Table of contents

Proteins interacting with Rac: Rac GEFs

unc-73 is required for cell migrations and axon guidance in C. elegans; it encodes overlapping isoforms of 283 and 189 kDa that are closely related to the vertebrate Trio and Kalirin proteins, respectively. These proteins are guanine nucleotide exchange facors (GEFs), which activate GTPases by stimulating exchange of GDP for GTP. UNC-73A contains, in order, eight spectrin-like repeats, a Dbl/Pleckstrin homology (DH/PH) element, an SH3-like domain, a second DH/PH element, an immunoglobulin domain, and a fibronectin type III domain. UNC-73B terminates just downstream of the SH3-like domain. The first DH/PH element specifically activates the Rac GTPase in vitro and stimulates actin polymerization when expressed in Rat2 cells. Both functions are eliminated by introducing the S1216F mutation of unc-73(rh40) into this DH domain. UNC-73 affects many cell and growth cone migrations in C. elegans. For example, the moderately uncoordinated cannonical unc-73(e936) allele has axon guidance defects in various touch receptor neurons and motoneurons. These results suggest that UNC-73 acts cell autonomously in a protein complex to regulate actin dynamics during cell and growth cone migrations. Trio (which likewise possesses an Ig domain) binds to the LAR receptor tyrosine phosphatase raising the possibility that the UNC-73 binds to a similar PTPase in C. elegans. Interestingly, the gene for the Drosophila LAR phosphatase is almost exclusively expressed by developing neurons; mutations in the gene cause defects in motor axon pathfinding. The spectrin-like, Ig, and FnIII domains of UNC-73 are probable mediators of interactions with unidentified proteins, whereas the PH domains likely mediate the association of proteins with specific membrane phosphoinositides (Steven, 1998).

The T-lymphoma and metastasis 1 (Tiam1) protein produces invasion and functions as a guanine nucleotide exchange factor (GEF) for the small GTPase Rac1. Differentiation-dependent expression of Tiam1 in the developing brain suggests a role for this GEF and its effector Rac1 in the control of neuronal morphology. Overexpression of Tiam1 induces cell spreading and affects neurite outgrowth in N1E-115 neuroblastoma cells. These effects are Rac-dependent and strongly promoted by laminin. Overexpression of Tiam1 recruits the alpha6beta1 integrin, a laminin receptor, to specific adhesive contacts at the cell periphery, which are different from focal contacts. Cells overexpressing Tiam1 no longer respond to lysophosphatidic acid- induced neurite retraction and cell rounding, processes mediated by Rho, suggesting that Tiam1-induced activation of Rac antagonizes Rho signaling. This inhibition can be overcome by coexpression of constitutively active RhoA, which may indicate that regulation occurs at the level of Rho or upstream. Conversely, neurite formation induced by Tiam1 or Rac1 is further promoted by inactivating Rho. These results demonstrate that Rac- and Rho-mediated pathways oppose each other during neurite formation and that a balance between these pathways determines neuronal morphology. Furthermore, the data underscore the potential role of Tiam1 as a specific regulator of Rac during neurite formation and illustrate the importance of reciprocal interactions between the cytoskeleton and the extracellular matrix during this process (Leeuwen, 1997).

Tiam1 encodes an exchange factor for the Rho-like guanosine triphosphatase Rac. Both Tiam1 and activated RacV12 promote invasiveness of T lymphoma cells. In epithelial Madin-Darby canine kidney (MDCK) cells, Tiam1 localizes to adherens junctions. Ectopic expression of Tiam1 or RacV12 inhibit hepatocyte growth factor-induced scattering by increasing E-cadherin-mediated cell-cell adhesion accompanied by actin polymerization at cell-cell contacts. In Ras-transformed MDCK cells, expression of Tiam1 or RacV12 restores E-cadherin-mediated adhesion, resulting in phenotypic reversion and loss of invasiveness. These data suggest an invasion-suppressor role for Tiam1 and Rac in epithelial cells (Hordijk, 1997).

Trio contains two functional guanine nucleotide exchange factor (GEF) domains for the Rho-like GTPases and a serine/threonine kinase domain. In vitro, GEF domain 1 (GEFD1) is specifically active on Rac1, while GEF domain 2 (GEFD2) targets RhoA. To determine whether Trio could activate Rac1 and RhoA in vivo, a study was carried out of the effects of Trio on mitogen activated protein kinase (MAPK) pathways and cytoskeletal rearrangement events mediated by the two GTPases. The following was observed:

  1. The GEFD1 domain of Trio triggers the MAPK pathway leading to Jun kinase (JNK) activation and the production of membrane ruffles.
  2. Co-expression of the TrioGEFD1 domain with a dominant-negative form of Rac blocks JNK induction, whereas a dominant-negative form of Cdc42 does not.
  3. A deletion mutant of TrioGEFD1 lacking a region important for exchange activity could not stimulate JNK activity.
  4. In contrast, the TrioGEFD2 domain does not stimulate JNK activity and induces the formation of stress fibers, as does activated RhoA.
  5. Co-expression of both GEF domains simultaneously induces the formation of ruffles and stress fibers.

Trio, therefore represents a unique member of the Rho-GEFs family possessing two functional domains of distinct specificities, that allow it to link Rho and Rac signaling pathway in vivo (Bellanger, 1997).

Lfc and Lsc are two recently identified oncoproteins that contain a Dbl homology domain in tandem with a pleckstrin homology domain and thus share sequence similarity with a number of other growth regulatory proteins, including Dbl, Tiam-1, and Lbc. Lfc and Lsc, like their closest relative, Lbc, are highly specific guanine nucleotide exchange factors (GEFs) for Rho, causing a >10-fold stimulation of [3H]GDP dissociation from Rho and a marked stimulation of GDP-GTPgammaS exchange. All three proteins (Lbc, Lfc, and Lsc) are able to act catalytically in stimulating the guanine nucleotide exchange activity, such that a single molecule of each of these oncoproteins can activate a number of molecules of Rho. Neither Lfc nor Lsc shows any ability to stimulate GDP dissociation from other related GTP-binding proteins such as Rac, Cdc42, or Ras. Thus Lbc, Lfc, and Lsc appear to represent a subgroup of Dbl-related proteins that function as highly specific GEFs toward Rho and can be distinguished from Dbl, Ost, and Dbs, which are less specific and show GEF activity toward both Rho and Cdc42. Consistent with these results, Lbc, Lfc, and Lsc each form tight complexes with the guanine nucleotide-depleted form of Rho and bind weakly to the GDP- and GTPgammaS-bound states. None of these oncoproteins are able to form complexes with Cdc42 or Ras. However, Lfc (but not Lbc nor Lsc) can bind to Rac, and this binding occurs equally well when Rac is nucleotide-depleted or is in the GDP- or GTPgammaS-bound state. These findings raise the possibility that in addition to acting directly as a GEF for Rho, Lfc may play other roles that influence the signaling activities of Rac and/or coordinate the activities of the Rac and Rho proteins (Glaven, 1996).

Ras and Rac are membrane-associated GTPases that function as molecular switches activating intracellular mitogen-activated protein kinase (MAPK) cascades and other effector pathways in response to extracellular signals. Activation of Ras and Rac into their GTP-bound conformations is directly controlled by specific guanine-nucleotide exchange factors (GEFs), which catalyze GDP release. Several Ras-specific GEFs that are related to the budding yeast protein Cdc25p have been described, whereas GEFs for Rac-related GTPases contain a region that is homologous to the oncoprotein DbI. The Ras-GRF1 and Ras-GRF2 proteins, which couple Ras activation to serpentine receptors and calcium signals, contain both Cdc25 and DbI homology (DH) regions. Ras-GRF2 is a bifunctional signaling protein that is able to bind and activate Ras and Rac, and thereby coordinate the activation of the extracellular-signal-regulated kinase (ERK) and stress-activated protein kinase (SAPK) pathways (Fan, 1998).

Small GTPases control key cellular events, including formation of cell-cell junctions and gene expression, and are regulated by activating and inhibiting factors. This study characterized the junctional protein paracingulin as a novel regulator of the activity of two small GTPases, Rac1 and RhoA, through the functional interaction with their respective activators, Tiam1 and GEF-H1. In confluent epithelial monolayers, paracingulin depletion leads to increased RhoA activity and increased expression of mRNA for the tight junction protein claudin-2. During tight junction assembly by the calcium-switch, Rac1 shows two transient peaks of activity, at earlier (10-20 min) and later (3-8 h) time points. Paracingulin depletion reduces such peaks of Rac1 activation in a Tiam1-dependent manner, resulting in a delay in junction formation. Paracingulin physically interacts with GEF-H1 and Tiam1 in vivo and in vitro, and it is required for their efficient recruitment to junctions, based on immunofluorescence and biochemical experiments. These results provide the first description of a junctional protein that interacts with GEFs for both Rac1 and RhoA, and identify a novel molecular mechanism whereby Rac1 is activated during junction formation (Guillemot, 2008).

Proteins interacting with Rac: Rac GAPs

In a search for new partners of the activated form of Rac GTPase, using a two-hybrid cloning procedure, a human cDNA encoding a new GTPase-activating protein (GAP) for Rho family GTPases has been isolated. A specific mRNA of 3.2 kilobases is detected in low abundance in many cell types and found highly expressed in testis. A protein of the predicted size 58 kDa, which is called MgcRacGAP, is detected in human testis as well as in germ cell tumor extracts by immunoblotting with antibodies specific to recombinant protein. In vitro, the GAP domain of MgcRacGAP strongly stimulates Rac1 and Cdc42 GTPase activity but is almost inactive on RhoA. N-terminal to its GAP domain, MgcRacGAP contains a cysteine-rich zinc finger-like motif characteristic of the Chimaerin family of RhoGAPs. The closest homolog of MgcRacGAP is RotundRacGAP, a product associated with the Drosophila rotund locus. In situ hybridization experiments in human testis demonstrate a specific expression of mgcRacGAP mRNA in spermatocytes similar to that of rotundRacGAP in Drosophila testis. Therefore, protein sequence similarity and analogous developmental and tissue specificities of gene expression support the hypothesis that RotundRacGAP and MgcRacGAP have equivalent functions in insect and mammalian germ cells. Since rotundRacGAP deletion leads to male sterility in the fruit fly, the mgcRacGAP gene may prove likewise to play a key role in mammalian male fertility (Toure, 1998).

n-Chimaerin in vitro is a GTPase-activating protein (GAP) primarily for Rac1 and less so for Cdc42Hs. The GAP activity of n-chimaerin is regulated by phospholipids and phorbol esters. Microinjection of Rac1 and Cdc42Hs into mammalian cells induces, respectively, formation of the actin-based structures lamellipodia and filopodia, the former being prevented by coinjection of the chimaerin GAP domain. Strikingly, microinjection of the full-length n-chimaerin into fibroblasts and neuroblastoma cells induces the simultaneous formation of lamellipodia and filopodia. These structures undergo cycles of dissolution and formation, resembling natural morphological events occurring at the leading edge of fibroblasts and neuronal growth cones. The effects of n-chimaerin on formation of lamellipodia and filopodia were inhibited by dominant negative Rac1(T17N) and Cdc42Hs(T17N), respectively. n-Chimaerin's effects were also inhibited by coinjection with Rho GDP dissociation inhibitor or by treatment with phorbol ester. A mutant n-chimaerin with no GAP activity and impaired p21 binding is ineffective in inducing morphological changes, while a mutant lacking GAP activity alone is effective. Microinjected n-chimaerin colocalizes in situ with F-actin. Taken together, these results suggest that n-chimaerin acts synergistically with Rac1 and Cdc42Hs to induce actin-based morphological changes and that this action involves Rac1 and Cdc42Hs binding but not GAP activity. Thus, GAPs may have morphological functions in addition to downregulation of GTPases (Kozma, 1996).

Activated forms of the GTPases, Rac and Cdc42, are known to stimulate formation of microfilament-rich lamellipodia and filopodia, respectively, but the underlying mechanisms have remained obscure. IQGAP1 is likely to mediate effects of these GTPases on microfilaments. Native IQGAP1 purified from bovine adrenal comprises two approximately 190-kD subunits per molecule plus substoichiometric calmodulin. IQGAP1 contains four potential calmodulin-binding IQ domains and a region homologous to catalytic domains of GTPase-activating proteins, or GAPs. Purified IQGAP1 binds directly to F-actin and cross-links the actin filaments into irregular, interconnected bundles that exhibited gel-like properties. Exogenous calmodulin partially inhibits binding of IQGAP1 to F-actin, and is more effective in the absence of calcium than in its presence.. Colocalization of IQGAP1 with cortical microfilaments is cytochalasin-D sensitve. These results, in conjunction with prior evidence that IQGAP1 binds directly to activated Rac and Cdc42, suggest that IQGAP1 serves as a direct molecular link between these GTPases and the actin cytoskeleton, and that the actin-binding activity of IQGAP1 is regulated by calmodulin (Bashour, 1997).

The Rho family GTPases are involved in a variety of cellular events: they act by changing the organization of actin cytoskeletal networks in response to extracellular signals. However, it is not clearly known how their activities are spatially and temporally regulated. A novel guanine nucleotide exchange factor for Rac1 (termed STEF for Sif and Tiam1-like exchange factor) has been identified. STEF is related in overall amino acid sequence and modular structure to mouse Tiam1 and Drosophila Still life (Sif) protein. The Drosophila sif gene was identified in a behavioral mutant screen. Sif protein is predominantly expressed in the nervous system and confined to the synaptic terminals. At the ultrastructural level, it is found in lateral regions of the active zones for neurotransmission. A loss-of-function sif mutation causes reduced motor activities, which can be rescued by expression of a sif minigene in the nervous system. Moreover, expression of a truncated Sif protein induces membrane ruffling with altered actin localization in human KB cells. These data suggest that Sif protein regulates the formation or maintenance of synapses, possibly by organizing the actin cytoskeleton through the activation of Rho family GTPases. Sif protein contains a DH domain, two pleckstrin homology (PH) domains, and a PDZ domain. The organization and amino acid sequences of these domains are highly related to those of mouse Tiam1 (Hoshino, 1999 and references).

Mammalian STEF protein contains two pleckstrin homology domains, a PDZ domain and a Dbl homology domain. The in vitro assay shows that STEF protein specifically enhances the dissociation of GDP from Rac1 but not that from either RhoA or Cdc42. Expression of a truncated STEF protein in culture cells induces membrane ruffling with altered actin localization, which implies that this protein also activates Rac1 in vivo. The stef transcript is observed in restricted parts of mice, including cartilaginous tissues and the cortical plate of the central nervous system during embryogenesis. These findings suggest that STEF protein participates in the control of cellular events in several developing tissues, possibly changing the actin cytoskeletal network by activating Rac1 (Hoshino, 1999).

Signaling from receptor tyrosine kinases (RTKs) requires the sequential activation of the small GTPases Ras and Rac. Son of sevenless (Sos-1), a bifunctional guanine nucleotide exchange factor (GEF), activates Ras in vivo and displays Rac-GEF activity in vitro, when engaged in a tricomplex with Eps8 and E3b1-Abi-1, a RTK substrate and an adaptor protein, respectively. A mechanistic understanding of how Sos-1 coordinates Ras and Rac activity is, however, still missing. This study demonstrate that (a) Sos-1, E3b1, and Eps8 assemble into a tricomplex in vivo under physiological conditions; (b) Grb2 and E3b1 bind through their SH3 domains to the same binding site on Sos-1, thus determining the formation of either a Sos-1-Grb2 (S/G) or a Sos-1-E3b1-Eps8 (S/E/E8) complex, endowed with Ras- and Rac-specific GEF activities, respectively; (c) the Sos-1-Grb2 complex is disrupted upon RTKs activation, whereas the S/E/E8 complex is not; and (d) in keeping with the previous result, the activation of Ras by growth factors is short-lived, whereas the activation of Rac is sustained. Thus, the involvement of Sos-1 at two distinct and differentially regulated steps of the signaling cascade allows for coordinated activation of Ras and Rac and different duration of their signaling within the cell (Innocenti, 2002).

Class I phosphoinositide 3-kinases (PI3Ks) are implicated in many cellular responses controlled by receptor tyrosine kinases (RTKs), including actin cytoskeletal remodeling. Within this pathway, Rac is a key downstream target/effector of PI3K. However, how the signal is routed from PI3K to Rac is unclear. One possible candidate for this function is the Rac-activating complex Eps8-Abi1-Sos-1, which possesses Rac-specific guanine nucleotide exchange factor (GEF) activity. Abi1 (also known as E3b1) recruits PI3K, via p85, into a multimolecular signaling complex that includes Eps8 and Sos-1. The recruitment of p85 to the Eps8-Abi1-Sos-1 complex and phosphatidylinositol 3, 4, 5 phosphate (PIP3), the catalytic product of PI3K, concur to unmask its Rac-GEF activity in vitro. Moreover, they are indispensable for the activation of Rac and Rac-dependent actin remodeling in vivo. On growth factor stimulation, endogenous p85 and Abi1 consistently colocalize into membrane ruffles, and cells lacking p85 fail to support Abi1-dependent Rac activation. These results define a mechanism whereby propagation of signals, originating from RTKs or Ras and leading to actin reorganization, is controlled by direct physical interaction between PI3K and a Rac-specific GEF complex (Innocetti, 2003).

NMDA-type glutamate receptors play a critical role in the activity-dependent development and structural remodeling of dendritic arbors and spines. However, the molecular mechanisms that link NMDA receptor activation to changes in dendritic morphology remain unclear. The Rac1-GEF Tiam1 is present in dendrites and spines and is required for their development. Tiam1 interacts with the NMDA receptor and is phosphorylated in a calcium-dependent manner in response to NMDA receptor stimulation. Blockade of Tiam1 function with either RNAi or dominant interfering mutants of Tiam1 suggests that Tiam1 mediates effects of the NMDA receptor on dendritic development by inducing Rac1-dependent actin remodeling and protein synthesis. Taken together, these findings define a molecular mechanism by which NMDA receptor signaling controls the growth and morphology of dendritic arbors and spines (Tolias, 2005).

The regulators of the Rho-family GTPases, GTPase-activating proteins (GAPs) and guanine exchange factors (GEFs), play important roles in axon guidance. By means of a functional genomic study of the Rho-family GEFs and GAPs in Drosophila, a Rho-family GAP, CrossGAP (CrGAP), has been identified that is involved in Roundabout (Robo) receptor-mediated repulsive axon guidance. CrGAP physically associates with the Robo receptor. Too much or too little CrGAP activity leads to defects in Robo-mediated repulsion at the midline choice point. The CrGAP gain-of-function phenotype mimics the loss-of-function phenotypes of both Robo and Rac. Dosage-sensitive genetic interactions among CrGAP, Robo, and Rac support a model in which CrGAP transduces signals downstream of Robo receptor to regulate Rac-dependent cytoskeletal changes (Hu, 2005; full text of article).

Neuronal network formation in the developing nervous system is dependent on the accurate navigation of nerve cell axons and dendrites, which is controlled by attractive and repulsive guidance cues. Ephrins and their cognate Eph receptors mediate many repulsive axonal guidance decisions by intercellular interactions resulting in growth cone collapse and axon retraction of the Eph-presenting neuron. This study shows that the Rac-specific GTPase-activating protein α2-chimaerin binds activated EphA4 and mediates EphA4-triggered axonal growth cone collapse. α-Chimaerin mutant mice display a phenotype similar to that of EphA4 mutant mice, including aberrant midline axon guidance and defective spinal cord central pattern generator activity. These results reveal an α-chimaerin-dependent signaling pathway downstream of EphA4, which is essential for axon guidance decisions and neuronal circuit formation in vivo (Wegmeyer, 2007).

Table of contents

Rac1: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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