Rho-family GTPases comprise a main branch of the
Ras superfamily of small (~21 kDa) GTPases .So far,
22 human members of the Rho family have been
identified and can be subdivided into 10 groups on
the basis of their identity to Cdc42, Rac1, RhoA,
mitochondrial Rho (Miro1/RhoT1) or Rho-related BTB-
domain-containing protein (RhoBTB).Like Ras,Rho
proteins function as bi-molecular switches by adopt-
ing different conformational states in response to
binding GDP or GTP. In contrast to Rho•GDP,
Rho•GTP actively transduces signals by interacting
with downstream effectors1,2.The activation of Rho-
family proteins is often mediated through various
cell-surface receptors,including the cytokine,tyro-
sine kinase and adhesion receptors, as well as the
G-PROTEIN-COUPLED RECEPTORS (GPCRs)3,4. Rho-family
proteins are regulators of the actin cytoskeleton,
cell-cycle progression and gene transcription, and
have been implicated in cellular processes such as
adhesion and migration, PHAGOCYTOSIS, CYTOKINESIS,
neurite extension and retraction, cellular morpho-
genesis and polarization,growth and cell survival5–8.
Furthermore, aberrant regulation of Rho-family
GTPases promotes malignant transformation and is
essential for the oncogenic properties of Ras and
Cycling between GDP- and GTP-bound states is
controlled primarily by two classes of regulatory mole-
cule (FIG.1):GTPase-activating proteins (GAPs),which
enhance the relatively slow intrinsic GTPase activity of
Rho proteins;and guanine nucleotide-exchange factors
(GEFs),which catalyse the exchange of GDP for GTP
in vivo.GAPs suppress Rho activity,whereas GEFs pro-
mote Rho activity.A third set ofregulatory proteins,the
guanine nucleotide-dissociation inhibitors (GDIs),
sequester GTPases in the cytosol in a GDP-bound state.
The mechanism of release of Rho GTPases from GDIs
remains to be fully elucidated.
The first mammalian Rho GEF was identified as a
transforming gene from diffuse B-cell-lymphoma cells,
and was therefore designated Dbl10,11.Dbl contains a
region of ~240 residues that is homologous to a region
in Saccharomyces cerevisiae Cdc24,which cooperates
with Cdc42 during S.cerevisiaebudding and polarity.
Dbl was subsequently shown to function as a GEF for
human Cdc42.So Dbl and Cdc24 represented the initial
members ofa new family ofGEFs12that specifically acti-
vate Rho GTPases.Since then,69 distinct members of
this Dbl family have been identified in humans (FIG.2).
The region of homology between Dbl and Cdc24
contains a ~200-residue Dbl homology (DH) domain
and an adjacent,C-terminal,~100-residue PLECKSTRIN
HOMOLOGY (PH) DOMAIN.Small GTPases have well-defined
GEF MEANS GO:TURNING ON
RHO GTPasesWITH GUANINE
Kent L.Rossman*‡,Channing J.Der*‡and John Sondek*‡§
Abstract | Guanine nucleotide-exchange factors (GEFs) are directly responsible for the activation
of Rho-family GTPases in response to diverse extracellular stimuli, and ultimately regulate
numerous cellular responses such as proliferation, differentiation and movement. With 69 distinct
homologues, Dbl-related GEFs represent the largest family of direct activators of Rho GTPases in
humans, and they activate Rho GTPases within particular spatio-temporal contexts. The failure
to do so can have significant consequences and is reflected in the aberrant function of Dbl-family
GEFs in some human diseases.
NATURE REVIEWS | MOLECULAR CELL BIOLOGY
VOLUME 6 | FEBRUARY 2005 | 167
and Biophysics,University of
North Carolina at Chapel
Correspondence to: C.J.D.
A seven-helix membrane-
spanning cell-surface receptor
that signals through
and -hydrolysing G proteins to
stimulate or inhibit the activity
of a downstream enzyme.
An actin-dependent process by
which cells engulf external
particulate material by extension
and fusion of pseudopods.
R E V I E W S
The separation of a cell into two,
which is marked by ingression of
the cleavage ‘furrow’between
two segregated masses of
PLECKSTRIN HOMOLOGY (PH)
A sequence of ~100 amino acids
that is present in many signalling
molecules and that binds to lipid
products of phosphatidylinositol
3-kinase.Pleckstrin is a protein
of unknown function that was
originally identified in platelets.
It is a principal substrate of
protein kinase C.
Regions of nucleotide-binding
proteins that have different
conformations in the
to the diphosphate-bound,state.
An element of protein secondary
structure in which hydrogen
bonds that lie along the
backbone of a single polypeptide
cause the chain to form a right-
168 | FEBRUARY 2005 | VOLUME 6
R E V I E W S
Structures ofDH domains.The three dimensional (3D)
structures of several DH domains, both free and in
complex with Rho GTPases,have been determined13–23.
The DH-domain fold is structurally distinct relative to the
domains of other GEFs and is consistently shown to be
helical (FIG.3).It comprises 10–15 α-HELICESand 310-HELICES
that are roughly arranged along six main axes to form an
oblong helical bundle that has been compared in appear-
ance to a chaise longue13,with the ‘seat back’created by a
U-shaped arrangement ofα-helices.
DH domains have three conserved regions
(CR1–CR3), which pack to form the domain core.
CR1 and CR3,along with conserved residues within
the C terminus of the domain (helix α6),form a con-
tiguous patch that constitutes the bulk of the GTPase-
binding surface. Amino-acid substitutions within
these regions typically adversely affect nucleotide-
exchange activity14,16 . The largest conformational
differences among different DH domains occur in the
length and orientation of the C-terminal helix;subtler
differences are found in the relative positions of the
seat backs.For example,the seat back within the struc-
ture of T-cell-lymphoma invasion and metastasis-1
(Tiam1) bound to Rac1 is more upright relative to the
DH domains of Son-of-sevenless-1 (Sos1) and β-Pix
(Pak-interacting exchange factor β; also known as
COOL1 and ARHGEF6)13and this difference might
be functionally important.
The interface between DH domains and Rho GTPases.
DH domains interact extensively with the switch
regions of Rho GTPases (FIG. 3). Switch 1 (residues
25–39 in Cdc42) interacts with CR1 and CR3;a highly
conserved glutamate (Glu639 in Dbl’s big sister
(Dbs)) in CR1 is crucial for complex formation and
nucleotide-exchange activity.Switch 2 (residues 57–75
in Cdc42) predominantly contacts CR3 and portions
of the C-terminal helix (α6) of the DH domain.
Conserved hydrophobic residues within switch 2
anchor it into a hydrophobic cleft on the surface of the
DH domain.Two residues in DH domains — a con-
served basic residue (Lys774 in Dbs) in CR3 and a
semi-conserved Asn (Asn810 in Dbs) — also make
significant interactions with switch 2 to contribute to
the exchange potential.
Furthermore, a significant portion of the Rho-
GEF–GTPase interface is mediated by interactions
between the seat-back region of the DH domain and
structural elements between the switch regions of the
GTPases — mainly within the β2- and β3-strands of
the GTPase. These interactions are highly variable
among different DH domains and GTPases,and they
mediate Dbl selectivity among the Rho-GTPase family.
Mechanism ofnucleotide exchange.DH domains cause
the remodelling of the switch regions to significantly
alter the nucleotide-binding pocket,while leaving the
remainder of the GTPase unperturbed (FIG. 4). The
switch regions are reconfigured into essentially identical
conformations between different Rho-GEF–GTPase
nucleotide- and Mg2+-binding pockets. The form of
nucleotide (GDP or GTP) that is bound modulates the
conformation of the SWITCH REGIONS, whereas Mg2+is
required for high-affinity binding ofguanine nucleotides
in Rho GTPases. DH domains are responsible for
catalysing the exchange of GDP for GTP within Rho
GTPases by promoting GTPase intermediates that are
devoid of nucleotide and Mg2+(FIG.1).In cells,GTP is
preferentially loaded into Rho GTPases during nucleotide
exchange because GTP is found at substantially higher
concentrations than GDP.
DH-associated PH domains,by binding phospho-
inositides,have been proposed to localize Dbl proteins
to plasma membranes,and to regulate their GEF activity
through allosteric mechanisms.Outside the DH–PH
domains,Dbl-family proteins show significant diver-
gence and typically contain other protein domains that
underlie the unique cellular functions of the different
In this review,the molecular details that control the
guanine nucleotide-exchange activity and selectivity of
Dbl-family proteins for Rho GTPases are outlined.In
addition,special attention is paid to intra- and intermol-
ecular mechanisms that regulate this exchange,with
particular emphasis on roles for DH-associated PH
domains. The review concludes by describing the
expanding set ofdiseases that arise from malfunctioning
Figure 1 | Regulating Rho-GTPase activity. Rho GTPases are considered functionally ‘primed’
when they are bound to GTP and essentially non-functional when they are GDP-bound. These two
nucleotide-bound states are tightly regulated. Guanine nucleotide-dissociation inhibitors (GDIs)
mainly bind the switch regions and the C-terminal isoprenyl moiety (orange wavy line) of Rho
GTPases to sequester them in the cytosol. The functional importance of GDI sequestration is
poorly understood, but it might be used to provide a large, stable pool of Rho GTPases that can be
easily mobilized on extensive Rho activation. Our knowledge of GDI release is also incomplete, but
this process is probably regulated, and is necessary before the engagement of guanine nucleotide-
exchange factors (GEFs), which also bind the switch regions. GEFs stabilize nucleotide-depleted
GTPases. However, owing to the relatively high concentration of intracellular GTP, nucleotide-
depleted complexes rapidly dissociate into GTP-bound GTPases and free GEFs. When they are
GTP-bound, Rho GTPases regulate the activity of their binding partners, or effectors (E), to
promote a host of cellular responses that usually influence the organization of the actin
cytoskeleton or the expression levels of various genes. GTPase-activating proteins (GAPs)
stimulate the intrinsic hydrolytic capacity of Rho GTPases to promote GDP-bound forms and
terminate signalling. Pi, inorganic phosphate.
NATURE REVIEWS | MOLECULAR CELL BIOLOGY
VOLUME 6 | FEBRUARY 2005 | 169
R E V I E W S
the face of the nucleotide-binding cleft,disrupting sev-
eral interactions with the bound nucleotide and the
required Mg2+cofactor (FIG.4a),while extensive remod-
elling of switch 2 occludes the Mg2+-binding site with
the equivalent ofAla59 in Cdc42 (FIG.4b).Similar switch 2
alterations occur within the structures of Sos1 (the
orthologue of which is known as Cdc25in S.cerevisiae)
bound to Ras24(FIG.4c),Mg2+-free RhoA bound to GDP27,
the bacterial SopEprotein bound to Cdc42 (REF.28),and
complexes,which is indicative of a highly conserved
mechanism of exchange despite low sequence identities
between DH domains.In contrast to GEFs for other
Ras-related GTPases24–26,residues within DH domains
are not directly inserted into the active site of their
cognate GTPases. Instead, formation of the Rho-
GEF–GTPase complexes disorganizes the nucleotide-
binding pocket and directly occludes the binding site for
the Mg2+cofactor.In particular,switch 1 movesalong
L27 MornPDZ PHRanGEFRasGEF RasGEFNRBD RGSRhoGAP Sec14SH2SH3Spec
Figure 2 | The Dbl family. The Dbl homology (DH) domains of the 69 unique Dbl proteins in humans have been aligned to produce
this phylogenetic tree. Dispersed around the tree branches are illustrations showing the domain composition and domain
organization for each member. Black arrowheads above a guanine nucleotide-exchange factor (GEF) indicate truncations that are
known to activate either the exchange activity or a related cellular function of the corresponding GEF. Black arrowheads with
brackets delineate similarly active fragments. Red brackets indicate regions of Dbl proteins with known three-dimensional
structures. The coloured spheres placed on the tree branches designate the reported Rho GTPase specificity of the corresponding
Dbl-family member. Note that the specificity summary is not complete for all of the 22 known human Rho GTPases, as the analyses
for most Dbl-family proteins has been restricted primarily to RhoA, Rac1 and Cdc42. Furthermore, some Rho GTPases — for
example, Rnd3/RhoE and TTF/RhoH — might be constitutively activated and not regulated by GEF activity. For descriptions of
domain abbreviations and functions, the reader is referred to the SMART website (see the online links box).
A tighter,less stable helix than
the α-helix,with three residues
per turn,which form hydrogen-
bonded loops of 10 atoms.
170 | FEBRUARY 2005 | VOLUME 6
R E V I E W S
patch at this interface yet differ significantly between
GTPases13.This patch,which mainly comprises strands
β1/β2,with single residues being contributed by the
N-terminal portion and switch 2,establishes an inter-
face with sequences in the seat-back region of the DH
domain.The seat back is also poorly conserved among
different DH domains,which implies that both sets of
proteins co-evolved the interface to dictate the spectrum
The importance of this non-conserved interface in
dictating specific Rho-GEF–GTPase pairings has
been verified in several biochemical studies. For
example, Rac1 with a Trp56Phe mutation is com-
pletely unresponsive to activation by Tiam1 (REF.30),
yet it is efficiently activated by intersectin-long
(ITSN-L), which is normally a Cdc42-specific GEF.
Similarly,substitutions in the seat-back region of the
DH domain of ITSN-L enable it to activate RhoA19,
whereas a single conservative Leu→Ile mutation in
the DH domain of Dbs confers on it the ability to
activate robustly Rac1,which is not normally a sub-
strate for Dbs31. Similarly, Dbs normally activates
Cdc42 and RhoA, but Whitehead et al.31altered the
preferences of Dbs by mutation to attribute its trans-
formation potential to the activation of RhoA.These
last results provide the precedent for deconvolving
complicated specificity profiles and in vivo responses.
Roles for DH-domain-associated PH domains
PH domains are invariably found C-terminal to DH
domains,and evidence is mounting that these linked
domains cooperate to facilitate the activation of Rho
GTPases.For example,several studies have concluded
that DH–PH fragments show greater nucleotide-
exchange activity than the respective DH domains
alone14,32.From a structural perspective,these roles are
the interaction of regulator of chromosome condensa-
tion-1 (Rcc1) with Ran26,which indicates that specific
aspects of nucleotide exchange are conserved among
various families of small GTPases.
The phosphate-binding loop or P-loop (residues
10–18 of Cdc42 and Rac1) mediates many of the
interactions with the α- and β-phosphates of guanine
nucleotides and is essentially undisturbed in the Rho-
GEF–GTPase complexes (FIG. 4a). The sole P-loop
alteration involves the movement of Cys18 out of
bond-forming distance of the α-phosphate and this is
crucial for the exchange mechanism29.The P-loop con-
formation is stabilized by the electrostatic interaction
between Lys16 and Glu62 from switch 2 and probably
promotes the efficient reloading ofnucleotide.
In general,for nucleotide exchange to occur,Rho
GEFs probably first interact with more rigid regions of
Rho GTPases,such as β2,β3 and portions ofswitch 2,in
a ‘lock and key’fashion,which is followed by ‘induced fit’
interactions that promote the restructuring of switch 1
(REF.13).The induced alterations in the GTPase encour-
age GDP and Mg2+dissociation. This leaves the
nucleotide-binding pocket within the binary complex
completely solvent-exposed to allow rebinding of
GTP•Mg2+in cells,which results in a more retracted state
ofthe switch regions and dissociation ofthe complex.
GTPase specificity among DH domains.Dbl-family pro-
teins show varied selectivity (FIG.2),and analyses have
mainly been carried out for Rac1, RhoA and Cdc42
(REF.12).Non-conserved residues that occur within the
interface between DH domains and GTPases are likely
candidates for determining specific coupling.Indeed,
the initial structure of Tiam1–Rac1,together with sub-
sequent similar structures, revealed nine positions
within the individual interfaces that form a contiguous
Figure 3 | Three-dimensional structures of DH–PH domains. a | The ribbon diagram of the Dbl homology (DH) domain (yellow)
and pleckstrin homology (PH) domain (blue) of Dbs bound to Cdc42 (green)18. PDB accession number 1KZ7. Conserved region
(CR)1 and CR3 (magenta) along with α6 of the DH domain are the primary sites of contact with the switch regions (S1 and S2) of
Cdc42 (red). The β3–β4 loop of the PH domain of Dbs also makes contacts with switch 2 of Cdc42, and these are essential for
catalysing nucleotide exchange by Dbs. Also highlighted is the ‘seat back’ region (grey outline) of the Dbs DH domain, which forms
a complementary interface with β-strands 1–3 of Rho GTPases. b | The structure of the Dbs DH–PH domain from the Dbs–Cdc42
complex (transparent) is superimposed on the GTPase-free form of Dbs20(PDB accession number 1RJ2) by superposition of the
DH domains from both structures. This comparison helps to illustrate the significant repositioning of the PH domain that occurs
when it binds the GTPase. c | The PH domain of Son-of-sevenless-1 (Sos1)15adopts a conformation that is distinct from the PH
domains of Dbs, T-cell-lymphoma invasion and metastasis-1 (Tiam1) and intersectin-long (ITSN-L). This unique conformation places
the Sos1 PH domain onto the GTPase-binding surface of the DH domain and effectively blocks binding and exchange of Rac1.
PDB accession number 1DBH. Dashed lines within the structures indicate disordered regions.
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R E V I E W S
Participation ofthe PH domain in the GTPase-binding
interface. The structure of Dbs–Cdc42 highlighted
direct interactions between Cdc42 and the DH-associ-
ated PH domain that are required for efficient activa-
tion of Rho GTPases and the associated cellular
responses18,33(FIG.4).Residues within the PH domain of
Dbs that contact Cdc42 are conserved among a subset
ofGEFs,including Trio,Dbl,Duo,obscurin and Unc-73,
which implies that they might also use their PH
domains similarly in the guanine nucleotide-exchange
process.Albeit differently from Dbs,the PH domain of
LARG also makes direct contacts to RhoA that are
important for nucleotide exchange22.However,within
the structures of Tiam1–Rac1 and ITSN-L–Cdc42,the
PH domains are significantly removed from the bound
GTPase such that it is difficult to imagine productive
interactions between them. Therefore, the limited
structural data do not point towards a universal role for
DH-associated PH domains in directly engaging
GTPases to facilitate nucleotide exchange.
The PH domain as a membrane anchor.Early studies
consistently found that deletion or destabilization of
Dbl-related PH domains resulted in the loss oftransfor-
mation by Dbl-family members34,35.For some members,
transformation could be restored by replacing their PH
domains with sequences that directed localization to
plasma membranes,which indicated a potential role for
these PH domains in membrane targeting,as has been
reported for other PH domains36. However, the PH
domains of Dbl-family members consistently bind
phospholipids with low affinity and little specificity,
which implies that these interactions are insufficient for
membrane localization37.Indeed,lipid binding to the
PH domains of Sos1,Dbs,Tiam1 and Vav1 does not
seem to be necessary for the localization of these Dbl-
family GEFs33,38,39and,not surprisingly,it is often found
that other domains/motifs control their cellular distrib-
Allosteric regulation ofGEF activity by phospholipids.
It is routinely speculated that the binding of phospho-
lipids to Dbl-related PH domains might modulate the
exchange activity of the adjacent DH domains through
ill-defined allosteric processes.However,there is limited
evidence for such regulatory mechanisms and this idea
remains highly controversial in light of several con-
flicting reports. For example, the binding of phos-
to Vav1 promoted a modest two-fold enhancement of
nucleotide exchange on Rac1,whereas PtdIns(4,5)P2
diminished this activity in vitro41.For Tiam1,one report
found that various fragments of Tiam1 activated
Rac1 only in the presence of phospholipids42,whereas
a second report concluded that various preparations
of phospholipids did not modulate the already robust
in vitroactivities of Tiam1,Dbs and ITSN-L towards
Rho GTPases37.By contrast,the activation of Cdc42 by
the DH–PH fragment of Dbl was inhibited by phos-
phoinositides43. The activation of Rac1 by P-Rex1
(PtdIns(3,4,5)P3-dependent Rac exchanger-1) was
not easily discerned,as the relative orientations of PH
domains to DH domains are different among the
known structures.The positioning of the PH domains
of Tiam1,Dbs,ITSN-L,Trio and LARG (leukaemia-
associated Rho GEF) are most similar as they pack
against the C terminus (helix α6) of the DH domain.
However,each PH domain is laterally shiftedand rotated
differentially relative to the DH domain and these differ-
ences have implications for nucleotide-exchange poten-
tial.For example,the PH domains of Dbs and LARG
directly contact the bound GTPases (FIGS 3,4),whereas
the PH domains of Tiam1 and ITSN-L do not interact
with DH-domain-bound GTPases.In the structure of
the DH–PH-domains from Sos1,the C-terminal helix
of the PH domain forms an interface with the seat-back
region of the DH domain and occludes the GTPase-
binding surface to effectively prohibit binding and
exchange of Rho GTPases.
Figure 4 |Key interactions viewed within the structure of Dbs–Cdc42.a| Conserved region 1
(CR1, yellow) of the Dbl homology (DH) domain of Dbs interacts with Cdc42 to reposition (arrows)
switch 1 (S1, red) and Cys18 within the P-loop (green) of Cdc42 (REF.18) relative to the GDP-
bound form of Cdc42 (translucent)29. These alterations disrupt interactions with the nucleotide
base, the α-phosphate and the Mg2+cofactor, which are mediated by Phe28, Cys18 and Thr35,
respectively, of Cdc42. b | A similar depiction of switch 2 (S2) highlights the occlusion of the Mg2+-
binding site of Cdc42 by the repositioning (arrow) of Ala59 supported by Glu62. c | Certain
aspects of the nucleotide exchange that is catalysed by guanine nucleotide-exchange factors
(GEFs) are conserved among small GTPases. Switch 2 (S2, red) of Ras, when bound to the
Cdc25 domain of Son-of-sevenless-1 (Sos1; PDB accession number 1BKD)24, is reconfigured
similarly to Cdc42 that is bound to Dbs. d | The pleckstrin homology (PH) domain (blue) of Dbs
participates with the DH domain (yellow) in binding Rho GTPases. In the structure, residues within
β1, β4 and the β3–β4 loop of the PH domain contact the switch 2 (S2, red) and α3b (green)
regions of Cdc42 (REF.18). A similar complement of interactions is preserved in the structure of
Dbs–RhoA19. In all panels, the dashed lines indicate hydrogen bonds.
172 | FEBRUARY 2005 | VOLUME 6
R E V I E W S
DH and PH domains to facilitate the productive
engagement of GTPases, as has been observed for
Dbs20. In one intriguing study, the N-terminal PH
domain of Trio was shown to bind to PtdIns(3,4)P2,but
only after engagement of the associated DH domain by
full-length RhoG, and not C-terminally truncated
RhoG46. This implies that the polybasic C-terminal
portion of RhoG participates with the PH domain in
binding phospholipids,and hypothetically ties the acti-
vation of RhoG by Trio to the signalling pathways that
generate specific phospholipids.However,these experi-
ments were done in the absence of lipid bilayers,which
might facilitate non-physiological interactions.
PH-domain–protein interactions.DH-associated PH
domains also function as docking sites for proteins that
are associated with signalling cascades that are regu-
lated by Rho GTPases.For example,the PH domain of
Dbl directly interacts with ezrin,a protein that links
the plasma membrane with the actin cytoskeleton and
that is activated by Rho GTPases47.Similarly,the PH
domain of the first DH–PH-domain array of Trio
directly interacts with filamin,a protein that crosslinks
filamentous actin48.The interaction of Trio with filamin
functions to localize Trio at actin filaments and to
facilitate actin-based ruffling,but does not enhance
the nucleotide-exchange activity of Trio.Similarly,the
first DH–PH-domain array of Trio also binds Tara49,
another protein that binds filamentous actin,to regu-
late the organization of the actin cytoskeleton. PH
domains are gaining widespread acceptance not just as
lipid-binding modules but also as protein-docking
sites.Undoubtedly,many more instances of interac-
tions between Dbl-related PH domains and diverse
proteins will be discovered,and these associations will
probably link the activation of Rho GTPases to distinct
cellular sites and signalling cascades.
DH domains without PH domains.The invariant need
for a PH domain immediately C-terminal to a DH
domain is questioned in some situations50.For example,
p164-RhoGEF has an easily identifiable DH domain,
but reportedly has no associated PH domain50.
However, HOMOLOGY MODELLINGidentifies with high confi-
dence a PH domain that is C-terminal to the DH
domain of p164-RhoGEF (FIG.2).The dynamin-binding
scaffold protein Tuba represents the only clear exception
and contains a BAR(Bin,Amphiphysin,Rvs),and not
PH, domain downstream of its DH domain. BAR
domains do not share the PH-domain tertiary fold but,
rather,are all α-helical.BAR domains are present in
numerous other proteins,interact with curved mem-
brane bilayers51, and therefore might functionally
replace a DH-domain-associated PH domain.
Rho GEFs as signal integrators
The regulation of Dbl proteins by regions outside the
conserved DH and PH domains remains poorly under-
stood despite intensive study.Unfortunately,no universal
mechanisms of regulation are apparent;however,some
general themes are emerging.
stimulated robustly by PtdIns(3,4,5)P3in vitro44.The
physiological significance and the mechanism of
phospholipid regulation of these Dbl proteins,as well
as the applicability of such regulation to other family
members,are issues that remain unresolved.
The role of lipid bilayers. The available in vivo data
highlight the importance of phosphoinositides in lipid
bilayers in promoting the activation of Rho GTPases
through Dbl-family proteins33,39,45.In live cells,most
data are consistent with Dbl-family proteins regulating
Rho GTPases that are attached to lipid membranes
through their prenyl moieties (FIG.5).The interaction of
the PH domains with membrane-resident phospho-
inositides could orientate the associated DH domain so
that it can properly engage Rho GTPases.Alternatively,
binding of phosphoinositides in membranes might
promote conformations between DH and PH domains
that result in enhanced GEF activity.For example,the
PH domain of Sos1 might be displaced from the DH
domain to uncover the GTPase-binding surface.The
engagement of lipid bilayers might also orientate linked
Prediction of the tertiary
structure of an unknown protein
using a known three-
dimensional structure of a
Basal stateNucleotide-depleted complexActive GTPase
Figure 5 | Model of PH-domain-assisted guanine nucleotide exchange. The local
accumulation of specific phosphoinositides and membrane-resident, GDP-bound Rho
proteins favour interactions of Rho GTPases with guanine nucleotide-exchange factors
(GEFs, as indicated by DH (Dbl homology) and PH (pleckstrin homology)) that are located
nearby (left). Interactions with phosphoinositides such as phosphoinositide-4,5-bisphosphate
(PtdIns(4,5)P2) and GEFs promote GDP and Mg2+ dissociation from Rho GTPases (centre)
and stabilize nucleotide-depleted complexes that are typified by the inset crystal structure.
The inset structure shows nucleotide-depleted Cdc42 bound to Dbs and modelled at a
membrane surface18. For Cdc42 that is bound to Dbs, both the DH and PH domains must
directly engage Cdc42 for maximal nucleotide exchange. The subsequent loading of
GTP•Mg2+results in the activation of Rho proteins (right)33. In this scenario, the DH and PH
domains of Dbl proteins would function as coincidence detectors that are designed to
integrate information regarding local fluctuations in the concentrations of Rho GTPases that
are released from guanine nucleotide-dissociation inhibitors (GDIs), as well as the membrane
composition for the tight regulation of GTPase activation.
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For instance,Tiam1 isphosphorylated on several differ-
ent sites by calcium/calmodulin-dependent (CaM)
kinase II (REF.54),protein kinase C (REF.55) and Src56,
with varying effects.Equally intriguing,Ras guanine
nucleotide-releasing factor (Ras-GRF) activates Rac
after phosphorylation by Src57,whereas its phosphory-
lation by cyclin-dependent kinase-5 (Cdk5) inhibits
Rac exchange58. At present, though, the molecular
details of activation of Rho GEFs by truncation or
phosphorylation are scarce and necessitate intensive
Subcellular sequestration.Regulation of the subcellu-
lar location of Dbl proteins is a common means of dic-
tating the spatio-temporal activation of Rho GTPases
in response to distinct stimuli.For instance,neuro-
epithelioma transforming gene-1 (Net1) is normally
sequestered in the nucleus,but its translocation to the
plasma membrane is associated with RhoA activa-
tion59.Similarly,epithelial-cell transforming gene-2
(Ect2) is localized within the nucleus during inter-
phase but is tightly associated with microtubules as
well as the CLEAVAGE FURROWduring subsequent phases of
cell division60.This dynamic movement ofEct2 is neces-
sary for the proper activation of Rho GTPases and cor-
rect cell division.Also,GEF-H1 interacts directly with
microtubules and this interaction inhibits the exchange
potential of GEF-H1 towards RhoA at the plasma
membrane61.Tubulin depolymerization in response to
specific stimuliupregulates RhoA activation by GEF-H1
and this process is exploited by enteropathogenic
Escherichia colito facilitate its pathogenicity62.Finally,
the activation of Rac1 by Tiam1 is dependent on the
redistribution of this GEF from the cytoplasm to the
plasma membrane after stimulation of cells with
serum63or platelet-derived growth factor64.
Effector-tethered modulation. Wu and colleagues65
highlighted a central role for α-Pixin coordinating the
activation of Cdc42 with GPCR-mediated signals that
are necessary for CHEMOTAXIS.The authors provide con-
vincing evidence that,on activation of chemoattractant
receptors,the liberated Gβγsubunit binds p21-activated
kinase (PAK) that is associated with α-Pix, which
results in enhanced activation of Cdc42 by α-Pix.GTP-
bound Cdc42 can then efficiently activate the high local
concentration of PAK.In essence,α-Pix functions as a
SCAFFOLDto integrate inputs that arise from GPCRs with
the activation of Cdc42 and the subsequent down-
stream activation of tethered PAK (FIG.6).The system
might even have built-in negative feedback, as data
indicate that activation of PAK inhibits its interaction
with Pix isoforms66,67.In a related example,β-Pix tethers
NADPH oxidase-1 for activation by Rac1, with the
resultant production of reactive oxygen species68,which
are used in various signal-transduction cascades and
for host defence.
ITSN-L shows Cdc42-specific nucleotide-exchange
activity that is enhanced by the binding of Wiskott–
Aldrich syndrome protein (WASP) to its SRC-HOMOLOGY-3
(SH3) DOMAIN69. As WASP is a downstream effector of
Activation by N-terminal truncation and phosphoryla-
tion.Many Rho GEFs are constitutively activated by the
N-terminal truncation of sequences that lie upstream of
the DH domain12.This implies that,despite consider-
able divergence in their sequence and domain architec-
ture,the N termini function as negative,intramolecular
regulators of DH-domain function.Perhaps the most
well-characterized mechanism of regulation of N-ter-
minal function is phosphorylation ofVav1 (REFS 17,52,53).
N-terminal regions of Vav1 that encompass Tyr174
interact with the DH domain to prevent access by
GTPases.Phosphorylation by various receptor-associ-
ated tyrosine kinases,such as Lck,of Tyr174 and proxi-
mal tyrosines opens the DH domain to GTPases.
Consequently,truncation of N-terminal regions that
surround Tyr174 abrogates this autoinhibition and
results in the constitutive activity ofVav1.
Unfortunately,the general application of such a sim-
ple model has proved more difficult,which is particu-
larly frustrating given that the N-terminal truncation of
many Rho GEFs results in their constitutive activation.
Similarly,although many Rho GEFs are phosphorylated
under various conditions,in most instances,the conse-
quences of phosphorylation are not well understood.
An invagination of the plasma
membrane in the division plane
of an animal cell that contains a
contractile ring,and that leads to
scission of the daughter cells.
A type of migration that is
stimulated by a gradient of a
chemical stimulant or
A protein that functions as a
support to assemble a
A protein module of ~80
amino acids that is present in a
range of proteins and that was
first identified in the protein
kinase Src.SH3 domains
interact with proline-rich
sequences that usually contain
a PXXXPXR motif (where X is
any amino acid).
a Scaffold modulation
c GTPase modulation
d Other examples
P-Rex, Pix Dbs, Tiam1, PixSos1 Dock180
Figure 6 | Rho GEFs as signalling nodes. a | Certain Dbl proteins are incorporated into
supramolecular complexes by specific scaffolding proteins (S1, S2 and S3) that also localize
distinct downstream effectors (E1 and E2). Such complexes increase the fidelity of signalling
cascades that can be activated by the same GTPase (G1). Examples include the scaffolding
functions of Tiam1 and Ras-GRF. In a similar vein, clustering (right-hand panel in part a) is a
unique form of scaffolding that enhances the activation of RhoA (G1) by ephexin while diminishing
the activation of Cdc42 (G2) and Rac1 (G3). b | Other Rho guanine nucleotide-exchange factors
(GEFs), such as the Pak-interacting exchange factor (Pix) isoforms and intersectin-long (ITSN-L),
tether the immediate downstream effectors of GTPases to increase the dynamic range and
efficiency of signal transduction. c | Cellular responses such as chemotaxis require the combined
action of heterotrimeric G proteins (Gα and Gβγ), Rho GTPases and other small GTPases.
Various Rho GEFs (such as LARG, p115-RhoGEF, PDZ-RhoGEF, P-Rex1, the Pix isoforms, Dbs
and Tiam1) are direct effectors or modulators of these proteins and function as nodes to integrate
these inputs with the activation of Rho GTPases. d | Such signalling circuits are mirrored in other
nucleotide-exchange systems. For example, the activation of Ras by the Cdc25-homology
domain of Son-of-sevenless-1 (Sos1) promotes a positive-feedback loop whereby Ras•GTP
binds to a second site within the Cdc25 domain to potentiate exchange122. In another case, a
dimer of ELMO and Dock180 activates Rac and this process is enhanced by the binding of
RhoG to ELMO123. DH, Dbl homology; RGS, regulator of G-protein signalling.
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GEF activities within this subfamily enhances the
‘operational selectivity’or ‘signal-to-noise ratio’of Rho
activation by GTP-bound Gα13subunits.More descrip-
tively,the RGS domains of p115-RhoGEF and LARG
would ensure a high concentration of GTP-bound Gα13
only under circumstances ofrapid and robust activation
of Gα13by GPCRs;low-grade or spurious activation of
Gα13would be eliminated effectively by the GAP activity;
consequent Rho activation would be finely controlled
and rapidly responsive to levels of GTP-bound Gα13.
Importantly, this type of antagonistic relationship
between GAP and effector functions within the same
protein — such as p115-RhoGEF or LARG — that
share a common G proteinwill also highly localize the
signalling outputs in a process that is known as ‘kinetic
scaffolding’or ‘signal focusing’79.
In another example,GPCR signalling through Gβγ
and PtdIns(3,4,5)P3synergistically stimulate the
capacity of P-Rex1 to activate Rac in vivo44.The two
GPCR-derived signals probably focus the location of
Rac activation as well as its response dynamics.
Coordinating GTPase cascades.Ephexin80and Vsm-
RhoGEF81are closely related nucleotide-exchange
factors that directly interact through their DH and PH
domains with the receptor tyrosine kinase EphA4 to
control the activation of RhoA in response to agonist-
mediated clustering of EphA4.Intriguingly,the cluster-
ing of activated EphA4 is reported to alter the exchange
profile of ephexin.In the absence of EphA4 clustering,
ephexin will strongly activate RhoA,weakly activate
Cdc42,and possibly also activate Rac;on clustering of
EphA4,the activation of Cdc42 and Rac is dampened
and RhoA activation is reinforced.Altering the exchange
profile of ephexin,and therefore the relative activities of
specific Rho GTPases,is an attractive mechanism for
shifting the balance between cellular extension (which
is associated with Rac activation and Rho inhibition)
and retraction (which is associated with Rho activa-
tion). This would be particularly useful for rapidly
adapting the response,for example,from GROWTH-CONE
extension to growth-cone collapse,which is associated
with RhoA activation and EphA4 ligation. In this
sense, EphA4 clustering can be viewed as a type of
scaffolding that is designed to reinforce and insulate
specific pathways over alternative routes.
Immediately N-terminal to the DH domain of
Tiam1 is a Ras-binding domain (RBD).Active Ras inter-
acts specifically,albeit weakly,with the RBD of Tiam1
and this interaction is sufficient to stimulate the ability
of Tiam1 to activate Rac in vivo82.In this way,and simi-
lar to previously discussed cases,Tiam1 functions to link
Ras-controlled signalling pathways with downstream
outputs that are controlled by active Rac.
Sos1 integrates the activation of Ras and Rac
through distinct Cdc25-homology and DH domains,
respectively83.Moreover,elegant structural and bio-
chemical experiments23highlight that active, GTP-
bound Ras binds to a site that is distal to the exchange
region of the Cdc25-homology domain of Sos1 to
enhance Ras activation in a positive-feedback loop.
activated Cdc42, Cdc42•GTP and ITSN-L probably
functionally compete for the available pool of WASP.
Titration ofWASP from ITSN-L by Cdc42•GTP would
therefore favour the basally repressed form of ITSN-L,
which shuts down further activation of Cdc42.In this
way, ITSN-L would function in a negative-feedback
loop to fine-tune the activation ofCdc42.
Rho GEFs as scaffolds.Tiam1 participates in supramolec-
ular complexes through the scaffold proteins JIP2 (JNK-
interacting protein-2 (where JNK is Jun N-terminal
kinase)) and spinophilin to direct the activation of Rac
towards specific signalling cascades that lead ultimately
to the activation ofp38 mitogen-activated protein kinase
(MAPK)70and p70 S6 kinase71, respectively. More
specifically,Tiam1 cooperatively interacts with JIP2 to
enhance the interaction of the Rac effector mixed-lin-
eage kinase-3 (MLK3) with MAPK-kinase kinase-3
(MKK3) and p38 MAPK — this promotes a kinase
cascade that enhances the activation of p38 MAPK.
Similarly, spinophilin simultaneously binds both
Tiam1 and p70 S6 kinase,another known effector of
Rac.The complex of Tiam1 and spinophilin enhances
the capacity of Tiam1 to activate p70 S6 kinases,pre-
sumably by concentrating active Rac in the vicinity of
Coordinating heterotrimeric G proteins with GTPases.
p115-RhoGEF (which is also known as Lsc) and the
related Dbl-family members LARG and PDZ-RhoGEF
are the main points of convergence between signalling
cascades that are controlled by HETEROTRIMERIC G PROTEINS
and those that are regulated by Rho GTPases.More
specifically, these Rho GEFs each contain a highly
divergent regulator of G-protein signalling (RGS)
domain,which is typically associated with enhancing
the intrinsic GTPase activity of heterotrimeric Gαsub-
units. Indeed, purified p115-RhoGEF72or LARG73
effectively increase the hydrolysis of GTP-bound Gα12
and GTP-bound Gα13to produce GDP-bound, and
therefore inactive, forms of these heterotrimeric
G proteins.Furthermore,purified p115-RhoGEF74and
LARG73can also activate RhoA by virtue of their DH
domains.Significantly,these exchange activities are
enhanced by activated Gα12 (REF.73) and Gα13 (REF.75).
For example,activated Gα13binds the DH–PH portion
of p115-RhoGEF and it seems likely that this second
site of interaction is essential for modulation of the
nucleotide-exchange potential76. In this way, p115-
RhoGEF and LARG are,simultaneously,downstream
effectors of GTP-bound Gα subunits,and GAPs that
are designed to turn off signalling by Gα subunits.
For these Rho GEFs,the GAP activity that is associ-
ated with the RGS domain opposes the capacity of
activated Gα13to enhance the nucleotide-exchange
activity of Rho through the DH and PH domains and,
at first glance, these functions seem unnecessarily
antagonistic.However,drawing from detailed experi-
mental and theoretical kinetic analyses of other signal-
transduction systems that use GAPs77–79, it seems
highly likely that the tight interconnection of GAP and
(Postsynaptic-density protein of
95 kDa,Discs large and Zona
occludens-1).A region that is
present in several scaffolding
proteins and that is named after
the founding members of this
protein family.PDZ domains
bind to specific short amino-
acid sequences that are found in
several proteins at,or outside,
HETEROTRIMERIC G PROTEIN
A complex of three proteins
(Gα,Gβand Gγ).Whereas Gβ
and Gγform a tight complex,
Gα is part of the complex in its
dissociates in its active,GTP-
bound,form.Both Gαand Gβγ
can transmit downstream
signals after activation.
Motile tip of the axon or
dendrite of a growing nerve cell,
which spreads out into a large
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and promotes focused concentrations of active Cdc42
in a positive-feedback loop that is required for sustain-
ing bud sites and mating projections89–91.Bem1 also
recruits Cla4,which hyperphosphorylates the com-
plex to attenuate the cascade86,92.In this way,Cdc24 is
an integral component of a large macromolecular
complex that is finely tuned for the highly dynamic
spatio-temporal activation of Cdc42 that is necessary
for regulated apical growth.
The regulation of Cdc24 highlights many common-
alities that are shared with other Dbl-family Rho GEFs.
These include: first,their subcellular sequestration
and stimuli-dependent recruitment; second, their
stabilization within large complexes; and, finally,
their signal amplification and attenuation using posi-
tive- and negative-feedback loops.Heterotrimeric G
proteins and small GTPases integrate and regulate
many of these processes (FIG.6).
Dbl-family proteins in disease and development
The correct functioning of Rho GTPases can be per-
turbed by various mechanisms that can be linked to a
diverse array of human diseases93.Such perturbations
include dysregulated GTPase expression and altered
function or expression of regulators (GAPs,GEFs and
GDIs) and effectors (for example, WASP) of Rho
GTPases.Following a briefoverview ofthe consequences
of perturbed Rho GEF expression in mouse studies,we
summarize perturbations in the function of Rho GEFs
that are associated with human disease.
Within the structure,this distal site is blocked by the
DH domain of Sos1 and,although it is unclear how this
blockage is relieved in vivo,these analyses indicate the
intimate coordination ofRac and Ras activation by Sos1
in the cell.
Finally,Dbl proteins might also function as effectors
of Rho GTPases.For example,the DH-domain-linked
PH domain of Dbs binds activated Rac1 and this com-
plex promotes the activation ofRhoA in vivo84.Scambio
operates similarly as a Rho GTPase GEF and effector85,
although the function ofScambio as an effector remains
to be understood.These recent studies highlight the
complex coordination of Rho GTPases that are directly
mediated by Dbl proteins.
The Cdc24 paradigm. It is becoming increasingly
clear that Rho GEFs integrate and modulate diverse
signalling inputs,which are often controlled by other
GTPases and heterotrimeric G proteins,to finely tune
the location and dynamics of the activation of Rho
GTPases. S. cerevisiae Cdc24 provides an excellent
example for understanding the spatio-temporal activa-
tion of Cdc42 according toits intracellular localization
and binding partners,which include Gβγ,other small
GTPases and the downstream effector Cla4. In this
case,Cdc24 is differentially and rapidly exported out of
the nucleus and recruited to distinct BUD SITES by the
small GTPase Bud1•GTP86,87,or to MATING PROJECTIONSby
Gβγ88.Initial activation of Cdc24 recruits the scaffold
protein Bem1,which stabilizes the location of Cdc24
Cell-wall sites where the yeast
reproduction by initiation of
A specialized structure that is
formed by vegetative S.cerevisiae
to initiate polarized cell growth,
and to allow polarized mating
cells to signal to one another.
Table 1 | Roles of Dbl GEFs in mouse development and tissue function
Widely expressed, high levels in
haematopoietic and nervous tissue
Widely expressed, high levels in
haematopoietic and nervous tissue
Brain, ovary and testes
Together with Bcr deficiency causes
cerebellar and vestibular defects
Defective dendrite elongation
Altered marginal zone B cell and other
haematopoietic cell functions
Long-term memory consolidation,
decreased body weight, hypoinsulinaemia
and glucose intolerance
Embryonic lethality, placental and heart defects
None, but impaired oncogenesis
Nervous tissue, pancreatic
Brain, spleen and lung
Widely expressed, highest levels
in brain and testes
Embryonic lethality, abnormal skeletal-muscle
and neural-tissue development
Partial defect in T-cell development and function,
normal B-lymphocyte development and function
Normal T- and B-cell development and function,
but B-cell defect in combination with Vav1 deficiency
Normal, but increased haematopoietic cell defects
when combined with loss of Vav1 and Vav2
Vav1 Haematopoietic cells 135–137
Vav2Widely expressed 95,96
Vav3Widely expressed, highest levels in
haematopoietic cells and brain
*Also possess distinct Rho GTPase-activating protein (GAP) catalytic domain; ‡also possess distinct Ras guanine nucleotide-exchange
factor (GEF) catalytic domain; §contains two distinct Dbl homology (DH)–pleckstrin homology (PH) tandem domains, as well as a
serine/threonine kinase domain. Abr, active Bcr-related; Bcr, breakpoint cluster region; Dbl, diffuse B-cell lymphoma; GRF, guanine
nucleotide-releasing factor; Sos, Son-of-sevenless; Tiam, T-cell-lymphoma invasion and metastasis.
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Although normal development occurred,restricted
tissue defects were also seen in mice that were deficient
in Dbl, Bcr, Abr (active Bcr-related gene) or Tiam1.
However,a deficiency in Trio caused embryonic lethality
in mice and was associated with abnormal development
ofskeletal muscle and neural tissues98.The tissue-specific
defects or embryonic lethality that are seen with some
GEFs are probably the result ofthe loss ofother catalytic
activities that are associated with these proteins — for
example,the Ras GEF activity of Sos1 or the Rho GAP
activities of Bcr and Abr.The multidomain nature of
most Dbl proteins,which is indicative of functions that
are independent of nucleotide exchange,will obviously
complicate the interpretation of Dbl-knockout studies.
Although a knock-in approach to create an endogenous
allele that encodes a missense-mutation-mediated loss
of function in the DH domain might seem attractive,it
cannot be ruled out that such GEF-deficient mutant
proteins would have a DOMINANT-NEGATIVEeffect.
Cancer. Although several Dbl GEFs were originally
identified as truncated,constitutively activated onco-
genes by EXPRESSION-LIBRARY SCREENINGusing DNA or RNA
that was isolated from human cancer cells,the activation
events were artefacts of experimental manipulation.
At present, only a handful of Dbl GEFs have been
found to be mutated in human cancers. Most well
characterized is the rearrangement of Bcr that is
caused by reciprocal chromosome translocation and
formation of the Philadelphia chromosome (Ph) in
human leukaemias (FIG. 7). Different lengths of the
N terminus of Bcr become fused with the Abl tyrosine
kinase,and all Bcr–Abl chimaeras possess constitu-
tively activated Abl kinase activity,which is essential for
oncogenesis99. Although an important role for the
N-terminal dimerization motif of Bcr in oncogenesis
has been established,the precise contribution of the
DH domain of Bcr to oncogenesis that is mediated by
Bcr–Abl is unknown.
A rearrangement of LARG and the mixed lineage
leukaemia (MLL) gene has been identified in acute
myelogenous leukemia100(FIG. 7). Formation of the
MLL–LARG chimeric protein results in the loss of
N-terminal sequences upstream of the DH domain of
LARG — an event that is known to cause constitutive
activation of other Dbl-family proteins. However,
N-terminal truncation of LARG alone is not sufficient
to activate its transforming activity,which indicates
that the presence of MLL sequences might be involved
in altering LARG GEF function.
Other examples of the involvement of GEFs in
cancer include a missense mutation in Tiam1 that is
derived from tumours and cell lines of renal cell carci-
noma101.This mutation (Ala441Gly),which is present in
the N-terminal PH domain that is important for the
association of Tiam1 with the membrane, induces
Tiam1 transforming activity by an unknown mecha-
nism.Alternative transcripts of the gene that encodes
the Rho GEF Tim, which lack sequences for the DH
domain,have been identified in breast cancers,and are
expected to show a loss offunction102.
Normal mouse development. So far,significant devel-
opmental consequences have not been observed
when mice with deficiencies in specific Dbl proteins
have been created (TABLE 1). Functional redundancy
and limited tissue expression of Dbl proteins might
account for the typically limited, tissue-restricted
effects that are seen.This is best demonstrated by the
analyses of mice that are deficient in Vav function.
Surprisingly,mice that lacked all three Vav genes were
viable,with no gross perturbations in organ develop-
ment.Closer examination of mice that were deficient
in Vav1 (REF.94),but not Vav2 (REFS 95,96)or Vav3 (REF.97),
showed them to have impaired T-cell development
and function,which indicated that these proteins have
non-redundant functions. By comparison, a defi-
ciency in both Vav1 and Vav2 was required to see
defects in B-cell function,which implies that Vav1 and
Vav2 share redundant roles in B-cell development and
function.So,Vav1 is significant for both T- and B-cell
function,and Vav2 and Vav3 seem to carry out more
A defective protein that retains
interaction capabilities and so
distorts or competes with
A genome-wide cloning strategy
that uses a biological gain-of-
function (for example,
uncontrolled growth) to isolate
and identify genes with specific
Morn PDZPHRBD RGS
C2 Coiled coilDH FYVEVps9
Figure 7 | Mutation of Rho GEFs in human diseases. A common mutation of breakpoint cluster
region (Bcr ) involves fusion of the Bcr gene on chromosome 22 with the Abl proto-oncogene on
chromosome 9. This results in the formation of Bcr–Abl chimaeras that express various portions of
N-terminal Bcr sequence fused to the Abl kinase domain: p185 is associated with 20–30% of
incidences of acute lymphocytic leukaemia, p210 with 90% of incidences of chronic myelogenous
leukaemia (CML), and p230 with a subset of patients that have chronic neutrophilic leukaemia.
A chimeric protein in which a C-terminal fragment of leukaemia-associated Rho guanine
nucleotide-exchange factor (LARG) is fused to the N-terminus of mixed lineage leukaemia (MLL)
has been identified in acute myelogenous leukemia. Ala441Gly is a missense mutation in Tiam1
(T-cell lymphoma invasion and metastasis-1) in renal cell cancer cell lines and tumours. Fgd1
mutations result in faciogenital dysplasia (or Aarskog–Scott) syndrome. Mutations include those
that introduce premature stop codons and missense mutations that are scattered throughout the
protein, with many involving deletion or mutation of the Dbl homology (DH) domain. Truncation
mutations within the amyotrophic lateral sclerosis-2 (ALS2) gene product, Alsin, result in the
premature truncation of Alsin and are thought to be causative for juvenile onset amyotrophic lateral
sclerosis. Indicated are the locations of breakpoints,missense mutations and sites of protein
truncations that result from nucleotide insertions or deletions. For descriptions of domain
abbreviations and functions, the reader is referred to the SMART website (see the online links box).
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receptor-1 (Par1) thrombin receptor — are found to be
overexpressed in cancers106.
Developmental and neurological disorders.The Dbl-
family member Fgd1 is deleted in patients that have
faciogenital dysplasia (FGDY)107,a developmental disor-
der that is characterized by the malformation ofspecific
skeletal structures.At least 16 distinct structural muta-
tions ofFgd1 have been described that co-segregate with
the disease — some of which include deletion or muta-
tion of the DH domain108(FIG.7)— which implies that
the loss of Fgd1 function is responsible for FGDY.
Several regulators of Rho-GTPase function have
been implicated in X-linked forms of mental retarda-
tion.For example,mutations in α-Pix have been found
— these include disruption of the gene that encodes
α-Pix as a result of reciprocal chromosome transloca-
tion,as well as the expression of a variant transcript
that has a 28-amino-acid deletion in the N-terminal
CALPONIN-HOMOLOGY DOMAIN.Mutation of Fgd1 has also
Perhaps indirect mechanisms that activate Rho
GEFs — for example, through perturbation of the
proteins that regulate their activation — will be more
commonly seen in human cancers. For example, as
described above, Tiam1 is an effector of Ras82and
facilitates Ras-mediated activation of Rac.Consistent
with the requirement for Rac function in Ras trans-
formation, Tiam1-deficient mice were impaired in
Ras-induced induction of SQUAMOUS CELL CARCINOMAS103.
Another example of the activation of Rho GEFs by
perturbed regulation involves the regulation by adeno-
matous polyposis coli (APC) ofAsef (APC-stimulated
GEF).APC is encoded by a tumour-suppressor gene
that is mutated in colorectal tumours104.The truncated
form of APC that is associated with tumour cells can
promote activation ofAsef GEF activity and the migra-
tion of colorectal cancer cells105.As mentioned above,
LARG and other RGS-box-containing Rho GEFs can be
activated by Gα12/13-coupled GPCRs,many of which —
for example, the Gα12/13-coupled protease-activated
Better known as Ca2+-dependent
in proteins such as conventional
protein kinase C isoforms and
synaptotagmin.The C2 domain
is another modular signalling
domain that can induce
after binding several Ca2+ions.
There are C2 domains that do
not bind Ca2+but constitutively
bind to a membrane,others that
might be involved in Ca2+-
interactions,and some that
might bind inositol
ARMADILLO (ARM) ARRAY
The armadillo repeat is an ~40-
repeated sequence motif that
was first identified in the
segment polarity gene armadillo.
SQUAMOUS CELL CARCINOMA
A carcinoma that develops from
the layers of thin,flat squamous
cells of the epithelium.
A protein domain,which is often
found tandemly arrayed,that
functions in the binding of actin.
PHC2 CC SH3ARM
Box 1 | Rho GEFs without the DH–PH domain structure
There are bona fide Rho guanine nucleotide-exchange factors (GEFs) that are unrelated in primary sequence to Dbl-
family proteins.Proteins that are related to Dock1 (which is also referred to as Dock180,see figure,left panel) lack
primary sequence identity to Dbl homology (DH) domains.Instead,they are characterized by two regions ofhigh
sequence conservation that are designated Dock-homology region-1 and -2 (DHR1 and DHR2).DHR2 is sometimes
sufficient for promoting guanine nucleotide exchange.On the basis ofhomology modelling,these motifs probably
encompass a C2 DOMAINand portions ofan ARMADILLO (ARM) ARRAY,as shown in the figure.C2 domains typically bind lipids,
and armadillo arrays form suprahelical structures that are used to engage other proteins,especially other ARM-array-
containing proteins.Certain members also have a Src-homology-3 (SH3) domain,which is typically used to bind
polyproline regions in other proteins,or a pleckstrin homology (PH) domain as shown in the figure.There are 11
proteins that are related to Dock1 in humans,which are clustered into four groups on the basis ofprimary-sequence
conservation (right panel).Dock1,Dock2 and Dock3 (which is also known as MOA) are Rac1-specific GEFs115,116,
whereas Dock9 (which is also known as zizimin1) is specific for Cdc42 (REF.117).The specificity ofother members is less
well characterized.SWAP70 and the related SLAT (SWAP70-like adaptor ofT cells)/IBP proteins contain a PH domain
and an adjacent,C-terminal coiled coil (CC) region,which has no statistically significant sequence identity to DH
domains118,119.SWAP70 shows Rac-specific nucleotide-exchange activity in vitroand phosphatidylinositol 3-kinase
(PI3K)-dependent Rac activation and membrane-ruffling activity in response to stimulation by epidermal growth factor.
Relative to Dbl proteins,these two classes ofnucleotide-exchange factor remain poorly characterized.
Although bacteria lack Rho GTPases,they do contain GEFs that act on mammalian Rho GTPases.For example,
Salmonella typhimurium injects SopE and the closely related SopE2 protein (62% identity)120into host cells to facilitate
invasion.SopE activates Cdc42 and Rac,whereas SopE2 specifically activates Cdc42 (REF.121).Structural analysis ofthe
SopE–Cdc42 heterodimer28shows that these proteins are distinct from DH domains,but nevertheless use a nearly
identical mechanism to promote nucleotide exchange.
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these PH domains can be essential for binding to Rho
GTPases,which hints,at least in vivo,at how lipid bind-
ing through the PH domain could regulate GEF activity
by promoting active conformations of DH–PH
domains.Experimental systems that study the function
of these PH domains using lipid vesicles and lipid-mod-
ified Rho GTPases should further our understanding of
their role in catalysing nucleotide exchange.
The remarkable diversity of protein domains and
motifs that flank DH–PH domains indicates the diverse
signalling pathways into which Dbl-family members are
integrated,and several themes are emerging that describe
how these GEFs focus many upstream signals through
Rho-GTPase activation to elicit cellular responses.In this
regard,Dbl-proteins have been shown to participate in
large scaffolding structures that are designed to modulate
the activity ofthe GEF or to restrict spatially their activity
near particular stimulus inputs, as well as near Rho
GTPases and their direct downstream effectors.Dbl pro-
teins themselves also recruit Rho effectors directly,
which efficiently transduces the activation ofGTPases to
their effectors.Often,the activated Rho GTPase or Rho-
effector molecule is thought to feed back on the circuit
to further potentiate the output signal or quell the input
signal.Some Dbl proteins are also downstream effectors
for specific Rho GTPases,which implies that Dbl pro-
teins will also function as nodes for the known interde-
pendent regulation of Rho-GTPase cascades.Future
studies will undoubtedly aim to determine the spatio-
temporal regulation of Dbl-family proteins,possibly by
the use of novel biosensors. Together with a more
detailed understanding of the molecular mechanisms
that are used by Rho GEFs to regulate the activation of
specific Rho GTPases,this information will essentially
allow the wiring diagrams that regulate the flow of
information through GEFs to GTPases to be plotted.
Together with structural information,it should then be
possible to effectively design small-molecule inhibitors
of GEFs to manipulate these pathways for research and
Our knowledge ofthe contribution ofaberrant Rho
GEF function to human diseases is limited,but there are a
few clear examples in which either the constitutive activa-
tion or inactivation ofa Dbl-family protein is a cause of
disease.The rate at which Rho GTPases have been impli-
cated in yet more cellular processes is remarkable,and a
full understanding ofthe function and regulation ofRho
GEFs will be crucial in furthering our understanding of
how various truncations and polymorphisms of GEF
genes might promote aberrant signalling and disease.To
this end,it will also be important to develop new animal
models that are designed to study GEFs in disease.
Finally,it is now known that Dbl-family GEFs are
not the sole activators of Rho GTPases.The number of
new molecules that function as nucleotide-exchange
factors,such as members of the Dock and SWAP fami-
lies,is growing and perhaps other classes of Rho GEFs
will be found.In what context each GEF,or family of
GEFs,is used and how they cooperate to regulate the
activation of Rho GTPases will certainly be an exciting
area of future research.
been linked to neurological disorders that are associated
A loss of function of Alsin is associated with a rare
form of a neurodegenerative disease, juvenile onset
amyotrophic lateral sclerosis (ALS2).Patients with ALS2
have mutations in a long form of Alsin,also known as
ALS2,that result in premature truncation of the protein
and a consequent loss ofthe DH domain109–111.
Viral and bacterial pathogenesis.Some Rho GEFs are tar-
geted to facilitate the pathogenic invasion ofmammalian
host cells.For example,several Dbl-family proteins are
regulated by viral proteins.T cells that are infected with
human T-cell-leukaemia virus I (HTLV-I) show altered
Vav phosphorylation112.The human immunodeficiency
virus type-1 (HIV-1) protein Nef,which is essential for
progression to AIDS, binds to, and activates, Vav113.
Similarly,the cytoplasmic domain of the HIV-1 trans-
membrane glycoprotein gp41,which is implicated in viral
replication and CYTOPATHICITY,inactivates p115-RhoGEF.
The resulting loss of RhoA function might facilitate
HIV-1pathogenicity by perturbing T-cell interactions
with other cells,T-cell migration and inhibition ofT-cell
proliferation114.Pathogenic bacteria have taken a different
approach to manipulating Rho GEFs to facilitate host
invasion.They have evolved proteins that do not share
sequence or domain similarity to Dbl proteins,but that
are structural and biochemical mimics of Dbl proteins
(BOX 1). For example, the bacterial SopE and SopE2
proteins are delivered by Salmonella typhimuriuminto
eukaryotic cells to promote invasion of their host
Conclusions and future perspectives
The remarkable number and structural diversity of Rho
GEFs,and the mechanisms by which they are regulated,
emphasize the crucial involvement of Rho GTPases in
normal cell physiology.A wealth of structural data has
led to key insights into the molecular mechanism that is
used by Dbl-family GEFs to promote the exchange of
GDP for GTP.Essentially,they do this by turning the
switch regions of the GTPases on themselves to eject
GDP and Mg2+.The structural data also allow for an
understanding,albeit preliminary,of how DH domains
show exquisite selectivity among various Rho proteins.
This selectivity is seen to arise from the varied sets of
interactions between two complementary,non-con-
served protein surfaces — one from the DH domain
and the other from the GTPase. It is imperative that
future structural,biochemical and biological studies aim
to identify the full complement of Rho-GTPase sub-
strates of each Dbl-family member.This will allow for a
detailed understanding of Dbl-family function,and to
ascertain whether all Rho GTPases are activated by Dbl
proteins,unconventional Rho GEFs,or perhaps require
no GEF intervention to be active signal transducers.
PH domains are (almost) always found C-terminal
to DH domains, but are, however, conformationally
diverse compared with their DH domains,which makes
it difficult to define a conserved structural role for the
linkage of DH and PH domains.However,it is clear that
The ability of certain viruses to
cause degenerative changes in
their host cells as a consequence
of viral invasion and
include changes in cell
morphology,cell lysis,cell death
and altered cell–cell interactions.
NATURE REVIEWS | MOLECULAR CELL BIOLOGY
VOLUME 6 | FEBRUARY 2005 | 179
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We would like to apologize for not being able to cite original work of
many colleagues due to space constraints. Our studies are sup-
ported by grants to C.J.D. and to J.S from the National Institutes of
Health. K.L.R. was supported by a fellowship from the National
Competing interests statement
The authors declare no competing financial interests.
The following terms in this article are linked online to:
SopE | SopE2
BAR domain | DH domain | PH domain
Saccharomyces genome database:
Cdc24 | Cdc25
Cdc42 | Dbl | Dbs | ITSN-L | LARG | Miro1 | p115-RhoGEF | PDZ-
RhoGEF | α-Pix | β-Pix | Rac1 | Ras | Rcc1 | RhoA | RhoBTB1 |
RhoD | RhoE | RhoF | RhoG | RhoH | RhoV | Sos1 | Tara | Tiam1 |
Trio | Vav1
SMART database: http://smart.embl-heidelberg.de
Protein Data Bank: http://www.rcsb.org/pdb/
Access to this links box is available online.