Intermediates in the Guanine Nucleotide Exchange Reaction of Rab8 Protein Catalyzed by Guanine Nucleotide Exchange Factors Rabin8 and GRAB

Small G-proteins of the Ras superfamily control the temporal and spatial coordination of intracellular signaling networks by acting as molecular on/off switches. Guanine nucleotide exchange factors (GEFs) regulate the activation of these G-proteins through catalytic replacement of GDP by GTP. During nucleotide exchange, three distinct substrate·enzyme complexes occur: a ternary complex with GDP at the start of the reaction (G-protein·GEF·GDP), an intermediary nucleotide-free binary complex (G-protein·GEF), and a ternary GTP complex after productive G-protein activation (G-protein·GEF·GTP). Here, we show structural snapshots of the full nucleotide exchange reaction sequence together with the G-protein substrates and products using Rabin8/GRAB (GEF) and Rab8 (G-protein) as a model system. Together with a thorough enzymatic characterization, our data provide a detailed view into the mechanism of Rabin8/GRAB-mediated nucleotide exchange.     

Multisite Phosphorylation of the Guanine Nucleotide Exchange Factor Cdc24 during Yeast Cell Polarization

Background

Cell polarization is essential for processes such as cell migration and asymmetric cell division. A common regulator of cell polarization in most eukaryotic cells is the conserved Rho GTPase, Cdc42. In budding yeast, Cdc42 is activated by a single guanine nucleotide exchange factor, Cdc24. The mechanistic details of Cdc24 activation at the onset of yeast cell polarization are unclear. Previous studies have suggested an important role for phosphorylation of Cdc24, which may regulate activity or function of the protein, representing a key step in the symmetry breaking process.

Methodology/Principal Findings

Here, we directly ask whether multisite phosphorylation of Cdc24 plays a role in its regulation. We identify through mass spectrometry analysis over thirty putative in vivo phosphorylation sites. We first focus on sites matching consensus sequences for cyclin-dependent and p21-activated kinases, two kinase families that have been previously shown to phosphorylate Cdc24. Through site-directed mutagenesis, yeast genetics, and light and fluorescence microscopy, we show that nonphosphorylatable mutations of these consensus sites do not lead to any detectable consequences on growth rate, morphology, kinetics of polarization, or localization of the mutant protein. We do, however, observe a change in the mobility shift of mutant Cdc24 proteins on SDS-PAGE, suggesting that we have indeed perturbed its phosphorylation. Finally, we show that mutation of all identified phosphorylation sites does not cause observable defects in growth rate or morphology.

Conclusions/Significance

We conclude that lack of phosphorylation on Cdc24 has no overt functional consequences in budding yeast. Yeast cell polarization may be more tightly regulated by inactivation of Cdc42 by GTPase activating proteins or by alternative methods of Cdc24 regulation, such as conformational changes or oligomerization.     

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.     

Dennd3 Functions as a Guanine Nucleotide Exchange Factor for Small GTPase Rab12 in Mouse Embryonic Fibroblasts

Small GTPase Rab12 regulates mTORC1 (mammalian target of rapamycin complex 1) activity and autophagy through controlling PAT4 (proton/amino acid transporter 4) trafficking from recycling endosomes to lysosomes, where PAT4 is degraded. However, the precise regulatory mechanism of the Rab12-mediated membrane trafficking pathway remained to be determined because a physiological Rab12-GEF (guanine nucleotide exchange factor) had yet to be identified. In this study we performed functional analyses of Dennd3, which has recently been shown to possess a GEF activity toward Rab12 in vitro. The results showed that knockdown of Dennd3 in mouse embryonic fibroblast cells caused an increase in the amount of PAT4 protein, the same as Rab12 knockdown did, and knockdown of Dennd3 and overexpression of Dennd3 were found to result in an increase and a decrease, respectively, in the intracellular amino acid concentration. Dennd3 overexpression was also found to reduce mTORC1 activity and promoted autophagy in a Rab12-dependent manner. Unexpectedly, however, Dennd3 knockdown had no effect on mTORC1 activity or autophagy despite increasing the intracellular amino acid concentration. Further study showed that Dennd3 knockdown reduced Akt activity, and the reduction in Akt activity is likely to have canceled out amino acid-induced mTORC1 activation through PAT4. These findings indicated that Dennd3 not only functions as a Rab12-GEF but also modulates Akt signaling in mouse embryonic fibroblast cells.     

The Rho-specific Guanine Nucleotide Exchange Factor Dbs Regulates Breast Cancer Cell Migration

Dbs is a Rho-specific guanine nucleotide exchange factor (RhoGEF) that regulates neurotrophin-3-induced cell migration in Schwann cells. Here we report that Dbs regulates cell motility in tumor-derived, human breast epithelial cells through activation of Cdc42 and Rac1. Cdc42 and Rac1 are activated in T47D cells that stably express onco- or proto-Dbs, and activation is dependent upon growth of the cells on collagen I. Transient suppression of expression of Cdc42 or Rac1 by small interfering RNAs attenuates Dbs-enhanced motility. Both onco- and proto-Dbs-enhanced motility correlates with an increase in tyrosine phosphorylation of focal adhesion kinase on Tyr-397 and p130^Cas on Tyr-410 and an increase in the abundance of the Crk·p130^Cas complex. Suppression of expression of Cdc42 or its effector, Ack1, reduces tyrosine phosphorylation of focal adhesion kinase and p130^Cas and disrupts the Crk·p130^Cas complex. We further determined that suppression of expression of Cdc42, Ack1, p130^Cas, or Crk reduces Rac1 activation and cell motility in Dbs-expressing cells to a level comparable with that in vector cells. Therefore, a cascade of activation of Cdc42 and Rac1 by Dbs through the Cdc42 effector Ack1 and the Crk·p130^Cas complex is established. Suppression of the expression of endogenous Dbs reduces cell motility in both T47D cells and MDA-MB-231 cells, which correlates with the down-regulation of Cdc42 activity. This suggests that Dbs activates Cdc42 in these two human breast cancer cell lines and that the normal function of Dbs may be required to support cell movement.Rho GTPases are a subfamily of the Ras superfamily of small signaling molecules that are widely expressed in mammalian cells (1). RhoA, Cdc42, and Rac1 are the most extensively studied members of the Rho GTPase family, and each plays a prominent and discrete role in cell migration (2, 3). Cdc42 promotes the formation of filopodia and is required to establish cell polarity (3–5); Rac1 promotes the formation of lamellipodia at the leading edge of motile cells (6), and RhoA promotes the formation of stress fibers which generate the traction forces needed to retract the cell tail and move the cell body beyond the leading edge (7, 8). Consistent with this important role in cell motility, RhoA, Cdc42, and Rac1 are often overexpressed in human tumors including breast, lung, and colon (9), and overexpression of constitutively active RhoA, Cdc42, or Rac1 increases cell migration and invasion (2, 10, 11).The spatiotemporal regulation of Rho GTPase activity is tightly controlled by three classes of proteins. Rho-specific guanine nucleotide exchange factors (RhoGEFs)2 activate Rho proteins by facilitating the exchange of GDP for GTP; Rho GTPase-activating proteins (RhoGAPs) stimulate the intrinsic rate of hydrolysis of Rho proteins, thus converting them into their inactive state; Rho-specific guanine nucleotide dissociation inhibitors (RhoGDIs) compete with RhoGEFs for binding to GDP-bound Rho proteins and sequester Rho in the inactive state (12).Dbs was identified in the screen for proteins whose overexpression cause malignant growth in murine fibroblasts (13, 14). The full-length Dbs protein (proto-Dbs) is a RhoGEF family member which contains multiple recognizable domains (Fig. 1A) including a Sec14-like domain, spectrin-like repeats, a RhoGEF domain (includes a DH and PH domain), and an SH3 domain (13). The original oncogenic version of Dbs that was identified (amino acid residues 525–1097; designated onco-Dbs) contains the RhoGEF domain alone. When expressed in murine fibroblasts, the transforming and catalytic activity of Dbs is subject to autoinhibition that is mediated by the NH₂-terminal Sec14 domain (15). Although the endogenous function of Dbs is not known, recent studies suggest that Dbs and the Rac-specific exchange factor Tiam1 regulate neurotrophin-stimulated cell migration in Schwann cells through activation of Cdc42 and Rac1, respectively (16, 17).Open in a separate windowFIGURE 1.Onco-Dbs and proto-Dbs induce cell migration in tumor-derived breast epithelial cells. A, domain structure of the onco-Dbs and proto-Dbs proteins (Sec14 = Sec14-like domain; Spec = Spectrin-like repeats; DH = Dbl homology domain; PH = pleckstrin homology domain; SH3 = Src homology 3 domain). B, stable expression of HA-epitope-tagged onco-Dbs (M_r = 65) and proto-Dbs (M_r = 129 kDa) was confirmed by Western blot using an anti-HA antibody. Three independent sets of cell lines were generated. C, T47D cells stably expressing vector (Vec), onco-Dbs, or proto-Dbs were compared in a transwell motility assay on filters pre-coated with collagen I. The motility of cells stably expressing onco-Dbs or proto-Dbs is expressed relative to that of cells stably expressing vector. Data are represented as the mean ± S.D. of three independent experiments performed in triplicate. D, T47D cells stably expressing vector, onco-Dbs, or proto-Dbs were cultured to monolayer on dishes coated with poly-l-lysine or collagen I, as indicated. Cells were serum-starved overnight, and then the surface of the plate was scraped. Migration of cells at the wound edge was monitored and photographed at 18 h. Representative images are shown. E, growth curves of T47D cells stably expressing vector, onco-Dbs, or proto-Dbs. Cells were cultured in triplicate on poly-l-lysine (filled symbols) or on dishes pre-coated with collagen I (open symbols) and counted on the indicated days. Data shown are representative of three independent experiments.Conversion of Rho proteins to their active GTP-bound state allows them to interact with effector signaling molecules. Ack1 is a nonreceptor-tyrosine kinase that binds to active Cdc42 but not Rac1 or RhoA (18, 19). Activated Ack1 is overexpressed in primary tumors and cancer cell lines and has been implicated in cancer metastasis (20). Recent studies have identified a signaling complex that regulates the motility of human breast epithelial cells that contains Cdc42, Ack1, p130^Cas, and Crk (21). Ack1 and p130^Cas interact through their respective SH3 domains, and Ack1 phosphorylates p130^Cas in a collagen I-dependent manner. p130^Cas was first identified as a hyperphosphorylated adapter protein in cells transformed by v-Src and v-Crk (22, 23). Further studies showed that p130^Cas is associated with both cellular Src and Crk in a tyrosine phosphorylation-dependent manner (24, 25). Focal adhesion kinase (FAK) binds to the NH₂ terminus of p130^Cas and phosphorylates the COOH terminus in a region that is involved in p130^Cas binding to Src (26). The binding of Crk to p130^Cas recruits binding partners to the SH3 domain of Crk, including C3G and DOCK180, which activate Rap1 and Rac1, respectively (27–31). Thus, formation of the Crk·p130^Cas complex is considered to be a molecular switch that can induce cell migration by activating Rac1 (32).Here we show that both proto-Dbs and onco-Dbs increase cell migration in human breast adenocarcinoma cells in a collagen I-dependent manner. Increased motility is dependent upon the activation of Rac1 and Cdc42 and is mediated by the assembly of Crk·p130^Cas complexes. Suppression of endogenous Dbs expression in human tumor-derived breast epithelial cells limits cell motility, suggesting that Dbs may be a critical regulator of cell behavior in breast cancer.     

The Atypical Guanine Nucleotide Exchange Factor Dock4 Regulates Neurite Differentiation through Modulation of Rac1 GTPase and Actin Dynamics

Precise regulation of neurite growth and differentiation determines accurate formation of synaptic connections, whose disruptions are frequently associated with neurological disorders. Dedicator of cytokinesis 4 (Dock4), an atypical guanine nucleotide exchange factor for Rac1, is found to be associated with neuropsychiatric diseases, including autism and schizophrenia. Nonetheless, the neuronal function of Dock4 is only beginning to be understood. Using mouse neuroblastoma (Neuro-2a) cells as a model, this study identifies that Dock4 is critical for neurite differentiation and extension. This regulation is through activation of Rac1 and modulation of the dynamics of actin-enriched protrusions on the neurites. In cultured hippocampal neurons, Dock4 regulates the establishment of the axon-dendrite polarity and the arborization of dendrites, two critical processes during neural differentiation. Importantly, a microdeletion Dock4 mutant linked to autism and dyslexia that lacks the GEF domain leads to defective neurite outgrowth and neuronal polarization. Further analysis reveals that the SH3 domain-mediated interaction of Dock4 is required for its activity toward neurite differentiation, whereas its proline-rich C terminus is not essential for this regulation. Together, our findings reveal an important role of Dock4 for neurite differentiation during early neuronal development.     

The Adenomatous Polyposis Coli-associated Guanine Nucleotide Exchange Factor Asef Is Involved in Angiogenesis

Mutation of the tumor suppressor adenomatous polyposis coli (APC) is a key early event in the development of most colorectal tumors. APC promotes degradation of β-catenin and thereby negatively regulates Wnt signaling, whereas mutated APCs present in colorectal tumor cells are defective in this activity. APC also stimulates the activity of the guanine nucleotide exchange factor Asef and regulates cell morphology and migration. Truncated mutant APCs constitutively activate Asef and induce aberrant migration of colorectal tumor cells. Furthermore, we have recently found that Asef and APC function downstream of hepatocyte growth factor and phosphatidylinositol 3-kinase. We show here that Asef is required for basic fibroblast growth factor- and vascular endothelial growth factor-induced endothelial cell migration. We further demonstrate that Asef is required for basic fibroblast growth factor- and vascular endothelial growth factor-induced microvessel formation. Furthermore, we show that the growth as well as vascularity of subcutaneously implanted tumors are markedly impaired in Asef^−/− mice compared with wild-type mice. Thus, Asef plays a critical role in tumor angiogenesis and may be a promising target for cancer chemotherapy.     

A Novel Guanine Nucleotide Exchange Factor,MYOGEF, is Required for Cytokinesis

The cleavage furrow is created by an actomyosin contractile ring that isregulated by small GTPase proteins such as Rac1 and RhoA. Guanine nucleotideexchange factors (GEFs) are positive regulators of the small GTPase proteins andhave been implicated as important factors in regulating cytokinesis. However, it isstill unclear how GEFs regulate the contractile ring during cytokinesis inmammalian cells. Here we report that a novel GEF, which is termed MyoGEF(myosin-interacting GEF), interacts with nonmuscle myosin II and exhibits activitytoward RhoA. MyoGEF and nonmuscle myosin II colocalize to the cleavage furrowin early anaphase cells. Disruption of MyoGEF expression in U2OS cells by RNAinterference (RNAi) results in the formation of multinucleated cells. These resultssuggest that MyoGEF, RhoA, and nonmuscle myosin II act as a functional unit atthe cleavage furrow to advance furrow ingression during cytokinesis.     

Regulation of Immature Dendritic Cell Migration by RhoA Guanine Nucleotide Exchange Factor Arhgef5

There are a large number of Rho guanine nucleotide exchange factors, most of which have no known functions. Here, we carried out a short hairpin RNA-based functional screen of Rho-GEFs for their roles in leukocyte chemotaxis and identified Arhgef5 as an important factor in chemotaxis of a macrophage phage-like RAW264.7 cell line. Arhgef5 can strongly activate RhoA and RhoB and weakly RhoC and RhoG, but not Rac1, RhoQ, RhoD, or RhoV, in transfected human embryonic kidney 293 cells. In addition, Gβγ interacts with Arhgef5 and can stimulate Arhgef5-mediated activation of RhoA in an in vitro assay. In vivo roles of Arhgef5 were investigated using an Arhgef-5-null mouse line. Arhgef5 deficiency did not affect chemotaxis of mouse macrophages, T and B lymphocytes, and bone marrow-derived mature dendritic cells (DC), but it abrogated MIP1α-induced chemotaxis of immature DCs and impaired migration of DCs from the skin to lymph node. In addition, Arhgef5 deficiency attenuated allergic airway inflammation. Therefore, this study provides new insights into signaling mechanisms for DC migration regulation.Leukocyte chemotaxis underlies leukocyte migration, infiltration, trafficking, and homing that are not only important for normal leukocyte functions, but also have a important role in inflammation-related diseases. Leukocyte chemotaxis is regulated by leukocyte chemoattractants that include bacterial by-products such as formylmethionylleucylphenylalanine, complement proteolytic fragments such as C5a, and the superfamily of chemotactic cytokines, chemokines. These chemoattractants bind to their specific cell G protein-coupled receptors and are primarily coupled to the G_i family of G proteins to regulate leukocyte chemotaxis. Previous studies have established that the Rho family of small GTPases regulates leukocyte migration (1, 2). Rac, Cdc42, and RhoA are the three best studied Rho small GTPases. In myeloid cells, Cdc42 regulates directionality by directing where F-actin and lamellipodia are formed, and Rac regulates F-actin formation in the lamellipodia, which provides a driving force for cell motility (3–6). On the other hand, RhoA regulates the formation and contractility of the actomyosin structure at the back that provides a pushing force (5, 7). Rho guanine nucleotide exchange factors (GEF)³ are key regulators for the activity of these small GTPases. GEFs activate small GTPases by promoting the loading of GTP to the small GTPases, a rate-limiting step in GTPase regulation (8–11). Previous biochemical and genetic studies have revealed how Cdc42 and Rac may be regulated by chemokine receptors in leukocytes. Chemokine receptors can regulate Cdc42 via a Rho-GEF PIXα, which is regulated by Gβγ from the G_i proteins via the interactions between Gβγ and Pak1 and between Pak1 and PIXα in myeloid cells 12. On the other hand, in neutrophils chemokine receptors regulate Rac2 via another Rho-GEF P-Rex1, which is directly regulated by Gβγ (13–15). Two Rho-GEFs have been implicated in regulation of RhoA in neutrophils. GEF115 was found in the leading edges of polarized mouse neutrophils, whereas PDZ Rho-GEF was found in the uropods of differentiated HL-60 cells. Both Rho-GEFs were believed to mediate pertussis toxin-resistant activation of RhoA in these cells. However, a significant portion of RhoA activity in leukocytes are pertussis toxin-sensitive, which is presumably regulated by the α and/or βγ subunits from the G_i proteins. The signaling mechanism for this pertussis toxin-sensitive RhoA regulation by chemokine receptors remains largely elusive.Molecular cloning and genomic sequencing have identified more than 70 Rho-GEFs in mammals (16–20). Many of these Rho-GEFs have been shown to activate RhoA in in vitro and overexpression assays (16–20). However, it is not known if any of them regulate RhoA in vivo, we have found that PIXα is a specific GEF for Cdcd42 in neutrophils (12) despite its potent activity on Rac in in vitro and overexpression assays (21, 22). Therefore, we used a siRNA-based loss of function screen in an attempt to identify the GEFs that regulate myeloid cell migration and RhoA activity. One of the candidates, Arhgef5, was found to be directly activated by Gβγ to regulate RhoA and has an important role in immature DC migration. In addition, Arhgef5 deficiency attenuated allergic airway inflammation in a mouse model.     

Structural Basis for the Dual Substrate Specificity of DOCK7 Guanine Nucleotide Exchange Factor

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Non-Muscle Myosin II Regulates Neuronal Actin Dynamics by Interacting with Guanine Nucleotide Exchange Factors

Background

Non-muscle myosin II (NM II) regulates a wide range of cellular functions, including neuronal differentiation, which requires precise spatio-temporal activation of Rho GTPases. The molecular mechanism underlying the NM II-mediated activation of Rho GTPases is poorly understood. The present study explored the possibility that NM II regulates neuronal differentiation, particularly morphological changes in growth cones and the distal axon, through guanine nucleotide exchange factors (GEFs) of the Dbl family.

Principal Findings

NM II colocalized with GEFs, such as βPIX, kalirin and intersectin, in growth cones. Inactivation of NM II by blebbistatin (BBS) led to the increased formation of short and thick filopodial actin structures at the periphery of growth cones. In line with these observations, FRET analysis revealed enhanced Cdc42 activity in BBS-treated growth cones. BBS treatment also induced aberrant targeting of various GEFs to the distal axon where GEFs were seldom observed under physiological conditions. As a result, numerous protrusions and branches were generated on the shaft of the distal axon. The disruption of the NM II–GEF interactions by overexpression of the DH domains of βPIX or Tiam1, or by βPIX depletion with specific siRNAs inhibited growth cone formation and induced slender axons concomitant with multiple branches in cultured hippocampal neurons. Finally, stimulation with nerve growth factor induced transient dissociation of the NM II–GEF complex, which was closely correlated with the kinetics of Cdc42 and Rac1 activation.

Conclusion

Our results suggest that NM II maintains proper morphology of neuronal growth cones and the distal axon by regulating actin dynamics through the GEF–Rho GTPase signaling pathway.     

Kinetic Analysis of the Guanine Nucleotide Exchange Activity of TRAPP, a Multimeric Ypt1p Exchange Factor

TRAPP complexes, which are large multimeric assemblies that function in membrane traffic, are guanine nucleotide exchange factors (GEFs) that activate the Rab GTPase Ypt1p. Here we measured rate and equilibrium constants that define the interaction of Ypt1p with guanine nucleotide (guanosine 5'-diphosphate and guanosine 5'-triphosphate/guanosine 5′-(β,γ-imido)triphosphate) and the core TRAPP subunits required for GEF activity. These parameters allowed us to identify the kinetic and thermodynamic bases by which TRAPP catalyzes nucleotide exchange from Ypt1p. Nucleotide dissociation from Ypt1p is slow (∼ 10^− 4 s^− 1) and accelerated > 1000-fold by TRAPP. Acceleration of nucleotide exchange by TRAPP occurs via a predominantly Mg²⁺-independent pathway. Thermodynamic linkage analysis indicates that TRAPP weakens nucleotide affinity by < 80-fold and vice versa, in contrast to most other characterized GEF systems that weaken nucleotide binding affinities by 4-6 orders of magnitude. The overall net changes in nucleotide binding affinities are small because TRAPP accelerates both nucleotide binding and dissociation from Ypt1p. Weak thermodynamic coupling allows TRAPP, Ypt1p, and nucleotide to exist as a stable ternary complex, analogous to strain-sensing cytoskeleton motors. These results illustrate a novel strategy of guanine nucleotide exchange by TRAPP that is particularly suited for a multifunctional GEF involved in membrane traffic.     

The PDZ Domain of the Guanine Nucleotide Exchange Factor PDZGEF Directs Binding to Phosphatidic Acid during Brush Border Formation

PDZGEF is a guanine nucleotide exchange factor for the small G protein Rap. It was recently found that PDZGEF contributes to establishment of intestinal epithelial polarity downstream of the kinase Lkb1. By binding to phosphatidic acid enriched at the apical membrane, PDZGEF locally activates Rap2a resulting in induction of brush border formation via a pathway that includes the polarity players TNIK, Mst4 and Ezrin. Here we show that the PDZ domain of PDZGEF is essential and sufficient for targeting PDZGEF to the apical membrane of polarized intestinal epithelial cells. Inhibition of PLD and consequently production of phosphatidic acid inhibitis targeting of PDZGEF to the plasma membrane. Furthermore, localization requires specific positively charged residues within the PDZ domain. We conclude that local accumulation of PDZGEF at the apical membrane during establishment of epithelial polarity is mediated by electrostatic interactions between positively charged side chains in the PDZ domain and negatively charged phosphatidic acid.     

The Guanine Nucleotide Exchange Factor ARNO mediates the activation of ARF and phospholipase D by insulin

Background  

Phospholipase D (PLD) is involved in many signaling pathways. In most systems, the activity of PLD is primarily regulated by the members of the ADP-Ribosylation Factor (ARF) family of GTPases, but the mechanism of activation of PLD and ARF by extracellular signals has not been fully established. Here we tested the hypothesis that ARF-guanine nucleotide exchange factors (ARF-GEFs) of the cytohesin/ARNO family mediate the activation of ARF and PLD by insulin.     

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.