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 www.sciencedirect.com 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 www.sciencedirect.com 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 www.sciencedirect.com 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 www.sciencedirect.com 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 www.sciencedirect.com through various growth factors, hormones and extracellular matrix molecules. 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