Dbl Family Guanine Nucleotide Exchange Research Articles

Deciphering the conformational dynamics of gephyrin-mediated collybistin activation

Efficient neuronal signaling depends on the proper assembly of the postsynaptic neurotransmitter machinery. The majority of inhibitory synapses feature γ-aminobutyric acid type A (GABAA) receptors. The function of these GABAergic synapses is controlled by the scaffolding protein gephyrin and collybistin, a Dbl family guanine nucleotide exchange factor and neuronal adaptor protein. Specifically, collybistin interacts with small GTPases, cell adhesion proteins, and phosphoinositides to recruit gephyrin and GABAA receptors to postsynaptic membrane specializations. Collybistin usually contains an N-terminal SH3 domain and exists in closed/inactive or open/active states. Here, we elucidate the molecular basis of the gephyrin-collybistin interaction with newly designed collybistin Förster resonance energy transfer (FRET) sensors. Using fluorescence lifetime-based FRET measurements, we deduce the affinity of the gephyrin-collybistin complex, thereby confirming that the C-terminal dimer-forming E domain binds collybistin, an interaction that does not require E domain dimerization. Simulations based on fluorescence lifetime and sensor distance distributions reveal at least a two-state equilibrium of the SH3 domain already in the free/unbound collybistin, thereby illustrating the accessible volume of the SH3 domain. Finally, our data provide strong evidence for a tightly regulated collybistin-gephyrin interplay, where, unexpectedly, switching of collybistin from closed/inactive to open/active states is efficiently triggered by gephyrin.

Structural/functional studies of Trio provide insights into its configuration and show that conserved linker elements enhance its activity for Rac1

Trio is a large and highly conserved metazoan signaling scaffold that contains two Dbl family guanine nucleotide exchange factor (GEF) modules, TrioN and TrioC, selective for Rac and RhoA GTPases, respectively. The GEF activities of TrioN and TrioC are implicated in several cancers, especially uveal melanoma. However, little is known about how these modules operate in the context of larger fragments of Trio. Here we show via negative stain electron microscopy that the N-terminal region of Trio is extended and could thus serve as a rigid spacer between the N-terminal putative lipid-binding domain and TrioN, whereas the C-terminal half of Trio seems globular. We found that regions C-terminal to TrioN enhance its Rac1 GEF activity and thus could play a regulatory role. We went on to characterize a minimal, well-behaved Trio fragment with enhanced activity, Trio1284–1959, in complex with Rac1 using cryo-electron microscopy and hydrogen-deuterium exchange mass spectrometry and found that the region conferring enhanced activity is disordered. Deletion of two different strongly conserved motifs in this region eliminated this enhancement, suggesting that they form transient intramolecular interactions that promote GEF activity. Because Dbl family RhoGEF modules have been challenging to directly target with small molecules, characterization of accessory Trio domains such as these may provide alternate routes for the development of therapeutics that inhibit Trio activity in human cancer.

More evidence on TRIO missense mutations in the spectrin repeat domain causing severe developmental delay and recognizable facial dysmorphism with macrocephaly.

TRIO is a Dbl family guanine nucleotide exchange factor (GEF) and an important regulator of neuronal development. Most truncating and missense variants affecting the Dbl homology domain of TRIO are associated with a neurodevelopmental disorder with microcephaly (MIM617061). Recently, de novo missense variants affecting the spectrin repeat region of TRIO were associated with a novel phenotype comprising severe developmental delay and macrocephaly (MIM618825). Here, we provide more evidence on this new TRIO-associated phenotype by reporting two severely affected probands with de novo missense variants in TRIO affecting the spectrin repeat region upstream of the typically affected GEF1 domain of the protein.

The Tiam1 guanine nucleotide exchange factor is auto-inhibited by its pleckstrin homology coiled-coil extension domain

T-cell lymphoma invasion and metastasis 1 (Tiam1) is a Dbl-family guanine nucleotide exchange factor (GEF) that specifically activates the Rho-family GTPase Rac1 in response to upstream signals, thereby regulating cellular processes including cell adhesion and migration. Tiam1 contains multiple domains, including an N-terminal pleckstrin homology coiled-coiled extension (PHn-CC-Ex) and catalytic Dbl homology and C-terminal pleckstrin homology (DH-PHc) domain. Previous studies indicate that larger fragments of Tiam1, such as the region encompassing the N-terminal to C-terminal pleckstrin homology domains (PHn-PHc), are auto-inhibited. However, the domains in this region responsible for inhibition remain unknown. Here, we show that the PHn-CC-Ex domain inhibits Tiam1 GEF activity by directly interacting with the catalytic DH-PHc domain, preventing Rac1 binding and activation. Enzyme kinetics experiments suggested that Tiam1 is auto-inhibited through occlusion of the catalytic site rather than by allostery. Small angle X-ray scattering and ensemble modeling yielded models of the PHn-PHc fragment that indicate it is in equilibrium between "open" and "closed" conformational states. Finally, single-molecule experiments support a model in which conformational sampling between the open and closed states of Tiam1 contributes to Rac1 dissociation. Our results highlight the role of the PHn-CC-Ex domain in Tiam1 GEF regulation and suggest a combinatorial model for GEF inhibition and activation of the Rac1 signaling pathway.

ARHGEF39 promotes gastric cancer cell proliferation and migration via Akt signaling pathway.

Dbl-family guanine nucleotide exchange factors (GEFs) can activate RhoGTPases by facilitating the exchange of GDP for GTP, the aberrant expression of which has been implicated in tumorigenicity and metastasis of human cancers. ARHGEF39, as a member of Dbl-family GEFs, was reported to be a potential oncogene in human hepatocellular carcinoma previously. However, the role of ARHGEF39 in gastric cancer (GC) remains unclear so far. In the current study, we demonstrated that ARHGEF39 expression was significantly upregulated in GC tissues compared with paired adjacent normal tissues by quantitative real-time PCR analysis. Functional analyses revealed that ARHGEF39 overexpression could promote proliferation, colony formation, and migration of GC cells in vitro, whereas ARHGEF39 knockdown markedly suppressed these phenotypes. Moreover, ARHGEF39 enhanced tumorigenicity and lung metastasis potential of GC cells in nude mice model. Mechanistically, we found that overexpressed ARHGEF39 significantly increased the phosphorylation level of Akt (p-Akt), and its effect on cell proliferation was attenuated by PI3K inhibitor LY294002. Thus, our findings suggest that ARHGEF39 may contribute to cell proliferation and migration in GC via a possible mechanism involving Akt signaling.

Mutations specific to the Rac-GEF domain of TRIO cause intellectual disability and microcephaly

BackgroundNeurodevelopmental disorders have challenged clinical genetics for decades, with over 700 genes implicated and many whose function remains unknown. The application of whole-exome sequencing is proving pivotal in closing the...

Insights into the Autoinhibition Mechanism of the Tiam1 Guanine Nucleotide Exchange Factor

The T-cell lymphoma invasion and metastasis 1 (Tiam1) is a Dbl-family guanine nucleotide exchange factor (GEF) specific for the Rho-family GTPase Rac1. Mutations or aberrant regulation of Tiam1 have been implicated in oncogenic transformation of cells and linked to several kinds of invasive and metastatic forms of cancer. In addition to the C-terminal catalytic Dbl-homology (DH) and pleckstrin homology (PH) bi-domain (DH-PHc), Tiam1 contains several N-terminal protein-protein interaction domains (PHn-CC-Ex, RBD and PDZ) that are thought to contribute to the regulation of its GEF activity through inter-domain interactions. The mechanism for how the Tiam1 GEF function is regulated, however, remains poorly understood. Here, we show that truncation of N-terminal domains activates Tiam1 catalytic GEF function, while addition of the PHn-CC-Ex domain inhibited the catalytic activity of the DH-PHc domain in vitro. In addition, enzymatic kinetics experiments revealed that auto-inhibition of Tiam1 GEF function occurs through a competitive inhibition mechanism whereby the N-terminus decreases the substrate (GTPase Rac1) binding affinity, but does not change the maximum velocity of catalytic activity. We will also present ongoing single-molecule total internal reflection fluorescence (TIRF) data that characterize the kinetics of binding between Rac1 and the activated DH-PHc and inhibited PHn-PHc fragments. Finally, we derived a structural model for the Tiam1 PHn-PHc fragment based on small-angle X-ray scattering (SAXS) data. The model reveals an auto-inhibited conformation in which the N-terminal PHn-CC-Ex domain and linker regions block access of Rac1 to the catalytic bi-domain. Together, these data elucidate the biochemical and structural mechanisms for Tiam1 GEF regulation.

Upregulated TRIO expression correlates with a malignant phenotype in human hepatocellular carcinoma.

Triple functional domain protein (TRIO) is an evolutionarily conserved Dbl family guanine nucleotide exchange factors (GEFs) involved in cell proliferation and progression of some types of cancer. However, the expression and prognostic role of TRIO in hepatocellular carcinoma (HCC) have not yet been determined. Therefore, we attempted to determine the impact of TRIO on the clinical outcome of HCC patients to further identify its role in HCC. TRIO expression was examined using quantitative real-time PCR (qRT-PCR) and Western blotting in nonmalignant liver cells, HCC cells, and 93 paired of HCC tissues and adjacent noncancerous tissues. Statistical analyses were used to assess associations between TRIO expression and clinicopathological and prognostic factors. Small interfering RNA (siRNA)-mediated TRIO inhibition was performed in Hep3B and Huh7 cells to elucidate its roles in HCC. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was employed to measure cell proliferation, and apoptosis assay was analyzed by flow cytometry, respectively. Adhesion and transwell invasion assay were performed to determine the invasion ability of HCC cells in vitro. TRIO was significantly upregulated in the HCC cell lines and tissues compared with the nonmalignant liver cells and adjacent noncancerous liver tissues. In addition, high TRIO expression level associated with lymph node metastasis (P = 0.0183), clinical tumor node metastasis (TNM) stage (P = 0.0.0106), and decrease in overall survival (OS) (P = 0.017). Knockdown of TRIO on Hep3B and Huh7 cell lines suppressed cell proliferation and migration and induced apoptosis. Furthermore, silencing TRIO expression led to decrease of ras-related C3 botulinum toxin substrate 1 (Rac1), p-P38, B cell lymphoma 2 (BCL-2), and matrix metallopeptidase 9 (MMP-9). Our results demonstrated that TRIO protein expression is elevated and associated with a worse over survival rates in patients with HCC. Aberrant expression of TRIO might play an important role in HCC through promoting cell proliferation and invasion, and TRIO may be a novel therapeutic target for the treatment of HCC.

Plekhg4 Is a Novel Dbl Family Guanine Nucleotide Exchange Factor Protein for Rho Family GTPases

Mutations in the PLEKHG4 (puratrophin-1) gene are associated with the heritable neurological disorder autosomal dominant spinocerebellar ataxia. However, the biochemical functions of this gene product have not been described. We report here that expression of Plekhg4 in the murine brain is developmentally regulated, with pronounced expression in the newborn midbrain and brainstem that wanes with age and maximal expression in the cerebellar Purkinje neurons in adulthood. We show that Plekhg4 is subject to ubiquitination and proteasomal degradation, and its steady-state expression levels are regulated by the chaperones Hsc70 and Hsp90 and by the ubiquitin ligase CHIP. On the functional level, we demonstrate that Plekhg4 functions as a bona fide guanine nucleotide exchange factor (GEF) that facilitates activation of the small GTPases Rac1, Cdc42, and RhoA. Overexpression of Plekhg4 in NIH3T3 cells induces rearrangements of the actin cytoskeleton, specifically enhanced formation of lamellopodia and fillopodia. These findings indicate that Plekhg4 is an aggregation-prone member of the Dbl family GEFs and that regulation of GTPase signaling is critical for proper cerebellar function.

Structural Basis for Autoinhibition of the Guanine Nucleotide Exchange Factor FARP2

FARP2 is a Dbl-family guanine nucleotide exchange factor (GEF) that contains a 4.1, ezrin, radixin and moesin (FERM) domain, a Dbl-homology (DH) domain and two pleckstrin homology (PH) domains. FARP2 activates Rac1 or Cdc42 in response to upstream signals, thereby regulating processes such as neuronal axon guidance and bone homeostasis. How the GEF activity of FARP2 is regulated remained poorly understood. We have determined the crystal structures of the catalytic DH domain and the DH-PH-PH domains of FARP2. The structures reveal an auto-inhibited conformation in which the GEF substrate-binding site is blocked collectively by the last helix in the DH domain and the two PH domains. This conformation is stabilized by multiple interactions among the domains and two well-structured inter-domain linkers. Our cell-based activity assays confirm the suppression of the FARP2 GEF activity by these auto-inhibitory elements.

C9orf100, a new member of the Dbl-family guanine nucleotide exchange factors, promotes cell proliferation and migration in hepatocellular carcinoma

Dbl-family guanine nucleotide exchange factors (GEFs) are important activators of Rho GTPases, which are significantly associated with tumorigenesis and metastasis. The catalytic ability of the Dbl-family GEFs to activate Rho GTPases depends on their Dbl-homology domain followed by a pleckstrin-homology domain. In the present study, we showed that C9orf100, a new member of the Dbl-family GEFs with a minimal catalytic unit, may contribute to hepatocellular carcinoma (HCC). Quantitative real-time PCR results demonstrated that C9orf100 was highly and widely upregulated in 42/44 (95.5%, >2‑fold) HCC specimens compared with adjacent non-cancerous livers, and this upregulation was correlated with intrahepatic metastasis and α-fetoprotein levels of HCC. Furthermore, the ectopic expression of C9orf100 promoted cell proliferation and colony formation in Huh-7 and YY-8103 cells, whereas silencing of C9orf100 resulted in the suppression of cell growth in MHCC-97H and PLC/PRF/5 cells. Flow cytometry confirmed this effect on MHCC-97H cell growth and indicated that C9orf100 may function in the G2/M phase. In addition, we showed that C9orf100 is involved in the positive regulation of HCC cell migration by a transwell chamber analysis. Our findings suggest that C9orf100 plays a potential oncogenic role in the development and metastasis of HCC.

Myosin II directly binds and inhibits Dbl family guanine nucleotide exchange factors

Cell migration requires the coordinated spatiotemporal regulation of actomyosin contraction and cell protrusion/adhesion. Nonmuscle myosin II (MII) controls Rac1 and Cdc42 activation, and cell protrusion and focal complex formation in migrating cells. However, these mechanisms are poorly understood. Here, we show that MII interacts specifically with multiple Dbl family guanine nucleotide exchange factors (GEFs). Binding is mediated by the conserved tandem Dbl homology‐pleckstrin homology module, the catalytic site of these GEFs, with dissociation constants of ~0.3 microM. Binding to the GEFs required assembly of the MII into filaments and actin‐stimulated ATPase activity. Binding of MII suppressed GEF activity. Accordingly, inhibition of MII ATPase activity caused release of GEFs and activation of Rho GTPases. Depletion of bPIX GEF in migrating NIH3T3 fibroblasts suppressed lamellipodial protrusions and focal complex formation induced by MII inhibition. The results elucidate a functional link between MII and Rac1/Cdc42 GTPases, which may regulate protrusion/adhesion dynamics in migrating cells.

Myosin II directly binds and inhibits Dbl family guanine nucleotide exchange factors: a possible link to Rho family GTPases

Cell migration requires the coordinated spatiotemporal regulation of actomyosin contraction and cell protrusion/adhesion. Nonmuscle myosin II (MII) controls Rac1 and Cdc42 activation, and cell protrusion and focal complex formation in migrating cells. However, these mechanisms are poorly understood. Here, we show that MII interacts specifically with multiple Dbl family guanine nucleotide exchange factors (GEFs). Binding is mediated by the conserved tandem Dbl homology-pleckstrin homology module, the catalytic site of these GEFs, with dissociation constants of approximately 0.3 microM. Binding to the GEFs required assembly of the MII into filaments and actin-stimulated ATPase activity. Binding of MII suppressed GEF activity. Accordingly, inhibition of MII ATPase activity caused release of GEFs and activation of Rho GTPases. Depletion of betaPIX GEF in migrating NIH3T3 fibroblasts suppressed lamellipodial protrusions and focal complex formation induced by MII inhibition. The results elucidate a functional link between MII and Rac1/Cdc42 GTPases, which may regulate protrusion/adhesion dynamics in migrating cells.

Regulation of mitotic spindle formation by the RhoA guanine nucleotide exchange factor ARHGEF10

BackgroundThe Dbl family guanine nucleotide exchange factor ARHGEF10 was originally identified as the product of the gene associated with slowed nerve-conduction velocities of peripheral nerves. However, the function of ARHGEF10 in mammalian cells is totally unknown at a molecular level. ARHGEF10 contains no distinctive functional domains except for tandem Dbl homology-pleckstrin homology and putative transmembrane domains.ResultsHere we show that RhoA is a substrate for ARHGEF10. In both G1/S and M phases, ARHGEF10 was localized in the centrosome in adenocarcinoma HeLa cells. Furthermore, RNA interference-based knockdown of ARHGEF10 resulted in multipolar spindle formation in M phase. Each spindle pole seems to contain a centrosome consisting of two centrioles and the pericentriolar material. Downregulation of RhoA elicited similar phenotypes, and aberrant mitotic spindle formation following ARHGEF10 knockdown was rescued by ectopic expression of constitutively activated RhoA. Multinucleated cells were not increased upon ARHGEF10 knockdown in contrast to treatment with Y-27632, a specific pharmacological inhibitor for the RhoA effector kinase ROCK, which induced not only multipolar spindle formation, but also multinucleation. Therefore, unregulated centrosome duplication rather than aberration in cytokinesis may be responsible for ARHGEF10 knockdown-dependent multipolar spindle formation. We further isolated the kinesin-like motor protein KIF3B as a binding partner of ARHGEF10. Knockdown of KIF3B again caused multipolar spindle phenotypes. The supernumerary centrosome phenotype was also observed in S phase-arrested osteosarcoma U2OS cells when the expression of ARHGEF10, RhoA or KIF3B was abrogated by RNA interference.ConclusionCollectively, our results suggest that a novel RhoA-dependent signaling pathway under the control of ARHGEF10 has a pivotal role in the regulation of the cell division cycle. This pathway is not involved in the regulation of cytokinesis, but instead may regulate centrosome duplication. The kinesin-like motor protein KIF3B may modulate the ARHGEF10-RhoA pathway through the binding to ARHGEF10.

Gαq Directly Activates p63RhoGEF and Trio via a Conserved Extension of the Dbl Homology-associated Pleckstrin Homology Domain

The coordinated cross-talk from heterotrimeric G proteins to Rho GTPases is essential during a variety of physiological processes. Emerging data suggest that members of the Galpha(12/13) and Galpha(q/11) families of heterotrimeric G proteins signal downstream to RhoA via distinct pathways. Although studies have elucidated mechanisms governing Galpha(12/13)-mediated RhoA activation, proteins that functionally couple Galpha(q/11) to RhoA activation have remained elusive. Recently, the Dbl-family guanine nucleotide exchange factor (GEF) p63RhoGEF/GEFT has been described as a novel mediator of Galpha(q/11) signaling to RhoA based on its ability to synergize with Galpha(q/11) resulting in enhanced RhoA signaling in cells. We have used biochemical/biophysical approaches with purified protein components to better understand the mechanism by which activated Galpha(q) directly engages and stimulates p63RhoGEF. Basally, p63RhoGEF is autoinhibited by the Dbl homology (DH)-associated pleckstrin homology (PH) domain; activated Galpha(q) relieves this autoinhibition by interacting with a highly conserved C-terminal extension of the PH domain. This unique extension is conserved in the related Dbl-family members Trio and Kalirin and we show that the C-terminal Rho-specific DH-PH cassette of Trio is similarly activated by Galpha(q).

The DH and PH Domains of Trio Coordinately Engage Rho GTPases for their Efficient Activation

Rho-family GTPases are activated by the exchange of bound GDP for GTP, a process that is catalyzed by Dbl-family guanine nucleotide exchange factors (GEFs). The catalytic unit of Dbl-family GEFs consists of a Dbl homology (DH) domain followed almost invariantly by a pleckstrin-homology (PH) domain. The majority of the catalytic interface forms between the switch regions of the GTPase and the DH domain, but full catalytic activity often requires the associated PH domain. Although PH domains are usually characterized as lipid-binding regions, they also participate in protein–protein interactions. For example, the DH-associated PH domain of Dbs must contact its cognate GTPases for efficient exchange. Similarly, the N-terminal DH/PH fragment of Trio, which catalyzes exchange on both Rac1 and RhoG, is fourfold more active in vitro than the isolated DH domain. Given continued uncertainty regarding functional roles of DH-associated PH domains, we have undertaken structural and functional analyses of the N-terminal DH/PH cassette of Trio. The crystal structure of this fragment of Trio bound to nucleotide-depleted Rac1 highlights the engagement of the PH domain with Rac1 and substitution of residues involved in this interface substantially diminishes activation of Rac1 and RhoG. Also, these mutations significantly reduce the ability of full-length Trio to induce neurite outgrowth dependent on RhoG activation in PC-12 cells. Overall, these studies substantiate a general role for DH-associated PH domains in engaging Rho GTPases directly for efficient guanine nucleotide exchange and support a parsimonious explanation for the essentially invariant linkage between DH and PH domains.

ASEF2 EXCHANGE ACTIVITY IS STIMULATED BY DYNAMIN2 AND PROMOTES TUMOR CELL POLARIZATION

Pancreatic cancer prognosis is poor largely due to the metastatic behavior of tumor cells associated with the disease; therefore, understanding the mechanisms which promote the disruption of cellular cohesion and stimulate migration are of fundamental importance. The Rho family of small GTPases and their associated proteins are central to this subject since they are essential for both the maintenance of cell-to-cell junctions, and activation of proteins involved in actin remodeling and cell migration. Dbl family guanine nucleotide exchange factors are of particular interest since these proteins act as a catalyst to promote GTP-loading and activation of Rho GTPases. In this regard, we have investigated a Cdc42-specific Rho family GEF called Asef2 which we have previously shown to interact with adenomatous polyposis coli (APC) through a discreet motif near the N-terminal region of Asef2. Herein, we describe a novel interaction with Asef2 and Dynamin2, a large GTPase involved in cortical actin remodeling as well as membrane vesicle endocytosis. The interaction between Dynamin2 and Asef2 is mediated through a canonical interaction with the SH3 domain of Asef2 and the proline-rich domain (PRD) of Dynamin2. Moreover, binding of the PRD to the SH3 domain of Asef2 stimulated in vitro exchange on Cdc42, indicating that Dynamin2 releases the auto-inhibition imposed by the SH3 domain. Binding of Asef2 with APC and Dynamin2 was not mutually exclusive, and a ternary complex of the three proteins was identified both in vitro using purified proteins and by co-immunoprecipitation of endogenous cellular protein. Furthermore the proteins were found to co-localize by confocal microscopy, and could be detected at the leading edge of scratch wounded cells. Since APC is known to bind to the microtubule filaments through EB1, and Dynamin2 has been linked to cortical actin through Cortactin, these findings suggest Asef2 acts as a critical linker protein between actin and microtubules during polarized cell migration.

Substrate Specificity and Recognition Is Conferred by the Pleckstrin Homology Domain of the Dbl Family Guanine Nucleotide Exchange Factor P-Rex2

Dbl family guanine nucleotide exchange factors (GEFs) are characterized by the presence of a catalytic Dbl homology domain followed invariably by a lipid-binding pleckstrin homology (PH) domain. To date, substrate recognition and specificity of this family of GEFs has been reported to be mediated exclusively via the Dbl homology domain. Here we report the novel and unexpected finding that, in the Dbl family Rac-specific GEF P-Rex2, it is the PH domain that confers substrate specificity and recognition. Moreover, the beta3beta4 loop of the PH domain of P-Rex2 is the determinant for Rac1 recognition, as substitution of the beta3beta4 loop of the PH domain of Dbs (a RhoA- and Cdc42-specific GEF) with that of P-Rex2 confers Rac1-specific binding capability to the PH domain of Dbs. The contact interface between the PH domain of P-Rex2 and Rac1 involves the switch loop and helix 3 of Rac1. Moreover, substitution of helix 3 of Cdc42 with that of Rac1 now enables the PH domain of P-Rex2 to bind this Cdc42 chimera. Despite having the ability to recognize this chimeric Cdc42, P-Rex2 is unable to catalyze nucleotide exchange on Cdc42, suggesting that recognition of substrate and catalysis are two distinct events. Thus substrate recognition can now be added to the growing list of functions that are being attributed to the PH domain of Dbl family GEFs.

Novel regulatory mechanisms for the Dbl family guanine nucleotide exchange factor Cool-2/alpha-Pix.

The Cool-2 (cloned-out of library-2) protein (identical to alpha-Pix for Pak-interactive exchange factor) has been implicated in various biological responses including chemoattractant signaling and in certain forms of mental retardation. We show that when Cool-2 exists as a dimer, it functions as a Rac-specific guanine nucleotide exchange factor (GEF). Dimerization of Cool-2 enables its Dbl (diffuse B-cell lymphoma) and pleckstrin homology domains to work together (in trans) to bind specifically to Rac-GDP. Dissociation of dimeric Cool-2 into its monomeric form allows it to act as a GEF for Cdc42 as well as for Rac. The binding of either PAK (p21-activated kinase) or Cbl (Casitas B-lymphoma) to the SH3 domain of monomeric Cool-2 is necessary for the functional interactions between GDP-bound Cdc42 or Rac and the Cool-2 monomer. The betagamma subunit complex of large GTP-binding proteins, by interacting with PAK, stimulates the dissociation of the Cool-2 dimer and activates its GEF activity for Cdc42. Overall, these findings highlight novel mechanisms by which extracellular signals can direct the specific activation of Rac versus Cdc42 by Cool-2/alpha-Pix.

Rational design and characterization of a Rac GTPase-specific small molecule inhibitor.

The signaling pathways mediated by Rho family GTPases have been implicated in many aspects of cell biology. The specificity of the pathways is achieved in part by the selective interaction between Dbl family guanine nucleotide exchange factors (GEFs) and their Rho GTPase substrates. Here, we report a first-generation small-molecule inhibitor of Rac GTPase targeting Rac activation by GEF. The chemical compound NSC23766 was identified by a structure-based virtual screening of compounds that fit into a surface groove of Rac1 known to be critical for GEF specification. In vitro it could effectively inhibit Rac1 binding and activation by the Rac-specific GEF Trio or Tiam1 in a dose-dependent manner without interfering with the closely related Cdc42 or RhoA binding or activation by their respective GEFs or with Rac1 interaction with BcrGAP or effector PAK1. In cells, it potently blocked serum or platelet-derived growth factor-induced Rac1 activation and lamellipodia formation without affecting the activity of endogenous Cdc42 or RhoA. Moreover, this compound reduced Trio or Tiam1 but not Vav, Lbc, Intersectin, or a constitutively active Rac1 mutant-stimulated cell growth and suppressed Trio, Tiam1, or Ras-induced cell transformation. When applied to human prostate cancer PC-3 cells, it was able to inhibit the proliferation, anchorage-independent growth and invasion phenotypes that require the endogenous Rac1 activity. Thus, NSC23766 constitutes a Rac-specific small-molecule inhibitor that could be useful to study the role of Rac in various cellular functions and to reverse tumor cell phenotypes associated with Rac deregulation.