Spatial control of Cdc42 activation determines cell width in fission yeast

The fission yeast Schizosaccharomyces pombe is a rod-shaped cell that grows by linear extension at the cell tips, with a nearly constant width throughout the cell cycle. This simple geometry makes it an ideal system for studying the control of cellular dimensions. In this study, we carried out a near-genome-wide screen for mutants wider than wild-type cells. We found 11 deletion mutants that were wider; seven of the deleted genes are implicated in the control of the small GTPase Cdc42, including the Cdc42 guanine nucleotide exchange factor (GEF) Scd1 and the Cdc42 GTPase-activating protein (GAP) Rga4. Deletions of rga4 and scd1 had additive effects on cell width, and the proteins localized independently of one another, with Rga4 located at the cell sides and Scd1 at the cell tips. Activated Cdc42 localization is altered in rga4Δ, scd1Δ, and scd2Δ mutants. Delocalization and ectopic retargeting experiments showed that the localizations of Rga4 and Scd1 are crucial for their roles in determining cell width. We propose that the GAP Rga4 and the GEF Scd1 establish a gradient of activated Cdc42 within the cellular tip plasma membrane, and it is this gradient that determines cell growth-zone size and normal cell width.     

Spatial Regulation of Cdc42 During Cytokinesis

Cdc42 GTPase plays a critical role in the establishment of cell polarity in most eukaryotic organisms. Cdc42 active state, as that of other GTPases, depends on the bound nucleotide. The protein with GTP is active, and only in this state can it interact with different target effector proteins. The spatio-temporal control of Cdc42 activity is therefore necessary to generate growth polarity. In fission yeast cells, Cdc42 mainly localizes to the division area, and also to the growing tips and to some internal membranes. While the role of Cdc42 in apical growth is well defined, no role has been described for Cdc42 in the process of cell division. Fission yeast Cdc42 activity is regulated by two specific guanidine nucleotide exchange factors (GEFs), Scd1, and Gef1. We discuss here how Hob3, a BAR domain containing protein similar to human BIN3 and S. cerevisiae Rsv161, may be required to recruit Cdc42 to the cell division site as well as for the activation of this GTPase mediated by Gef1. We also discuss the possible role of Cdc42 in the contraction of the actomyosin ring necessary for cytokinesis.     

Activation of the Cdc42p GTPase by cyclin-dependent protein kinases in budding yeast

Cyclin-dependent kinases (CDKs) trigger essential cell cycle processes including critical events in G1 phase that culminate in bud emergence, spindle pole body duplication, and DNA replication. Localized activation of the Rho-type GTPase Cdc42p is crucial for establishment of cell polarity during G1, but CDK targets that link the Cdc42p module with cell growth and cell cycle commitment have remained largely elusive. Here, we identify the GTPase-activating protein (GAP) Rga2p as an important substrate related to the cell polarity function of G1 CDKs. Overexpression of RGA2 in the absence of functional Pho85p or Cdc28p CDK complexes is toxic, due to an inability to polarize growth. Mutation of CDK consensus sites in Rga2p that are phosphorylated both in vivo and in vitro by Pho85p and Cdc28p CDKs results in a loss of G1 phase-specific phosphorylation. A failure to phosphorylate Rga2p leads to defects in localization and impaired polarized growth, in a manner dependent on Rga2p GAP function. Taken together, our data suggest that CDK-dependent phosphorylation restrains Rga2p activity to ensure appropriate activation of Cdc42p during cell polarity establishment. Inhibition of GAPs by CDK phosphorylation may be a general mechanism to promote proper G1-phase progression.     

Up-regulation of the Cdc42 GTPase limits the replicative life span of budding yeast

Cdc42, a conserved Rho GTPase, plays a central role in polarity establishment in yeast and animals. Cell polarity is critical for asymmetric cell division, and asymmetric cell division underlies replicative aging of budding yeast. Yet how Cdc42 and other polarity factors impact life span is largely unknown. Here we show by live-cell imaging that the active Cdc42 level is sporadically elevated in wild type during repeated cell divisions but rarely in the long-lived bud8 deletion cells. We find a novel Bud8 localization with cytokinesis remnants, which also recruit Rga1, a Cdc42 GTPase activating protein. Genetic analyses and live-cell imaging suggest that Rga1 and Bud8 oppositely impact life span likely by modulating active Cdc42 levels. An rga1 mutant, which has a shorter life span, dies at the unbudded state with a defect in polarity establishment. Remarkably, Cdc42 accumulates in old cells, and its mild overexpression accelerates aging with frequent symmetric cell divisions, despite no harmful effects on young cells. Our findings implicate that the interplay among these positive and negative polarity factors limits the life span of budding yeast.     

Pom1 DYRK regulates localization of the Rga4 GAP to ensure bipolar activation of Cdc42 in fission yeast

BACKGROUND: In the fission yeast Schizosaccharomyces pombe, cell growth takes place exclusively at both ends of the cylindrical cell. During this highly polarized growth, microtubules are responsible for the placement of the cell-end marker proteins, the Tea1-Tea4/Wsh3 complex, which recruits the Pom1 DYRK-family protein kinase. Pom1 is required for proper positioning of growth sites, and the Deltapom1 mutation brings about monopolar cell growth. RESULTS: Pom1 kinase physically interacts with Rga4, which has a GAP (GTPase-activating protein) domain for Rho-family GTPase. Genetic and biochemical evidence indicates that Rga4 functions as GAP for the Cdc42 GTPase, an evolutionarily conserved regulator of F-actin. CRIB (Cdc42/Rac interactive binding)-GFP microscopy has revealed that GTP-bound, active Cdc42 is concentrated to growing cell ends accompanied by developed F-actin structures, where the Rga4 GAP is excluded. The monopolar Deltapom1 mutant fails to eliminate Rga4 from the nongrowing cell end, resulting in monopolar distribution of GTP-Cdc42 to the growing cell end. However, mutational inactivation of Rga4 allows Cdc42 to be active at both ends of Deltapom1 cells, suggesting that mislocalization of Rga4 in the Deltapom1 mutant contributes to its monopolar phenotype. CONCLUSIONS: Pom1 kinase recruited to cell ends by the Tea1-Tea4/Wsh3 complex is essential for proper localization of a GAP for Cdc42, Rga4, which ensures bipolar localization of GTP-bound, active Cdc42. Because of the established role of Cdc42 in F-actin formation, these observations provide a new insight into how the microtubule system achieves localized formation of F-actin to generate cell polarity.     

Polarization of Diploid Daughter Cells Directed by Spatial Cues and GTP Hydrolysis of Cdc42 in Budding Yeast

Cell polarization occurs along a single axis that is generally determined by a spatial cue. Cells of the budding yeast exhibit a characteristic pattern of budding, which depends on cell-type-specific cortical markers, reflecting a genetic programming for the site of cell polarization. The Cdc42 GTPase plays a key role in cell polarization in various cell types. Although previous studies in budding yeast suggested positive feedback loops whereby Cdc42 becomes polarized, these mechanisms do not include spatial cues, neglecting the normal patterns of budding. Here we combine live-cell imaging and mathematical modeling to understand how diploid daughter cells establish polarity preferentially at the pole distal to the previous division site. Live-cell imaging shows that daughter cells of diploids exhibit dynamic polarization of Cdc42-GTP, which localizes to the bud tip until the M phase, to the division site at cytokinesis, and then to the distal pole in the next G1 phase. The strong bias toward distal budding of daughter cells requires the distal-pole tag Bud8 and Rga1, a GTPase activating protein for Cdc42, which inhibits budding at the cytokinesis site. Unexpectedly, we also find that over 50% of daughter cells lacking Rga1 exhibit persistent Cdc42-GTP polarization at the bud tip and the distal pole, revealing an additional role of Rga1 in spatiotemporal regulation of Cdc42 and thus in the pattern of polarized growth. Mathematical modeling indeed reveals robust Cdc42-GTP clustering at the distal pole in diploid daughter cells despite random perturbation of the landmark cues. Moreover, modeling predicts different dynamics of Cdc42-GTP polarization when the landmark level and the initial level of Cdc42-GTP at the division site are perturbed by noise added in the model.     

Gef1p and Scd1p,the Two GDP-GTP exchange factors for Cdc42p,form a ring structure that shrinks during cytokinesis in Schizosaccharomyces pombe

Fission yeast Cdc42p, a small GTPase of the Rho family, is essential for cell proliferation and maintenance of the rod-like cell morphology. Scd1/Ral1p is a GDP-GTP exchange factor (GEF) for Cdc42p. This study and a parallel study by others establish that Gef1p is another GEF for Cdc42p. Deletions of gef1 and scd1 are synthetically lethal, generating round dead cells, and hence mimic the phenotype of cdc42 deletion. Gef1p is localized mainly to the cell division site. Scd1p is also there, but it is also detectable in other parts of the cell, including the nucleus, growing ends, and the tips of conjugation tubes. Gef1p and Scd1p form a ring structure at the cell division site, which shrinks during cytokinesis following the contraction of the actomyosin ring. Formation of the Gef1p/Scd1p ring apparently depends on the integrity of the actomyosin ring. In turn, recruitment of Cdc42p to the cell division site follows the shrinking Gef1p/Scd1p ring; the Cdc42p accumulates like a closing iris. These observations suggest that Gef1p and Scd1p may have a role in mediating between contraction of the actomyosin ring and formation of the septum, by recruiting active Cdc42p to the septation site.     

A Highlights from MBoC Selection: Rga6 is a fission yeast Rho GAP involved in Cdc42 regulation of polarized growth

Active Cdc42 is essential for the establishment of polarized growth. This GTPase is negatively regulated by the GTPase-activating proteins (GAPs), which are important for the spatial specificity of Cdc42 function. Rga4 is the only GAP described as negative regulator of fission yeast Cdc42. We report here that Rga6, another fission yeast Cdc42 GAP, shares some functions with Rga4. Cells lacking Rga6 are viable but slightly shorter and broader than wild type, and cells lacking Rga6 and Rga4 simultaneously are rounded. In these cells, active Cdc42 is observed all around the membrane. These additive effects indicate that both GAPs collaborate in the spatial regulation of active Cdc42. Rga6 localizes to the plasma membrane, forming clusters different from those formed by Rga4. A polybasic region at the Rga6 C-terminus is responsible for its membrane localization. Rga6-GFP fluorescence decreases considerably at the growing tips, and this decrease is dependent on the actin cables. Of note, in the absence of Rga6, the amplitude of active Cdc42 oscillations at the tips decreases, and less GTP-Cdc42 accumulates at the new end of the cells. We propose that Rga6 collaborates with Rga4 to spatially restrict active Cdc42 at the cell tips and maintain cell dimensions.     

The guanine-nucleotide-exchange factor Cdc24p is targeted to the nucleus and polarized growth sites.

Generation of cellular asymmetry or cell polarity plays a critical role in cell-cycle-regulated morphogenetic processes involving the actin cytoskeleton. The GTPase Cdc42 regulates actin rearrangements and signal transduction pathways in all eukaryotic cells [1], and the temporal and spatial regulation of Cdc42p depends on the activity and targeting of its guanine-nucleotide exchange factor (GEF). Cdc24p, the Saccharomyces cerevisiae GEF for Cdc42p, is found in a particulate fraction and localizes to the plasma membrane [2] [3] at sites of polarized growth [4]. We show that Cdc24p labeled with green fluorescent protein (GFP-Cdc24p) was targeted to pre-bud sites, the tips and sides of enlarging buds, and mating projections in pheromone-treated cells. Unexpectedly, GFP-Cdc24p also localized to the nucleus and GFP-Cdc24p levels diminished before nuclear division followed by its reappearance in divided nuclei and mother-bud necks during cytokinesis. The Cdc24p amino-terminal 283 amino acids were necessary and sufficient for nuclear localization, which depended on the cyclin-dependent-kinase inhibitor Far1p. The Cdc24p carboxy-terminal 289 amino acids were necessary and sufficient for targeting to the pre-bud site, bud, mother-bud neck, and mating projection. Targeting was independent of the Cdc24p-binding proteins Far1p, the GTPase Rsr1p/Bud1p, the scaffold protein Bem1p, and the G(beta) subunit Ste4p. These data are consistent with a temporal and spatial regulation of Cdc24p-dependent activation of Cdc42p during the cell cycle.     

Phosphorylation of Rga2, a Cdc42 GAP, by CDK/Hgc1 is crucial for Candida albicans hyphal growth

Cyclin-dependent kinases (CDKs) control yeast morphogenesis, although how they regulate the polarity machinery remains unclear. The dimorphic fungus Candida albicans uses Cdc28/Hgc1, a CDK/cyclin complex, to promote persistent actin polarization for hyphal growth. Here, we report that Rga2, a GTPase-activating protein (GAP) of the central polarity regulator Cdc42, undergoes Hgc1-dependent hyperphosphorylation. Using the analog-sensitive Cdc28as mutant, we confirmed that Cdc28 controls Rga2 phosphorylation in vitro and in vivo. Deleting RGA2 produced elongated yeast cells without apparent effect on hyphal morphogenesis. However, deleting it or inactivating its GAP activity restored hyphal growth in hgc1Delta mutants, suggesting that Rga2 represses hyphal development and Cdc28/Hgc1 inactivates it upon hyphal induction. We provide evidence that Cdc28/Hgc1 may act to prevent Rga2 from localizing to hyphal tips, leading to localized Cdc42 activation for hyphal extension. Rga2 also undergoes transient Cdc28-dependent hyperphosphorylation at bud emergence, suggesting that regulating a GAP(s) of Cdc42 by CDKs may play an important role in governing different forms of polarized morphogenesis in yeast. This study reveals a direct molecular link between CDKs and the polarity machinery.     

Cdc42 GTPase-activating proteins (GAPs) regulate generational inheritance of cell polarity and cell shape in fission yeast

The highly conserved small GTPase Cdc42 regulates polarized cell growth and morphogenesis from yeast to humans. We previously reported that Cdc42 activation exhibits oscillatory dynamics at cell tips of Schizosaccharomyces pombe cells. Mathematical modeling suggests that this dynamic behavior enables a variety of symmetric and asymmetric Cdc42 activation distributions to coexist in cell populations. For individual wild-type cells, however, Cdc42 distribution is initially asymmetrical and becomes more symmetrical as cell volume increases, enabling bipolar growth activation. To explore whether different patterns of Cdc42 activation are possible in vivo, we examined S. pombe rga4∆ mutant cells, lacking the Cdc42 GTPase-activating protein (GAP) Rga4. We found that monopolar rga4∆ mother cells divide asymmetrically leading to the emergence of both symmetric and asymmetric Cdc42 distributions in rga4∆ daughter cells. Motivated by different hypotheses that can mathematically reproduce the unequal fate of daughter cells, we used genetic screening to identify mutants that alter the rga4∆ phenotype. We found that the unequal distribution of active Cdc42 GTPase is consistent with an unequal inheritance of another Cdc42 GAP, Rga6, in the two daughter cells. Our findings highlight the crucial role of Cdc42 GAP localization in maintaining consistent Cdc42 activation and growth patterns across generations.     

Cellular signaling for activation of Rho GTPase Cdc42

The Rho family GTPase Cdc42 regulates cytoskeletal organization and membrane trafficking in physiological processes such as cell proliferation, motility and polarity. Aberrant activation of Cdc42 results in pathogenesis, such as tumorigenesis and tumor progression, cardiovascular diseases, diabetes, and neuronal degenerative diseases. The activation of Cdc42 in response to upstream signals is mediated by guanine nucleotide exchange factors (GEFs), which converse GDP-bound inactive form to the GTP-bound active form of Cdc42. The activated Cdc42 transduces signals to downstream effectors and generates cellular effects. This review will discuss the molecular mechanism of activation of Cdc42 and postulate that signaling specificity of Cdc42 is conferred by the GEF/GTPase/Effector (GGE) complexes in response to external stimuli.     

Gef1p, a new guanine nucleotide exchange factor for Cdc42p, regulates polarity in Schizosaccharomyces pombe

Schizosaccharomyces pombe cdc42(+) regulates cell morphology and polarization of the actin cytoskeleton. Scd1p/Ral1p is the only described guanine nucleotide exchange factor (GEF) for Cdc42p in S. pombe. We have identified a new GEF, named Gef1p, specifically regulating Cdc42p. Gef1p binds to inactive Cdc42p but not to other Rho GTPases in two-hybrid assays. Overexpression of gef1(+) increases specifically the GTP-bound Cdc42p, and Gef1p is capable of stimulating guanine nucleotide exchange of Cdc42p in vitro. Overexpression of gef1(+) causes changes in cell morphology similar to those caused by overexpression of the constitutively active cdc42G12V allele. Gef1p localizes to the septum. gef1(+) deletion is viable but causes a mild cell elongation and defects in bipolar growth and septum formation, suggesting a role for Gef1p in the control of cell polarity and cytokinesis. The double mutant gef1delta scd1delta is not viable, indicating that they share an essential function as Cdc42p activators. However, both deletion and overexpression of either gef1(+) or scd1(+) causes different morphological phenotypes, which suggest different functions. Genetic evidence revealed a link between Gef1p and the signaling pathway of Shk1/Orb2p and Orb6p. In contrast, no genetic interaction between Gef1p and Shk2p-Mkh1p pathway was observed.     

PAR-6-PAR-3 mediates Cdc42-induced Rac activation through the Rac GEFs STEF/Tiam1

A polarity complex of PAR-3, PAR-6 and atypical protein kinase C (aPKC) functions in various cell-polarization events, including neuron specification. The small GTPase Cdc42 binds to PAR-6 and regulates cell polarity. However, little is known about the downstream signals of the Cdc42-PAR protein complex. Here, we found that PAR-3 directly interacted with STEF/Tiam1, which are Rac-specific guanine nucleotide-exchange factors, and that STEF formed a complex with PAR-3-aPKC-PAR-6-Cdc42-GTP. Cdc42 induces lamellipodia in a Rac-dependent manner in N1E-115 neuroblastoma cells. Disruption of Cdc42-PAR-6 or PAR-3-STEF binding inhibited Cdc42-induced lamellipodia but not filopodia. The isolated STEF-binding PAR-3 fragment was sufficient to induce lamellipodia independently of Cdc42 and PAR-6. PAR-3 is required for Cdc42-induced Rac activation, but is not essential for lamellipodia formation itself. In cultured hippocampal neurons, STEF accumulated at the tip of the growing axon and colocalized with PAR-3. The spatio-temporal activation and signalling of Cdc42-PAR-6-PAR-3-STEF/Tiam1-Rac seem to be involved in neurite growth and axon specification. We propose that the PAR-6-PAR-3 complex mediates Cdc42-induced Rac activation by means of STEF/Tiam1, and that this process seems to be required for the establishment of neuronal polarity.     

GTPase-activating proteins for Cdc42

The Rho-type GTPase, Cdc42, has been implicated in a variety of functions in the yeast life cycle, including septin organization for cytokinesis, pheromone response, and haploid invasive growth. A group of proteins called GTPase-activating proteins (GAPs) catalyze the hydrolysis of GTP to GDP, thereby inactivating Cdc42. At the time this study began, there was one known GAP, Bem3, and one putative GAP, Rga1, for Cdc42. We identified another putative GAP for Cdc42 and named it Rga2 (Rho GTPase-activating protein 2). We confirmed by genetic and biochemical criteria that Rga1, Rga2, and Bem3 act as GAPs for Cdc42. A detailed characterization of Rga1, Rga2, and Bem3 suggested that they regulate different subsets of Cdc42 function. In particular, deletion of the individual GAPs conferred different phenotypes. For example, deletion of RGA1, but not RGA2 or BEM3, caused hyperinvasive growth. Furthermore, overproduction or loss of Rga1 and Rga2, but not Bem3, affected the two-hybrid interaction of Cdc42 with Ste20, a p21-activated kinase (PAK) kinase required for haploid invasive growth. These results suggest Rga1, and possibly Rga2, facilitate the interaction of Cdc42 with Ste20 to mediate signaling in the haploid invasive growth pathway. Deletion of BEM3 resulted in cells with severe morphological defects not observed in rga1Δ or rga2Δ strains. These data suggest that Bem3 and, to a lesser extent, Rga1 and Rga2 facilitate the role of Cdc42 in septin organization. Thus, it appears that the GAPs play a role in modulating specific aspects of Cdc42 function. Alternatively, the different phenotypes could reflect quantitative rather than qualitative differences in GAP activity in the mutant strains.     

Transbilayer phospholipid flipping regulates Cdc42p signaling during polarized cell growth via Rga GTPase-activating proteins

An important problem in polarized morphogenesis is how polarized transport of membrane vesicles is spatiotemporally regulated. Here, we report that a local change in the transbilayer phospholipid distribution of the plasma membrane regulates the axis of polarized growth. Type 4 P-type ATPases Lem3p-Dnf1p and -Dnf2p are putative heteromeric phospholipid flippases in budding yeast that are localized to polarized sites on the plasma membrane. The lem3Delta mutant exhibits prolonged apical growth due to a defect in the switch to isotropic bud growth. In lem3Delta cells, the small GTPase Cdc42p remains polarized at the bud tip where phosphatidylethanolamine remains exposed on the outer leaflet. Intriguingly, phosphatidylethanolamine and phosphatidylserine stimulate GTPase-activating protein (GAP) activity of Rga1p and Rga2p toward Cdc42p, whereas PI(4,5)P(2) inhibits it. We propose that a redistribution of phospholipids to the inner leaflet of the plasma membrane triggers the dispersal of Cdc42p from the apical growth site, through activation of GAPs.     

Adjacent positioning of cellular structures enabled by a Cdc42 GTPase-activating protein-mediated zone of inhibition

Cells of the budding yeast Saccharomyces cerevisiae are born carrying localized transmembrane landmark proteins that guide the subsequent establishment of a polarity axis and hence polarized growth to form a bud in the next cell cycle. In haploid cells, the relevant landmark proteins are concentrated at the site of the preceding cell division, to which they recruit Cdc24, the guanine nucleotide exchange factor for the conserved polarity regulator Cdc42. However, instead of polarizing at the division site, the new polarity axis is directed next to but not overlapping that site. Here, we show that the Cdc42 guanosine triphosphatase–activating protein (GAP) Rga1 establishes an exclusion zone at the division site that blocks subsequent polarization within that site. In the absence of localized Rga1 GAP activity, new buds do in fact form within the old division site. Thus, Cdc42 activators and GAPs establish concentric zones of action such that polarization is directed to occur adjacent to but not within the previous cell division site.     

Independence of symmetry breaking on Bem1-mediated autocatalytic activation of Cdc42

The ability to break symmetry and polarize through self-organization is a fundamental feature of cellular systems. A prevailing theory in yeast posits that symmetry breaking occurs via a positive feedback loop, wherein the adaptor protein Bem1 promotes local activation and accumulation of Cdc42 by directly tethering Cdc42^GTP with its guanine nucleotide exchange factor (GEF) Cdc24. In this paper, we find that neither Bem1 nor the ability of Bem1 to bind Cdc42^GTP is required for cell polarization. Instead, Bem1 functions primarily by boosting GEF activity, a role critical for polarization without actin filaments. In the absence of actin-based transport, polarization of Cdc42 is accomplished through Rdi1, the Cdc42 guanine nucleotide dissociation inhibitor. A mathematical model is constructed describing cell polarization as a product of distinct pathways controlling Cdc42 activation and protein localization. The model predicts a nonmonotonic dependence of cell polarization on the concentration of Rdi1 relative to that of Cdc42.     

Hob3p, the fission yeast ortholog of human BIN3, localizes Cdc42p to the division site and regulates cytokinesis

Cdc42 GTPase is required for polarization in eukaryotic cells, but its spatial regulation is poorly understood. In Schizosaccharomyces pombe, Cdc42p is activated by Scd1p and Gef1p, two guanine-nucleotide exchange factors. Two-hybrid screening identified Hob3p as a Gef1p binding partner. Hob3p is a BAR domain-containing protein ortholog of human Bin3. Hob3p also interacts directly with Cdc42p independently of Gef1p. Hob3p, Cdc42p and Gef1p form a complex, and Hob3p facilitates Gef1p-Cdc42p interaction and activation. Hob3p forms a ring in the division area, similar to that of Gef1p. This localization requires actin polymerization and Cdc15p but is independent of the septation initiation network. Hob3p is required for the concentration of Cdc42p to the division area. The actomyosin ring contraction is slower in hob3Delta than in wild-type cells, and this contributes to its cytokinesis defect. Moreover, this report extends previous evidence that human Bin3 suppresses the cytokinesis phenotype of hob3Delta cells, showing that Bin3 can partially recover the GTP-Cdc42p level and its localization. These results suggest that Hob3p is required to recruit and activate Cdc42p at the cell division site and that this function might be conserved in other eukaryotes.     

A novel connection between the yeast Cdc42 GTPase and the Slt2-mediated cell integrity pathway identified through the effect of secreted Salmonella GTPase modulators

Modulation of host cellular GTPases through the injection of the effector proteins SopE2 and SptP is essential for Salmonella typhimurium to enter into non-phagocytic cells. Here we show that expression of the guanine nucleotide exchange factor for Cdc42 SopE2 in Saccharomyces cerevisiae leads to the activation of Fus3 and Kss1 MAPKs, which operate in the mating and filamentation pathways, causing filamentous growth in haploid yeast cells. Furthermore, it promotes the activation of the cell integrity MAPK Slt2. Cdc42 activation by removal of its putative intrinsic GTPase-activating proteins (GAPs), Rga1, Rga2, and Bem3, also results in the phosphorylation of Kss1, Fus3, and Slt2 MAPKs. These data support the role of these GAP proteins as negative regulators of Cdc42, confirm the modulating effect of this GTPase on the filamentation and mating pathways and point to a novel connection between Cdc42 and the cell integrity pathway. Cdc42-induced activation of Slt2 occurs in a mating and filamentation pathway-dependent manner, but it does not require the function of Rho1, which is the GTPase that operates in the cell integrity pathway. Moreover, we report that Salmonella SptP can act as a GAP for Cdc42 in S. cerevisiae, down-regulating MAPK-mediated signaling. Thus, yeast provides a useful system to study the interaction of bacterial pathogenic proteins with eukaryotic signaling pathways. Furthermore, these proteins can be used as a tool to gain insight into the mechanisms that regulate MAPK-mediated signaling in eukaryotes.