A novel role for RhoGDI as an inhibitor of GAP proteins.

RhoGDI inhibits guanine nucleotide dissociation from post-translationally processed Rho and Rac proteins but its biochemical role in vivo is unknown. We show here that N-terminal effector site mutations in the Rac protein do not compromise its interaction with RhoGDI and that, whilst geranylgeranylation and -AAX proteolysis of the C-terminal CAAX motif of Rac1 and RhoA are required for efficient interaction with RhoGDI, methylesterification of the C-terminal cysteine residue is not required. In vitro, RhoGDI can form stable complexes with Rho and Rac proteins in both the GTP and GDP bound states. Furthermore the Rac-GTP--RhoGDI complex is resistent to the action of recombinant RhoGAP and recombinant BCR. Thus GDI, by complexing with Rac-GTP and preventing GAP stimulated GTP hydrolysis, may allow transit of the activated form of the Rac protein between physically separated activator and effector proteins in the cell.     

p190RhoGAP has cellular RacGAP activity regulated by a polybasic region

p190RhoGAP is a GTPase-activating protein (GAP) known to regulate actin cytoskeleton dynamics by decreasing RhoGTP levels through activation of the intrinsic GTPase activity of Rho. Although the GAP domain of p190RhoGAP stimulates the intrinsic' GTPase activity of several Rho family members (Rho, Rac, Cdc42) under in vitro conditions, p190RhoGAP is generally regarded as a GAP for RhoA in the cell. The cellular RacGAP activity of the protein has not been proven directly. We have previously shown that the in vitro RacGAP and RhoGAP activity of p190RhoGAP was inversely regulated through a polybasic region of the protein. Here we provide evidence that p190RhoGAP shows remarkable GAP activity toward Rac also in the cell. The cellular RacGAP activity of p190RhoGAP requires an intact polybasic region adjacent to the GAP domain whereas the RhoGAP activity is inhibited by the same domain. Our data indicate that through its alternating RacGAP and RhoGAP activity, p190RhoGAP plays a more complex role in the Rac–Rho antagonism than it was realized earlier.     

The role of Gln61 and Glu63 of Ras GTPases in their activation by NF1 and Ras GAP.

Two distinct GAPs of 120 and 235 kDa called GAP1 and NF1 serve as attenuators of Ras, a member of GTP-dependent signal transducers, by stimulating its intrinsic guanosine triphosphatase (GTPase) activity. The GAP1 (also called Ras GAP) is highly specific for Ras and does not stimulate the intrinsic GTPase activity of Rap1 or Rho. Using GAP1C, the C-terminal GTPase activating domain (residues 720-1044) of bovine GAP1, we have shown previously that the GAP1 specificity is determined by the Ras domain (residues 61-65) where Gln61 plays the primary role. The corresponding domain (residues 1175-1531) of human NF1 (called NF1C), which shares only 26% sequence identity with the GAP1C, also activates Ras GTPases. In this article, we demonstrate that the NF1C, like the GAP1C, is highly specific for Ras and does not activate either Rap1 or Rho GTPases. Furthermore, using a series of chimeric Ras/Rap1 and mutated Ras GTPases, we show that Gln at position 61 of the GTPases primarily determines that NF1C as well as GAP1C activates Ras GTPases, but not Rap1 GTPases, and Glu at position 63 of the GTPases is required for maximizing the sensitivity of Ras GTPases to both NF1C and GAP1C. Interestingly, replacement of Glu63 of c-HaRas by Lys reduces its intrinsic GTPase activity and abolishes the GTPase activation by both NF1C and GAP1C. Thus, the potentiation of oncogenicity by Lys63 mutation of c-HaRas appears primarily to be due to the loss of its sensitivity to the two major Ras signal attenuators (NF1 and GAP1).     

The N-terminal region of GAP regulates cytoskeletal structure and cell adhesion.

Ras GTPase activating protein (GAP) possesses a C-terminal domain that interacts with GTP-bound Ras, and an N-terminal region containing two SH2 domains and an SH3 domain. In addition to its association with Ras, GAP binds stably to autophosphorylated beta PDGF receptors, and to two cytoplasmic phosphoproteins: p62, an RNA binding protein, and p190, which possesses GAP activity towards small guanine nucleotide binding proteins in the Rho/Rac family. To define the region of GAP that mediates these interactions with cellular phosphoproteins, and to investigate the biological significance of these complexes, a truncated GAP polypeptide (GAP-N) containing residues 1-445 was stably expressed in Rat-2 fibroblasts. GAP-N contains the SH2 and SH3 domains, but lacks the Ras GTPase activating domain. Stimulation of cells expressing GAP-N with PDGF induced association of GAP-N with the beta PDGF receptor, and phosphorylation of GAP-N on tyrosine, consistent with the notion that GAP SH2 domains direct binding to the autophosphorylated beta PDGF receptor in vivo. GAP-N bound constitutively to p190 in both serum-deprived and growth factor-stimulated cells. This GAP-N-p190 complex had Rho GAP activity in vitro. The expression of GAP-N in Rat-2 cells correlated with changes in the cytoskeleton and in cell adhesion, typified by the disruption of action stress fibres, a reduction in focal contacts, and an impaired ability to adhere to fibronectin. These results suggest that the N-terminal domain of GAP can direct interactions with cellular phosphoproteins in vivo, and thereby exert an effector function which modulates the cytoskeleton and cell adhesion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Arabidopsis RopGAPs are a novel family of rho GTPase-activating proteins that require the Cdc42/Rac-interactive binding motif for rop-specific GTPase stimulation

The plant-specific Rop subfamily of Rho GTPases, most closely related to the mammalian Cdc42 and Rac GTPases, plays an important role in the regulation of calcium-dependent pollen tube growth, H(2)O(2)-mediated cell death, and many other processes in plants. In a search for Rop interactors using the two-hybrid method, we identified a family of Rho GTPase-activating proteins (GAP) from Arabidopsis, termed RopGAPs. In addition to a GAP catalytic domain, RopGAPs contain a Cdc42/Rac-interactive binding (CRIB) motif known to allow Cdc42/Rac effector proteins to bind activated Cdc42/Rac. This novel combination of a GAP domain with a CRIB motif is widespread in higher plants and is unique to the regulation of the Rop GTPase. A critical role for CRIB in the regulation of in vitro RopGAP activity was demonstrated using point and deletion mutations. Both types of mutants have drastically reduced capacities to stimulate the intrinsic Rop GTPase activity and to bind Rop. Furthermore, RopGAPs preferentially stimulate the GTPase activity of Rop, but not Cdc42 in a CRIB-dependent manner. In vitro binding assays show that the RopGAP CRIB domain interacts with GTP- and GDP-bound forms of Rop, as well as the transitional state of Rop mimicked by aluminum fluoride. The CRIB domain also promotes the association of the GAP domain with the GDP-bound Rop, as does aluminum fluoride. These results reveal a novel CRIB-dependent mechanism for the regulation of the plant-specific family of Rho GAPs. We propose that the CRIB domain facilitates the formation of or enhanced GAP-mediated stabilization of the transitional state of the Rop GTPase.     

Autoinhibition of p50 Rho GTPase-activating protein (GAP) is released by prenylated small GTPases

Interaction of p50 Rho GTPase-activating protein (p50RhoGAP) with Rho family small GTPases was investigated in a yeast two-hybrid system, by radioactive GAP assay, and in a Rac-regulated enzymatic reaction, through superoxide production by the phagocytic NADPH oxidase. The yeast two-hybrid system revealed an interaction between the C-terminal GAP domain and the N-terminal part of p50RhoGAP. The first 48 amino acids play a special role both in the stabilization of the intramolecular interaction and in recognition of the prenyl tail of small GTPases. The GAP assay and the NADPH oxidase activity indicate that the GTPase-activating effect of full-length p50RhoGAP is lower on non-prenylated than on prenylated small GTPase. Removal of amino acids 1-48 and 169-197 of p50RhoGAP increases the GAP effect on non-prenylated Rac, whereas prenylated Rac reacts equally well with the full-length and the truncated proteins. We suggest that p50RhoGAP is in an autoinhibited conformation stabilized by the stretches 1-48 and 169-197 and the prenyl group of the small GTPase plays a role in releasing this intramolecular restraint.     

How the Pseudomonas aeruginosa ExoS toxin downregulates Rac

Pseudomonas aeruginosa is an opportunistic bacterial pathogen. One of its major toxins, ExoS, is translocated into eukaryotic cells by a type III secretion pathway. ExoS is a dual function enzyme that affects two different Ras-related GTP binding proteins. The C-terminus inactivates Ras through ADP ribosylation, while the N-terminus inactivates Rho proteins through its GTPase activating protein (GAP) activity. Here we have determined the three-dimensional structure of a complex between Rac and the GAP domain of ExoS in the presence of GDP and AlF3. Composed of approximately 130 residues, this ExoS domain is the smallest GAP hitherto described. The GAP domain of ExoS is an all-helical protein with no obvious structural homology, and thus no recognizable evolutionary relationship, with the eukaryotic RhoGAP or RasGAP fold. Similar to other GAPs, ExoS downregulates Rac using an arginine finger to stabilize the transition state of the GTPase reaction, but the details of the ExoS-Rac interaction are unique. Considering the intrinsic resistance of P. aeruginosa to antibiotics, this might open up a new avenue towards blocking its pathogenicity.     

Rac downregulates Rho activity: reciprocal balance between both GTPases determines cellular morphology and migratory behavior

Using biochemical assays to determine the activation state of Rho-like GTPases, we show that the guanine nucleotide exchange factor Tiam1 functions as a specific activator of Rac but not Cdc42 or Rho in NIH3T3 fibroblasts. Activation of Rac by Tiam1 induces an epithelial-like morphology with functional cadherin-based adhesions and inhibits migration of fibroblasts. This epithelial phenotype is characterized by Rac-mediated effects on Rho activity. Transient PDGF-induced as well as sustained Rac activation by Tiam1 or V12Rac downregulate Rho activity. We found that Cdc42 also downregulates Rho activity. Neither V14Rho or N19Rho affects Rac activity, suggesting unidirectional signaling from Rac towards Rho. Downregulation of Rho activity occurs independently of Rac- induced cytoskeletal changes and cell spreading. Moreover, Rac effector mutants that are defective in mediating cytoskeleton changes or Jun kinase activation both downregulate Rho activity, suggesting that neither of these Rac signaling pathways are involved in the regulation of Rho. Restoration of Rho activity in Tiam1-expressing cells by expression of V14Rho results in reversion of the epithelioid phenotype towards a migratory, fibroblastoid morphology. We conclude that Rac signaling is able to antagonize Rho activity directly at the GTPase level, and that the reciprocal balance between Rac and Rho activity determines cellular morphology and migratory behavior in NIH3T3 fibroblasts.     

Rsr1 and Rap1 GTPases are activated by the same GTPase-activating protein and require threonine 65 for their activation

The Rsr1 protein of Saccharomyces cerevisiae has been shown to be essential for bud site selection (Bender, A., and Pringle, J. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 9976-9980). This protein of 272 amino acids shares approximately 50% sequence identity with both Ras and Rap GTPases. However, neither GTP binding nor GTPase activity of the Rsr1 protein has been reported. The Rsr1 protein shares with human Rap1 GTPases the four specific motifs, i.e. Gly-12, residues 32-40, Ala-59, and residues 64-70, that are required for GAP3-dependent activation of the Rap1 GTPases. In this paper we demonstrate that the intrinsic GTPase activity of the Rsr1 protein is stimulated by GAP3 purified from bovine brain cytosol. The Rsr1 GTPase is not activated by either GAP1 or GAP2 which are specific for the Ras and Rho GTPases, respectively. Thus, it appears that the Rsr1 GTPase is a new member of the Rap1 GTPase family. Replacement of Gly-12 by Val in the Rsr1 GTPase completely abolishes the GAP3-dependent activation. The chimeric GTPases, Ras(1-60)/Rsr1(61-168) and Rsr1(1-65)/Ras(66-189), are activated by GAP3 but not by GAP1. Replacement of Thr-65 by Ser in the latter chimeric GTPase completely abolishes the GAP3-dependent activation, indicating that Thr-65 is required for distinguishing GAP3 from GAP1. We have previously shown that Gln-61 and Ser-65 are sufficient to determine the GAP1 specificity. Replacement of Thr-35 by Ala in the common effector domain (residues 32-40) of the chimeric Ras/Rsr1 GTPases completely abolishes GAP3-dependent activation.     

A dual functional signal mediator showing RhoGAP and phospholipase C-delta stimulating activities.

We have cloned a novel regulator protein, p122, in the PLC-delta signalling pathway by screening a rat brain expression library with antiserum raised against purified phospholipase C-delta 1 (PLC-delta 1). This novel p122-RhoGAP binds to PLC-delta 1 and activates the phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolyzing activity of PLC-delta 1. As suggested by the deduced amino acid sequence, this regulator protein shows a similarity to the GTPase activating protein (GAP) homology region of Bcr and possesses GAP activity for RhoA, but not for Rac1; no guanine nucleotide exchange activity for RhoA and Rac1 was detected. These findings suggest that this novel RhoGAP is involved in the Rho signalling pathway, probably downstream of Rho activation, and mediates the stimulation of PLC-delta, which leads to actin-related cytoskeletal changes through the hydrolysis of PIP2, which binds to actin binding proteins such as gelsolin and profilin.     

Stimulation of phospholipase C-beta2 by the Rho GTPases Cdc42Hs and Rac1.

Neutrophils contain a soluble guanine-nucleotidebinding protein, made up of two components with molecular masses of 23 and 26 kDa, that mediates stimulation of phospholipase C-beta2 (PLCbeta2). We have identified the two components of the stimulatory heterodimer by amino acid sequencing as a Rho GTPase and the Rho guanine nucleotide dissociation inhibitor LyGDI. Using recombinant Rho GTPases and LyGDI, we demonstrate that PLCbeta2 is stimulated by guanosine 5'-O-(3-thiotriphosphate) (GTP[S])-activated Cdc42HsxLyGDI, but not by RhoAxLyGDI. Stimulation of PLCbeta2, which was also observed for GTP[S]-activated recombinant Rac1, was independent of LyGDI, but required C-terminal processing of Cdc42Hs/Rac1. Cdc42Hs/Rac1 also stimulated PLCbeta2 in a system made up of purified recombinant proteins, suggesting that this function is mediated by direct protein-protein interaction. The Cdc42Hs mutants F37A and Y40C failed to stimulate PLCbeta2, indicating that the Cdc42Hs effector site is involved in this interaction. The results identify PLCbeta2 as a novel effector of the Rho GTPases Cdc42Hs and Rac1, and as the first mammalian effector directly regulated by both heterotrimeric and low-molecular-mass GTP-binding proteins.     

Rap-afadin axis in control of Rho signaling and endothelial barrier recovery

Activation of the Rho GTPase pathway determines endothelial cell (EC) hyperpermeability after injurious stimuli. To date, feedback mechanisms of Rho down-regulation critical for barrier restoration remain poorly understood. We tested a hypothesis that Rho down-regulation and barrier recovery of agonist-stimulated ECs is mediated by the Ras family GTPase Rap1. Thrombin-induced EC permeability driven by rapid activation of the Rho GTPase pathway was followed by Src kinase–dependent phosphorylation of the Rap1-specific guanine nucleotide exchange factor (GEF) C3G, activation of Rap1, and initiation of EC barrier recovery. Knockdown experiments showed that Rap1 activation was essential for down-regulation of Rho signaling and actin stress fiber dissolution. Rap1 activation also enhanced interaction between adherens junction (AJ) proteins VE-cadherin and p120-catenin and stimulated AJ reannealing mediated by the Rap1 effector afadin. This mechanism also included Rap1-dependent membrane translocation of the Rac1-specific GEF Tiam1 and activation of Rac1-dependent peripheral cytoskeletal dynamics, leading to resealing of intercellular gaps. These data demonstrate that activation of the Rap1-afadin axis is a physiological mechanism driving restoration of barrier integrity in agonist-stimulated EC monolayers via negative-feedback regulation of Rho signaling, stimulation of actin peripheral dynamics, and reestablishment of cell–cell adhesive complexes.     

Nadrin GAP activity is isoform- and target-specific regulated by tyrosine phosphorylation

Cytoskeletal reorganization is crucial for platelet adhesion and thrombus formation to avoid excessive bleeding. Major regulators of cytoskeletal dynamics are small GTPases of the Rho family. Rho GTPases become activated by G-protein coupled receptor activation, downstream of immunoreceptor tyrosine-based activation motif (ITAM)-coupled receptors and by outside-in signaling of integrins. They act as molecular switches and cycle between active and inactive states. GTPase activating proteins (GAPs) stimulate the hydrolysis of GTP to GDP to terminate Rho signaling. Nadrin is a RhoGAP that was recently identified in platelets. Five Nadrin isoforms are known consisting of a unique GAP and an N-terminal BAR domain responsible for the selective regulation of RhoA, Cdc42 and Rac1. Besides BAR domain mediated regulation of Nadrin GAP activity nothing is known about the regulation of Nadrin and the impact on cytoskeletal reorganization. Here we show that Nadrin becomes tyrosine phosphorylated upon platelet activation. We found Src family proteins (Src, Lyn, Fyn) to be responsible to control Nadrin GAP activity by phosphorylation. Interestingly, phosphorylation of Nadrin leads to tightly regulated Rho activation that was found to be Nadrin isoform- and (Rho) target-specific. Src-phosphorylation of Nadrin5 mediated inactivation of Cdc42 while RhoA and Rac1 became activated upon Src-mediated phosphorylation of Nadrin2. Our results suggest a critical role for spatial and temporal regulation of Nadrin and thus for the control of Rho GTPases in platelets.     

Targeting and activation of Rac1 are mediated by the exchange factor beta-Pix

Rho guanosine triphosphatases (GTPases) are critical regulators of cytoskeletal dynamics and control complex functions such as cell adhesion, spreading, migration, and cell division. It is generally accepted that localized GTPase activation is required for the proper initiation of downstream signaling events, although the molecular mechanisms that control targeting of Rho GTPases are unknown. In this study, we show that the Rho GTPase Rac1, via a proline stretch in its COOH terminus, binds directly to the SH3 domain of the Cdc42/Rac activator beta-Pix (p21-activated kinase [Pak]-interacting exchange factor). The interaction with beta-Pix is nucleotide independent and is necessary and sufficient for Rac1 recruitment to membrane ruffles and to focal adhesions. In addition, the Rac1-beta-Pix interaction is required for Rac1 activation by beta-Pix as well as for Rac1-mediated spreading. Finally, using cells deficient for the beta-Pix-binding kinase Pak1, we show that Pak1 regulates the Rac1-beta-Pix interaction and controls cell spreading and adhesion-induced Rac1 activation. These data provide a model for the intracellular targeting and localized activation of Rac1 through its exchange factor beta-Pix.     

Activation of Rac1 by Rho-guanine nucleotide dissociation inhibitor-beta with defective isoprenyl-binding pocket

Rho-guanine nucleotide dissociation inhibitor-beta (RhoGDIbeta), a regulator for Rho GTPases, is implicated in cancer cell progression. We reported that C-terminal truncated RhoGDIbeta (DeltaC(166-201)-RhoGDIbeta) promoted metastasis through activating Rac1 signaling pathway in ras-transformed fibroblast cells. To better understand the mechanism of Rac1 activation by DeltaC(166-201)-RhoGDIbeta during metastasis, the amount of GTP-bound Rac1 was measured as the activation level of Rac1 in cells expressing various mutant RhoGDIbeta with sequential C-terminal deletions. Three C-terminal hydrophobic amino acid residues (Trp191, Leu193, and Ile195) supposed to interact with isoprenyl groups of Rac1, was indispensable for a proper regulation of Rac1 activation/inhibition. Deletion of this region led RhoGDIbeta to continuously associate with GTP-bound Rac1, provoking constitutive activation of Rac1. Thus, impaired interaction of RhoGDIbeta with Rac1 isoprenyl groups possibly makes RhoGDIbeta function as a positive regulator for Rac1 during metastasis.     

The RhoGAP domain of CYK-4 has an essential role in RhoA activation

Cytokinesis in animal cells is mediated by a cortical actomyosin-based contractile ring. The GTPase RhoA is a critical regulator of this process as it activates both nonmuscle myosin and a nucleator of actin filaments [1]. The site at which active RhoA and its effectors accumulate is controlled by the microtubule-based spindle during anaphase [2]. ECT-2, the guanine nucleotide exchange factor (GEF) that activates RhoA during cytokinesis, is regulated by phosphorylation and subcellular localization [3-5]. ECT2 localization depends on interactions with CYK-4/MgcRacGAP, a Rho GTPase-activating protein (GAP) domain containing protein [5, 6]. Here we show that, contrary to expectations, the Rho GTPase-activating protein (GAP) domain of CYK-4 promotes activation of RhoA during cytokinesis. Furthermore, we show that the primary phenotype caused by mutations in the GAP domain of CYK-4 is not caused by ectopic activation of CED-10/Rac1 and ARX-2/Arp2. However, inhibition of CED-10/Rac1 and ARX-2/Arp2 facilitates ingression of weak cleavage furrows. These results demonstrate that?a GAP domain can contribute to activation of a small GTPase. Furthermore, cleavage furrow ingression is sensitive to the balance of contractile forces and cortical tension.     

A C-terminal domain of GAP is sufficient to stimulate ras p21 GTPase activity.

The cDNA for bovine ras p21 GTPase activating protein (GAP) has been cloned and the 1044 amino acid polypeptide encoded by the clone has been shown to bind the GTP complexes of both normal and oncogenic Harvey (Ha) ras p21. To identify the regions of GAP critical for the catalytic stimulation of ras p21 GTPase activity, a series of truncated forms of GAP protein were expressed in Escherichia coli. The C-terminal 343 amino acids of GAP (residues 702-1044) were observed to bind Ha ras p21-GTP and stimulate Ha ras p21 GTPase activity with the same efficiency (kcat/KM congruent to 1 x 10(6) M-1 s-1 at 24 degrees C) as GAP purified from bovine brain or full-length GAP expressed in E. coli. Deletion of the final 61 amino acid residues of GAP (residues 986-1044) rendered the protein insoluble upon expression in E. coli. These results define a distinct catalytic domain at the C terminus of GAP. In addition, GAP contains amino acid similarity with the B and C box domains conserved among phospholipase C-II, the crk oncogene product, and the non-receptor tyrosine kinase oncogene products. This homologous region is located in the N-terminal half of GAP outside of the catalytic domain that stimulates ras p21 GTPase activity and may constitute a distinct structural or functional domain within the GAP protein.     

The activity of the GTPase-activating protein CdGAP is regulated by the endocytic protein intersectin.

The Rho GTPases RhoA, Rac1, and Cdc42 play a major role in regulating the reorganization of the actin cytoskeleton. We recently identified CdGAP, a novel GTPase-activating protein with activity toward Rac1 and Cdc42. CdGAP consists of a N-terminal GAP domain, a central domain, and a C-terminal proline-rich domain. Here we show that through a subset of its Src homology 3 domains, the endocytic protein intersectin interacts with CdGAP. In platelet-derived growth factor-stimulated Swiss 3T3 cells, intersectin co-localizes with CdGAP and inhibits its GAP activity toward Rac1. Intersectin-Src homology 3 also inhibits CdGAP activity in GAP assays in vitro. Although the C-terminal proline-rich domain of CdGAP is required for the regulation of its GAP activity by intersectin both in vivo and in vitro, it is not necessary for CdGAP-intersectin interaction. Our data suggest that the central domain of CdGAP is required for CdGAP-intersectin interaction. Thus, we propose a model in which intersectin binding results in a change of CdGAP conformation involving the proline-rich domain that leads to the inhibition of its GAP activity. These observations provide the first demonstration of a direct regulation of RhoGAP activity through a protein-protein interaction and suggest a function for intersectin in Rac1 regulation and actin dynamics.