Protection from Clostridium difficile toxin B-catalysed Rac1/Cdc42 glucosylation by tauroursodeoxycholic acid-induced Rac1/Cdc42 phosphorylation

Toxin A (TcdA) and toxin B (TcdB) are the major virulence factors of Clostridium difficile-associated diarrhoea (CDAD). TcdA and TcdB mono-glucosylate small GTPases of the Rho family, thereby causing actin re-organisation in colonocytes, resulting in the loss of colonic barrier function. The hydrophilic bile acid tauroursodeoxycholic acid (TUDCA) is an approved drug for the treatment of cholestasis and biliary cirrhosis. In this study, TUDCA-induced activation of Akt1 is presented to increase cellular levels of pS71-Rac1/Cdc42 in human hepatocarcinoma (HepG2) cells, showing for the first time that bile acid signalling affects the activity of Rho proteins. Rac1/Cdc42 phosphorylation, in turn, protects Rac1/Cdc42 from TcdB-catalysed glucosylation and reduces the TcdB-induced cytopathic effects in HepG2 cells. The results of this study indicate that TUDCA may prove useful as a therapeutic agent for the treatment of CDAD.     

Regulation of anoikis by Cdc42 and Rac1

Ras family small GTPases play a critical role in malignant transformation, and Rho subfamily members contribute significantly to this process. Anchorage-independent growth and the ability to avoid detachment-induced apoptosis (anoikis) are hallmarks of transformed epithelial cells. In this study, we have demonstrated that constitutive activation of Cdc42 inhibits anoikis in Madin-Darby canine kidney (MDCK) epithelial cells. We showed that activated Cdc42 stimulates the ERK, JNK, and p38 MAPK pathways in suspension condition; however, inhibition of these signaling does not affect Cdc42-stimulated cell survival. However, we demonstrated that inhibition of phosphatidylinositol 3-kinase (PI3K) pathway abolishes the protective effect of Cdc42 on anoikis. Taking advantage of a double regulatory expression system, we also showed that Cdc42-stimulated cell survival in suspension condition is, at least in part, mediated by Rac1. We also provide evidence for a positive feedback loop involving Rac1 and PI3K. In addition, we show that the survival functions of both constitutively active Cdc42 and Rac1 GTPases are abrogated by Latrunculin B, an actin filament-depolymerizing agent, implying an important role for the actin cytoskeleton in mediating survival signaling activated by Cdc42 and Rac1. Together, our results indicate a role for Cdc42 in anchorage-independent survival of epithelial cells. We also propose that this survival function depends on a positive feedback loop involving Rac1 and PI3K.     

Adiponectin promotes migration activities of endothelial progenitor cells via Cdc42/Rac1

Adiponectin has anti-atherosclerotic effects through its direct actions on vascular cells. The present study investigates the molecular mechanisms of adiponectin in the migration of endothelial progenitor cells (EPCs) which play an important role in neovascularization and re-endothelization. The phosphorylation of Akt and the activations of Cdc42 and Rac1 were significantly increased by adiponectin. Adiponectin increased the migration activity of EPCs, which was completely inhibited by a PI3-kinase inhibitor. siRNA of Cdc42 or Rac1 completely inhibited the adiponectin-induced migration, but siRNA of Akt had no effects, indicating that adiponectin promotes the migration activities of EPCs mainly through PI3-kinase/Cdc42/Rac1.

Structured summary

MINT-7217629: PAK1 (uniprotkb:Q13153) physically interacts (MI:0914) with CDC42 (uniprotkb:P60953) by pull down (MI:0096)MINT-7217644: PAK1 (uniprotkb:Q13153) physically interacts (MI:0914) with Rac1 (uniprotkb:P63000) by pull down (MI:0096)     

Inhibition of the calcium release-activated calcium (CRAC) current in Jurkat T cells by the HIV-1 envelope protein gp160.

The HIV-1 envelope glycoprotein gp120/160 has pleiotropic effects on T cell function. We investigated whether Ca(2+) signaling, a crucial step for T cell activation, was altered by prolonged exposure of Jurkat T cells to gp160. Microfluorometric measurements showed that Jurkat cells incubated with gp160 had smaller (approximately 40%) increases in [Ca(2+)](i) in response to phytohemagglutinin and had a reduced Ca(2+) influx (approximately 25%). gp160 had similar effects on Jurkat cells challenged with thapsigargin. We used the patch clamp technique to record the Ca(2+) current, which is responsible for Ca(2+) influx and has properties of the calcium release-activated Ca(2+) current (I(CRAC)). gp160 reduced I(CRAC) by approximately 40%. The inhibitory effects of gp160 were antagonized by staurosporine (0.1 microm), an inhibitor of protein-tyrosine kinases and protein kinase Cs (PKCs), and by G? 6976 (5 microm), an inhibitor acting especially on PKC alpha and PKC beta I. 12-O-Tetradecanoyl phorbol 13-acetate (16 nm), a PKC activator, reproduced the effects of gp160 in untreated cells. A Western blotting analysis of PKC isoforms alpha, beta I, delta, and zeta showed that only the cellular distribution of PKC alpha and -beta I were significantly modified by gp160. In addition, gp160 was able to modify the subcellular distribution of PKC alpha and PKC beta I caused by phytohemagglutinin. Therefore the reduction in I(CRAC) caused by prolonged incubation with gp160 is probably mediated by PKC alpha or -beta I.     

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.     

Akt mediates Rac/Cdc42-regulated cell motility in growth factor-stimulated cells and in invasive PTEN knockout cells

Growth factors promote cell survival and cell motility, presumably through the activation of Akt and the Rac and Cdc42 GTPases, respectively. Because Akt is dispensable for Rac/Cdc42 regulation of actin reorganization, it has been assumed that Rac and Cdc42 stimulate cell motility independent of Akt in mammalian cells. However, in this study we demonstrate that Akt is essential for Rac/Cdc42-regulated cell motility in mammalian fibroblasts. A dominant-negative Akt inhibits cell motility stimulated by Rac/Cdc42 or by PDGF treatment, without affecting ruffling membrane-type actin reorganization. We have confirmed a previous report that Akt is activated by expression of Rac and Cdc42 and also observed colocalization of endogenous phosphorylated Akt with Rac and Cdc42 at the leading edge of fibroblasts. Importantly, expression of active Akt but not the closely related kinase SGK is sufficient for increasing cell motility. This effect of Akt is cell autonomous and not mediated by inhibition of GSK3. Finally, we found that dominant-negative Akt but not SGK reverses the increased cell motility phenotype of fibroblasts lacking the PTEN tumor suppressor gene. Taken together, these results suggest that Akt promotes cell motility downstream of Rac/Cdc42 in growth factor-stimulated cells and in invasive PTEN-deficient cells.     

Lovastatin-induced apoptosis in macrophages through the Rac1/Cdc42/JNK pathway

Statins, inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase, have been used successfully in the treatment of hypercholesterolemia for more than a decade. Statins also exhibit overall clinical benefits on cardiovascular diseases independent of their effects on lowering serum cholesterol levels. These beneficial effects of statin therapy are believed to be due, at least in part, to the anti-inflammatory and immunomodulatory roles of statins. Statin treatment reduces the levels of inflammatory markers, decreases the activation and recruitment of immune cells, and delays the progression of atherosclerosis, a chronic inflammatory disease. However, little is known about the direct impact of statins on immune cells, particularly on macrophages. We report that lovastatin, a member of the statin family, effectively induces apoptosis in macrophages. Further investigation of the molecular mechanism has revealed that Rac1 and Cdc42, the small GTPase family members, may play an important role in lovastatin-induced macrophage apoptosis. Moreover, the activation of the JNK pathway may contribute to this event. Our findings provide a better understanding of the molecular basis underlying the anti-inflammatory clinical benefits of statin therapy in cardiovascular diseases.     

Small GTPase Rin induces neurite outgrowth through Rac/Cdc42 and calmodulin in PC12 cells

The novel Ras-like small GTPase Rin is expressed prominently in adult neurons, and binds calmodulin (CaM) through its COOH-terminal-binding motif. It might be involved in calcium/CaM-mediated neuronal signaling, but Rin-mediated signal transduction pathways have not yet been elucidated. Here, we show that expression of Rin induces neurite outgrowth without nerve growth factor or mitogen-activated protein kinase activation in rat pheochromocytoma PC12 cells. Rin-induced neurite outgrowth was markedly inhibited by coexpression with dominant negative Rac/Cdc42 protein or CaM inhibitor treatment. We also found that expression of Rin elevated the endogenous Rac/Cdc42 activity. Rin mutant proteins, in which the mutation disrupted association with CaM, failed to induce neurite outgrowth irrespective of Rac/Cdc42 activation. Disruption of endogenous Rin function inhibited the neurite outgrowth stimulated by forskolin and extracellular calcium entry through voltage-dependent calcium channel evoked by KCl. These findings suggest that Rin-mediated neurite outgrowth signaling requires not only endogenous Rac/Cdc42 activation but also Rin-CaM association, and that endogenous Rin is involved in calcium/CaM-mediated neuronal signaling pathways.     

CD151蛋白激活Rac/Cdc42通路并促进血管生成

目的研究CD151及其突变体CD151-AAA194-196对人脐静脉内皮细胞(HUVEC)增殖及激活Rac/cdc42通路的影响,探讨CD151促血管生成的机制。方法构建pAAV-CD151及其突变体pAAV-CD151-AAA194-196(整合素结合缺陷突变体),测序成功后分别转染入HUVEC。CCK-8法测定pAAV-CD151及其突变体pAAV-CD151-AAA194-196在HUVEC中增殖的能力,Western Blot方法检测CD151及突变体转染后CD151蛋白的表达及对激活Rac/Cdc42通路的影响。结果 pAAV-CD151组的CD151蛋白表达高于正常对照组、pAAV-GFP组,但pAAV-CD151组及pAAV-CD151-AAA194-196组之间CD151蛋白表达没有统计学意义(P>0.05)。CCK-8检测结果示:Control组、pAAV-GFP组、pAAV-CD151组、pAAV-CD151-AAA194-196组OD值分别为1.491±1.780、1.403±1.776、2.742±2.49和1.66±1.845。pAAV-CD151组较pAAV-GFP组和Control组细胞增殖能力明显增强(P<0.01),pAAV-CD151-AAA194-196组较pAAV-CD151组细胞增殖能力减弱(P<0.05)。此外,pAAV-CD151组中P-Rac/cdc42表达较pAAV-GFP组和正常对照组明显增加(P<0.01),pAAV-CD151-AAA194-196组较pAAV-CD151组P-Rac/cdc42表达降低(P<0.05)。结论 CD151通过与整合素形成功能性复合体机制促进Rac/Cdc42通路的激活,进而促进血管生成作用。上述机制可能为CD151促血管生成的重要机制。     

Conformation of a Cdc42/Rac interactive binding peptide in complex with Cdc42 and analysis of the binding interface.

Most of the putative effectors for the Rho-family small GTPases Cdc42 and Rac share a common sequence motif referred to as the Cdc42/Rac interactive binding (CRIB) motif. This sequence, with a consensus of I-S-x-P-(x)2-4-F-x-H-x-x-H-V-G [Burbelo, P. D., et al. (1995) J. Biol. Chem. 270, 29071-29074], has been shown to be essential for the functional interactions between these effector proteins and Cdc42. We have characterized the interactions of a 22-residue CRIB peptide derived from human PAK2 [PAK2(71-92)] with Cdc42 using proton and heteronuclear NMR spectroscopy. This CRIB peptide binds to GTP-gammaS-loaded Cdc42 in a saturable manner, with an apparent Kd of 0.6 microM, as determined by fluorescence titration using sNBD-labeled Cdc42. Interaction of the 22-residue peptide PAK2(71-92) with GTP-gammaS-loaded Cdc42 causes resonance perturbations in the 1H-15N HSQC spectrum of Cdc42 that are similar to those observed for a longer (46-amino acid) CRIB-containing protein fragment [Guo, W., et al. (1998) Biochemistry 37, 14030-14037]. Proton NMR studies of PAK2(71-92) demonstrate structuring of PAK2(71-92) in the presence of GTP-gammaS-loaded Cdc42, through the observation of many nonsequential transferred NOEs. Structure calculations based on the observed transferred NOEs show that the central portion of the Cdc42-bound CRIB peptide assumes a loop conformation in which the side chains of consensus residues Phe80, His82, Ile84, His85, and Val86 are brought into proximity. The CRIB motif may therefore represent a minimal interfacial region in the complexes between Cdc42 and its effector proteins.     

Polarized trafficking of E-cadherin is regulated by Rac1 and Cdc42 in Madin-Darby canine kidney cells

E-cadherin is a major cell-cell adhesion protein of epithelia that is trafficked to the basolateral cell surface in a polarized fashion. The exact post-Golgi route and regulation of E-cadherin transport have not been fully described. The Rho GTPases Cdc42 and Rac1 have been implicated in many cell functions, including the exocytic trafficking of other proteins in polarized epithelial cells. These Rho family proteins are also associated with the cadherin-catenin complexes at the cell surface. We have used functional mutants of Rac1 and Cdc42 and inactivating toxins to demonstrate specific roles for both Cdc42 and Rac1 in the post-Golgi transport of E-cadherin. Dominant-negative mutants of Cdc42 and Rac1 accumulate E-cadherin at a distinct post-Golgi step. This accumulation occurs before p120^ctn interacts with E-cadherin, because p120^ctn localization was not affected by the Cdc42 or Rac1 mutants. Moreover, the GTPase mutants had no effect on the trafficking of a targeting mutant of E-cadherin, consistent with the selective involvement of Cdc42 and Rac1 in basolateral trafficking. These results provide a new example of Rho GTPase regulation of basolateral trafficking and demonstrate novel roles for Cdc42 and Rac1 in the post-Golgi transport of E-cadherin. Rho family GTPases; catenin; polarity; sorting; actin     

MEK kinases are regulated by EGF and selectively interact with Rac/Cdc42.

MEK kinases (MEKKs) 1, 2, 3 and 4 are members of sequential kinase pathways that regulate MAP kinases including c-Jun NH2-terminal kinases (JNKs) and extracellular regulated kinases (ERKs). Confocal immunofluorescence microscopy of COS cells demonstrated differential MEKK subcellular localization: MEKK1 was nuclear and in post-Golgi vesicular-like structures; MEKK2 and 4 were localized to distinct Golgi-associated vesicles that were dispersed by brefeldin A. MEKK1 and 2 were activated by EGF, and kinase-inactive mutants of each MEKK partially inhibited EGF-stimulated JNK activity. Kinase-inactive MEKK1, but not MEKK2, 3 or 4, strongly inhibited EGF-stimulated ERK activity. In contrast to MEKK2 and 3, MEKK1 and 4 specifically associated with Rac and Cdc42 and kinase-inactive mutants blocked Rac/Cdc42 stimulation of JNK activity. Inhibitory mutants of MEKK1-4 did not affect p21-activated kinase (PAK) activation of JNK, indicating that the PAK-regulated JNK pathway is independent of MEKKs. Thus, in different cellular locations, specific MEKKs are required for the regulation of MAPK family members, and MEKK1 and 4 are involved in the regulation of JNK activation by Rac/Cdc42 independent of PAK. Differential MEKK subcellular distribution and interaction with small GTP-binding proteins provides a mechanism to regulate MAP kinase responses in localized regions of the cell and to different upstream stimuli.     

Cdc42/Rac1-mediated activation primes PAK2 for superactivation by tyrosine phosphorylation

The involvement of p21-activated kinases (PAKs) in important cellular processes such as regulation of the actin skeleton morphology, transduction of signals controlling gene expression, and execution of programmed cell death has directed attention to the regulation of the activity of these kinases. Here we report that activation of PAK2 by p21 GTPases can be strongly potentiated by cellular tyrosine kinases. PAK2 became tyrosine phosphorylated in its N-terminal regulatory domain, where Y130 was identified as the major phosphoacceptor site. Tyrosine phosphorylation-mediated superactivation of PAK2 could be induced by overexpression of different Src kinases or by inhibiting cellular tyrosine phosphatases with pervanadate and could be blocked by the Src kinase inhibitor PP1 or by mutating the Y130 residue. Analysis of PAK2 mutants activated by amino acid changes in the autoinhibitory domain or the catalytic domain indicated that GTPase-induced conformational changes, rather than catalytic activation per se, rendered PAK2 a target for tyrosine phosphorylation. Thus, PAK activation represents a potentially important point of convergence of tyrosine kinase- and p21 GTPase-dependent signaling pathways.     

Monoglucosylation of low-molecular-mass GTP-binding Rho proteins by clostridial cytotoxins

Rho proteins, which are involved in recepto-mediated regulation of the actin cytoskeleton, are substrates for ADP-ribosylation by Clostridium botulinum C3 toxins. Recently, it was shown that Rho and other members of the Rho subfamily of low-molecular-mass GTP-binding proteins are glucosylated by C. difficile toxins A and B. Glucosylation occurs at threonine-37, which is a crucial amino acid residue for the regulatory functions of the small GTP-binding proteins. These toxins should prove useful as tools for studying the functions of Rho proteins.     

RhoG signals in parallel with Rac1 and Cdc42

RhoG is a member of the Rho family of small GTPases and shares high sequence identity with Rac1 and Cdc42. Previous studies suggested that RhoG mediates its effects through activation of Rac1 and Cdc42. To further understand the mechanism of RhoG signaling, we studied its potential activation pathways, downstream signaling properties, and functional relationship to Rac1 and Cdc42 in vivo. First, we determined that RhoG was regulated by guanine nucleotide exchange factors that also activate Rac and/or Cdc42. Vav2 (which activates RhoA, Rac1, and Cdc42) and to a lesser degree Dbs (which activates RhoA and Cdc42) activated RhoG in vitro. Thus, RhoG may be activated concurrently with Rac1 and Cdc42. Second, some effectors of Rac/Cdc42 (IQGAP2, MLK-3, PLD1), but not others (e.g. PAKs, POSH, WASP, Par-6, IRSp53), interacted with RhoG in a GTP-dependent manner. Third, consistent with this differential interaction with effectors, activated RhoG stimulated some (JNK and Akt) but not other (SRF and NF-kappaB) downstream signaling targets of activated Rac1 and Cdc42. Finally, transient transduction of a tat-tagged Rac1(17N) dominant-negative fusion protein inhibited the induction of lamellipodia by the Rac-specific activator, Tiam1, but not by activated RhoG. Together, these data argue that RhoG function is mediated by signals independent of Rac1 and Cdc42 activation and instead by direct utilization of a subset of common effectors.     

RhoA/ROCK-mediated switching between Cdc42- and Rac1-dependent protrusion in MTLn3 carcinoma cells

Rho GTPases are versatile regulators of cell shape that act on the actin cytoskeleton. Studies using Rho GTPase mutants have shown that, in some cells, Rac1 and Cdc42 regulate the formation of lamellipodia and filopodia, respectively at the leading edge, whereas RhoA mediates contraction at the rear of moving cells. However, recent reports have described a zone of RhoA/ROCK activation at the front of cells undergoing motility. In this study, we use a FRET-based RhoA biosensor to show that RhoA activation localizes to the leading edge of EGF-stimulated cells. Inhibition of Rho or ROCK enhanced protrusion, yet markedly inhibited cell motility; these changes correlated with a marked activation of Rac-1 at the cell edge. Surprisingly, whereas EGF-stimulated protrusion in control MTLn3 cells is Rac-independent and Cdc42-dependent, the opposite pattern is observed in MTLn3 cells after inhibition of ROCK. Thus, Rho and ROCK suppress Rac-1 activation at the leading edge, and inhibition of ROCK causes a switch between Cdc42 and Rac-1 as the dominant Rho GTPase driving protrusion in carcinoma cells. These data describe a novel role for Rho in coordinating signaling by Rac and Cdc42.     

Slit3 regulates cell motility through Rac/Cdc42 activation in lipopolysaccharide-stimulated macrophages

Three slit genes, slit1 to slit3, have been cloned to date. Slit1 and slit2 act as chemorepellent factors for axon guidance. Slit3 is involved in the formation of the diaphragm and kidney during embryogenesis. However, its molecular function remains unclear. We found that slit3 expression was induced by lipopolysaccharide (LPS)-stimulation in macrophages and that it was localized in the mitochondria and along the plasma membrane. Silencing of slit3 expression by RNA interference reduced cell motility and Rac/Cdc42 activation. These results suggest that slit3 functions as an intracellular signaling molecule for cell motility as part of the LPS-induced signaling cascade.     

An essential role for Rac/Cdc42 GTPases in cerebellar granule neuron survival

Rho family GTPases are critical molecular switches that regulate the actin cytoskeleton and cell function. In the current study, we investigated the involvement of Rho GTPases in regulating neuronal survival using primary cerebellar granule neurons. Clostridium difficile toxin B, a specific inhibitor of Rho, Rac, and Cdc42, induced apoptosis of granule neurons characterized by c-Jun phosphorylation, caspase-3 activation, and nuclear condensation. Serum and depolarization-dependent survival signals could not compensate for the loss of GTPase function. Unlike trophic factor withdrawal, toxin B did not affect the antiapoptotic kinase Akt or its target glycogen synthase kinase-3beta. The proapoptotic effects of toxin B were mimicked by Clostridium sordellii lethal toxin, a selective inhibitor of Rac/Cdc42. Although Rac/Cdc42 GTPase inhibition led to F-actin disruption, direct cytoskeletal disassembly with Clostridium botulinum C2 toxin was insufficient to induce c-Jun phosphorylation or apoptosis. Granule neurons expressed high basal JNK and low p38 mitogen-activated protein kinase activities that were unaffected by toxin B. However, pyridyl imidazole inhibitors of JNK/p38 attenuated c-Jun phosphorylation. Moreover, both pyridyl imidazoles and adenoviral dominant-negative c-Jun attenuated apoptosis, suggesting that JNK/c-Jun signaling was required for cell death. The results indicate that Rac/Cdc42 GTPases, in addition to trophic factors, are critical for survival of cerebellar granule neurons.     

p21-activated kinase links Rac/Cdc42 signaling to merlin.

The neurofibromatosis type 2 tumor suppressor gene, NF2, is mutated in the germ line of NF2 patients and predisposes affected individuals to intracranial and spinal tumors. Moreover, somatic mutations of NF2 can occur in the sporadic counterparts of these neurological tumor types as well as in certain neoplasms of non-neuroectodermal origin, such as malignant mesothelioma and melanoma. NF2 encodes a 595-amino acid protein, merlin, which exhibits significant homology to the ezrin-radixin-moesin family of proteins. However, the mechanism by which merlin exerts its tumor suppressor activity is not well understood. In this investigation, we show that merlin is phosphorylated in response to expression of activated Rac and activated Cdc42 in mammalian cells. Furthermore, we demonstrate that merlin phosphorylation is mediated by p21-activated kinase (Pak), a common downstream target of both Rac and Cdc42. Both in vivo and in vitro kinase assays demonstrated that Pak can directly phosphorylate merlin at serine 518, a site that affects merlin activity and localization. These biochemical investigations provide insights into the regulation of merlin function and establish a framework for elucidating tumorigenic mechanisms involved in neoplasms associated with merlin inactivation.     

Cdc42 and Rac stimulate exocytosis of secretory granules by activating the IP(3)/calcium pathway in RBL-2H3 mast cells

We have expressed dominant-active and dominant-negative forms of the Rho GTPases, Cdc42 and Rac, using vaccinia virus to evaluate the effects of these mutants on the signaling pathway leading to the degranulation of secretory granules in RBL-2H3 cells. Dominant-active Cdc42 and Rac enhance antigen-stimulated secretion by about twofold, whereas the dominant-negative mutants significantly inhibit secretion. Interestingly, treatment with the calcium ionophore, A23187, and the PKC activator, PMA, rescues the inhibited levels of secretion in cells expressing the dominant-negative mutants, implying that Cdc42 and Rac act upstream of the calcium influx pathway. Furthermore, cells expressing the dominant-active mutants exhibit elevated levels of antigen-stimulated IP(3) production, an amplified antigen-stimulated calcium response consisting of both calcium release from internal stores and influx from the extracellular medium, and an increase in aggregate formation of the IP(3) receptor. In contrast, cells expressing the dominant-negative mutants display the opposite phenotypes. Finally, we are able to detect an in vitro interaction between Cdc42 and PLCgamma1, the enzyme immediately upstream of IP(3) formation. Taken together, these findings implicate Cdc42 and Rac in regulating the exocytosis of secretory granules by stimulation of IP(3) formation and calcium mobilization upon antigen stimulation.