Oligomerization of DH domain is essential for Dbl-induced transformation

The dbl oncogene product (onco-Dbl) is the prototype member of a family of guanine nucleotide exchange factors (GEFs) for Rho GTPases. The Dbl homology (DH) domain of onco-Dbl is responsible for the GEF catalytic activity, and the DH domain, together with the immediately adjacent pleckstrin homology (PH) domain, constitutes the minimum module bearing transforming function. In the present study, we demonstrate that the onco-Dbl protein exists in oligomeric form in vitro and in cells. The oligomerization is mostly homophilic in nature and is mediated by the DH domain. Mutagenesis studies mapped the region involved in oligomerization to the conserved region 2 of the DH domain, which is located at the opposite side of the Rho GTPase interacting surface. Residue His556 of this region, in particular, is important for this activity, since the H556A mutant retained the GEF catalytic capability and the binding activity toward Cdc42 and RhoA in vitro but was deficient in oligomer formation. Consequently, the Rho GTPase activating potential of the H556A mutant was significantly reduced in cells. The focus-forming and anchorage-independent growth activities of onco-Dbl were completely abolished by the His556-to-Ala mutation, whereas the abilities to stimulate cell growth, activate Jun N-terminal kinase, and cause actin cytoskeletal changes were retained by the mutant. The ability of onco-Dbl to oligomerize allowed multiple Rho GTPases to be recruited to the same signaling complex, and such an ability is defective in the H556A mutant. Taken together, these results suggest that oligomerization of onco-Dbl through the DH domain is essential for cellular transformation by providing the means to generate a signaling complex that further augments and/or coordinates its Rho GTPase activating potential.     

Requirement for C-terminal sequences in regulation of Ect2 guanine nucleotide exchange specificity and transformation

Ect2 was identified originally as a transforming protein and a member of the Dbl family of Rho guanine nucleotide exchange factors (GEFs). Like all Dbl family proteins, Ect2 contains a tandem Dbl homology (DH) and pleckstrin homology (PH) domain structure. Previous studies demonstrated that N-terminal deletion of sequences upstream of the DH domain created a constitutively activated, transforming variant of Ect2 (designated DeltaN-Ect2 DH/PH/C), indicating that the N terminus served as a negative regulator of DH domain function in vivo. The role of sequences C-terminal to the DH domain has not been established. Therefore, we assessed the consequences of mutation of C-terminal sequences on Ect2-transforming activity. Surprisingly, in contrast to observations with other Dbl family proteins, we found that mutation of the invariant tryptophan residue in the PH domain did not impair DeltaN-Ect2 DH/PH/C transforming activity. Furthermore, although the sequences C-terminal to the PH domain lack any known functional domains or motifs, deletion of these sequences (DeltaN-Ect2 DH/PH) resulted in a dramatic reduction in transforming activity. Whereas DeltaN-Ect2 caused formation of lamellipodia, DeltaN-Ect2 DH/PH enhanced actin stress fiber formation, suggesting that C-terminal sequences influenced Ect2 Rho GTPase specificity. Consistent with this possibility, we determined that DeltaN-Ect2 DH/PH activated RhoA, but not Rac1 or Cdc42, whereas DeltaN-Ect2 DH/PH/C activated all three Rho GTPases in vivo. Taken together, these observations suggest that regions of Ect2 C-terminal to the DH domain alter the profile of Rho GTPases activated in vivo and consequently may contribute to the enhanced transforming activity of DeltaN-Ect2 DH/PH/C.     

Regulation of the Dbl proto-oncogene by heat shock cognate protein 70 (Hsc70)

The dbl oncogene product is the defining member of a family of onco-proteins known as Dbl guanine nucleotide exchange factors (GEFs) that facilitate the activation of the small GTP-binding proteins Cdc42, Rac, and Rho. Oncogenic activation of proto-Dbl occurs through loss of the amino-terminal 497 residues, rendering the protein constitutively active. Because both onco- and proto-Dbl contain the structural elements required for GEF activity (i.e. the Dbl homology (DH) and pleckstrin homology (PH) domains), it is thought that the amino terminus of proto-Dbl somehow inhibits the biochemical activity of the protein. To better understand the molecular basis of this regulation, we set forth to identify cellular proteins that preferentially bind the proto-oncogenic form of Dbl. We identified the molecular chaperone heat shock cognate protein (Hsc70) as a binding partner that preferentially interacts with the proto-oncogenic form of Dbl. Dbl is complexed with Hsc70 in transfected cells, as well as in native mouse brain extracts. The interaction between Hsc70 and proto-Dbl is mediated by at least two regions in Dbl, the aminoterminal spectrin homology domain (residues 224-417) and the pleckstrin homology domain (residues 711-808). Overexpression of a dominant negative Hsc70 mutant leads to activation of proto-Dbl GEF activity, indicating that the chaperone negatively regulates proto-Dbl function in vivo. We propose that Hsc70 attenuates Dbl activity by maintaining an inactive conformation in which the amino terminus is "folded over" the catalytic DH-PH domain.     

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.     

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.     

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.     

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.     

The Dbs PH domain contributes independently to membrane targeting and regulation of guanine nucleotide-exchange activity

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.     

Critical role of the pleckstrin homology and cysteine-rich domains in Vav signaling and transforming activity

Vav family proteins are members of the Dbl family of guanine nucleotide exchange factors and activators of Rho family small GTPases. In addition to the Dbl homology (DH) domain important for guanine nucleotide exchange factor catalytic function, all Dbl family proteins contain an adjacent pleckstrin homology (PH) domain that serves to regulate DH domain activity. Although the role of the PH domain in Vav function has been evaluated extensively, its precise role and whether it serves a distinct role in different Vav proteins remain unresolved. Additionally, the precise role of an adjacent cysteine-rich domain (CRD) in regulating DH domain function is also unclear. In this study, we evaluated the contribution of these putative protein-protein or protein-lipid interaction domains to Vav signaling and transforming activity. In contrast to previous observations, we found that the PH domain is critical for Vav transforming activity. Similarly, the CRD was also essential and served a function distinct from that of the PH domain. Although mutation of either domain reduced Vav membrane association, addition of plasma membrane targeting sequences to either the CRD or PH domain mutant proteins did not restore Vav transforming activity. This result contrasts with other Dbl family proteins, where a membrane targeting sequence alone was sufficient to restore the loss of function caused by mutation of the PH domain. Furthermore, green fluorescent protein fusion proteins containing the PH domain or CRD, or both, failed to target to the plasma membrane, suggesting that these two domains also serve regulatory functions independent of promoting membrane localization. Finally, we found that phosphatidylinositol 3-kinase activation may promote Vav membrane association via phosphatidylinositol 3,4,5-triphosphate binding to the PH domain.     

Inhibition of PI3K induces Rac Activation and Membrane Ruffling in Proto-Dbl Expressing Cells

Proto-Dbl protein, a guanine nucleotide exchange factor (GEF) for Rho GTPases, is tightly regulated by a combination of mechanisms that involve intra- and intermolecular interaction and N- and C-terminal domain-dependent turnover of the protein. Moreover, the interaction of the PH domain of proto-Dbl with phosphoinositides regulates its subcellular localization and biological activity. Here we show that inhibition of the phosphatidylinositol 3-kinase (PI3K) by molecular and pharmacological inhibitors causes a strong inhibition of proto-Dbl-induced cell proliferation and transformation. Conversely, inhibition of PI3K results in the translocation of proto-Dbl to the plasma membrane, Rac activation and increased membrane ruffles and cell motility. Furthermore, we investigated the signaling molecules involved in proto-Dbl-induced cell transformation and motility and observed that inhibition of PI3K in proto-Dbl expressing cells induces an increase in p38 activity and a decrease in ERK phosphorylation. Our results suggest that proto-Dbl activates distinct downstream effectors to induce morphological changes and cell transformation.     

Structural Insights into the Activation of the RhoA GTPase by the Lymphoid Blast Crisis (Lbc) Oncoprotein

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 ¹H,¹³C,¹⁵N-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.     

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.     

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.     

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.     

Modulation of a GEF switch: autoinhibition of the intrinsic guanine nucleotide exchange activity of p115-RhoGEF

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 α 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 Gα subunits activate p115 in part by relieving autoinhibition imposed by the linker region.     

Critical role of the pleckstrin homology domain in Dbs signaling and growth regulation

Dbl family proteins act as guanine nucleotide exchange factors and positive regulators of Rho GTPase function by stimulating formation of the active, GTP-bound state. All Dbl family Rho guanine nucleotide exchange factors possess an invariant tandem domain structure consisting of a Dbl homology (DH) catalytic domain followed by a pleckstrin homology (PH) regulatory domain. We determined previously that the PH domain of Dbs was critical for the intrinsic catalytic activity of the DH domain in vitro and for Dbs transformation in vivo. In this study, we evaluated the role of phosphoinositide binding to the PH domain in regulating the DH domain function of Dbs in vitro and in vivo. We determined that mutation of basic amino acids located within the beta1-beta2 and beta3-beta4 loops of the PH domain resulted in impaired phospholipid binding in vitro, yet full guanine nucleotide exchange activity in vitro was retained for RhoA and Cdc42. Surprisingly, these mutants were compromised in their ability to activate Rho GTPases in vivo and to cause transformation of NIH 3T3 cells. However, Dbs subcellular localization was impaired by these PH domain mutations, supporting a role for phospholipid interactions in facilitating membrane association. Despite the importance of phospholipid binding for Dbs function in vivo, we found that Dbs signaling and transforming activity was not stimulated by phosphatidylinositol 3-kinase activation. We suggest that the PH domain of Dbs facilitates two distinct roles in the regulation of DH domain function, one critical for GTPase association and activation in vitro and one critical for phosphoinositide binding and GTPase interaction in vivo, that together promote Dbs association with membranes.     

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.     

Identification of a Novel Actin-Binding Domain within the Rho Guanine Nucleotide Exchange Factor TEM4

Spatio-temporal activation of Rho GTPases is essential for their function in a variety of biological processes and is achieved in part by regulating the localization of their activators, the Rho guanine nucleotide exchange factors (RhoGEFs). In this study, we provide the first characterization of the full-length protein encoded by RhoGEF TEM4 and delineate its domain structure, catalytic activity, and subcellular localization. First, we determined that TEM4 can stimulate guanine nucleotide exchange on RhoA and the related RhoB and RhoC isoforms. Second, we determined that TEM4, like other Dbl RhoGEFs, contains a functional pleckstrin homology (PH) domain immediately C-terminal to the catalytic Dbl homology (DH) domain. Third, using immunofluorescence analysis, we showed that TEM4 localizes to the actin cytoskeleton through sequences in the N-terminus of TEM4 independently of the DH/PH domains. Using site-directed mutagenesis and deletion analysis, we identified a minimal region between residues 81 and 135 that binds directly to F-actin and has an ～90-fold higher affinity for ATP-loaded F-actin. Finally, we demonstrated that a single point mutation (R130D) within full-length TEM4 abolishes actin binding and localization of TEM4 to the actin cytoskeleton, as well as dampens the in vivo activity of TEM4 towards RhoC. Taken together, our data demonstrate that TEM4 contains a novel actin binding domain and binding to actin is essential for TEM4 subcellular localization and activity. The unique subcellular localization of TEM4 suggests a spatially-restricted activity and expands the diversity of mechanisms by which RhoGEF function can be regulated.