Opinion
TRENDS in Cell Biology
Vol.15 No.7 July 2005
GDIs: central regulatory molecules in
Rho GTPase activation
Céline DerMardirossian and Gary M. Bokoch
Departments of Immunology and Cell Biology, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, CA 92037, USA
The GDP dissociation inhibitors (GDIs) are pivotal
regulators of Rho GTPase function. GDIs control the
access of Rho GTPases to regulatory guanine nucleotide
exchange factors and GTPase-activating proteins, to
effector targets and to membranes where such effectors
reside. We discuss here our current understanding of
how Rho GTPase–GDI complexes are regulated by
various proteins, lipids and enzymes that exert GDI
displacement activity. We propose that phosphorylation
mediated by diverse kinases might provide a means of
controlling and coordinating Rho GTPase activation.
The low-molecular weight Rho GTPases are involved in
the regulation of a plethora of biological pathways [1].
These GTPases function as molecular switches in cell
signaling, alternating between an inactive, primarily
cytosolic, GDP-bound state, and an active GTP-bound
state usually associated with membranes, where effector
targets reside (see Box 1). Because of the presence of
high concentrations of cytosolic free Mg2C that prevent
spontaneous guanine nucleotide exchange, conversion of
inactive GTPases to an active form requires the action of
guanine nucleotide exchange factors (GEFs) that catalyze
the exchange of bound GDP for ambient GTP. GTPase
inactivation involves the catalysis of GTP hydrolysis,
which is intrinsically slow, through the action of GAPs
(GTPase-activating proteins) that convert the GTPase to
the inactive GDP-bound state.
GEFs and GAPs are thus important determinants of lowmolecular-weight GTPase activity. However, an additional
level of regulation exists for GTPases of the Rho and Rab
subfamilies owing to their association with a third class of
protein – the GDP dissociation inhibitors (GDIs). So named
because of their ability to inhibit the dissociation of bound
guanine nucleotide (usually GDP) from their partner
GTPases [2], GDIs have other functions as well, as described
below. Although Rab GDIs differ substantially at the
structural level from Rho GDIs, they exhibit very similar
biological regulatory activities (reviewed in [3–5]). Recognized mechanisms for the regulation of Rab GDI functional
responses thus provide a useful paradigm for discussion of
Rho GDI regulation, which is the focus of this article.
Biological activities of Rho GDIs
Three human Rho GDIs have been identified: the ubiquitously expressed RhoGDI (or GDIa/GDI1) [2,6], the
Corresponding author: Bokoch, G.M. (bokoch@scripps.edu).
Available online 24 May 2005
hematopoietic cell-selective Ly/D4GDI (or GDIb/GDI2)
[7,8] and RhoGDIg (or GDI3), specifically expressed in
lung, brain and testis [9,10]. Both RhoGDI and D4GDI
(hereafter referred to collectively as GDIs) are cytosolic
and form 1:1 complexes with Rho family GTPases,
although several Rho family members have been reported
to not effectively bind to GDIs (Table 1). By contrast,
RhoGDIg is associated with vesicular membranes and
exhibits specificity for interactions with RhoB and RhoG
[10] The expression levels of GDIs have been reported to
be up- or down-regulated in certain cancers [11–13] and in
other pathological conditions (e.g. [14]). D4GDI, but not
RhoGDI, is proteolyzed in apoptotic T cells through cleavage by caspases at sites (Asp19 and Asp55) that might
render it functionally inactive, potentially modifying the
activity of Rho GTPases under such conditions [15,16].
Three distinct biochemical activities have been
described for Rho GDIs (reviewed in [17,18]). First, they
inhibit the dissociation of GDP from Rho proteins,
maintaining the GTPase in an inactive form and preventing GTPase activation by GEFs (Box 1). Second, they are
able to interact with the GTP-bound form of the Rho
GTPase to inhibit GTP hydrolysis, blocking both intrinsic
Table 1. Association of Rho family GTPases with GDIs
Rho
GTPase
Cdc42
TC10
TCL
Chp
Wrch-1
Rac1
Rac2
Rac3
RhoG
Rac1b
RhoA
RhoB
RhoC
Rnd1
Rnd2
Rnd3/RhoE
RhoD
Rif
RhoH/TTF
RhoBTB1
RhoBTB2
Miro-1
RhoGDI
(GDI-1, a)
C
K
?
?
?
C
C
?
C
K
C
K
C
C
?
?
C/K
?
?
Weak
?
?
?
D4GDI
(GDI-2, b)a
C
?
?
?
?
C
C
?
?
?
C
?
?
?
?
?
?
?
?
?
?
?
?
RhoGDIg
(GDI-3)
K
?
?
?
?
K
K
K
C
K
K
C
K
K
?
?
?
?
?
?
?
?
?
Refs
[78,79]
[38]
–
–
–
[80,81]
[80,81]
–
[9,82]
[83]
[2]
[9,38]
[82]
[82]
–
–
[70,84]
–
–
[82]
–
–
–
a
In Ref. [64], the authors reported that none of these GTPases (RhoA, Rac1, Rac2,
Cdc42) was found in complex with D4/LyGDI in U937 cells.
www.sciencedirect.com 0962-8924/$ - see front matter Q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.tcb.2005.05.001
Opinion
TRENDS in Cell Biology
Box 1. Regulation of Rho GTPases
Rho GTPases act as molecular switches to regulate downstream
biological responses. To perform this function, they must cycle
between GDP-bound inactive states and GTP-bound active states.
This regulatory cycle of GTP binding and hydrolysis is controlled
overall through the action of three classes of regulatory protein
(see Figure I).
† Guanine nucleotide exchange factors (GEFs) catalyze the release
of bound GDP, resulting in the formation of the GTP-bound active
protein in the cytosol, where GTP levels are relatively high.
† GTPase-activating proteins (GAPs) stimulate the intrinsically
low GTP hydrolytic activity of the Rho GTPases, resulting in their
conversion to the inactive GDP state.
† GDP dissociation inhibitors (GDIs: RhoGDI and D4GDI) sequester
the inactive GTPase, preventing the dissociation of GDP and
interactions with regulatory and effector molecules. This inhibitory
action of GDIs requires that they be dissociated from their partner
GTPases for the GTPases to become activated and elicit their
biological effects. Such dissociation might be regulated by various
types of GDI dissociation factor (GDF) activities (see text). GDIs also
regulate membrane-to-cytosol cycling of Rho family GTPases, as
described more fully in the text.
The many regulatory and effector partners of Rho GTPases have
been shown to physically interact with several regions on Rho
GTPases, shown schematically in Figure II: the switch I and switch II
domains are highly conserved in all GTPases and in general undergo
conformational changes upon conversion of the GTPase from the
inactive GDP- to the active GTP-bound state [73]. These structural
changes mediate the GTP-dependent regulation of effector activities.
The C-terminus of the Rho GTPases contains a highly variable
‘polybasic domain’ (PB) adjacent to the terminal ‘CAAX motif’ that
becomes modified by prenylation (wavy line), proteolysis and
carboxymethylation [74,75]. Finally, Rho family GTPases contain a
unique ‘insert domain’ that provides an additional solvent-exposed
protein-interaction surface [76,77]. The interaction of GEFs, GAPs,
GDIs, and effectors with each region is indicated in the figure.
GDI
GDF
GEF
GDP
GTPase
GTPase
GTP
GAP
Inactive
Active
GDF
GDI
TRENDS in Cell Biology
Figure I.
Effectors
GAPs
GEFs
GDI
Effectors
GAPs
GEFs
GDI
Effectors
Switch I
Switch II
Insert
Effectors
GEFs?
GDI
PB
GTPase
TRENDS in Cell Biology
Figure II.
and GAP-catalyzed GTPase activity and preventing interactions with effector targets. Both of these two activities
prevent the resulting biological effects of Rho GTPase
activation, emphasizing the crucial importance of GDIs in
modulating Rho GTPase function. Indeed, the exogenous
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Vol.15 No.7 July 2005
357
introduction of GDIs into cells has been shown to inhibit
numerous Rho GTPase-dependent processes, ranging
from cytoskeletal dynamics and motility to phagocyte
NADPH oxidase activity to gene expression (see [17]).
While the strength of the binding of RhoGDI to GTPbound GTPase has been reported to vary from weak [19] to
of equal affinity [20,21] with binding to GDP-bound
GTPase, it remains unclear under which circumstances
an interaction with GTP-bound GTPase might take place
and what the biological consequences of this interaction
are (e.g. could it serve to translocate active GTPases
within the cell?). These biochemical effects of GDI binding
stem from physical interaction of the N-terminal regulatory portion of GDIs with the GTP-regulated binding
domains (switch I and switch II regions, see Box 1) of their
partner Rho GTPases [22–25].
A third biochemical activity of GDIs is to modulate the
cycling of Rho GTPases between cytosol and membranes
(Figure 1). GDIs maintain Rho GTPases as soluble
cytosolic proteins by forming high-affinity complexes in
which the geranylgeranyl membrane-targeting moiety
present at the C-terminus of the Rho GTPases is shielded
from the solvent by its insertion into the hydrophobic
pocket formed by the immunoglobulin-like b sandwich of
the GDI [22–27]. When Rho proteins are released from
GDIs, they are able to insert into the lipid bilayer of the
plasma membrane through their isoprenylated C-terminus.
In this uncomplexed form, the Rho GTPases can then
interact with, and are activated by, membrane-associated
GEFs, thereby initiating the association with effector
targets at the membrane [28]. Extraction from the membrane through re-association with GDI, possibly initiated
by GTP hydrolysis, is postulated to induce recycling of the
GTPase back into the cytosol [17,29–31]. GDIs thus assist
in the termination of GTPase signaling at the membrane
and serve to replenish the pool of cytosolic GTPases.
As the binding of GDIs to Rho GTPases overlaps
with effector interaction sites, and the affinity of GDI
binding to Rho GTPase is in the low nanomolar range
(Kdw1.6–30 nM [20,26]), comparable or exceeding that for
GTPase binding to most effector targets (e.g. [32]), the
complex of a Rho GTPase with a GDI is biologically inert
(unless the complex itself has activity, as proposed in
Refs [33,34]). A similar antagonism occurs with Dbl family
GEFs: interaction of GDI with the side-chain of the essential Thr35 residue in switch I of the GTPase stabilizes
Mg2C coordination, thereby stabilizing nucleotide binding
and preventing the GEF-catalyzed nucleotide exchange
reaction [22–24]. Biochemical studies generally support
this view: the binding of Rho GTPases to GDI has been
shown to dramatically reduce the action of Dbl [35] and
Tiam1 [28] GEFs to catalyze nucleotide exchange (see also
[29,30]). The GDI–GTPase complex is thus a major point
of regulation of Rho GTPase activity and function. In this
regard, it is somewhat surprising that targeted knockout
of RhoGDI or D4GDI results in viable mice, with selective
defects in kidney and reproductive organs (RhoGDI [36])
or in immune responses (D4GDI [37]) being the major
physiological consequences. The lack of a more severe
phenotype in the RhoGDIK/K mouse could be due to
compensatory upregulation of D4 GDI activity or to
358
Opinion
TRENDS in Cell Biology
Vol.15 No.7 July 2005
GEFs
s?
se ?
a
s
n
Ki ipid ?
L DF
(1)
G
GDP
Rac
(3)
Effector
Rac
(2)
GDP
Rac
Rac
GTP
?
Rac
GAPs
GTP
GDI
GTP
(4)
Rac
Kinases? GDP
GDI
GDI
GDI
(5)
GDP
(6)
Rac
GDI
Other
regulatory
proteins?
TRENDS in Cell Biology
Figure 1. A model of the Rho GTPase–GDI cycle. Based largely on biochemical and in vitro evidence, GDP dissociation inhibitor (GDI) is proposed to regulate both the
interactions of Rho GTPases with regulators and effector targets, as well as cytosol-to-membrane cycling. In resting cells, Rho GTPase–GDI complexes are cytosolic and noninteractive. In response to cell stimulation, Rho GTPases are induced to dissociate from GDI through the actions of GDI displacement factors (GDFs), lipids with complex
dissociative activity and/or through kinase-mediated phosphorylation of GDI (Step 1). This results in the membrane association of the released Rho GTPase (in this example,
Rac) through the insertion of the C-terminal isoprenyl group into the membrane bilayer. It is here that an interaction with guanine nucleotide exchange factors (GEFs) that are
also activated by cell stimulation takes place (Step 2). The GDI-free GTPase is converted to the active GTP-bound form, which is then able to bind to and activate various
effectors modulated by that particular GEF–GTPase pair (Step 3). The possible reassociation of Rac–GTP with GDI has been suggested (see text). The interaction of GTPase
with effector is terminated by the GTPase-activating protein (GAP)-mediated conversion of the Rho GTPase to the GDP form (Step 4). Its release from the membrane requires
re-association with GDI in a two-component reaction that is kinetically separable into a rapid step involving GDI binding to the GTPase (Step 5), followed by a slow step
involving transfer of the prenylated C-terminus of the GTPase from the lipid bilayer to the hydrophobic binding pocket in GDI (Step 6). These steps might require additional
protein regulatory factors and might be enhanced by phosphorylation of the C-terminal polybasic domain of the Rho GTPase.
changes in the normal regulation of Rho GTPases in the
deficient cells. It will be of interest to evaluate the
phenotype of the double GDI knockout, which has not
yet been reported.
Quantitation of RhoGDI levels in distinct cell types has
revealed that the molar amount of RhoGDI is in excess of
any single Rho GTPase, but roughly equal to the total
levels of the RhoA, Rac1 and Cdc42 GTPases in these cells
[38]. In human neutrophils, RhoA, Rac1/Rac2 and Cdc42
are also equimolar with overall GDI (RhoGDI and D4GDI)
levels, and exist largely as cytosolic GDI complexes, with
no apparent pools of free GTPase [39]. However, the level
of uncomplexed versus GDI-associated Rho GTPases
seems to exhibit wide variations in different cell types
[40]. In certain cells, there might thus exist significant
pools of Rho GTPases not complexed with GDIs that are
available for immediate activation in response to external
stimuli. The ability of hormonal stimuli to specifically
activate individual members of the Rho GTPase family is
well documented [41–44]. It is likely that specific mechanisms exist to dissociate individual members of the Rho
GTPase family from cytosolic RhoGDI complexes to
facilitate the activation process. This dissociation would
likely be tightly coupled to GEF-mediated GDP–GTP
exchange and membrane association of the activated
GTPase, resulting in effector binding and functional
responses (Figure 1). We will now briefly consider the
relatively well-defined mechanisms for regulation of Rab
GTPase–GDI interactions as a comparative model for a
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discussion of the mechanisms regulating Rho GTPase
release from GDIs.
Rab–RabGDI: precedence for regulated dissociation by
GDI displacement factors
The reversible control of membrane versus cytosolic
portioning of Rab GTPases is crucial to their function as
regulators of vesicular trafficking. This function is served
by RabGDI, which is structurally distinct from, but
functionally analogous to, the Rho GDIs. The existence
of proteins that regulate the rapid dissociation of Rab
GTPases from RabGDI (termed GDI displacement factors
or GDFs) has been established as an integral part of the
Rab regulatory cycle [3,4,45,46].
Early experiments suggested that the membrane
translocation of Rab GTPases preceded guanine nucleotide exchange, and that this translocation was initiated by
the dissociation of Rab from cytosolic RabGDI complexes.
The mammalian Ypt-interacting protein 3 (Yip3), also
called prenylated Rab acceptor 1 (Pra1) [47], was shown to
be a GDF for Rab9, catalyzing the dissociation of Rab9
from RabGDI in vitro and promoting Rab9 recruitment
onto membranes [46]. This activity exhibited selectivity
for endosomal Rab5 and Rab9 versus secretory pathway
Rab1 and Rab2, raising the possibility that the large
family of Yip proteins exert Rab-specific GDF activities.
The heat-shock protein 90 (HSP90) chaperone complex on
synaptic membranes was shown to be a Rab3A-selective
GDF during neurotransmitter release [48]. The regulation
Opinion
TRENDS in Cell Biology
of Rab GTPase cycling between membrane and cytosol by
RabGDIs might also require a functionally defined protein
factor that facilitates the re-association of membranebound Rab GTPase with RabGDI [49].
GDFs and regulation of the Rho GTPase–RhoGDI cycle
By analogy with the regulation of the Rab GTPase–RabGDI
interaction, the action of proteins able to stimulate GTPase
dissociation from RhoGDI was suggested by the observed
protease sensitivity of this process in in vitro studies using
human neutrophil fractions [31]. Subsequently, RhoGDI
displacement activity resident in members of the ERM
family [50], the tyrosine kinase Etk [51] and the p75
neurotropin receptor (p75NTR) [52] has been described to
induce the release of RhoA from RhoGDI. However, the
ERM proteins might act to physically sequester RhoGDI and
other inhibitory signaling molecules after cell activation, as
in the ‘distal pole complex’ of T cells [53]. As yet, no common
structural features of these putative GDFs has been
identified to account for their GDI dissociative activity.
Collectively, these observations suggest the possible existence of multiple GDF proteins, perhaps exhibiting both
cell-type-specific and context-specific functions.
Lipids as GDFs for Rho GTPases
Interestingly, several biologically relevant lipids have
been reported to have the ability to decrease the affinity
of RhoGDI for Rho and Rac [39,54]. These include several
saturated and unsaturated fatty acids, phosphatidic acids
and phosphoinositides. Effects of the latter were observed
at possibly ‘physiologic’ concentrations, from 1 to 10 mM.
Faure et al. [55] showed that phosphoinositides enhanced
GDP–GTP exchange in complexes consisting of prenylated
RhoA with RhoGDI. They suggested that these lipids
induced a partial disruption of the GTPase–GDI complex,
opening the complex enough to promote interaction with
membrane-associated GEFs and nucleotide exchange.
This model is consistent with the separate roles of the
two primary RhoGDI–GTPase-interaction interfaces suggested by the ability of point mutations in these interfaces
to distinguish RhoGDI inhibitory activities from complex
formation [56]. It is also supported by the biphasic membrane extraction of Cdc42 by RhoGDI, possibly accounted
for by the formation of kinetically distinct GTPase–GDI
and lipid–GDI interfaces [57].
It is interesting in this regard that a fraction of
Rac1–RhoGDI in rat brain homogenates was found in a
complex with both diacylglycerol kinase (DGK) and type I
phosphatidylinositol 4-phosphate 5-kinase [58]. It is
tempting to speculate that this might represent a
regulatory complex in which lipid products (i.e. phosphatidic acid and PtdIns(4,5)P2) capable of dissociating Rac1
from RhoGDI are generated in close proximity during cell
activation, thus enhancing GTPase activation through
nucleotide exchange factors.
Regulation of GTPase–GDI complexes by
phosphorylation
Phosphorylation of RabGDI
Evidence obtained in the Rab–RabGDI system suggests
that phosphorylation also plays important roles in
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Vol.15 No.7 July 2005
359
modulating the association of Rab GTPases with RabGDI.
A cytosolic phosphoprotein co-precipitating with Rab5 was
tentatively identified as RabGDI [59] and the Drosophila
homolog of RabGDI was shown to exhibit a basic isoelectric point shift in the developmental mutant quartet,
consistent with phosphorylation [60]. Mutation of Tyr249
in RabGDI-2 induced a gain-of-function phenotype
towards specific Rab GTPases, and pharmacologically
induced tyrosine phosphorylation of RabGDI-2 stimulated
a pronounced increase in cytosolic complexes of the
phosphorylated RabGDI and Rab4 [61]. Gruenberg and
colleagues [62] used a biochemical approach to identify
p38 MAPK as an enhancer of Rab5–RabGDI complex
formation. p38 MAPK phosphorylated RabGDI on Ser121,
a surface residue opposing the Rab binding site and
predicted to interact with putative membrane-associated
RabGDI receptor. Overall, these observations suggest the
importance of multiple kinases in the regulation of Rab
binding to RabGDI.
Phosphorylation of Rho GDIs
Bourmeyster and Vignais [63] have provided evidence that
the activity of kinases modulating the phosphorylation
state of RhoGDIs might also be important regulatory
elements. They showed that RhoGDI was constitutively
phosphorylated in resting neutrophils and that dephosphorylation of RhoGDI by treatment with exogenously
added phosphatases resulted in a decreased affinity for
RhoA. These data suggested the existence of an unidentified RhoGDI-directed kinase that increased RhoGDI
binding to RhoA through an undefined mechanism.
Several additional studies have demonstrated differentially charged species of RhoGDI and D4GDI in cells,
consistent with the existence of phosphorylated forms of
GDI [63,64]. D4GDI is phosphorylated in response to cell
stimulation by phorbol esters [8,64] and by T-cell receptor
stimulation [65]. PKC-dependent phosphorylation of
RhoGDI in human umbilical vein endothelial cells has
been reported, accompanied by the activation of RhoA
[66]. Unfortunately, this study neither established a direct
effect of phosphorylation of RhoGDI on its ability to bind
to Rho GTPases, nor did it determine the sites of RhoGDI
phosphorylation.
DerMardirossian et al. [67] recently described the
binding and phosphorylation of RhoGDI, both in vitro
and in vivo, by p21-activated kinase 1 (Pak1), a downstream effector of Rac and Cdc42. This phosphorylation
occurred on two sites (Ser101 and Ser174) in RhoGDI on
the external surface of the hydrophobic cleft in which the
GTPase prenyl group binds. Both of these sites lie
adjacent to hydrophobic residues that directly line the
RhoGDI geranylgeranyl-binding pocket. Phosphorylation
of these two sites resulted in the selective release of Rac1,
but not RhoA, from the GDI complex, leading to its subsequent activation by exchange factors. We have observed
significant cooperative structural changes induced by the
phosphorylation of both Ser101 and Ser174 (G.M. Bokoch,
C. DerMardirossian and L-Y. Lian, unpublished), and,
given the resulting selectivity for Rac1 dissociation, it
is tempting to speculate that the differing polybasic
C-terminal domains in Rac1, RhoA and Cdc42 might
360
Opinion
TRENDS in Cell Biology
play important roles in these interactions [25,67]. Interestingly, mutation of the nearby Ile177 to Asn induced an
w20-fold decrease in the affinity of Cdc42 binding to
RhoGDI [68]. Rac1 dissociation from RhoGDI and subsequent Rac1 activation induced by the growth factors
EGF and PDGF required phosphorylation of S101 and
S174 by Pak1. The phosphorylation of RhoGDI by Pak1
might serve as a positive feed-forward mechanism to
account for sustained Rac activation during processes
such as cell motility.
Phosphorylation of Rho GTPase
Phosphorylation of the Rho GTPase itself has also been
observed to affect binding affinity for RhoGDI. In most
(all?) cases, this results in an increase in complex
formation [69–71]. The cAMP-dependent protein kinase
A (PKA)-mediated phosphorylation of RhoA on Ser188
within the RhoA C-terminus has been described both
in vitro and in vivo in cytotoxic T lymphocytes [69].
Phosphorylation affected neither nucleotide binding nor
intrinsic GTPase activity but led to the preferential
extraction of phosphorylated RhoA from membranes by
RhoGDI and enhancement of the cytosolic pool of RhoA.
Notably, manipulations that modified cellular cAMP levels
and PKA activity caused morphological changes consistent with RhoA inhibition, suggesting that PKA phosphorylation of RhoA inhibits its activity by promoting
formation of a RhoA–RhoGDI complex.
Subsequent work by Beliveau and coworkers showed
that PKA might also enhance Cdc42–GDI interactions as
Cdc42 is also phosphorylated at Ser188 by PKA [70].
Interestingly, Rac GTPases lack a serine or threonine
residue at position 188 and thus do not serve as PKA
substrates at this position. Ser188 lies within a consensus
PKA phosphorylation motif in RhoA and Cdc42 that is
also within the GTPase C-terminal polybasic domain. The
positively charged polybasic domain interacts with a
negatively charged acidic patch in RhoGDI, which might
provide a competitive binding surface contributing to
extraction of the membrane-associated GTPase from
acidic phospholipids in the membrane bilayer [22]. The
addition of a negatively charged phosphate group to the
GTPase polybasic domain could be postulated to facilitate
GTPase extraction from membranes by GDI.
The EGF-dependent phosphorylation of Cdc42 at Tyr64
within the switch II domain through Src tyrosine kinase
has been reported and is associated with enhanced Cdc42
binding to RhoGDI. Tu et al. [71] noted that the X-ray
structure for the Cdc42–RhoGDI complex shows that
Tyr64 of Cdc42 is in close proximity to lysine residues
43 and 52 of RhoGDI and that the negative charge
accompanying the phosphorylation of Tyr64 might stabilize the two positive charges provided by these basic
residues. However, these authors went on to show that
this simple hypothesis was insufficient, as changing Tyr64
to a negatively charged glutamic acid residue did not
enhance RhoGDI binding. The mechanism responsible for
the increase in RhoGDI affinity induced by this covalent
modification is thus not yet clear. It extends, however, the
range of kinases that can regulate the interaction of Rho
GTPases with RhoGDI, and we note that this tyrosine
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Vol.15 No.7 July 2005
residue is conserved in Rac1 and RhoA. Of interest,
Cerione et al. showed that the enhanced association of
Cdc42 with RhoGDI induced by phosphorylation of Tyr64
was necessary for Cdc42 to induce cell transformation,
which they proposed might be due to a role for RhoGDI in
subcellular relocalization of Cdc42 activity.
.phosphorylation of GTPases and
GDIs provides a flexible, yet simple,
mechanism for coordinating Rho
GTPase action in response to cell
activation
Phosphorylation – a general mechanism for regulation
of Rho GTPase–GDI interactions by diverse stimuli?
Although the existence of GDF proteins for Rho GTPases
modulating their release from GDI seems likely, few
examples of such proteins have been identified. As noted
above, however, there is increasing evidence that kinases,
acting on either GDIs or Rho GTPases themselves, act to
regulate formation of complexes. These kinases are
diverse, with PKA, PKCa, Pak1, Src and several other,
unidentified, kinases reported to either enhance or
decrease GTPase–GDI binding affinity by phosphorylation of the GTPase or GDI, respectively. We suggest that
the regulation of Rho GTPase association with GDI by
phosphorylation (Figure 2) has several attractive features:
Specificity of GTPase activation
Diverse receptor-initiated signals can stimulate Rho
GTPase activity, and many of these receptors also activate
kinase cascades. These kinases might act to promote the
release of Rho GTPases from GDI complexes, allowing
them to interact with GEFs to become activated and
mediate receptor-initiated cellular responses. The activation of certain kinases (e.g. Pak1) might cause the
release of a specific GTPase (i.e. Rac), whereas other
kinases might selectively induce the dissociation of other
Rho GTPases. Alternatively, some of these phosphorylation events might serve as a general release mechanism
for Rho GTPases. It is well known that the ability of
receptors to activate single, or multiple, Rho GTPases
varies widely. We hypothesize that this is partially
dependent on the repertoire of kinases activated by each
individual receptor.
Coincidence detection
The need for phosphorylation of RhoGDI on multiple sites
to induce GTPase release (e.g. in the case of Pak1-induced
Rac release) provides a mechanism for ‘coincidence
detection’ of signal input. While Pak1 phosphorylated
both Ser101 and Ser174 in RhoGDI, PKA only phosphorylated Ser101, and this in itself was not sufficient to
dissociate the induced Rac–RhoGDI complex [67]. Thus,
individual receptors acting through distinct kinases might
phosphorylate individual sites on RhoGDI, leading to
Opinion
TRENDS in Cell Biology
Vol.15 No.7 July 2005
361
Pak
Kinase X
GDP
GTPase
Kinase 1
GDP
RhoGDI
S101
GTPase
S174
Kinase 2
Lipids?
GDF?
Lipids?
GDP
+
RhoGDI
S101
RhoGDI
GTP
GTPase
S174
GTPase
Cell
activation
GEFs
Lipids?
?
Kinases?
TRENDS in Cell Biology
Figure 2. Proposed models for coordinated regulation of Rho GTPase activation through kinase-mediated phosphorylation of GDP dissociation inhibitor (GDI). The
dissociation of Rac GTPase from RhoGDI can be induced by the p21-activated kinase (Pak)-mediated phosphorylation of Ser101 and Ser174 on RhoGDI. It is possible that
other kinases might individually phosphorylate these two sites to induce dissociation of the complex. It is also likely that other kinase phosphorylation sites on RhoGDI
(indicated by ‘Kinase X’) exist that are able to regulate GTPase release. Thus, different signaling pathways resulting from cell stimulation might combine to induce Rho
GTPase release from GDI complexes. Additionally, release might be initiated through the action of lipid mediators and specific GDI displacement factors (GDFs). The release
of the Rho GTPase from GDI might be coordinated with guanine nucleotide exchange factor (GEF) activation by the same combinations of signaling pathways, resulting in the
biological activities of the Rho GTPase being manifest in cell activation. See text for details. (The jagged line represents the GTPase isoprenyl group.)
dissociation of the complex only when multiple sites are
phosphorylated.
Coordination of GTPase release with activation
Regulation by phosphorylation could allow the coordination of GEF activation with availability of free (uncomplexed) Rho GTPase. It is well established that the activity
of many Rho GEFs is controlled by their phosphorylation
state (see [72]). Similarly, phosphorylation-induced
changes in GDI binding might go hand-in-hand with the
generation of lipid mediators capable of modulating the
affinity of GTPase binding to GDI (see above). Both signals
might be necessary for full dissociation and, significantly,
both might act to modulate GEF activity subsequent to
GTPase release [72] (Figure 2).
Signal termination
Finally, it is apparent that phosphorylation of the Rho
GTPases themselves seems to invariably lead to enhanced
association with GDI. This provides a mechanism for
specific termination of the Rho GTPase signal. Alternatively, this might modify active GTPase localization
through GDI binding, as in the case cited by Cerione and
colleagues [71].
Concluding remarks
While still largely speculative, we suggest that modulation of the dynamics of Rho GTPase–GDI complex
formation by phosphorylation of GTPases and GDIs
provides a flexible, yet simple, mechanism for coordinating Rho GTPase action in response to cell activation
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through various growth factors, hormones and extracellular matrix molecules. Kinases might act in concert with
regulatory phosphatases, the formation of lipid mediators
and, potentially, other covalent modifications to control
the specificity and dynamics of Rho GTPase action. The
molecular and structural basis for reduced affinity of the
GTPase–GDI complex will require additional investigation of the structural changes accompanying the
regulatory phosphorylation of Rho GTPase–GDI complexes. How specific kinases, phosphatases and lipid
mediators contribute to specific hormonal pathways in
individual cells is an important goal of future investigations. Finally, studies to elicit the mechanisms by which
GDI regulation is coordinated with the closely linked
action of GEFs and GAPs to modulate Rho GTPase
activity will provide novel insights into how the action of
these important ‘molecular switches’ is regulated to
control cellular behavior.
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