MgcRacGAP, a new human GTPase-activating protein for Rac and Cdc42 similar to Drosophila rotundRacGAP gene product, is expressed in male germ cells

In a search for new partners of the activated form of Rac GTPase, we have isolated through a two-hybrid cloning procedure a human cDNA encoding a new GTPase-activating protein (GAP) for Rho family GTPases. A specific mRNA of 3.2 kilobases was detected in low abundance in many cell types and found highly expressed in testis. A protein of the predicted size 58 kDa, which we call MgcRacGAP, was 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 of 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.     

An SH3 domain-containing GTPase-activating protein for Rho and Cdc42 associates with focal adhesion kinase

The integrin family of cell surface receptors mediates cell adhesion to components of the extracellular matrix (ECM). Integrin engagement with the ECM initiates signaling cascades that regulate the organization of the actin-cytoskeleton and changes in gene expression. The Rho subfamily of Ras-related low-molecular-weight GTP-binding proteins and several protein tyrosine kinases have been implicated in mediating various aspects of integrin-dependent alterations in cell homeostasis. Focal adhesion kinase (FAK or pp125FAK) is one of the tyrosine kinases predicted to be a critical component of integrin signaling. To elucidate the mechanisms by which FAK participates in integrin-mediated signaling, we have used expression cloning to identify cDNAs that encode potential FAK-binding proteins. We report here the identification of a cDNA that encodes a new member of the GTPase-activating protein (GAP) family of GTPase regulators. This GAP, termed Graf (for GTPase regulator associated with FAK), binds to the C-terminal domain of FAK in an SH3 domain-dependent manner and preferentially stimulates the GTPase activity of the GTP-binding proteins RhoA and Cdc42. Subcellular localization studies using Graf-transfected chicken embryo cells indicates that Graf colocalizes with actin stress fibers, cortical actin structures, and focal adhesions. Graf mRNA is expressed in a variety of avian tissues and is particularly abundant in embryonic brain and liver. Graf represents the first example of a regulator of the Rho family of small GTP-binding proteins that exhibits binding to a protein tyrosine kinase. We suggest that Graf may function to mediate cross talk between the tyrosine kinases such as FAK and the Rho family GTPase that control steps in integrin-initiated signaling events.     

Regulation of dendritic growth and remodeling by Rho, Rac, and Cdc42

The acquisition of cell type-specific morphologies is a central feature of neuronal differentiation and has important consequences for nervous system function. To begin to identify the underlying molecular mechanisms, we have explored the role of Rho-related GTPases in the dendritic development of cortical neurons. Expression of dominant negative mutants of Rac or Cdc42, the Rho-inhibitory molecule C3 transferase, or the GTPase-activating protein RhoGAP p190 causes a marked reduction in the number of primary dendrites in nonpyramidal (multipolar) neurons and in the number of basal dendrites in neurons with pyramidal morphologies. Conversely, the expression of constitutively active mutants of Rho, Rac, or Cdc42 leads to an increase in the number of primary and basal dendrites. In cortical cultures, as in vivo, dendritic remodeling leads to an apparent transformation from pyramidal to nonpyramidal morphologies over time. Strikingly, this shift in favor of nonpyramidal morphologies is also inhibited by the expression of dominant negative mutants of Cdc42 and Rac and by RhoGAP p190. These observations indicate that Rho, Rac, and Cdc42 play a central role in dendritic development and suggest that differential activation of Rho-related GTPases may contribute to the generation of morphological diversity in the developing cortex.     

Activation of Rac and Cdc42 by integrins mediates cell spreading

Adhesion to ECM is required for many cell functions including cytoskeletal organization, migration, and proliferation. We observed that when cells first adhere to extracellular matrix, they spread rapidly by extending filopodia-like projections and lamellipodia. These structures are similar to the Rac- and Cdc42-dependent structures observed in growth factor-stimulated cells. We therefore investigated the involvement of Rac and Cdc42 in adhesion and spreading on the ECM protein fibronectin. We found that integrin-dependent adhesion led to the rapid activation of p21-activated kinase, a downstream effector of Cdc42 and Rac, suggesting that integrins activate at least one of these GTPases. Dominant negative mutants of Rac and Cdc42 inhibit cell spreading in such a way as to suggest that integrins activate Cdc42, which leads to the subsequent activation of Rac; both GTPases then contribute to cell spreading. These results demonstrate that initial integrin-dependent activation of Rac and Cdc42 mediates cell spreading.     

RhoG GTPase controls a pathway that independently activates Rac1 and Cdc42Hs

RhoG is a member of the Rho family of GTPases that shares 72% and 62% sequence identity with Rac1 and Cdc42Hs, respectively. We have expressed mutant RhoG proteins fused to the green fluorescent protein and analyzed subsequent changes in cell surface morphology and modifications of cytoskeletal structures. In rat and mouse fibroblasts, green fluorescent protein chimera and endogenous RhoG proteins colocalize according to a tubular cytoplasmic pattern, with perinuclear accumulation and local concentration at the plasma membrane. Constitutively active RhoG proteins produce morphological and cytoskeletal changes similar to those elicited by a simultaneous activation of Rac1 and Cdc42Hs, i.e., the formation of ruffles, lamellipodia, filopodia, and partial loss of stress fibers. In addition, RhoG and Cdc42Hs promote the formation of microvilli at the cell apical membrane. RhoG-dependent events are not mediated through a direct interaction with Rac1 and Cdc42Hs targets such as PAK-1, POR1, or WASP proteins but require endogenous Rac1 and Cdc42Hs activities: coexpression of a dominant negative Rac1 impairs membrane ruffling and lamellipodia but not filopodia or microvilli formation. Conversely, coexpression of a dominant negative Cdc42Hs only blocks microvilli and filopodia, but not membrane ruffling and lamellipodia. Microtubule depolymerization upon nocodazole treatment leads to a loss of RhoG protein from the cell periphery associated with a reversal of the RhoG phenotype, whereas PDGF or bradykinin stimulation of nocodazole-treated cells could still promote Rac1- and Cdc42Hs-dependent cytoskeletal reorganization. Therefore, our data demonstrate that RhoG controls a pathway that requires the microtubule network and activates Rac1 and Cdc42Hs independently of their growth factor signaling pathways.     

The p21Rac/Cdc42-activated kinases (PAKs)

PURPOSE: Purely subjective events, identified as "seizures" by patients but not accompanied by any clinical or EEG changes, commonly occur during evaluation of patients for intractable epilepsy. We wished to determine the significance of such events. METHODS: We analyzed the frequency of subjective episodes in 379 adults who underwent EEG video monitoring as part of their evaluation for intractable epilepsy and the relationship of these experiences to epileptic and psychogenic nonepileptic (NES) seizures. We examined the results of tests of mental ability and of emotional, psychosocial, and quality of life (QOL) adjustment to determine if individuals with subjective events alone could be distinguished from those with epileptic seizures only, NES only, or no events, by these measures. RESULTS: We noted subjective events in 39% (147 of 379) of the patients. Of patients with subjective episodes, 52% also had epileptic seizures, 7% had NES, 1% had both epileptic seizures and NES, and 40% had subjective events alone. As a group, patients with subjective events alone were slightly more intelligent and less impaired neuropsychologically than people in the other groups, but they performed more poorly on several important measures of adjustment. CONCLUSIONS: The identification of persons with purely subjective episodes apart from all other episodes is important both phenomenologically and from a practical viewpoint.     

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.     

Cloning and characterization of a novel Cdc42-associated tyrosine kinase, ACK-2, from bovine brain

Cdc42 plays an important role in intracellular signaling pathways that influence cell morphology and motility and stimulate DNA synthesis. In attempts to determine whether nonreceptor tyrosine kinases play a fundamental role in Cdc42 signaling, we have cloned and biochemically characterized a new Cdc42-associated tyrosine kinase (ACK) from bovine brain. This tyrosine kinase, named ACK-2, has a calculated molecular mass of 83 kDa and shares a number of primary structural domains with the 120-kDa ACK (ACK-1). The main differences between the primary structures of ACK-2 and ACK-1 occur in the amino- and carboxyl-terminal regions. Like ACK-1, ACK-2 binds exclusively to activated (GTP-bound) Cdc42 and does not bind to its closest homologs, e.g. activated Rac. ACK-2 could not be activated by addition of glutathione S-transferase (GST)-Cdc42(Q61L), a GTPase-defective mutant, or by GTPgammaS-loaded GST-Cdc42 in in vitro kinase assays. However, ACK-2 was activated when cotransfected with wild type Cdc42 or Cdc42(Q61L) and stably associated with Cdc42(Q61L) in vivo, indicating that ACK-2 interacts with active Cdc42 in cells. Furthermore, the tyrosine kinase activity of ACK-2 was stimulated both by epidermal growth factor and bradykinin, suggesting that ACK-2 may play a role in the signaling actions of both receptor tyrosine kinases or heterotrimeric G-protein-coupled receptors.     

Role of IQGAP1, a target of the small GTPases Cdc42 and Rac1, in regulation of E-cadherin- mediated cell-cell adhesion

The small guanosine triphosphatases (GTPases) Cdc42 and Rac1 regulate E-cadherin-mediated cell-cell adhesion. IQGAP1, a target of Cdc42 and Rac1, was localized with E-cadherin and beta-catenin at sites of cell-cell contact in mouse L fibroblasts expressing E-cadherin (EL cells), and interacted with E-cadherin and beta-catenin both in vivo and in vitro. IQGAP1 induced the dissociation of alpha-catenin from a cadherin-catenin complex in vitro and in vivo. Overexpression of IQGAP1 in EL cells, but not in L cells expressing an E-cadherin-alpha-catenin chimeric protein, resulted in a decrease in E-cadherin-mediated cell-cell adhesive activity. Thus, IQGAP1, acting downstream of Cdc42 and Rac1, appears to regulate cell-cell adhesion through the cadherin-catenin pathway.     

WAVE, a novel WASP-family protein involved in actin reorganization induced by Rac

Rac is a Rho-family small GTPase that induces the formation of membrane ruffles. However, it is poorly understood how Rac-induced reorganization of the actin cytoskeleton, which is essential for ruffle formation, is regulated. Here we identify a novel Wiskott-Aldrich syndrome protein (WASP)-family protein, WASP family Verprolin-homologous protein (WAVE), as a regulator of actin reorganization downstream of Rac. Ectopically expressed WAVE induces the formation of actin filament clusters that overlap with the expressed WAVE itself. In this actin clustering, profilin, a monomeric actin-binding protein that has been suggested to be involved in actin polymerization, was shown to be essential. The expression of a dominant-active Rac mutant induces the translocation of endogenous WAVE from the cytosol to membrane ruffling areas. Furthermore, the co-expression of a deltaVPH WAVE mutant that cannot induce actin reorganization specifically suppresses the ruffle formation induced by Rac, but has no effect on Cdc42-induced actin-microspike formation, a phenomenon that is also known to be dependent on rapid actin reorganization. The deltaVPH WAVE also suppresses membrane-ruffling formation induced by platelet-derived growth factor in Swiss 3T3 cells. Taken together, we conclude that WAVE plays a critical role downstream of Rac in regulating the actin cytoskeleton required for membrane ruffling.     

Identification of a novel Rac3-interacting protein C1D

The actin cytoskeleton is an important contributor to the integrity of cellular shape and responses in neurons. However, the molecular mechanisms associated with functional interactions between the actin cytoskeleton and neuronal ion channels are largely unknown. Whole-cell and single channel recording techniques were thus applied to identified retinal bipolar neurons of the tiger salamander (Ambystoma tigrinum) to assess the role of acute changes in actin-based cytoskeleton dynamics in the regulation of voltage-gated ion channels. Disruption of endogenous actin filaments after brief treatment (20-30 min) with cytochalasin D (CD) activated voltage-gated K+ currents in bipolar cells, which were largely prevented by intracellular perfusion with the actin filament-stabilizer agent, phalloidin. Either CD treatment under cell-attached conditions or direct addition of actin to excised, inside-out patches of bipolar cells activated and/or increased single K+ channels. Thus, acute changes in actin-based cytoskeleton dynamics regulate voltage-gated ion channel activity in bipolar cells.     

Fibroblast transformation by Fps/Fes tyrosine kinases requires Ras, Rac, and Cdc42 and induces extracellular signal-regulated and c-Jun N-terminal kinase activation

The small GTP-binding proteins Ras, Rac, and Cdc42 link protein-tyrosine kinases with mitogen-activated protein kinase (MAPK) signaling cascades. Ras controls the activation of extracellular signal-regulated kinases (ERKs), while Rac and Cdc42 regulate the c-Jun N-terminal kinases (JNKs). In this study, we investigated whether small G protein/MAPK cascades contribute to signal transduction by transforming variants of c-Fes, a nonreceptor tyrosine kinase implicated in cytokine signaling and myeloid differentiation. First, we investigated the effects of dominant-negative small G proteins on Rat-2 fibroblast transformation by a retroviral homolog of c-Fes (v-Fps) and by c-Fes activated via N-terminal addition of the v-Src myristylation signal (Myr-Fes). We observed that dominant-negative Ras, Rac, and Cdc42 inhibited v-Fps- and Myr-Fes-induced growth of Rat-2 cells in soft agar, indicating that activation of these small GTP-binding proteins is required for fibroblast transformation by Fps/Fes tyrosine kinases. To determine whether MAPK pathways are activated downstream of these small G proteins, we measured ERK and JNK activity in the v-Fps- and Myr-Fes-transformed Rat-2 cells. Both ERK and JNK activities were elevated in the transformed cells, suggesting that these pathways are involved in cellular transformation. Dominant-negative mutants of Ras (but not Rac or Cdc42) specifically inhibited ERK activation by v-Fps and Myr-Fes, demonstrating that ERK activation occurs exclusively downstream of Ras. All three dominant-negative small G proteins inhibited JNK activation by v-Fps and Myr-Fes, indicating that JNK activation by these tyrosine kinases requires both Ras and Rho family GTPases. These data demonstrate that multiple small G protein/MAPK cascades are involved in downstream signal transduction by Fps/Fes tyrosine kinases.     

Structures of Cdc42 bound to the active and catalytically compromised forms of Cdc42GAP

The Rho-related small GTP-binding protein Cdc42 has a low intrinsic GTPase activity that is significantly enhanced by its specific GTPase-activating protein, Cdc42GAP. In this report, we present the tertiary structure for the aluminum fluoride-promoted complex between Cdc42 and a catalytically active domain of Cdc42GAP as well as the complex between Cdc42 and the catalytically compromised Cdc42GAP(R305A) mutant. These structures, which mimic the transition state for the GTP hydrolytic reaction, show the presence of an AIF3 molecule, as was seen for the corresponding Ras-p120RasGAP complex, but in contrast to what has been reported for the Rho-Cdc42GAP complex or for heterotrimeric G protein alpha subunits, where AIF4- was observed. The Cdc42GAP stabilizes both the switch I and switch II domains of Cdc42 and contributes a highly conserved arginine (Arg 305) to the active site. Comparison of the structures for the wild type and mutant Cdc42GAP complexes provides important insights into the GAP-catalyzed GTP hydrolytic reaction.     

The small GTPases Cdc42Hs, Rac1 and RhoG delineate Raf-independent pathways that cooperate to transform NIH3T3 cells

BACKGROUND: Ras-mediated transformation of mammalian cells has been shown to activate multiple signalling pathways, including those involving mitogen-activated protein kinases and the small GTPase Rho. Members of the Rho family affect cell morphology by controlling the formation of actin-dependent structures: specifically, filopodia are induced by Cdc42Hs, lamellipodia and ruffles by Rac, and stress fibers by RhoA. In addition, Rho GTPases are involved in progression through the G1 phase of the cell cycle, and Rac1 and RhoA have recently been directly implicated in the morphogenic and mitogenic responses to transformation by oncogenic Ras. In order to examine the cross-talk between Ras and Rho proteins, we investigated the effects on focus-forming activity and cell growth of the Rho-family members Cdc42Hs, Rac1 and RhoG by expressing constitutively active or dominant-negative forms in NIH3T3 cells. RESULTS: Expression of Rac1 or RhoG modulated the saturation density to which the cells grew, probably by affecting the level of contact inhibition. Although all three GTPases were required for cell transformation mediated by Ras but not by constitutively active Raf, the selective activation of each GTPase was not sufficient to induce the formation of foci. The coordinated activation of Cdc42Hs, RhoG and Rac1, however, elicited a high focus-forming activity, independent of the mitogen-activated ERK and JNK protein kinase pathways. CONCLUSIONS: Ras-mediated transformation induces extensive changes in cell morphology which require the activity of members of the Rho family of GTPases. Our data show that the pattern of coordinated Rho family activation that elicits a focus-forming activity in NIH3T3 cells is distinct from the regulatory cascade that has been proposed for the control of actin-dependent structures in Swiss 3T3 cells.     

Characterization of the association of the actin-binding protein, IQGAP, and activated Cdc42 with Golgi membranes

IQGAP is a recently identified actin-binding protein, which is a putative target for the Cdc42 and Rac GTP-binding proteins. Cdc42 was localized to the Golgi (Erickson, J. W., Zhang, C., Kahn, R. A., Evans, T., and Cerione, R. A. (1996) J. Biol. Chem. 271, 26850-26854), and here we show by immunofluorescence that IQGAP has a perinuclear localization, that it can be co-immunoprecipitated with Cdc42 from Golgi-enriched fractions, and that purified Golgi membranes are recognized by specific antibodies raised against IQGAP and Cdc42 in negative-stain immunogold electron microscopy experiments. Addition of activated, recombinant Cdc42 or solubilization of endogenous Cdc42 from Golgi membranes by the Rho-GDP dissociation inhibitor protein fails to solubilize IQGAP, suggesting that it associates with these membranes in a Cdc42-independent manner. Detergent solubilization of Golgi membranes leaves IQGAP and actin in an insoluble pellet but releases Cdc42 to the supernatant, whereas treatments that release actin from this detergent-insoluble pellet also release IQGAP. Addition of the COOH-terminal half of the IQGAP protein, which contains the Cdc42-binding domain, removes Cdc42 from Golgi membranes in a dose-dependent manner. These data suggest that IQGAP and Cdc42 are part of a cytoskeletal complex in Golgi membranes that may mediate Cdc42-regulated effects on the actin cytoskeleton in these membranes.     

A role for Cdc42 in macrophage chemotaxis

Grammatical morphemes, such as articles and auxiliary verbs, provide potentially useful information to language learners. However, children with specific language impairment (SLI) frequently fail to produce grammatical morphemes, raising questions about their sensitivity to them. To address this issue, two experiments were conducted in which 3- to 5-year-old children with SLI and with normally developing language (NL) heard sentences asking them to identify a picture corresponding to a named target word. The target occurred in either a grammatical sentence or one with an incorrectly used grammatical morpheme. In Experiment 1, the picture representing the target occurred with three unrelated distractor pictures. In Experiment 2, distractor sets included pictures that were semantically related to the target. In both studies, the SLI group chose fewer correct pictures when the target followed an incorrectly used morpheme. In Experiment 2, the SLI group chose more semantically related than unrelated distractors. These results suggest that children with SLI are sensitive to grammatical morphemes and that their incorrect picture choices may reflect a failure to maintain the target in memory.     

Agents that inhibit Rho, Rac, and Cdc42 do not block formation of actin pedestals in HeLa cells infected with enteropathogenic Escherichia coli

Enteropathogenic Escherichia coli (EPEC) induces formation of actin pedestals in infected host cells. Agents that inhibit the activity of Rho, Rac, and Cdc42, including Clostridium difficile toxin B (ToxB), compactin, and dominant negative Rho, Rac, and Cdc42, did not inhibit formation of actin pedestals. In contrast, treatment of HeLa cells with ToxB inhibited EPEC invasion. Thus, Rho, Rac, and Cdc42 are not required for assembly of actin pedestals; however, they may be involved in EPEC uptake by HeLa cells.     

PAK4, a novel effector for Cdc42Hs, is implicated in the reorganization of the actin cytoskeleton and in the formation of filopodia

The GTPases Rac and Cdc42Hs control diverse cellular functions. In addition to being mediators of intracellular signaling cascades, they have important roles in cell morphogenesis and mitogenesis. We have identified a novel PAK-related kinase, PAK4, as a new effector molecule for Cdc42Hs. PAK4 interacts only with the activated form of Cdc42Hs through its GTPase-binding domain (GBD). Co-expression of PAK4 and the constitutively active Cdc42HsV12 causes the redistribution of PAK4 to the brefeldin A-sensitive compartment of the Golgi membrane and the subsequent induction of filopodia and actin polymerization. Importantly, the reorganization of the actin cytoskeleton is dependent on PAK4 kinase activity and on its interaction with Cdc42Hs. Thus, unlike other members of the PAK family, PAK4 provides a novel link between Cdc42Hs and the actin cytoskeleton. The cellular locations of PAK4 and Cdc42Hs suggest a role for the Golgi in cell morphogenesis.