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.     

The Cool-2/alpha-Pix protein mediates a Cdc42-Rac signaling cascade

BACKGROUND: Cloned-out of library-2 (Cool-2)/PAK-interactive exchange factor (alpha-Pix) was identified through its ability to bind the Cdc42/Rac target p21-activated kinase (PAK) and has been implicated in certain forms of X-linked mental retardation as well as in growth factor- and chemoattractant-coupled signaling pathways. We recently found that the dimeric form of Cool-2 is a specific guanine nucleotide exchange factor (GEF) for Rac, whereas monomeric Cool-2 is a GEF for Cdc42 as well as Rac. However, unlike many GEFs, Cool-2 binds to activated forms of Cdc42 and Rac. Thus, we have investigated the functional consequences of these interactions. RESULTS: We show that the binding of activated Cdc42 to the Cool-2 dimer markedly enhances its ability to associate with GDP bound Rac1, resulting in a significant activation of Rac-GEF activity. While the Rac-specific GEF activity of Cool-2 is mediated through the Dbl homology (DH) domain from one monomer and the Pleckstrin homology domain from the other, activated Cdc42 interacts with the DH domain, most likely opposite the DH domain binding site for GDP bound Rac. Activated Rac also binds to Cool-2; however, it strongly inhibits the GEF activity of dimeric Cool-2. CONCLUSIONS: We provide evidence for novel mechanisms of allosteric regulation of the Rac-GEF activity of the Cool-2 dimer, involving stimulatory effects by Cdc42 and feedback inhibition by Rac. These findings demonstrate that by serving as a target for GTP bound Cdc42 and a GEF for Rac, Cool-2 mediates a GTPase cascade where the activation of Cdc42 is translated into the activation of Rac.     

Monoclonal antibodies against the Androctonus australis hector scorpion neurotoxin I: characterisation and use for venom neutralisation

Several guanine nucleotide exchange factors (GEFs) for Rho-GTPases have been identified, all of them containing a Dbl homology (DH) and pleckstrin homology (PH) domain, but exhibiting different specificities to the Rho family members, Rho, Rac and Cdc42. We report here that KIAA0380, a protein with a tandem DH/PH domain, an amino-terminal PDZ domain and a regulator of G protein signalling (RGS) homology domain, is a specific GEF for RhoA, but not for Rac1 and Cdc42, as determined by GDP release, guanosine 5'-O-(3-thio)triphosphate (GTPgammaS) binding and protein binding assays. When expressed in J82 cells, DH/PH domain-containing forms of KIAA0380 induced actin stress fibers, whereas expression of the RGS homology domain prevented lysophosphatidic acid (LPA)-induced stress fiber formation.     

Modulation of oncogenic DBL activity by phosphoinositol phosphate binding to pleckstrin homology domain

The Dbl family guanine nucleotide exchange factors (GEFs) contain a region of sequence similarity consisting of a catalytic Dbl homology (DH) domain in tandem with a pleckstrin homology (PH) domain. PH domains are involved in the regulated targeting of signaling molecules to plasma membranes by protein-protein and/or protein-lipid interactions. Here we show that Dbl PH domain binding to phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3,4,5-triphosphate results in the inhibition of Dbl GEF activity on Rho family GTPase Cdc42. Phosphatidylinositol 4,5-bisphosphate binding to the PH domain significantly inhibits the Cdc42 interactive activity of the DH domain suggesting that the DH domain is subjected to the PH domain modulation under the influence of phosphoinositides (PIPs). We generated Dbl mutants unable to interact with PIPs. These mutants retained GEF activity on Cdc42 in the presence of PIPs and showed a markedly enhanced activating potential for both Cdc42 and RhoA in vivo while displaying decreased cellular transforming activity. Immunofluorescence analysis of NIH3T3 transfectants revealed that whereas the PH domain localizes to actin stress fibers and plasma membrane, the PH mutants are no longer detectable on the plasma membrane. These results suggest that modulation of PIPs in both the GEF catalytic activity and the targeting to plasma membrane determines the outcome of the biologic activity of Dbl.     

Molecular basis for Rac1 recognition by guanine nucleotide exchange factors.

Rho GTPases are activated by a family of guanine nucleotide exchange factors (GEFs) known as Dbl family proteins. The structural basis for how GEFs recognize and activate Rho GTPases is presently ill defined. Here, we utilized the crystal structure of the DH/PH domains of the Rac-specific GEF Tiam1 in complex with Rac1 to determine the structural elements of Rac1 that regulate the specificity of this interaction. We show that residues in the Rac1 beta2-beta3 region are critical for Tiam1 recognition. Additionally, we determined that a single Rac1-to-Cdc42 mutation (W56F) was sufficient to abolish Rac1 sensitivity to Tiam1 and allow recognition by the Cdc42-specific DH/PH domains of Intersectin while not impairing Rac1 downstream activities. Our findings identified unique GEF specificity determinants in Rac1 and provide important insights into the mechanism of DH/PH selection of GTPase targets.     

Mechanistic insights into specificity, activity, and regulatory elements of the regulator of G-protein signaling (RGS)-containing Rho-specific guanine nucleotide exchange factors (GEFs) p115, PDZ-RhoGEF (PRG), and leukemia-associated RhoGEF (LARG)

The multimodular guanine nucleotide exchange factors (GEFs) of the Dbl family mostly share a tandem Dbl homology (DH) and pleckstrin homology (PH) domain organization. The function of these and other domains in the DH-mediated regulation of the GDP/GTP exchange reaction of the Rho proteins is the subject of intensive investigations. This comparative study presents detailed kinetic data on specificity, activity, and regulation of the catalytic DH domains of four GEFs, namely p115, p190, PDZ-RhoGEF (PRG), and leukemia-associated RhoGEF (LARG). We demonstrate that (i) these GEFs are specific guanine nucleotide exchange factors for the Rho isoforms (RhoA, RhoB, and RhoC) and inactive toward other members of the Rho family, including Rac1, Cdc42, and TC10. (ii) The DH domain of LARG exhibits the highest catalytic activity reported for a Dbl protein till now with a maximal acceleration of the nucleotide exchange by 10(7)-fold, which is at least as efficient as reported for GEFs specific for Ran or the bacterial toxin SopE. (iii) A novel regulatory region at the N terminus of the DH domain is involved in its association with GDP-bound RhoA monitored by a fluorescently labeled RhoA. (iv) The tandem PH domains of p115 and PRG efficiently contribute to the DH-mediated nucleotide exchange reaction. (v) In contrast to the isolated DH or DH-PH domains, a p115 fragment encompassing both the regulator of G-protein signaling and the DH domains revealed a significantly reduced GEF activity, supporting the proposed models of an intramolecular autoinhibitory mechanism for p115-like RhoGEFs.     

PH domain-mediated membrane targeting of Asef

The APC-associated guanine nucleotide exchange factor (GEF) Asef regulates cell morphology and migration. Asef contains a pleckstrin homology (PH) domain in addition to Dbl homology (DH), APC-binding (ABR), and Src homology 3 (SH3) domains. Here we show that the PH domain of Asef binds to phosphatidylinositol 3,4,5-trisphophate [PtdIns(3,4,5)P3] and targets Asef to the cell-cell adhesion sites in MDCK II cells. Furthermore, we demonstrate that overexpression of Asef in MDCK II cells results in increases in the amounts of E-cadherin and the actin filaments at the sites of cell-cell contact. These results suggest that Asef is targeted via its PH domain to the cell-cell adhesion sites and is involved in the regulation of cell adhesion.     

Crystal structure of the rac activator, Asef, reveals its autoinhibitory mechanism

The Rac-specific guanine nucleotide exchange factor (GEF) Asef is activated by binding to the tumor suppressor adenomatous polyposis coli mutant, which is found in sporadic and familial colorectal tumors. This activated Asef is involved in the migration of colorectal tumor cells. The GEFs for Rho family GTPases contain the Dbl homology (DH) domain and the pleckstrin homology (PH) domain. When Asef is in the resting state, the GEF activity of the DH-PH module is intramolecularly inhibited by an unidentified mechanism. Asef has a Src homology 3 (SH3) domain in addition to the DH-PH module. In the present study, the three-dimensional structure of Asef was solved in its autoinhibited state. The crystal structure revealed that the SH3 domain binds intramolecularly to the DH domain, thus blocking the Rac-binding site. Furthermore, the RT-loop and the C-terminal region of the SH3 domain interact with the DH domain in a manner completely different from those for the canonical binding to a polyproline-peptide motif. These results demonstrate that the blocking of the Rac-binding site by the SH3 domain is essential for Asef autoinhibition. This may be a common mechanism in other proteins that possess an SH3 domain adjacent to a DH-PH module.     

Endocytic protein intersectin-l regulates actin assembly via Cdc42 and N-WASP

Intersectin-s is a modular scaffolding protein regulating the formation of clathrin-coated vesicles. In addition to the Eps15 homology (EH) and Src homology 3 (SH3) domains of intersectin-s, the neuronal variant (intersectin-l) also has Dbl homology (DH), pleckstrin homology (PH) and C2 domains. We now show that intersectin-l functions through its DH domain as a guanine nucleotide exchange factor (GEF) for Cdc42. In cultured cells, expression of DH-domain-containing constructs cause actin rearrangements specific for Cdc42 activation. Moreover, in vivo studies reveal that stimulation of Cdc42 by intersectin-l accelerates actin assembly via N-WASP and the Arp2/3 complex. N-WASP binds directly to intersectin-l and upregulates its GEF activity, thereby generating GTP-bound Cdc42, a critical activator of N-WASP. These studies reveal a role for intersectin-l in a novel mechanism of N-WASP activation and in regulation of the actin cytoskeleton.     

A crystallographic view of interactions between Dbs and Cdc42: PH domain-assisted guanine nucleotide exchange

Dbl-related oncoproteins are guanine nucleotide exchange factors (GEFs) specific for Rho guanosine triphosphatases (GTPases) and invariably possess tandem Dbl (DH) and pleckstrin homology (PH) domains. While it is known that the DH domain is the principal catalytic subunit, recent biochemical data indicate that for some Dbl-family proteins, such as Dbs and Trio, PH domains may cooperate with their associated DH domains in promoting guanine nucleotide exchange of Rho GTPases. In order to gain an understanding of the involvement of these PH domains in guanine nucleotide exchange, we have determined the crystal structure of a DH/PH fragment from Dbs in complex with Cdc42. The complex features the PH domain in a unique conformation distinct from the PH domains in the related structures of Sos1 and Tiam1.Rac1. Consequently, the Dbs PH domain participates with the DH domain in binding Cdc42, primarily through a set of interactions involving switch 2 of the GTPase. Comparative sequence analysis suggests that a subset of Dbl-family proteins will utilize their PH domains similarly to Dbs.     

Specific recognition of Rac2 and Cdc42 by DOCK2 and DOCK9 guanine nucleotide exchange factors

Recognition of cognate Rho GTPases by guanine-nucleotide exchange factors (GEF) is fundamental to Rho GTPase signaling specificity. Two main GEF families use either the Dbl homology (DH) or the DOCK homology region 2 (DHR-2) catalytic domain. How DHR-2-containing GEFs distinguish between the GTPases Rac and Cdc42 is not known. To determine how these GEFs specifically recognize the two Rho GTPases, we studied the amino acid sequences in Rac2 and Cdc42 that are crucial for activation by DOCK2, a Rac-specific GEF, and DOCK9, a distantly related Cdc42-specific GEF. Two elements in the N-terminal regions of Rac2 and Cdc42 were found to be essential for specific interactions with DOCK2 and DOCK9. One element consists of divergent amino acid residues in the switch 1 regions of the GTPases. Significantly, these residues were also found to be important for GTPase recognition by Rac-specific DOCK180, DOCK3, and DOCK4 GEFs. These findings were unexpected because the same residues were shown previously to interact with GTPase effectors rather than GEFs. The other element comprises divergent residues in the beta3 strand that are known to mediate specific recognition by DH domain containing GEFs. Remarkably, Rac2-to-Cdc42 substitutions of four of these residues were sufficient for Rac2 to be specifically activated by DOCK9. Thus, DOCK2 and DOCK9 specifically recognize Rac2 and Cdc42 through their switch 1 as well as beta2-beta3 regions and the mode of recognition via switch 1 appears to be conserved among diverse Rac-specific DHR-2 GEFs.     

Critical but distinct roles for the pleckstrin homology and cysteine-rich domains as positive modulators of Vav2 signaling and transformation

Vav2, like all Dbl family proteins, possesses tandem Dbl homology (DH) and pleckstrin homology (PH) domains and functions as a guanine nucleotide exchange factor for Rho family GTPases. Whereas the PH domain is a critical positive regulator of DH domain function for a majority of Dbl family proteins, the PH domains of the related Vav and Vav3 proteins are dispensable for DH domain activity. Instead, Vav proteins contain a cysteine-rich domain (CRD) critical for DH domain function. We evaluated the contribution of the PH domain and the CRD to Vav2 guanine nucleotide exchange, signaling, and transforming activity. Unexpectedly, we found that mutations of the PH domain impaired Vav2 signaling, transforming activity, and membrane association. However, these mutations do not influence exchange activity on Rac and only slightly affect exchange on RhoA and Cdc42. We also found that the CRD was critical for the exchange activity in vitro and contributed to Vav2 membrane localization. Finally, we found that phosphoinositol 3-kinase activation synergistically enhanced Vav2 transforming and signaling activity by stimulating exchange activity but not membrane association. In conclusion, the PH domain and CRD are mechanistically distinct, positive modulators of Vav2 DH domain function in vivo.     

Role of the pleckstrin homology domain in intersectin-L Dbl homology domain activation of Cdc42 and signaling

Intersectin-long (ITSN-L) contains the invariant Dbl homology (DH) and pleckstrin homology (PH) domain structure characteristic of the majority of Dbl family proteins. This strict domain topography suggests that the PH domain serves an essential, conserved function in the regulation of the intrinsic guanine nucleotide exchange activity of the DH domain. We evaluated the role of the PH domain in regulating the DH domain function of ITSN-L. Surprisingly, we found that the PH domain was dispensable for guanine nucleotide exchange activity on Cdc42 in vitro, yet the PH domain enhanced the ability of the DH domain to activate Cdc42 signaling in vivo. PH domains can interact with phosphoinositide substrates and products of phosphatidylinositol 3-kinase (PI3K). However, PI3K activation did not modulate ITSN-L DH domain function in vivo.     

Loss of phosphatidylinositol 3-phosphate binding by the C-terminal Tiam-1 pleckstrin homology domain prevents in vivo Rac1 activation without affecting membrane targeting

Dbl family guanine nucleotide exchange factors (GEFs) for Rho family small GTPases invariably contain a pleckstrin homology (PH) domain that immediately follows their Dbl homology (DH) domain. Although the DH domain is responsible for GEF activity, the role of the PH domain is less clear. We previously reported that PH domains from several Dbl family members bind phosphoinositides with very low affinity (K(d) values in the 10 microM range). This suggests that, unlike several other PH domains, those from Dbl proteins will not function as independent membrane-targeting modules. To determine the functional relevance of low affinity phosphoinositide binding, we mutated the corresponding PH domain from Tiam-1 to abolish its weak, specific binding to phosphatidylinositol 3-phosphate. We first confirmed in vitro that phosphoinositide binding by the isolated DH/PH domain was impaired by the mutations but that intrinsic GEF activity was unaffected. We then introduced the PH domain mutations into full-length Tiam-1 and found that its ability to activate Rac1 or serum response factor in vivo was abolished. Immunofluorescence studies showed that membrane targeting of Tiam-1 was essentially unaffected by mutations in the C-terminal PH domain. Our studies therefore indicate that low affinity phosphatidylinositol 3-phosphate binding by the C-terminal PH domain may be critical for in vivo regulation and activity of Tiam-1 but that the PH domain exerts its regulatory effects without altering membrane targeting. We suggest instead that ligand binding to the PH domain induces conformational and/or orientational changes at the membrane surface that are required for maximum exchange activity of its adjacent DH domain.     

Structural basis for the recognition of Asef by adenomatous polyposis coli

Adenomatous polyposis coli (APC) regulates cell-cell adhesion and cell migration through activating the APC-stimulated guanine nucleotide-exchange factor (GEF; Asef), which is usually autoinhibited through the binding between its Src homology 3 (SH3) and Dbl homology (DH) domains. The APC-activated Asef stimulates the small GTPase Cdc42, which leads to decreased cell-cell adherence and enhanced cell migration. In colorectal cancers, truncated APC constitutively activates Asef and promotes cancer cell migration and angiogenesis. Here, we report crystal structures of the human APC/Asef complex. We find that the armadillo repeat domain of APC uses a highly conserved surface groove to recognize the APC-binding region (ABR) of Asef, conformation of which changes dramatically upon binding to APC. Key residues on APC and Asef for the complex formation were mutated and their importance was demonstrated by binding and activity assays. Structural superimposition of the APC/Asef complex with autoinhibited Asef suggests that the binding between APC and Asef might create a steric clash between Asef-DH domain and APC, which possibly leads to a conformational change in Asef that stimulates its GEF activity. Our structures thus elucidate the molecular mechanism of Asef recognition by APC, as well as provide a potential target for pharmaceutical intervention against cancers.     

Functional analysis of the Caenorhabditis elegans UNC-73B PH domain demonstrates a role in activation of the Rac GTPase in vitro and axon guidance in vivo

The Caenorhabditis elegans UNC-73B protein regulates axon guidance through its ability to act as a guanine nucleotide exchange factor (GEF) for the CeRAC/MIG-2 GTPases. Like other GEFs for Rho family GTPases, UNC-73B has a Dbl homology (DH) catalytic domain, followed by a C-terminal pleckstrin homology (PH) domain. We have explored whether the PH domain cooperates with the adjacent DH domain to promote UNC-73B GEF activity and axonal pathfinding. We show that the UNC-73B PH domain binds preferentially to monophosphorylated phosphatidylinositides in vitro. Replacement of residues Lys1420 and Arg1422 with Glu residues within the PH domain impaired this phospholipid binding but did not affect the in vitro catalytic activity of the DH domain. In contrast, a mutant UNC-73B protein with a Trp1502-to-Ala substitution in the PH domain still interacted with phosphorylated phosphatidylinositides but had lost its GEF activity. UNC-73B minigenes containing these mutations were microinjected into C. elegans and transferred to unc-73(e936) mutant worms. Unlike the wild-type protein, neither PH domain mutant was able to rescue the unc-73 axon guidance defect. These results suggest that the UNC-73B PH domain plays distinct roles in targeting and promoting GEF activity towards the Rac GTPase, both of which are important for the directed movements of motorneurons in vivo.     

Identification of Rho GTPase-dependent sites in the Dbl homology domain of oncogenic Dbl that are required for transformation

The Dbl family guanine-nucleotide exchange factors (GEFs) for Rho GTPases share the structural array of a Dbl homology (DH) domain in tandem with a Pleckstrin homology (PH) domain. For oncogenic Dbl, the DH domain is responsible for the GEF activity, and the DH-PH module constitutes the minimum structural unit required for cellular transformation. To understand the structure-function relationship of the DH domain, we have investigated the role of specific residues of the DH domain of Dbl in interaction with Rho GTPases and in Dbl-induced transformation. Alanine substitution mutagenesis identified a panel of DH mutants made in the alpha1, alpha6, and alpha9 regions and the PH junction site that suffer complete or partial loss of GEF activity toward Cdc42 and RhoA. Kinetic and binding analysis of these mutants revealed that although most displayed decreased k(cat) values in the GEF reaction, the substrate binding activities of T506A and R634A were significantly reduced. E502A, Q633A, and N673A/D674A, on the other hand, retained the binding capability to the Rho GTPases but lost the GEF catalytic activity. In general, the in vitro GEF activity of the DH mutants correlated with the in vivo Cdc42- and RhoA-activating potential, and the GEF catalytic efficiency mirrored the transforming activity in NIH 3T3 cells. Moreover, the N673A/D674A mutant exhibited a potent dominant-negative effect on serum-induced cell growth and caused retraction of actin structures. These studies identify important sites of the DH domain involved in binding or catalysis of Rho proteins and demonstrate that maintaining a threshold of GEF catalytic activity, in addition to the Rho GTPase binding activity, is essential for efficient transformation by oncogenic Dbl.     

A minimal Rac activation domain in the unconventional guanine nucleotide exchange factor Dock180

Guanine nucleotide exchange factors (GEFs) activate Rho GTPases by catalyzing the exchange of bound GDP for GTP, thereby resulting in downstream effector recognition. Two metazoan families of GEFs have been described: Dbl-GEF family members that share conserved Dbl homology (DH) and Pleckstrin homology (PH) domains and the more recently described Dock180 family members that share little sequence homology with the Dbl family and are characterized by conserved Dock homology regions 1 and 2 (DHR-1 and -2, respectively). While extensive characterization of the Dbl family has been performed, less is known about how Dock180 family members act as GEFs, with only a single X-ray structure having recently been reported for the Dock9-Cdc42 complex. To learn more about the mechanisms used by the founding member of the family, Dock180, to act as a Rac-specific GEF, we set out to identify and characterize its limit functional GEF domain. A C-terminal portion of the DHR-2 domain, composed of approximately 300 residues (designated as Dock180(DHR-2c)), is shown to be necessary and sufficient for robust Rac-specific GEF activity both in vitro and in vivo. We further show that Dock180(DHR-2c) binds to Rac in a manner distinct from that of Rac-GEFs of the Dbl family. Specifically, Ala(27) and Trp(56) of Rac appear to provide a bipartite binding site for the specific recognition of Dock180(DHR-2c), whereas for Dbl family Rac-GEFs, Trp(56) of Rac is the sole primary determinant of GEF specificity. On the basis of our findings, we are able to define the core of Dock180 responsible for its Rac-GEF activity as well as highlight key recognition sites that distinguish different Dock180 family members and determine their corresponding GTPase specificities.     

Signaling to the Rho GTPases: networking with the DH domain

The Dbl homology (DH) domain was first identified in the Dbl oncogene product as the limit region required for mediating guanine nucleotide exchange on the Rho family GTPase Cdc42. Since the initial biochemical characterization of the DH domain, this conserved motif has been identified in a large family of proteins. In each case, a pleckstrin homology (PH) domain immediately follows the DH domain and this tandem DH-PH module is the signature motif of the Dbl family of guanine nucleotide exchange factors (GEFs). Recent structural studies have provided significant insight into the molecular basis of guanine nucleotide exchange by Dbl family GEFs, opening the door for understanding the specificity of the DH/GTPase interaction as well as providing a starting point for understanding how the exchange activity of these proteins is modulated to achieve specific biological outcomes in the cell.