Catalytic Dbl Homology Domain Research Articles

RhoGEF Tiam2 Regulates Glutamatergic Synaptic Transmission in Hippocampal CA1 Pyramidal Neurons.

Glutamatergic synapses exhibit significant molecular diversity, but circuit-specific mechanisms that underlie synaptic regulation are not well characterized. Prior reports show that Rho-guanine nucleotide exchange factor (RhoGEF) Tiam1 regulates perforant path→dentate gyrus granule neuron synapses. In the present study, we report Tiam1's homolog Tiam2 is implicated in glutamatergic neurotransmission in CA1 pyramidal neurons. We find that Tiam2 regulates evoked excitatory glutamatergic currents via a postsynaptic mechanism mediated by the catalytic Dbl-homology domain. Overall, we present evidence for RhoGEF Tiam2's role in glutamatergic synapse function at Schaffer collateral→CA1 pyramidal neuron synapses.

New insights into regulation of P‐Rex1 autoinhibition

Phosphatidylinositol 3,4,5‐trisphosphate (PIP3)–dependent Rac exchanger 1 (P‐Rex1) is a guanine nucleotide exchange factor for Rho GTPases downstream of G protein coupled receptors (GPCRs) and tyrosine kinases. It is primarily expressed in hematopoietic cells and brain. Its ectopic expression is reported in different cancers including breast, prostrate, skin, and ovarian cancers, making it a potential therapeutic target. P‐Rex1 is a multi‐domain protein consisting of a catalytic Dbl‐homology (DH) domain, followed by a PH, two DEP, two PDZ, and an inositolpolyphosphate‐4 phosphatase‐like (IP4P) domain. P‐Rex1 is regulated by PIP3, Gbg binding and phosphorylation via PKC and PKA. In unstimulated cells, P‐Rex1 is autoinhibited by intradomain interactions with its catalytic DH domain. Previously, our lab had shown that first DEP (DEP1) domain plays a key role in inhibition of DH/PH‐DEP1 exchange activity. However, there is no information available as how these domains are engaged in regulation of full length P‐Rex1. In our current study, we addressed this gap by employing single particle cryo‐electron microscopy (cryo‐EM) complemented by SAXS, HDX‐MS, and functional enzymatic assays to understand P‐Rex1 regulation. A structure with the head group of PIP3 (IP4) bound to the PH domain revealed interactions between DH and DEP1 domain that would block GTPase access to DH domain by coercing a novel configuration of the DH‐PH module. In addition, we observed that the IP4P domain was anchored to PH domain via a previously unrecognized FAT domain. Mutagenesis of residues involved in the DH–DEP1 interface led to activation, suggestive of inhibitory role of this interface. IP4 is a soluble second messenger signaling molecule which is reported to compete with PIP3 binding site in other proteins. Therefore, we tested whether it could regulate P‐Rex1 in a similar manner and found that IP4 competes with PIP3 and negatively regulates P‐Rex1 activity. Based on these observations, we propose that in unstimulated cell, P‐Rex1 is autoinhibited by intradomain interactions between the DEP1 and DH and between the FAT and PH domains, and that inositol phosphate levels may add an additional layer of desensitization by opposing P‐Rex1 membrane translocation and favoring a closed autoinhibited state.

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.

Insights into the Molecular Activation Mechanism of the RhoA-specific Guanine Nucleotide Exchange Factor, PDZRhoGEF

PDZRhoGEF (PRG) belongs to a small family of RhoA-specific nucleotide exchange factors that mediates signaling through select G-protein-coupled receptors via Gα(12/13) and activates RhoA by catalyzing the exchange of GDP to GTP. PRG is a multidomain protein composed of PDZ, regulators of G-protein signaling-like (RGSL), Dbl-homology (DH), and pleckstrin-homology (PH) domains. It is autoinhibited in cytosol and is believed to undergo a conformational rearrangement and translocation to the membrane for full activation, although the molecular details of the regulation mechanism are not clear. It has been shown recently that the main autoregulatory elements of PDZRhoGEF, the autoinhibitory "activation box" and the "GEF switch," which is required for full activation, are located directly upstream of the catalytic DH domain and its RhoA binding surface, emphasizing the functional role of the RGSL-DH linker. Here, using a combination of biophysical and biochemical methods, we show that the mechanism of PRG regulation is yet more complex and may involve an additional autoinhibitory element in the form of a molten globule region within the linker between RGSL and DH domains. We propose a novel, two-tier model of autoinhibition where the activation box and the molten globule region act synergistically to impair the ability of RhoA to bind to the catalytic DH-PH tandem. The molten globule region and the activation box become less ordered in the PRG-RhoA complex and dissociate from the RhoA-binding site, which may constitute a critical step leading to PRG activation.

The Minimal Autoinhibited Unit of the Guanine Nucleotide Exchange Factor Intersectin

Intersectin-1L is a member of the Dbl homology (DH) domain guanine nucleotide exchange factors (GEF) which control Rho-family GTPase signaling. Intersectin-1L is a GEF that is specific for Cdc42. It plays an important role in endocytosis, and is regulated by several partners including the actin regulator N-WASP. Intact intersectin-1L shows low Cdc42 exchange activity, although the isolated catalytic DH domain shows high activity. This finding suggests that the molecule is autoinhibited. To investigate the mechanism of autoinhibition we have constructed a series of domain deletions. We find that the five SH3 domains of intersectin are important for autoinhibition, with the fifth domain (SH3(E)) being sufficient for the bulk of the autoinhibitory effect. This SH3 domain appears to primarily interact with the DH domain. We have determined the crystal structure of the SH3(E)-DH domain construct, which shows a domain swapped arrangement in which the SH3 from one monomer interacts with the DH domain of the other monomer. Analytical ultracentrifugation and gel filtration, however, show that under biochemical concentrations, the construct is fully monomeric. Thus we propose that the actual autoinhibited structure contains the related intramolecular SH3(E)-DH interaction. We propose a model in which this intramolecular interaction may block or distort the GTPase binding region of the DH domain.

Oligomerization Regulates the Localization of Cdc24, the Cdc42 Activator in Saccharomyces cerevisiae

Guanine nucleotide exchange factor activation of Rho G-proteins is critical for cytoskeletal reorganization. In the yeast Saccharomyces cerevisiae, the sole guanine nucleotide exchange factor for the Rho G-protein Cdc42p, Cdc24p, is essential for its site-specific activation. Several mammalian exchange factors have been shown to oligomerize; however, the function of this homotypic interaction is unclear. Here we show that Cdc24p forms oligomers in yeast via its catalytic Dbl homology domain. Mutation of residues critical for Cdc24p oligomerization also perturbs the localization of this exchange factor yet does not alter its catalytic activity in vitro. Chemically induced oligomerization of one of these oligomerization-defective mutants partially restored its localization to the bud tip and nucleus. Furthermore, chemically induced oligomerization of wild-type Cdc24p does not affect in vitro exchange factor activity, yet it results in a decrease of activated Cdc42p in vivo and the presence of Cdc24p in the nucleus at all cell cycle stages. Together, our results suggest that Cdc24p oligomerization regulates Cdc42p activation via its localization.

Vav1 Acidic Region Tyrosine 174 Is Required for the Formation of T Cell Receptor-induced Microclusters and Is Essential in T Cell Development and Activation

Vav proteins are multidomain signaling molecules critical for mediating signals downstream of several surface receptors, including the antigen receptors of T and B lymphocytes. The catalytic guanine nucleotide exchange factor (GEF) activity of the Vav Dbl homology (DH) domain is thought to be controlled by an intramolecular autoinhibitory mechanism involving an N-terminal extension and phosphorylation of tyrosine residues in the acidic region (AC). Here, we report that the sequences surrounding the Vav1 AC: Tyr(142), Tyr(160), and Tyr(174) are evolutionarily conserved, conform to consensus SH2 domain binding motifs, and bind several proteins implicated in TCR signaling, including Lck, PI3K p85alpha, and PLCgamma1, through direct interactions with their SH2 domains. In addition, the AC tyrosines regulate tyrosine phosphorylation of Vav1. We also show that Tyr(174) is required for the maintenance of TCR-signaling microclusters and for normal T cell development and activation. In this regard, our data demonstrate that while Vav1 Tyr(174) is essential for maintaining the inhibitory constraint of the DH domain in both developing and mature T cells, constitutively activated Vav GEF disrupts TCR-signaling microclusters and leads to defective T cell development and proliferation.

Acidic Region Tyrosines Provide Access Points for Allosteric Activation of the Autoinhibited Vav1 Dbl Homology Domain

Autoinhibited proteins serve key roles in many signal transduction pathways, and therefore proper regulation of these proteins is critical for normal cellular function. Proto-oncogene Vav1 is an autoinhibited guanine nucleotide exchange factor (GEF) for Rho family GTPases. The core autoinhibitory module of Vav1 consists of the catalytic Dbl homology (DH) domain bound through its active site to an alpha helix centered about Tyr174 in the Acidic (Ac) region of the protein. Phosphorylation of Tyr174 and two other tyrosines in the Ac region, Tyr142 and Tyr160, relieves autoinhibition and activates the catalytic DH domain. In this study, we use biochemical and structural analyses of the Vav1 Ac and DH domains to examine the kinetic and thermodynamic properties of Vav1 activation by the Src family kinase, Lck, and the role of the Lck SH2 domain in this process. We find that in the Ac-DH fragment of Vav1, Tyr174, but not Tyr142 or Tyr160, is protected from phosphorylation by interactions with the DH domain. Binding of the Lck SH2 domain to phosphorylated Tyr142 increases kcat/KM for Tyr174 by 4-fold, likely because the kinase domain can act on the substrate effectively in an intramolecular fashion. These studies of the autoinhibited Ac-DH module provide the foundation for a quantitative structural and thermodynamic understanding of the regulation of full length Vav1. Moreover, kinetic pathways involving initial interactions with exposed sites or "access points", as observed here for Vav1, may be generally important in the regulation of many autoinhibited proteins.

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.

β3 Tyrosine Phosphorylation and αvβ3-mediated Adhesion Are Required for Vav1 Association and Rho Activation in Leukocytes

Integrin alpha(v)beta(3)-mediated adhesion of hematopoietic cells to vitronectin results in activation of the Rho GTPases. Mutation of beta(3) tyrosine residue 747, previously shown to disrupt cell adhesion, results in sustained activation of Cdc42 and diminished Rac and Rho activity. We investigated the role of the hematopoietically restricted guanine nucleotide exchange factor Vav1 in alpha(v)beta(3)-mediated adhesion. We find that Vav1, a guanine nucleotide exchange factor for Rac and Rho, associates with alpha(v)beta(3) upon cell adhesion to vitronectin and that this association requires beta(3) tyrosine phosphorylation. Expression of exogenous Vav1 demonstrates that Y160F, but not wild type or the Vav1Y174F mutant, inhibits Rac and Rho activation during alpha(v)beta(3)-mediated cell adhesion to vitronectin. Cells expressing Vav1Y160F exhibit a sustained Cdc42 activation similar to nonphosphorylatable beta(3) mutants. In addition, cytoskeletal reorganization and cell adhesion are severely suppressed in Vav1Y160F-transfected cells, and Vav1Y160F fails to associate with beta(3) integrins. Furthermore, Vav1 itself is selectively phosphorylated upon tyrosine 160 after alpha(v)beta(3)-mediated adhesion, and the association between Vav1 and beta(3) occurs in specific response to adhesion to substrate. These studies describe a phosphorylation-dependent association between beta(3) integrin and Vav1 which is essential for cell progression to a Rho-dominant phenotype during cell adhesion.

Regulation of P-Rex1 by Phosphatidylinositol (3,4,5)-Trisphosphate and Gβγ Subunits

P-Rex1 is a guanine-nucleotide exchange factor (GEF) for the small GTPase Rac. We have investigated here the mechanisms of stimulation of P-Rex1 Rac-GEF activity by the lipid second messenger phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P3) and the Gbetagamma subunits of heterotrimeric G proteins. We show that a P-Rex1 mutant lacking the PH domain (DeltaPH) cannot be stimulated by PtdIns(3,4,5)P3, which implies that the PH domain confers PtdIns(3,4,5)P3 regulation of P-Rex1 Rac-GEF activity. Consistent with this, we found that PtdIns(3,4,5)P3 binds to the PH domain of P-Rex1 and that the DH/PH domain tandem is sufficient for PtdIns(3,4,5)P3-stimulated P-Rex1 activity. The Rac-GEF activities of the DeltaPH mutant and the DH/PH domain tandem can both be stimulated by Gbetagamma subunits, which infers that Gbetagamma subunits regulate P-Rex1 activity by binding to the catalytic DH domain. Deletion of the DEP, PDZ, or inositol polyphosphate 4-phosphatase homology domains has no major consequences on the abilities of either PtdIns(3,4,5)P3 or Gbetagamma subunits to stimulate P-Rex1 Rac-GEF activity. However, the presence of any of these domains impacts on the levels of basal and/or stimulated P-Rex1 Rac-GEF activity, suggesting that there are important functional interactions between the DH/PH domain tandem and the DEP, PDZ, and inositol polyphosphate 4-phosphatase homology domains of P-Rex1.

Autoinhibition mechanism of proto-Dbl.

The dbl oncogene encodes a prototype member of the Rho GTPase guanine nucleotide exchange factor (GEF) family. Oncogenic activation of proto-Dbl occurs through truncation of the N-terminal 497 residues. The C-terminal half of proto-Dbl includes residues 498 to 680 and 710 to 815, which fold into the Dbl homology (DH) domain and the pleckstrin homology (PH) domain, respectively, both of which are essential for cell transformation via the Rho GEF activity or cytoskeletal targeting function. Here we have investigated the mechanism of the apparent negative regulation of proto-Dbl imposed by the N-terminal sequences. Deletion of the N-terminal 285 or C-terminal 100 residues of proto-Dbl did not significantly affect either its transforming activity or GEF activity, while removal of the N-terminal 348 amino acids resulted in a significant increase in both transformation and GEF potential. Proto-Dbl displayed a mostly perinuclear distribution pattern, similar to a polypeptide derived from its N-terminal sequences, whereas onco-Dbl colocalized with actin stress fibers, like the PH domain. Coexpression of the N-terminal 482 residues with onco-Dbl resulted in disruption of its cytoskeletal localization and led to inhibition of onco-Dbl transforming activity. The apparent interference with the DH and PH functions by the N-terminal sequences can be rationalized by the observation that the N-terminal 482 residues or a fragment containing residues 286 to 482 binds specifically to the PH domain, limiting the access of Rho GTPases to the catalytic DH domain and masking the intracellular targeting function of the PH domain. Taken together, our findings unveiled an autoinhibitory mode of regulation of proto-Dbl that is mediated by the intramolecular interaction between its N-terminal sequences and PH domain, directly impacting both the GEF function and intracellular distribution.