins, no furtherdomains are found in the other family members. Ingeneral CZH proteins are less versatile than theRhoGEF-containing proteins in their domain composi-tion. Some CZH proteins, probably only those of theDock180 subfamily, function in complex with ELMO(engulfment and cell motility protein), although theexact role of this protein is controversial (Lu et al.,2005). Dictyostelium has 6 putative ELMO orthologsbased on the presence of a conserved central regioncharacteristic of proteins of the ELMO family.

In addition to the RhoGEFs of the two familiesdescribed above, darlin, an armadillo-like protein

related to the mammalian SmgGDS has beed describedin Dictyostelium (Vithalani et al., 1998). VertebrateSmgGDS displays weak GEF activity for a widespectrum of GTPases, not only those of the Rho family.Darlin binds to RacE and RacC, however a GEFactivity has not yet been demonstrated. The role ofdarlin remains obscure, and disruption of the gene onlyelicited a moderate defect in early aggregation.

RhoGAPs

The RhoGAP family is defined by the presence of aconserved RhoGAP domain. This domain consists of

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about 150 amino acids that fold into nine a-helices. Oneof the loops contains a highly conserved arginine residueimportant for catalytic activity. Interestingly, theRasGAP domain presents a similar folding andGTPase-activating mechanism, suggesting a sharedancestry (Gambling and Smerdon, 1998). In Dictyost-

elium, 46 genes encode proteins with a RhoGAP domain(Fig. 2; for details see Supplementary Fig. 2), which isabout half the number found in the human genome(Moon and Zheng, 2003). As in the case of the RhoGEFdomains, sequence comparison reveals a high degree ofhomogeneity across the RhoGAP domains, and withone exception (see below) well-defined clades cannot berecognized. Even proteins with similar domain composi-tion, like for example those carrying an N-terminal FCHdomain, group together only very loosely. Twenty-oneRhoGAPs do not present any recognizable additionaldomain. The rest display a high diversity of domainarchitectures, in most cases only found in Dictyostelium.In several proteins the RhoGAP domain appears incombination with other signaling domains, like Ras-GEF (in RasGEF-D and two RasGEF-related proteins;Wilkins et al., 2005), RhoGEF or a serine/threoninekinase.

Some of the Dictyostelium RhoGAPs have apparentrelatives in other organisms (Peck and Burbelo, 2002;Moon and Zheng, 2003). A group of four proteins withan N-terminal FCH domain is related to the Slit-RoboGAPs (srGAPs), although srGAPs have an additionalSH3 domain downstream of the RhoGAP domain that isabsent both in the Dictyostelium proteins and in the yeasthomolog Rgd1p. srGAPs become activated upon inter-action with the Robo receptor and inactivate Cdc42.These proteins are involved in neuronal migration (Wonget al., 2001). The yeast relative, Rgd1p is involved in low-pH survival (Gatti et al., 2005). DDB0204640 is related top50RhoGAP, the first RhoGAP protein identified. It istargeted to endosomes by virtue of the N-terminal Sec14domain, which interacts with Rab (Sirokmany et al.,2006). DDB0189888 has an N-terminal inositol polypho-sphate 5-phosphatase domain, and is related to humanOCRL-1, which appears mutated in oculocerebrorenalsyndrome and may be implicated in Golgi vesiculartransport (Suchy et al., 1995).

Finally, three RhoGAPs have an SH3 domainimmediately after the RhoGAP domain. Interestingly,the RhoGAP domains of two of these proteins,RacGAP1 and DB204400, are the only ones thatconstitute a significant clade. RacGAP1 is to date theonly protein whose GAP activity has been assayed: it isactive on Rac1a, RacC and RacE, as well as on RabD(Knetsch et al., 2001). DDB0189127 has the domainstructure PH-RhoGAP-SH3 that is found in severalmammalian proteins of the GRAF family, first identi-fied as RhoGAPs associated with focal adhesion kinase(Hildebrand et al., 1996).

RhoGDIs

RhoGDIs are characterized by a short N-terminaldomain that adopts an a-helical hairpin structure uponinteraction with the GTPase, and a C-terminal domainthat adopts an immunoglobulin folding. A hydrophobicpocket in the C-terminal domain accomodates theisoprenyl moiety characteristic of Rho GTPases. Thislipid modification is essential for interaction withRhoGDI. RhoGDIs are widespread regulators of RhoGTPases initially thought to play a passive rolepreventing nucleotide exchange and membrane associa-tion. More recent evidence place RhoGDIs as importantplayers able to participate in multimolecular complexesthat regulate correct targeting and interaction of RhoGTPases with RhoGEFs and effectors. They are alsosubjected to regulation through phosphorylation byPAK kinases, another family of Rho-regulated signalingcomponents (see Dovas and Couchman, 2005; Dransartet al., 2005, for recent reviews).

Two RhoGDI homologs had been identified pre-viously in Dictyostelium, RhoGDI1 and RhoGDI2(Rivero et al., 2002). RhoGDI1 is a typical RhoGDIand interacts with several Rho GTPases (Rac1a/b/c,RacB, RacC and RacE) whereas RhoGDI2, which isexpressed at late stages of development, is moredivergent, lacks the N-terminal regulatory arm that isresponsible for most of the interactions with the GTPaseand consequently does not interact with Rho GTPases.RhoGDI2, which is unique to Dictyostelium, might beinvolved in protein–protein interactions independentlyof binding to Rho GTPases.

Effectors and other Rho-binding proteins

Effectors are defined by their ability to interact withthe GTP-bound form of the GTPase. The list of knowneffectors of Rho GTPases is very large and is stillgrowing, therefore the inventory presented below willnecessarily be incomplete at present. In addition, someeffectors display very unspecific sequence features orcarry very common non-informative domains, andmight therefore have escaped identification. Such is thecase of kinectin, IRSp53, POSH, PAR6 and kinases likep70S6, MEKK, PKN, ROCK, rhotekin and rhophilin.Direct search of the genome using the blast tool has notrevealed reliable homologs of these proteins and otherslike POR1 (arfaptin 2) and citron kinase.

Proteins with CRIB domain and related effectors

Among the best well-known effectors of Rho GTPasesare those bearing a Cdc and Rac interactive binding(CRIB) domain, also known as PBD (p21-binding

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domain). In general, these effectors exist in a foldedinactive conformation in which an autoinhibitory domainoverlaps with the CRIB domain. This interaction isdisrupted upon binding of the activated GTPase, bring-ing about an open conformation that allows the effectorto establish further interactions or display catalyticactivity (Bokoch, 2003; Bompard and Caron, 2004).The CRIB domain is found in Dictyostelium in two majorfamilies: Scar/WASP (Wiscott-Aldrich syndrome protein)and PAK (p21-activated kinase). In addition, the genomehas revealed a gene (gnrC) encoding a unique gelsolinhomolog with two N-terminal CRIB domains. Thisunusual domain architecture suggests that this putativeactin-binding protein might be subject to direct regula-tion by small GTPases.

Members of the WASP/Scar family are well-char-acterized activators of the Arp2/3 complex in metazoaand fungi and have been recently described also inplants (Frank et al., 2004). These proteins share acentral proline-rich region and a C-terminal regioncomposed of one or two WH2 (WASP-homology 2)domains that bind actin monomers followed by oneacidic region that interacts with the Arp2/3 complex.The proline-rich region binds profilin as well as SH3domains from a variety of proteins (Miki and Takena-wa, 2003). In Dictyostelium, this family has fourmembers: Scar, WASP and two WASP-related proteins(DDB0232169 and DDB0232170). Scar is the onlymember of the family that lacks a CRIB domain, whileWASP is the only member that possesses a WH1(WASP-homology 1) domain binding poly prolinehelices. DDB0232170 has a long central region withpredicted coiled-coil structure.

Both Scar and WASP are positive regulators of actinpolymerization in Dictyostelium (Seastone et al., 2001;Myers et al., 2005). WASP and WASP-related proteinsinteract with several Dictyostelium Rac proteins (ourunpublished observations). Although Scar lacks a CRIBdomain, the mammalian homolog, WAVE (WASPfamily verprolin homology protein), is subject toregulation by Rac. Scar/WAVE exists as a multi-molecular complex, and the components of this com-plex, PIR121, Nap125, Abi2 and HSP300, are alsofound in the genome of Dictyostelium, each encoded bya single gene (Blagg et al., 2003). PIR121 is thecomponent involved in interaction with the RhoGTPase, but this has not been demonstrated inDictyostelium yet.

PAKs are Ser/Thr protein kinases whose activity isstimulated by binding of active Rho GTPases, althoughGTPase-independent mechanisms of activation havebeen described. In PAK kinases the catalytic domain isplaced typically at the C-terminus and is preceded by aregulatory domain that harbors the CRIB domain.PAKs act on a large list of substrates and regulate notonly cytoskeletal dynamics, but signal cascades involved

in gene transcription, apoptosis and cell cycle progres-sion as well (Bokoch, 2003; Zhao and Manser, 2005).The role of three Dictyostelium PAKs, PAKa, PAKb(formerly MIHCK, myosin I heavy chain kinase) andPAKc in polarity and chemotaxis are well established,and the spectrum of interactions with Dictyostelium RhoGTPases has been determined recently (Rivero andSomesh, 2002; Weeks et al., 2005).

Inspection of the Dictyostelium genome has revealed atotal of 9 genes encoding 8 PAK kinases, PAKa–PAKh.The gene encoding PAKh exists as two copies onchromosome 2, one of them placed in a largechromosomal duplication characteristic of the se-quenced strain AX3. Based on the catalytic domainthese proteins can be grouped in two distinct clades(PAKa–PAKd vs. PAKe–PAKh) but the significance ofthis is unknown. PAKc has a PH domain immediatelyupstream of the CRIB domain, and is therefore relatedto the fungal Cla4p-like PAKs known to be involved inseptin ring formation at the mother-bud neck (Verseleand Thorner, 2004). PAKd is unusual in that it bears aCH domain at the very N-terminus, and a C1 domainimmediately upstream of the CRIB domain. Finally,PAKf, PAKg and PAKh have atypical C-terminalextensions after the catalytic domain that in PAKhend with a single transmembrane region.

Formins

Formins have emerged as important regulators ofactin dynamics, and consequently they are implicated ina wide range of actin-based processes (Wallar andAlberts, 2003). Formins are multidomain proteinspresent in all eukaryotes and are defined by the presenceof the FH2 (formin homology 2) domain, capable offorming a ring-shaped flexible dimer that caps thebarbed end and allows processive elongation of the actinfilament. The Dictyostelium genome harbors 10 genesencoding formins (for A–J) that, with the exception ofForI and ForC, share the GBD–FH1–FH2–DADdomain structure present in almost all fungal andmetazoan formins and are thus clearly divergent fromplant formins (see Rivero et al. (2005) for a detailedphylogenetic and gene expression analysis). The FH1(formin homology 1) domain is a proline-rich regionthat interacts with profilin-actin. Formins are able tointeract with activated Rho GTPases through an N-terminal Rho GTPase-binding domain (GBD). Bindingof GTPases unleashes the intramolecular inhibitoryinteraction between the GBD and a C-terminal Dia-phanous autoregulatory domain (DAD) and renders theprotein active (Lammers et al., 2005). Based on sequenceanalysis, Dictyostelium formins might be functional interms of actin nucleation activity and, with the excep-tion of ForI, that lacks recognizable GBD and DAD

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domains, might also be regulated by Rho GTPases.These aspects have been investigated so far only in ForH(dDia2): this formin interacts with Rac1a (but not withRacC or RacE) and promotes actin polymerization(Schirenbeck et al., 2005).

IQGAP

IQGAP proteins constitute a conserved family ofscaffolding proteins that interact with cytoskeletal andsignal transduction proteins. They are composed of a CHdomain (probably implicated in binding to F-actin), atandem of coiled-coil repeats, an SH3-mimicking WWdomain, one to four IQ repeats (responsible for binding tocalmodulin) and a RasGAP homology domain (GRD)followed by RasGAP C-terminal domain (RGCT) (Ma-teer et al., 2003). The GRD domain does not exhibitRasGAP activity, but interacts with activated RhoGTPases. Two IQGAP-related proteins were identifiedand characterized previously in Dictyostelium, DGAP1/DdRasGAP1 and GAPA, that play important roles incytokinesis and other actin-based processes (reviewed inRivero and Somesh, 2002). DGAP1 binds preferentiallyactivated Rac1a but not RacC or RacE. Two moremembers of this family have been revealed in the genome,DDB0233055 and DDB0232202. All four IQGAPs consistof a GRD-RGCT region preceded by some weaklyconserved IQ repeats, and with the exception ofDDB0232202 they lack further N-terminal domainscharacteristic of mammalian IQGAPs. Interestingly,DDB0232202 has two CH domains related to the firstactin-binding domain of fimbrin, but not the CH domaincharacteristic of IQGAP and other signaling molecules.

PCH family

PCH (pombe Cdc15 homology) proteins consist of anN-terminal FCH domain and one or two C-terminalSH3 domains. The FCH domain binds to microtubules,while the SH3 domain interacts with WASP (Tian et al.,2000). Binding to Rho GTPases has been documented inseveral PCH proteins, like CIP4 (Cdc42 interactingprotein 4), rapostlin and Toca-1 (transducer of Cdc42-dependent actin assembly), and the Rho GTPase-binding region has been mapped to an HR1 (proteinkinase C-related kinase homology region 1) domain,which was originally identified as a Rho-interactivemodule in several RhoA-binding proteins (Ho et al.,2004). In Dictyostelium a total of six proteins harbor anN-terminal FCH domain, four of these have beendescribed above among the RhoGAPs. Two more,DDB0203245 and DDB0168480, have respectively oneor two SH3 domains at the C-terminus, and thereforemight be considered CIP4/Toca-1 homologs, althoughthis point needs to be confirmed experimentally.

Lipid kinases and phospholipases

Class I phosphatidylinositol (PI) 3-kinases play pivotalroles in chemotaxis, both in leukocytes and Dictyostelium.These enzymes consist of a catalytic and a regulatorysubunit each existing as several isoforms in mammaliancells (Foster et al., 2003). Rho GTPases have been putforward as key factors in a positive feedback loop.Activation of the PI 3-kinase leads to accumulation of 3-phosphoinositides, which in turn results in translocation ofPH domain-containing proteins, like RhoGEFs, andactivation of Rho GTPases. Cdc42 and Rac can bind tothe p85 regulatory subunit and stimulate the catalyticactivity of the PI-3 kinase (Procko andMcColl, 2005). Thep85 subunit has a characteristic domain structure consist-ing of an SH3 domain followed by a RhoGAP-likedomain and two SH2 domains, and is involved in signalingfrom tyrosine kinase receptors. In Dictyostelium six genesencode class I PI 3-kinases with the characteristic domainarchitecture of metazoan p110 subunits (Ras-binding, C2-like, accessory and catalytic domain), and only one has anadditional N-terminal PH domain. Three PI 3-kinaseshave been extensively characterized and their roles inchemotaxis and other processes are well established (seeMerlot and Firtel (2003) for a review). There areapparently no proteins in Dictyostelium that structurallyresemble the p85 subunit, consistent with the absence oftyrosine kinase receptors in this organism. Interestingly,inspection of the genome does not reveal direct homo-logues of the p101 regulatory subunit that mediatessignaling from G-protein-coupled receptors. Consideringthat the Ras-binding domain of the catalytic subunits isconserved, it is possible that Ras, not p101, is the mainregulator of p110. It remains to be established whetherthere is a direct regulation of the PI 3-kinase by RhoGTPases in Dictyostelium and through which mechanisms.

Two further classes of lipid kinases have been foundto directly interact with Rho GTPases: phosphatidyl-inositol-4-phosphate-5-kinases (PIP5K) and diacyl-glycerol kinases (DGK). RhoA, Rac1 and Cdc42stimulate the activity of PIP5K isoforms, and a directinteraction of Rac1 and RhoA has been demonstrated,although this interaction is nucleotide-independent(Weernink et al., 2004). An interaction between PIP5Kand Rop proteins has been reported also in plants (Kostet al., 1999). In mammalian cells some isotypes of DGKare found associated with Rho GTPases, and in C.

elegans binding of Rho to the DAG kinase DGK-1 isrequired for activation of neurotransmitter release (vanBlitterswijk and Houssa, 2000). In Dictyostelium 8 genesencode PIP5-kinases and 5 genes encode proteins with thecatalytic domain of DGKs, but the role of Rho GTPasesin their regulation, if any, remains to be investigated.

There is also evidence linking phospholipase C andphospholipase D (PLD) to Rho signaling (Fukami, 2002;Powner and Wakelam, 2002). In Dictyostelium 9 genes

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encode predicted PLD proteins. Of these, four displaysignificant similarity with the C-terminus of mammalianPLD1, which is the isoform that interacts with RhoGTPases. The C-terminus harbors the catalytic domainand a poorly characterized Rho-binding region. Immedi-ately preceding the catalytic domain, PLD1 and relatedenzymes of plants and fungi have a PH and a PX domainboth involved in binding of phosphoinositides. Only twoof the Dictyostelium PLD1-related enzymes (DDB0204320and DDB0205794) carry a recognizable PH domain,and all apparently lack a PX domain. Interestingly,DDB0205794 has two EF hands, a unique feature thatimplies a direct regulation of this protein by calcium ions.Although the roles of each of these Dictyostelium PLDshave not been reported yet, PLD activity was found to berequired for actin localization, since specific inhibition ofthe enzyme activity with butan-1-ol resulted in clusteringof actin and impairment of actin-dependent processes(Zouwail et al., 2005). The single phospholipase C gene ofDictyostelium, pipA, was identified as a chemotaxis-relatedgene in a microarray phenotyping study (Booth et al.,2005). As in the case of the lipid kinases, a role for RhoGTPases in the regulation of these phospholipases awaitsexperimental verification.

NADPH oxidase

The NADPH oxidase is responsible for superoxideanion production, which in turn gives rise to reactiveoxygen species (ROS) generation. In phagocytes ROScontribute to destruction of ingested bacteria, andconstitute an essential component of the innate immuneresponse of phagocytes. The NADPH is a multisubunitenzyme consisting of two membranous components,gp91 and p22, that constitute the cytochrome b558, andfour cytosolic components, p47, p67, p40 and Rac.During leukocyte activation Rac dissociates fromRhoGDI, becomes activated and translocates to thephagosome membrane along with the other cytosoliccomponents, but how the complex assembles and theexact role of Rac are not clear (see Bokoch, 2005, for adetailed review). p67 is the subunit that binds activatedRac through a tetratricopeptide at the N-terminus. Inaddition, the insert region of Rac interacts with thecytochrome. Several components of the NADPHoxidase complex of Dictyostelium have been character-ized recently, including three gp91 (NoxA, NoxB andNoxC) and one p22 subunits. Mutation of any of thesegenes resulted in aberrant developmental phenotype(Lardy et al., 2005). A p67 homolog has also beenidentified that shows the characteristic N-terminaltetratricopeptide, a central PB1 (Phox and Bem1p)region and a C-terminal WW domain that may functionlike the C-terminal SH3 domain of the mammalian p67orthologs. Interestingly, a 20-amino-acid insertion

between two of the tetratricopetides that is involved inthe interface between Rac and p67 is also conserved.The cytosolic components p40 and p47 might be absentin Dictyostelium or so divergent that they have escapedidentification. Considering that Dictyostelium is aprofessional phagocyte and that signaling through Racis conserved, we anticipate that NADPH oxidaseactivity may be regulated by Rac in this organism too.

The exocyst

The exocyst has been identified as an effector forsmall GTPases of the Rab, Rho and Ras families. It wasinitially described in Saccharomyces cerevisiae, where itis essential for exocytosis, but it has subsequently beenidentified in other eukaryotes. The exocyst is an eight-subunit complex involved in docking of exocyticvesicles. It localizes at regions of active secretion andcell growth, like the bud tip in yeast, growth cones andtips of growing neurites of neurons, and near tightjunctions of epithelial cells (Hsu et al., 2004). Theexocyst components are: Sec3, Sec5, Sec6, Sec8, Sec10,Sec15, Exo70 and Exo84. Two components, Sec3 andExo70, undergo interactions with activated RhoGTPases, although the exact mechanism is unknown.In yeast Rho1 and Cdc42 interact with Sec3, whereasRho3 interacts with Exo70 (Lipschutz and Mostov,2002). In mammalian cells TC10 interacts with Exo70and is required for exocyst-dependent GLUT-4 translo-cation to the plasma membrane in response to insulin(Inoue et al., 2003). In Dictyostelium each member of theexocyst complex is encoded apparently by a single gene.It is very likely that interactions of exocyst componentswith Rho GTPases also take place in Dictyostelium, butthere is no experimental evidence so far.

LIS1

Dictyostelium LIS1 has been identified recently as abinding partner of Rac. LIS1 is a WD40-repeat proteinthat binds to microtubules. The function on microtubuledynamics has been attributed to an N-terminal LisH(Lissencephaly type-1-like homology) domain. LIS1interacts directly with Rac1a, both in the GDP and inthe GTP form, and therefore cannot be considered aneffector (Rehberg et al., 2005). However, it may becontributing to targeting of Rac to its site of action. Inneurons LIS1 promotes Cdc42 activation throughinteraction with IQGAP1 and contributes to tetheringmicrotubule ends to the cortical actin cytoskeleton(Kholmanskikh et al., 2006). In Dictyostelium inter-ference of LIS1 function not only disrupts microtubuledynamics, but also results in reduced F-actin contentand altered actin dynamics (Rehberg et al., 2005). Thereare additional proteins, both in Dictyostelium and in

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other organisms, with the LisH-WD40 architecture, butfor these a link to Rho signaling has not been reported.

LimE

LimE (initially described as DdLim) is a 199-amino-acid protein related to members of the mammalian CRP(cystein-rich protein) family of LIM proteins, whichreportedly associate with the cytoskeleton. In pull-downexperiments with GST-fused Rho proteins LimE wasshown to interact preferentially with the activated formof Rac1a (Prassler et al., 1998). However, it has not beeninvestigated whether the interaction of LimE with theGTPases is direct or takes place indirectly throughformation of a multiprotein complex.

Conclusion

Perhaps the most dramatic feature of the inventorypresented above is the contrast between the complexityof Rho signaling components and the apparent sim-plicity of the organism. In general, the repertoire of Rhosignaling components is clearly more similar to metazoaand fungi than it is to plants, which confirms the statusof Dictyostelium as an ideal organism for determiningthe biological role of many of those components.Nevertheless, there are many instances of proteinswith unique domain architectures. This is particularlyevident for the RhoGEF and RhoGAP families andmay represent specific adaptations to the way of life anddevelopmental cycle of Dictyostelium.

Many studies dealing with signaling during chemotaxisand polarity have used traditionally Dictyostelium as amodel. A comparison of signaling pathways, in particularthose that involve Rho GTPases, during chemotaxis hasshown that although the details may differ, the principlesof organization and several components are shared byDictyostelium and leukocytes (Parent, 2004). However, itbecomes clear from our inventory how much remains tobe investigated. Further efforts will be necessary to char-acterize every one of the components listed in Table 1,determine their structure, define how and under whichcircumstances they interact with each other and elucidatetheir physiological role. Listing the parts alone certainlydoes not provide the answers, but knowing what is thereand what is missing will help us in choosing the rightapproaches to address these questions.

Acknowledgments

This work would not have been possible without theeffort of the teams of the genome and EST sequencingprojects and the dedication of the team of curators at

DictyBase. Work in the authors’ lab is supported bygrants from the Deutsche Forschungsgemeinschaft, theKoln Fortune Program of the Medical Faculty of theUniversity of Cologne and the Center for MolecularMedicine Cologne.

Appendix A. Supplementary material

Supplementary data associated with this article can befound in the online version at doi:10.1016/j.ejcb.2006.04.011.

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