Family Of Guanine Nucleotide Exchange Factors Research Articles

Polarizing Neurons

Early in development, neurons form neurites; one neurite elongates to become an axon and the others then become dendrites (see Pinheiro and Gertler). But what "primes" that neurite to become the axon? Localized activation of the small guanosine triphosphatase (GTPase) Rac, which regulates the actin cytoskeleton and microtubule dynamics, has been implicated in axon formation, leading Watabe-Uchida et al . to search for upstream regulators involved in establishing neuronal polarity. A yeast two-hybrid screen of a human fetal cDNA brain library with a dominant-negative Rac1 mutant identified DOCK7 (dedicator of cytokinesis 7), one of a family of unconventional Rho family guanine nucleotide exchange factors, as a binding partner. DOCK7 was highly expressed in rat brain and was most abundant during late embryonic and early postnatal development. In primary cultures of hippocampal neurons, DOCK7 was most abundant at the time axons appeared and was preferentially expressed in one of the early neurites and then in the developing axon. DOCK7 overexpression did not affect the total number of neurites but increased the number of axons; a mutant form of DOCK7 that did not activate Rac failed to induce additional axons (and sometimes prevented formation of any). DOCK7 knockdown with RNA interference blocked neuronal polarization and axon formation. Rac-dependent phosphorylation of Oncoprotein 18 (Op18 or stathmin) on serine-16 (Op18-S16-P) inhibits destabilization of microtubules by Op18. DOCK7 knockdown decreased Op18-S16-P in developing neurons, whereas DOCK7 overexpression enhanced Op18-S16-P. Op18-S16-P was enhanced in developing axons in vitro, and expression of a nonphosphorylatable mutant inhibited axon formation, as well as DOCK7-dependent induction of multiple axons. Thus, the authors propose that DOCK 7 regulates axon formation by means of Op18 regulation of microtubule dynamics. M. Watabe-Uchida, K. A. John, J. A. Janas, S. E. Newey, L. Van Aelst, The Rac activator DOCK7 regulates neuronal polarity through local phosphorylation of stathmin/Op18. Neuron 51 , 727-739 (2006). [PubMed] E. M. Pinheiro, F. B. Gertler, Nervous Rac: DOCK7 regulation of axon formation. Neuron 51 , 674-676 (2006). [PubMed]

Intersectin-1L nucleotide exchange factor regulates secretory granule exocytosis by activating Cdc42

Rho GTPases are key regulators of the actin cytoskeleton in membrane trafficking events. We previously reported that Cdc42 facilitates exocytosis in neuroendocrine cells by stimulating actin assembly at docking sites for secretory granules. These findings raise the question of the mechanism activating Cdc42 in exocytosis. The neuronal guanine nucleotide exchange factor, intersectin-1L, which specifically activates Cdc42 and is at an interface between membrane trafficking and actin dynamics, appears as an ideal candidate to fulfill this function. Using PC12 and chromaffin cells, we now show the presence of intersectin-1 at exocytotic sites. Moreover, through an RNA interference strategy coupled with expression of various constructs encoding the guanine nucleotide exchange domain, we demonstrate that intersectin-1L is an essential component of the exocytotic machinery. Silencing of intersectin-1 prevents secretagogue-induced activation of Cdc42 revealing intersectin-1L as the factor integrating Cdc42 activation to the exocytotic pathway. Our results extend the current role of intersectin-1L in endocytosis to a function in exocytosis and support the idea that intersectin-1L is an adaptor that coordinates exo-endocytotic membrane trafficking in secretory cells.

Essential Role of Vav Family Guanine Nucleotide Exchange Factors in EphA Receptor-Mediated Angiogenesis

Angiogenesis, the process by which new blood vessels are formed from preexisting vasculature, is critical for vascular remodeling during development and contributes to the pathogenesis of diseases such as cancer. Prior studies from our laboratory demonstrate that the EphA2 receptor tyrosine kinase is a key regulator of angiogenesis in vivo. The EphA receptor-mediated angiogenic response is dependent on activation of Rho family GTPase Rac1 and is regulated by phosphatidylinositol 3-kinase. Here we report the identification of Vav2 and Vav3 as guanine nucleotide exchange factors (GEFs) that link the EphA2 receptor to Rho family GTPase activation and angiogenesis. Ephrin-A1 stimulation recruits the binding of Vav proteins to the activated EphA2 receptor. The induced association of EphA receptor and Vav proteins modulates the activity of Vav GEFs, leading to activation of Rac1 GTPase. Overexpression of either Vav2 or Vav3 in primary microvascular endothelial cells promotes Rac1 activation, cell migration, and assembly in response to ephrin-A1 stimulation. Conversely, loss of Vav2 and Vav3 GEFs inhibits Rac1 activation and ephrin-A1-induced angiogenic responses both in vitro and in vivo. In addition, embryonic fibroblasts derived from Vav2-/- Vav3-/- mice fail to spread on an ephrin-A1-coated surface and exhibit a significant decrease in the formation of ephrin-A1-induced lamellipodia and filopodia. These findings suggest that Vav GEFs serve as a molecular link between EphA2 receptors and the actin cytoskeleton and provide an important mechanism for EphA2-mediated angiogenesis.

Rho Family Guanine Nucleotide Exchange Factor Brx Couples Extracellular Signals to the Glucocorticoid Signaling System

Glucocorticoids regulate many crucial biologic functions through their cytoplasmic/nuclear glucocorticoid receptors (GR). Excess, deficiency, or alteration in tissue sensitivity to glucocorticoids has been associated with major causes of human morbidity and mortality. Brx, a cytoplasmic Rho family guanine nucleotide exchange factor, binds to and influences the activity of several nuclear hormone receptors. We examined the functional and molecular interactions between GR and Brx. The glucocorticoid sensitivity of lymphocytes obtained from mice haplo-insufficient for Brx was significantly decreased. Conversely, GR-mediated transcriptional activity of a glucocorticoid response element (GRE)-mediated glucocorticoid-responsive promoter was enhanced by Brx in a guanine nucleotide exchange factor domain-dependent fashion. Brx interacted with GR, forming a ternary complex with RhoA. In a chromatin immunoprecipitation assay, Brx and RhoA were co-precipitated with GREs only in the presence of ligand-activated GR. Extracellularly administered lysophosphatidic acid, which activates its signaling cascade through a specific membrane GTP-binding protein (G-protein)-coupled receptor in a G-protein alpha(13)-, Brx-, and RhoA-dependent fashion, enhanced GR transcriptional activity, whereas depletion of endogenous Brx attenuated this effect. These findings suggest that glucocorticoid signaling and, hence, the tissue sensitivity to glucocorticoids, may be coupled to extracellular signals via Brx and small G-proteins. Nuclear Brx might act as a local GRE-GR-transcriptosome activator by mediating the effect of small G-proteins on glucocorticoid-regulated genes.

Identification of novel neuronal isoforms of the Rho‐GEF Trio

The large family of GEFs (guanine nucleotide-exchange factors) for Rho GTPases activate the GTPases by accelerating their GDP/GTP exchange. The multidomain protein Trio is the founding member of an intriguing subfamily of Rho-GEFs exhibiting two Rho-GEF and numerous additional domains. The members of the Trio family play an important role in neuronal physiology, and their structural organization is very well conserved through evolution. It has previously been shown that all the members, except mammalian Trio, display several isoforms, the functions of which have been well established. In this study, we have identified, by a combination of different approaches, novel Trio isoforms that have been generated by alternative splicing, giving rise to proteins that exhibit one or two Rho-GEF domains (GEFDs). These isoforms are specifically expressed in the nervous system, at a higher level than the full-length Trio, which is ubiquitously expressed. In addition, we show that all the GEFD1-containing isoforms induce neurite outgrowth in neuroblastoma cells. We have identified neuronal specific isoforms of Trio which could be essential for Trio function in neuronal morphology.

Members of a Novel Class of Arabidopsis Rho Guanine Nucleotide Exchange Factors Control Rho GTPase-Dependent Polar Growth

Rho family small GTPases are signaling switches controlling many eukaryotic cellular processes. Conversion from the GDP- to GTP-bound form is catalyzed by guanine nucleotide exchange factors (GEFs). Rho GEFs in animals fall into two structurally distinct classes containing DH and DOCKER catalytic domains. Using a plant Rho GTPase (ROP1) as bait in yeast two-hybrid screens, we identified a family of Rho GEFs, named RopGEFs. The Arabidopsis thaliana RopGEF family of 14 members contains a conserved central domain, the domain of unknown function 315 (DUF315), and variable N- and C-terminal regions. In vitro GEF assays show that DUF315 but not the full-length version of RopGEF1 has high GEF activity toward ROP1. Our data suggest that the variable regions of RopGEF1 are involved in regulation of RopGEF through an autoinhibitory mechanism. RopGEF1 overexpression in pollen tubes produced growth depolarization, as does a constitutively active ROP1 mutant. The RopGEF1 overexpression phenotype was suppressed by expression of a dominant-negative mutant of ROP1, probably by trapping RopGEF1. Deletion mutant analysis suggested a requirement of RopGEF activity for the function of RopGEF1 in polar growth. Green fluorescent protein-tagged RopGEF1 was localized to the tip of pollen tubes where ROP1 is activated. These results provide strong evidence that RopGEF1 activates ROP1 in control of polar growth in pollen tubes.

Modulation of Muscle Regeneration, Myogenesis, and Adipogenesis by the Rho Family Guanine Nucleotide Exchange Factor GEFT

Rho family guanine nucleotide exchange factors (GEFs) regulate diverse cellular processes including cytoskeletal reorganization, cell adhesion, and differentiation via activation of the Rho GTPases. However, no studies have yet implicated Rho-GEFs as molecular regulators of the mesenchymal cell fate decisions which occur during development and repair of tissue damage. In this study, we demonstrate that the steady-state protein level of the Rho-specific GEF GEFT is modulated during skeletal muscle regeneration and that gene transfer of GEFT into cardiotoxin-injured mouse tibialis anterior muscle exerts a powerful promotion of skeletal muscle regeneration in vivo. In order to molecularly characterize this regenerative effect, we extrapolate the mechanism of action by examining the consequence of GEFT expression in multipotent cell lines capable of differentiating into a number of cell types, including muscle and adipocyte lineages. Our data demonstrate that endogenous GEFT is transcriptionally upregulated during myogenic differentiation and downregulated during adipogenic differentiation. Exogenous expression of GEFT promotes myogenesis of C2C12 cells via activation of RhoA, Rac1, and Cdc42 and their downstream effector proteins, while a dominant-negative mutant of GEFT inhibits this process. Moreover, we show that GEFT inhibits insulin-induced adipogenesis in 3T3L1 preadipocytes. In summary, we provide the first evidence that the Rho family signaling pathways act as potential regulators of skeletal muscle regeneration and provide the first reported molecular mechanism illustrating how a mammalian Rho family GEF controls this process by modulating mesenchymal cell fate decisions.

ASEF2 EXCHANGE ACTIVITY IS STIMULATED BY DYNAMIN2 AND PROMOTES TUMOR CELL POLARIZATION

Pancreatic cancer prognosis is poor largely due to the metastatic behavior of tumor cells associated with the disease; therefore, understanding the mechanisms which promote the disruption of cellular cohesion and stimulate migration are of fundamental importance. The Rho family of small GTPases and their associated proteins are central to this subject since they are essential for both the maintenance of cell-to-cell junctions, and activation of proteins involved in actin remodeling and cell migration. Dbl family guanine nucleotide exchange factors are of particular interest since these proteins act as a catalyst to promote GTP-loading and activation of Rho GTPases. In this regard, we have investigated a Cdc42-specific Rho family GEF called Asef2 which we have previously shown to interact with adenomatous polyposis coli (APC) through a discreet motif near the N-terminal region of Asef2. Herein, we describe a novel interaction with Asef2 and Dynamin2, a large GTPase involved in cortical actin remodeling as well as membrane vesicle endocytosis. The interaction between Dynamin2 and Asef2 is mediated through a canonical interaction with the SH3 domain of Asef2 and the proline-rich domain (PRD) of Dynamin2. Moreover, binding of the PRD to the SH3 domain of Asef2 stimulated in vitro exchange on Cdc42, indicating that Dynamin2 releases the auto-inhibition imposed by the SH3 domain. Binding of Asef2 with APC and Dynamin2 was not mutually exclusive, and a ternary complex of the three proteins was identified both in vitro using purified proteins and by co-immunoprecipitation of endogenous cellular protein. Furthermore the proteins were found to co-localize by confocal microscopy, and could be detected at the leading edge of scratch wounded cells. Since APC is known to bind to the microtubule filaments through EB1, and Dynamin2 has been linked to cortical actin through Cortactin, these findings suggest Asef2 acts as a critical linker protein between actin and microtubules during polarized cell migration.

The regulation of membrane to cytosol partitioning of signalling proteins by phosphoinositides and their soluble headgroups

Inositol phospholipids [PIs (phosphoinositides)] represent a group of membrane-tethered signalling molecules which differ with respect to the number and distribution of monoester phosphate groups around the inositol ring. They function by binding to proteins which possess one of several domains that bind a particular PI species, often with high affinity and specificity. PH (pleckstrin homology) domains for example possess ligand-binding pockets that are often lined with positively charged residues and which bind PIs with varying degrees of specificity. Several PH domains bind not only PIs, but also their cognate headgroups, many of which occur naturally in cells as relatively abundant cytosolic inositol phosphates. The subcellular distributions of proteins possessing such PH domains are therefore determined by the relative levels of competing membrane-bound and soluble ligands. A classic example of the latter is the PH domain of phospholipase Cdelta1, which binds both phosphatidylinositol 4,5-bisphosphate and inositol 1,4,5-trisphosphate. We have shown that the N-terminal PH domain of the Rho family guanine nucleotide-exchange factor, Tiam 1, binds PI ligands promiscuously allowing multiple modes of regulation. We also recently analysed the ligand-binding specificity of the PH domain of PI-dependent kinase 1 and found that it could bind abundant inositol polyphosphates such as inositol hexakisphosphate. This could explain the dual distribution of this key signalling component, which needs to access substrates at both the plasma membrane and in the cytosol.

Drosophila Ric-8 regulates Gαi cortical localization to promote Gαi-dependent planar orientation of the mitotic spindle during asymmetric cell division

Localization and activation of heterotrimeric G proteins have a crucial role during asymmetric cell division. The asymmetric division of the Drosophila sensory precursor cell (pl) is polarized along the antero-posterior axis by Frizzled signalling and, during this division, activation of Galphai depends on Partner of Inscuteable (Pins). We establish here that Ric-8, which belongs to a family of guanine nucleotide-exchange factors for Galphai, regulates cortical localization of the subunits Galphai and Gbeta13F. Ric-8, Galphai and Pins are not necessary for the control of the anteroposterior orientation of the mitotic spindle during pl cell division downstream of Frizzled signalling, but they are required for maintainance of the spindle within the plane of the epithelium. On the contrary, Frizzled signalling orients the spindle along the antero-posterior axis but also tilts it along the apico-basal axis. Thus, Frizzled and heterotrimeric G-protein signalling act in opposition to ensure that the spindle aligns both in the plane of the epithelium and along the tissue polarity axis.

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 Rho-Specific GEF Lfc Interacts with Neurabin and Spinophilin to Regulate Dendritic Spine Morphology

Neurabin and spinophilin are homologous protein phosphatase 1 and actin binding proteins that regulate dendritic spine function. A yeast two-hybrid analysis using the coiled-coil domain of neurabin revealed an interaction with Lfc, a Rho GEF. Lfc was highly expressed in brain, where it interacted with either neurabin or spinophilin. In neurons, Lfc was largely found in the shaft of dendrites in association with microtubules but translocated to spines upon neuronal stimulation. Moreover, expression of Lfc resulted in reduction in spine length and size. Both the translocation and the effect on spine morphology depended on the coiled-coil domain of Lfc. Coexpression of neurabin or spinophilin with Lfc resulted in their clustering together with F-actin, a process that depended on Rho activity. Thus, interaction between Lfc and neurabin/spinophilin selectively regulates Rho-dependent organization of F-actin in spines and is a link between the microtubule and F-actin cytoskeletons in dendrites.

GEFs Ensure EPHective Axon Steering

During development of the nervous system, neurons and growth cones migrate toward their targets by responding to environmental cues. Interactions between cell surface receptors and these extracellular signals elicit changes in the neuronal cytoskeleton that cause a growth cone to turn either away from a repellant or toward an attractant molecule. The Eph family of receptor tyrosine kinases and ephrins, their membrane-bound ligands, guide axons during formation of complex neuronal circuitry. These receptors alter the dynamics of the actin cytoskeleton by controlling the activity of the small GTP-binding proteins RhoA, Rac, and Cdc42. Sahin et al. report that in the absence of ephrin stimulation, EphA4 is associated with ephexin1, a guanine nucleotide exchange factor (GEF) located in growth cones that activates RhoA, Rac1, and Cdc42 to promote axon outgrowth. Upon activation with ligand, EphA4 induced phosphorylation of ephexin1 by a Src family tyrosine kinase. The modification enhanced ephexin's exchange activity on RhoA, resulting in growth cone collapse and axon repulsion in cultured neurons. Neurons from transgenic mice lacking ephexin1 had shorter axons and did not exhibit the repulsive response to ephrin. Cowan et al. also report that Vav2, another GEF located in growth cones that acts on RhoA, Rac, and Cdc42, is important for Eph-mediated axon guidance. Eph receptor-ephrin interaction mediates cell-cell contact. However, upon activation by ligand, activated Eph receptors are internalized in a Rac-dependent manner, disrupting cell-cell associations and promoting the repulsive response. Activated Eph receptors associated with Vav2. The exchange factor became phosphorylated on tyrosine, increasing its activity. This corresponded with endocytosis of Eph receptors. Vav-deficient neurons showed abnormal axon projections, and stimulation with ephrin molecules failed to induce endocytosis of Eph receptors or growth cone collapse. The studies suggest that by engaging specific GEFs to coordinate different intracellular processes, Eph receptors can promote highly localized changes in growth cones to facilitate accurate axon pathfinding. M. Sahin, P. L. Greer, M. Z. Lin, H. Poucher, J. Eberhart, S. Schmidt, T. M. Wright, S. M. Shamah, S. O'Connell, C. W. Cowan, L. Hu, J. L. Goldberg, A. Debant, G. Corfas, C. E. Krull, M. E. Greenberg, Eph-dependent tyrosine phosphorylation of ephexin1 modulates growth cone collapse. Neuron 46 , 191-204 (2005). [Online Journal] C. W. Cowan, Y. R. Shao, M. Sahin, S. M. Shamah, M. Z. Lin, P. L. Greer, S. Gao, E. C. Griffith, J. S. Brugge, M. E. Greenberg, Vav family GEFs link activated Ephs to endocytosis and axon guidance. Neuron 46 , 205-217 (2005). [Online Journal]

Novel intermediate of Rac GTPase activation by guanine nucleotide exchange factor

The biochemical role of guanine nucleotide exchange factors (GEFs) in catalyzing small GTPase GDP–GTP exchange is thought to be twofold: stimulation of GDP dissociation and stabilization of a nucleotide-free GTPase intermediate. Here we report that TrioN, a Dbl family GEF, activates Rac1 by facilitating GTP binding to, as well as stimulating GDP dissociation from, Rac1. The TrioN-catalyzed GDP dissociation is dependent upon the structural nature and the concentration of free nucleotide, and nucleotide binding serves as the rate-limiting step of the GEF reaction. The TrioN-stimulated nucleotide exchange may undergo a novel two nucleotide-one G-protein intermediate involving two cryptic subsites on Rac1 induced by the GEF, with one subsite contributing to the recognition of the β/γ phosphates of the incoming GTP and another to the binding of the guanine base of the leaving GDP. We propose that the Rac GEF reaction may proceed by competitive displacement of bound GDP by GTP through a transient intermediate of GEF–[GTP–Rac–GDP].

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.

Vav3 regulates osteoclast function and bone mass

Osteoporosis, a leading cause of morbidity in the elderly, is characterized by progressive loss of bone mass resulting from excess osteoclastic bone resorption relative to osteoblastic bone formation. Here we identify Vav3, a Rho family guanine nucleotide exchange factor, as essential for stimulated osteoclast activation and bone density in vivo. Vav3-deficient osteoclasts show defective actin cytoskeleton organization, polarization, spreading and resorptive activity resulting from impaired signaling downstream of the M-CSF receptor and alpha(v)beta3 integrin. Vav3-deficient mice have increased bone mass and are protected from bone loss induced by systemic bone resorption stimuli such as parathyroid hormone or RANKL. Moreover, we provide genetic and biochemical evidence for the role of Syk tyrosine kinase as a crucial upstream regulator of Vav3 in osteoclasts. Thus, Vav3 is a potential new target for antiosteoporosis therapy.

Dynamin 2 regulates T cell activation by controlling actin polymerization at the immunological synapse

Actin reorganization at the immunological synapse is required for the amplification and generation of a functional immune response. Using small interfering RNA, we show here that dynamin 2 (Dyn2), a large GTPase involved in receptor-mediated internalization, did not alter antibody-mediated T cell receptor internalization but considerably affected T cell receptor-stimulated T cell activation by regulating multiple biochemical signaling pathways and the accumulation of F-actin at the immunological synapse. Moreover, Dyn2 interacted directly with the Rho family guanine nucleotide exchange factor Vav1, and this interaction was required for T cell activation. These data identify a functionally important interaction between Dyn2 and Vav1 that regulates actin reorganization and multiple signaling pathways in T lymphocytes.

A Steric-Inhibition Model for Regulation of Nucleotide Exchange via the Dock180 Family of GEFs

CDM (CED-5, Dock180, Myoblast city) family members have been recently identified as novel, evolutionarily conserved guanine nucleotide exchange factors (GEFs) for Rho-family GTPases . They regulate multiple processes, including embryonic development, cell migration, apoptotic-cell engulfment, tumor invasion, and HIV-1 infection, in diverse model systems . However, the mechanism(s) of regulation of CDM proteins has not been well understood. Here, our studies on the prototype member Dock180 reveal a steric-inhibition model for regulating the Dock180 family of GEFs. At basal state, the N-terminal SH3 domain of Dock180 binds to the distant catalytic Docker domain and negatively regulates the function of Dock180. Further studies revealed that the SH3:Docker interaction sterically blocks Rac access to the Docker domain. Interestingly, ELMO binding to the SH3 domain of Dock180 disrupted the SH3:Docker interaction, facilitated Rac access to the Docker domain, and contributed to the GEF activity of the Dock180/ELMO complex. Additional genetic rescue studies in C. elegans suggested that the regulation of the Docker-domain-mediated GEF activity by the SH3 domain and its adjoining region is evolutionarily conserved. This steric-inhibition model may be a general mechanism for regulating multiple SH3-domain-containing Dock180 family members and may have implications for a variety of biological processes.

Expression of Trio, a member of the Dbl family of Rho GEFs in the developing rat brain

Trio, a member of the Dbl family of guanine nucleotide exchange factors (GEFs), has a series of spectrin repeats, two GEF domains, protein interaction domains, and a putative kinase domain, potentially important in neuronal axon guidance and cell migration. Most knowledge about Trio is based on studies of Caenorhabditis elegans and Drosophila, while the function of Trio in vertebrates is unclear. The aim of these experiments was to establish the patterns of Trio expression in the postnatal rat brain. During postnatal (P) development, high levels of Trio mRNA are found in the cerebral cortex, hippocampus, thalamus, caudate/putamen, and olfactory bulb, with lower levels in the septal nucleus, nucleus accumbens, amygdala, and hypothalamus. Except for the cerebellum, Trio mRNA in major brain areas is highest at P1, decreasing gradually during development, with low but detectable levels at P30. In P14 cerebral cortex, pyramidal neurons with strongly staining soma and dendrites are observed primarily in layer 5. In hippocampus, strong staining is observed in pyramidal neurons, granule cells, and isolated interneurons. Cerebellar Purkinje neurons exhibit intense staining in the soma and in extensive dendritic arbors at P7 and P14. Levels of Trio mRNA and the intensity of Trio immunostaining in cerebellar Purkinje cells increase from P1 to P30. Consistent with the in situ hybridization pattern, Western blot analyses show that Trio levels in the hippocampus and cortex are high at P1, decreasing until P30. The data suggest that Trio plays an important role during neuronal development.

Endothelin 1 Induces β1Pix Translocation and Cdc42 Activation via Protein Kinase A-dependent Pathway

p21-activated kinase (Pak)-interacting exchange factor (Pix), a Rho family guanine nucleotide exchange factor (GEF), has been shown to co-localize with Pak and form activated Cdc42- and Rac1-driven focal complexes. In this study we have presented evidence that treatment of human mesangial cells (HMC) with endothelin 1 (ET-1) and stimulation of adenylate cyclase with either forskolin or with the cAMP analog 8-Br-cAMP activated the GTP loading of Cdc42. Transient expression of constitutively active G alpha(s) also stimulated Cdc42. In addition, overexpression of beta(1)Pix enhanced ET-1-induced Cdc42 activation, whereas the expression of beta(1)Pix SH3m(W43K), which lacks the ability to bind Pak, and beta(1)PixDHm(L238R/L239S), which lacks GEF activity, decreased ET-1-induced Cdc42 activation. Furthermore, ET-1 stimulation induced beta(1)Pix translocation to focal complexes. Interestingly, pretreatment of HMC with protein kinase A (PKA) inhibitors blocked both Cdc42 activation and beta(1)Pix translocation induced by ET-1, indicating the involvement of the PKA pathway. Through site-directed mutagenesis studies of consensus PKA phosphorylation sites and in vitro PKA kinase assay, we have shown that beta(1)Pix is phosphorylated by PKA. Using purified recombinant beta(1)Pix(wt) and beta(1)Pix mutants, we have identified Ser-516 and Thr-526 as the major phosphorylation sites by PKA. beta(1)Pix(S516A/T526A), in which both phosphorylation sites are replaced by alanine, blocks beta(1)Pix translocation and Cdc42 activation. Our results have provided evidence that stimulation of PKA pathway by ET-1 or cAMP analog results in beta(1)Pix phosphorylation, which in turn controls beta(1)Pix translocation to focal complexes and Cdc42 activation.