The Dbs PH domain contributes independently to membrane targeting and regulation of guanine nucleotide-exchange activity.
ABSTRACT: Dbl family GEFs (guanine nucleotide-exchange factors) for the Rho GTPases almost invariably contain a PH (pleckstrin homology) domain adjacent to their DH (Dbl homology) domain. The DH domain is responsible for GEF activity, and the PH domain plays a regulatory role that remains poorly understood. We demonstrated previously that Dbl family PH domains bind phosphoinositides with low affinity and cannot function as independent membrane targeting modules. In the present study, we show that dimerization of a Dbs (Dbl's big sister) DH/PH domain fragment is sufficient to drive it to the plasma membrane through a mechanism involving PH domain-phosphoinositide interactions. Thus, the Dbs PH domain could play a significant role in membrane targeting if it co-operates with other domains in the protein. We also show that mutations that prevent phosphoinositide binding by the Dbs PH domain significantly impair cellular GEF activity even in chimaeric proteins that are robustly membrane targeted by farnesylation or by the PH domain of phospholipase C-delta1. This finding argues that the Dbs PH domain plays a regulatory role that is independent of its ability to aid membrane targeting. Thus, we suggest that the PH domain plays dual roles, contributing independently to membrane localization of Dbs (as part of a multi-domain interaction) and allosteric regulation of the DH domain.
Project description: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.
Project description: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.
Project description: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.
Project description:The RhoGEF (Rho GTPase guanine-nucleotide-exchange factor) domain of AKAP-Lbc (A-kinase-anchoring protein-Lbc, also known as AKAP13) catalyses nucleotide exchange on RhoA and is involved in the development of cardiac hypertrophy. The RhoGEF activity of AKAP-Lbc has also been implicated in cancer. We have determined the X-ray crystal structure of the complex between RhoA-GDP and the AKAP-Lbc RhoGEF [DH (Dbl-homologous)-PH (pleckstrin homology)] domain to 2.1 Å (1 Å = 0.1 nm) resolution. The structure reveals important differences compared with related RhoGEF proteins such as leukaemia-associated RhoGEF. Nucleotide-exchange assays comparing the activity of the DH-PH domain to the DH domain alone showed no role for the PH domain in nucleotide exchange, which is explained by the RhoA-AKAP-Lbc structure. Comparison with a structure of the isolated AKAP-Lbc DH domain revealed a change in conformation of the N-terminal 'GEF switch' region upon binding to RhoA. Isothermal titration calorimetry showed that AKAP-Lbc has only micromolar affinity for RhoA, which combined with the presence of potential binding pockets for small molecules on AKAP-Lbc, raises the possibility of targeting AKAP-Lbc with GEF inhibitors.
Project description:The small GTPase RhoA promotes deregulated signaling upon interaction with lymphoid blast crisis (Lbc), the oncogenic form of A-kinase anchoring protein 13 (AKAP13). The onco-Lbc protein is a hyperactive Rho-specific guanine nucleotide exchange factor (GEF), but its structural mechanism has not been reported despite its involvement in cardiac hypertrophy and cancer causation. The pleckstrin homology (PH) domain of Lbc is located at the C-terminal end of the protein and is shown here to specifically recognize activated RhoA rather than lipids. The isolated dbl homology (DH) domain can function as an independent activator with an enhanced activity. However, the DH domain normally does not act as a solitary Lbc interface with RhoA-GDP. Instead it is negatively controlled by the PH domain. In particular, the DH helical bundle is coupled to the structurally dependent PH domain through a helical linker, which reduces its activity. Together the two domains form a rigid scaffold in solution as evidenced by small angle x-ray scattering and (1)H,(13)C,(15)N-based NMR spectroscopy. The two domains assume a "chair" shape with its back possessing independent GEF activity and the PH domain providing a broad seat for RhoA-GTP docking rather than membrane recognition. This provides structural and dynamical insights into how DH and PH domains work together in solution to support regulated RhoA activity. Mutational analysis supports the bifunctional PH domain mediation of DH-RhoA interactions and explains why the tandem domain is required for controlled GEF signaling.
Project description: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.
Project description:Xpln is a guanine nucleotide-exchange factor (GEF) for Rho GTPases. A Dbl homology (DH) domain followed by a pleckstrin homology (PH) domain is a widely adopted GEF-domain architecture. The Xpln structure solely comprises these two domains. Xpln activates RhoA and RhoB, but not RhoC, although their GTPase sequences are highly conserved. The molecular mechanism of the selectivity of Xpln for Rho GTPases is still unclear. In this study, the crystal structure of the tandemly arranged DH-PH domains of mouse Xpln, with a single molecule in the asymmetric unit, was determined at 1.79?Å resolution by the multiwavelength anomalous dispersion method. The DH-PH domains of Xpln share high structural similarity with those from neuroepithelial cell-transforming gene 1 protein, PDZ-RhoGEF, leukaemia-associated RhoGEF and intersectins 1 and 2. The crystal structure indicated that the ?4-?5 loop in the DH domain is flexible and that the DH and PH domains interact with each other intramolecularly, thus suggesting that PH-domain rearrangement occurs upon RhoA binding.
Project description:p115-RhoGEF (p115) belongs to the family of RGS-containing guanine nucleotide exchange factors for Rho GTPases (RGS-RhoGEFs) that are activated by G12 class heterotrimeric G protein ? subunits. All RGS-RhoGEFs possess tandemly linked Dbl-homology (DH) and plekstrin-homology (PH) domains, which bind and catalyze the exchange of GDP for GTP on RhoA. We have identified that the linker region connecting the N-terminal RGS-homology (RH) domain and the DH domain inhibits the intrinsic guanine nucleotide exchange (GEF) activity of p115, and determined the crystal structures of the DH/PH domains in the presence or absence of the inhibitory linker region. An N-terminal extension of the canonical DH domain (the GEF switch), which is critical to GEF activity, is well folded in the crystal structure of DH/PH alone, but becomes disordered in the presence of the linker region. The linker region is completely disordered in the crystal structure and partially disordered in the molecular envelope calculated from measurements of small angle x-ray scattering (SAXS). It is possible that G? subunits activate p115 in part by relieving autoinhibition imposed by the linker region.
Project description:The guanine nucleotide exchange factor (GEF) Vav1 plays an important role in T-cell activation and tumorigenesis. In the GEF superfamily, Vav1 has the ability to interact with multiple families of Rho GTPases. The structure of the Vav1 DH-PH-CRD/Rac1 complex to 2.6 A resolution reveals a unique intramolecular network of contacts between the Vav1 cysteine-rich domain (CRD) and the C-terminal helix of the Vav1 Dbl homology (DH) domain. These unique interactions stabilize the Vav1 DH domain for its intimate association with the Switch II region of Rac1 that is critical for the displacement of the guanine nucleotide. Small angle x-ray scattering (SAXS) studies support this domain arrangement for the complex in solution. Further, mutational analyses confirms that the atypical CRD is critical for maintaining both optimal guanine nucleotide exchange activity and broader specificity of Vav family GEFs. Taken together, the data outline the detailed nature of Vav1's ability to contact a range of Rho GTPases using a novel protein-protein interaction network.
Project description: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.