Cdc42 GTPase-activating Protein Research Articles

Cdc42 mobility and membrane flows regulate fission yeast cell shape and survival

Polarized exocytosis induced by local Cdc42 GTPase activity results in membrane flows that deplete low-mobility membrane-associated proteins. A reaction-diffusion particle model comprising Cdc42 positive feedback activation, hydrolysis by GTPase-activating proteins (GAPs), and flow-induced displacement by exo/endocytosis shows that flow-induced depletion of low mobility GAPs promotes polarization. We modified Cdc42 mobility in Schizosaccharomyces pombe by replacing its prenylation site with 1, 2 or 3 repeats of the Rit C-terminal membrane-binding domain (ritC), yielding alleles with progressively lower mobility and increased flow-coupling. While Cdc42-1ritC cells are viable and polarized, Cdc42-2ritC polarize poorly and Cdc42-3ritC are inviable, in agreement with model’s predictions. Deletion of Cdc42 GAPs restores viability to Cdc42-3ritC cells, verifying the model’s prediction that GAP deletion increases Cdc42 activity at the expense of polarization. Our work demonstrates how membrane flows are an integral part of Cdc42-driven pattern formation and require Cdc42-GTP to turn over faster than the surface on which it forms.

CdGAP is a talin-binding protein and a target of TGF-β signaling that promotes HER2-positive breast cancer growth and metastasis

Epithelial-to-mesenchymal transition (EMT) plays a crucial role in metastasis, which is the leading cause of death in breast cancer patients. Here, we show that Cdc42 GTPase-activating protein (CdGAP) promotes tumor formation and metastasis to lungs in the HER2-positive (HER2+) murine breast cancer model. CdGAP facilitates intravasation, extravasation, and growth at metastatic sites. CdGAP depletion in HER2+ murine primary tumors mediates crosstalk with a Dlc1-RhoA pathway and is associated with a transforming growth factor β (TGF-β)-induced EMT transcriptional signature. CdGAP is positively regulated by TGF-β signaling during EMT and interacts with the adaptor talin to modulate focal adhesion dynamics and integrin activation. Moreover, HER2+ breast cancer patients with high CdGAP mRNA expression combined with a high TGF-β-EMT signature are more likely to present lymph node invasion. Our results suggest CdGAP as a candidate therapeutic target for HER2+ metastatic breast cancer by inhibiting TGF-β and integrin/talin signaling pathways.

The Cdc42 GAP Rga6 promotes monopolar outgrowth of spores.

The molecular mechanisms underlying the establishment of the monopolar growth of fission yeast spores have been less characterized. Here, we report that the Cdc42 GTPase-activating protein (GAP) Rga6 is required for promoting monopolar growth during spore germination. The absence of Rga6 increases the number of spores that grow in a bipolar fashion. Rga6 decorates the non-growing cortical region, binds phosphatidylinositol 4,5-bisphosphate, and colocalizes with the phosphatidylinositol 4,5-bisphosphate-binding protein Opy1. Overexpression of Opy1 diminishes the cortical localization of Rga6. The characteristic localization of Rga6 on the cell cortex depends on the C-terminal PBR region of Rga6. Moreover, engineered chimera composed of the Rga6 C-terminal PBR region fused to the GAP domain of Rga3 or Rga4 are sufficient to rescue the spore growth phenotype caused by the absence of Rga6. Hence, our work establishes a paradigm in which the lipid composition of the plasma membrane directs polarized cell growth by specifying the cortical localization of a GAP protein.

CDC42 Regulates Cell Proliferation and Apoptosis in Bladder Cancer via the IQGAP3-Mediated Ras/ERK Pathway.

Bladder cancer (BC) is the most common malignant tumour of the urinary system. The current conventional treatments for BC have certain limitations. It is very urgent and necessary to find new treatment strategies for BC. Our study elucidated the underlying regulatory mechanisms of cell division control protein 42 homologue (CDC42) to regulate the development of BC. Quantitative real-time polymerase chain reaction, Western blot, immunofluorescence and immunohistochemistry were used to assess the expression of CDC42 and IQ motif-containing GTPase-activating protein 3 (IQGAP3) in BC tissues and BC cells. We induced the knockdown or overexpression by transfecting sh-CDC42 or oe-IQGAP3 into BC cells. In addition, cell proliferation and apoptosis were evaluated by cell counting kit-8 and flow cytometry assays, respectively. Moreover, proteins involved in the rat sarcoma (Ras)/extracellular regulated protein kinase (ERK) pathway were determined by Western blot. The expression of CDC42 and IQGAP3 was markedly upregulated in both BC tissues and BC cells. CDC42 silencing downregulated the expression of IQGAP3 and suppressed the Ras/ERK pathway. In addition, CDC42 silencing markedly promoted apoptosis and inhibited proliferation in BC cells. Further experiments showed that overexpression of IQGAP3 dramatically abolished the bioeffects mediated by CDC42 silencing on the proliferation and apoptosis of BC cells. All our results suggested that CDC42 promoted the Ras/ERK pathway by regulating IQGAP3, thus enhancing cell proliferation and suppressing cell apoptosis in BC cells and ultimately participating in the pathogenesis of BC.

First person – Shengnan Zheng

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping early-career researchers promote themselves alongside their papers. Shengnan Zheng is first author on ‘ The Cdc42 GTPase-activating protein Rga6 promotes the cortical localization of septin’, published in JCS. Shengnan is a PhD student in the lab of Chuanhai Fu at University of Science and Technology of China, Hefei, Anhui, China, investigating how septin and microtubule cytoskeletons regulate cell septation and cell polarity.

The Cdc42 GTPase-activating protein Rga6 promotes the cortical localization of septin.

Septins are a family of filament-forming GTP-binding proteins that regulate fundamental cellular activities, such as cytokinesis and cell polarity. In general, septin filaments function as barriers and scaffolds on the cell cortex. However, little is known about the mechanism that governs the recruitment and localization of the septin complex to the cell cortex. Here, we identified the Cdc42 GTPase-activating protein Rga6 as a key protein involved in promoting the localization of the septin complex to the cell cortex in the fission yeast Schizosaccharomyces pombe. Rga6 interacts with the septin complex and partially colocalizes with the septin complex on the cell cortex. Live-cell microscopy analysis further showed septin enrichment at the cortical regions adjacent to the growing cell tip. The septin enrichment likely plays a crucial role in confining active Cdc42 to the growing cell tip. Hence, our findings support a model whereby Rga6 regulates polarized cell growth partly through promoting targeted localization of the septin complex on the cell cortex. This article has an associated First Person interview with the first author of the paper.

Stress-dependent inhibition of polarized cell growth through unbalancing the GEF/GAP regulation of Cdc42.

Cdc42 GTPase rules cell polarity and growth in fission yeast. It is negatively and positively regulated by GTPase-activating proteins (GAPs) and guanine nucleotide exchange factors (GEFs), respectively. Active Cdc42-GTP localizes to the poles, where it associates with numerous proteins constituting the polarity module. However, little is known about its downregulation. We describe here that oxidative stress causes Sty1-kinase-dependent Cdc42 inactivation at cell poles. Both the amount of active Cdc42 at tips and cell length inversely correlate with Sty1 activity, explaining the elongated morphology of Δsty1 cells. We have created stress-blinded cell poles either by eliminating two Cdc42 GAPs or through the constitutive tethering of Gef1 to cell tips, and we biochemically demonstrate that the GAPs Rga3/6 and the GEF Gef1 are direct substrates of Sty1. We propose that phosphorylation of Rga3/6 and Gef1 mediates the Sty1-dependent inhibition of Cdc42 at cell tips, halting polarized growth during stress adaptation.

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.

Mechanism of Rac and Cdc42 Synchronization at the Cell Edge by ARHGAP39-Dependent Signaling and the Impact on Protrusion Dynamics

Compartmentalization of GTPase regulators into signaling nodules dictates specific GTPase signaling pathway selection. Rac and Cdc42 are synchronized at the cell edge for effective protrusion in motile cells, but how their activity is coordinated remains elusive. Here, we report that ARHGAP39, a Rac and Cdc42 GTPase-activating protein, sequentially interacts with WAVE and mDia2 to control lamellipodia and filopodia protrusions, respectively. Mechanistically, ARHGAP39 binds WAVE, whereupon phosphorylation by Src promotes Rac inactivation and leads to Cdc42-induced filopodia formation. Using an optimized FRET biosensor, we detected active Cdc42 at the filopodia tips that controls filopodia extension. ARHGAP39 is transported to filopodia tips by Myosin-X where it binds mDia2 and inactivates Cdc42, leading to filopodia retraction. Failure in lamellipodia-to-filopodia switch by defective ARHGAP39 impairs cell invasion and metastasis. Our study reveals that compartmentalization of ARHGAP39 within Rac/Cdc42 signaling nodules orchestrates the synchronization of lamellipodia and filopodia and highlights the intricate regulation of leading edge dynamics in migrating cells.

ADP-ribosylation factor-like 4A interacts with Robo1 to promote cell migration by regulating Cdc42 activation.

Cell migration is a highly regulated event that is initiated by cell membrane protrusion and actin reorganization. Robo1, a single-pass transmembrane receptor, is crucial for neuronal guidance and cell migration. ADP-ribosylation factor (Arf)–like 4A (Arl4A), an Arf small GTPase, functions in cell morphology, cell migration, and actin cytoskeleton remodeling; however, the molecular mechanisms of Arl4A in cell migration are unclear. Here, we report that the binding of Arl4A to Robo1 modulates cell migration by promoting Cdc42 activation. We found that Arl4A interacts with Robo1 in a GTP-dependent manner and that the Robo1 amino acid residues 1394–1398 are required for this interaction. The Arl4A-Robo1 interaction is essential for Arl4A-induced cell migration and Cdc42 activation but not for the plasma membrane localization of Robo1. In addition, we show that the binding of Arl4A to Robo1 decreases the association of Robo1 with the Cdc42 GTPase-activating protein srGAP1. Furthermore, Slit2/Robo1 binding down-regulates the Arl4A-Robo1 interaction in vivo, thus attenuating Cdc42-mediated cell migration. Therefore, our study reveals a novel mechanism by which Arl4A participates in Slit2/Robo1 signaling to modulate cell motility by regulating Cdc42 activity.

Differential GAP requirement for Cdc42-GTP polarization during proliferation and sexual reproduction.

The formation of a local zone of Cdc42 GTPase activity, which governs cell polarization in many cell types, requires not only local activation but also switch-off mechanisms. In this study, we identify Rga3, a paralog of Rga4, as a novel Cdc42 GTPase-activating protein (GAP) in the fission yeast Schizosaccharomyces pombe Contrary to Rga4, Rga3 localizes with Cdc42-GTP to sites of polarity. Rga3 is dispensable for cell polarization during mitotic growth, but it limits the lifetime of unstable Cdc42-GTP patches that underlie cell pairing during sexual reproduction, masking a partly compensatory patch-wandering motion. In consequence, cells lacking rga3 hyperpolarize and lose out in mating competition. Rga3 synergizes with the Cdc42 GAPs Rga4 and Rga6 to restrict Cdc42-GTP zone sizes during mitotic growth. Surprisingly, triple-mutant cells, which are almost fully round, retain pheromone-dependent dynamic polarization of Cdc42-GTP, extend a polarized projection, and mate. Thus, the requirement for Cdc42-GTP hydrolysis by GAPs is distinct during polarization by intrinsic or extrinsic cues.

Local and global Cdc42 guanine nucleotide exchange factors for fission yeast cell polarity are coordinated by microtubules and the Tea1-Tea4-Pom1 axis.

ABSTRACTThe conserved Rho-family GTPase Cdc42 plays a central role in eukaryotic cell polarity. The rod-shaped fission yeast Schizosaccharomyces pombe has two Cdc42 guanine nucleotide exchange factors (GEFs), Scd1 and Gef1, but little is known about how they are coordinated in polarized growth. Although the microtubule cytoskeleton is normally not required for polarity maintenance in fission yeast, we show here that when scd1 function is compromised, disruption of microtubules or the polarity landmark proteins Tea1, Tea4 or Pom1 leads to disruption of polarized growth. Instead, cells adopt an isotropic-like pattern of growth, which we term PORTLI growth. Surprisingly, PORTLI growth is caused by spatially inappropriate activity of Gef1. Although most Cdc42 GEFs are membrane associated, we find that Gef1 is a broadly distributed cytosolic protein rather than a membrane-associated protein at cell tips like Scd1. Microtubules and the Tea1–Tea4–Pom1 axis counteract inappropriate Gef1 activity by regulating the localization of the Cdc42 GTPase-activating protein Rga4. Our results suggest a new model of fission yeast cell polarity regulation, involving coordination of ‘local’ (Scd1) and ‘global’ (Gef1) Cdc42 GEFs via microtubules and microtubule-dependent polarity landmarks.

Structure of the tandem PX-PH domains of Bem3 from Saccharomyces cerevisiae.

The structure of the tandem lipid-binding PX and pleckstrin-homology (PH) domains of the Cdc42 GTPase-activating protein Bem3 from Saccharomyces cerevisiae (strain S288c) has been determined to a resolution of 2.2 Å (Rwork = 21.1%, Rfree = 23.4%). It shows that the domains adopt a relative orientation that enables them to simultaneously bind to a membrane and suggests possible cooperativity in membrane binding.

CdGAP/ARHGAP31 is regulated by RSK phosphorylation and binding to 14-3-3β adaptor protein.

Cdc42 GTPase-activating protein (CdGAP, also named ARHGAP31) is a negative regulator of the GTPases Rac1 and Cdc42. Associated with the rare developmental disorder Adams-Oliver Syndrome (AOS), CdGAP is critical for embryonic vascular development and VEGF-mediated angiogenesis. Moreover, CdGAP is an essential component in the synergistic interaction between TGFβ and ErbB-2 signaling pathways during breast cancer cell migration and invasion, and is a novel E-cadherin transcriptional co-repressor with Zeb2 in breast cancer. CdGAP is highly phosphorylated on serine and threonine residues in response to growth factors and is a substrate of ERK1/2 and GSK-3. Here, we identified Ser1093 and Ser1163 in the C-terminal region of CdGAP, which are phosphorylated by RSK in response to phorbol ester. These phospho-residues create docking sites for binding to 14-3-3 adaptor proteins. The interaction between CdGAP and 14-3-3 proteins inhibits the GAP activity of CdGAP and sequesters CdGAP into the cytoplasm. Consequently, the nucleocytoplasmic shuttling of CdGAP is inhibited and CdGAP-induced cell rounding is abolished. In addition, 14-3-3β inhibits the ability of CdGAP to repress the E-cadherin promoter and to induce cell migration. Finally, we show that 14-3-3β is unable to regulate the activity and subcellular localization of the AOS-related mutant proteins lacking these phospho-residues. Altogether, we provide a novel mechanism of regulation of CdGAP activity and localization, which impacts directly on a better understanding of the role of CdGAP as a promoter of breast cancer and in the molecular causes of AOS.

Fine-tuning the orientation of the polarity axis by Rga1, a Cdc42 GTPase-activating protein.

In yeast and animal cells, signaling pathways involving small guanosine triphosphatases (GTPases) regulate cell polarization. In budding yeast, selection of a bud site directs polarity establishment and subsequently determines the plane of cell division. Rga1, a Cdc42 GTPase-activating protein, prevents budding within the division site by inhibiting Cdc42 repolarization. A protein complex including Nba1 and Nis1 is involved in preventing rebudding at old division sites, yet how these proteins and Rga1 might function in negative polarity signaling has been elusive. Here we show that Rga1 transiently localizes to the immediately preceding and older division sites by interacting with Nba1 and Nis1. The LIM domains of Rga1 are necessary for its interaction with Nba1, and loss of this interaction results in premature delocalization of Rga1 from the immediately preceding division site and, consequently, abnormal bud-site selection in daughter cells. However, such defects are minor in mother cells of these mutants, likely because the G1 phase is shorter and a new bud site is established prior to delocalization of Rga1. Indeed, our biphasic mathematical model of Cdc42 polarization predicts that premature delocalization of Rga1 leads to more frequent Cdc42 repolarization within the division site when the first temporal step in G1 is assumed to last longer. Spatial distribution of a Cdc42 GAP in coordination with G1 progression may thus be critical for fine-tuning the orientation of the polarity axis in yeast.

PTEN controls glandular morphogenesis through a juxtamembrane β-Arrestin1/ARHGAP21 scaffolding complex.

PTEN controls three-dimensional (3D) glandular morphogenesis by coupling juxtamembrane signaling to mitotic spindle machinery. While molecular mechanisms remain unclear, PTEN interacts through its C2 membrane-binding domain with the scaffold protein β-Arrestin1. Because β-Arrestin1 binds and suppresses the Cdc42 GTPase-activating protein ARHGAP21, we hypothesize that PTEN controls Cdc42 -dependent morphogenic processes through a β-Arrestin1-ARHGAP21 complex. Here, we show that PTEN knockdown (KD) impairs β-Arrestin1 membrane localization, β-Arrestin1-ARHGAP21 interactions, Cdc42 activation, mitotic spindle orientation and 3D glandular morphogenesis. Effects of PTEN deficiency were phenocopied by β-Arrestin1 KD or inhibition of β-Arrestin1-ARHGAP21 interactions. Conversely, silencing of ARHGAP21 enhanced Cdc42 activation and rescued aberrant morphogenic processes of PTEN-deficient cultures. Expression of the PTEN C2 domain mimicked effects of full-length PTEN but a membrane-binding defective mutant of the C2 domain abrogated these properties. Our results show that PTEN controls multicellular assembly through a membrane-associated regulatory protein complex composed of β-Arrestin1, ARHGAP21 and Cdc42.

The Cdc42/Rac1 regulator CdGAP is a novel E-cadherin transcriptional co-repressor with Zeb2 in breast cancer.

The loss of E-cadherin causes dysfunction of the cell-cell junction machinery, which is an initial step in epithelial-to-mesenchymal transition (EMT), facilitating cancer cell invasion and the formation of metastases. A set of transcriptional repressors of E-cadherin (CDH1) gene expression, including Snail1, Snail2 and Zeb2 mediate E-cadherin down-regulation in breast cancer. However, the molecular mechanisms underlying the control of E-cadherin expression in breast cancer progression remain largely unknown. Here, by using global gene expression approaches, we uncover a novel function for Cdc42 GTPase-activating protein (CdGAP) in the regulation of expression of genes involved in EMT. We found that CdGAP used its proline-rich domain to form a functional complex with Zeb2 to mediate the repression of E-cadherin expression in ErbB2-transformed breast cancer cells. Conversely, knockdown of CdGAP expression led to a decrease of the transcriptional repressors Snail1 and Zeb2, and this correlated with an increase in E-cadherin levels, restoration of cell-cell junctions, and epithelial-like morphological changes. In vivo, loss of CdGAP in ErbB2-transformed breast cancer cells impaired tumor growth and suppressed metastasis to lungs. Finally, CdGAP was highly expressed in basal-type breast cancer cells, and its strong expression correlated with poor prognosis in breast cancer patients. Together, these data support a previously unknown nuclear function for CdGAP where it cooperates in a GAP-independent manner with transcriptional repressors to function as a critical modulator of breast cancer through repression of E-cadherin transcription. Targeting Zeb2-CdGAP interactions may represent novel therapeutic opportunities for breast cancer treatment.

Calcineurin, the Ca2+-dependent phosphatase, regulates Rga2, a Cdc42 GTPase-activating protein, to modulate pheromone signaling

Calcineurin, the conserved Ca2+/calmodulin-activated phosphatase, is required for viability during prolonged exposure to pheromone and acts through multiple substrates to down-regulate yeast pheromone signaling. Calcineurin regulates Dig2 and Rod1/Art4 to inhibit mating-induced gene expression and activate receptor internalization, respectively. Recent systematic approaches identified Rga2, a GTPase-activating protein (GAP) for the Cdc42 Rho-type GTPase, as a calcineurin substrate. Here we establish a physiological context for this regulation and show that calcineurin dephosphorylates and positively regulates Rga2 during pheromone signaling. Mating factor activates the Fus3/MAPK kinase, whose substrates induce gene expression, cell cycle arrest, and formation of the mating projection. Our studies demonstrate that Fus3 also phosphorylates Rga2 at inhibitory S/TP sites, which are targeted by Cdks during the cell cycle, and that calcineurin opposes Fus3 to activate Rga2 and decrease Cdc42 signaling. Yeast expressing an Rga2 mutant that is defective for regulation by calcineurin display increased gene expression in response to pheromone. This work is the first to identify cross-talk between Ca2+/calcineurin and Cdc42 signaling and to demonstrate modulation of Cdc42 activity through a GAP during mating.

Roles of an N-terminal coiled-coil-containing domain in the localization and function of Bem3, a Rho GTPase-activating protein in budding yeast

GTPase-activating proteins (GAPs) play critical roles in the spatial and temporal control of small GTPases. The budding yeast Bem3 is a GAP for Cdc42, a Rho GTPase crucial for actin and septin organization. Bem3 localizes to the sites of polarized growth. However, the amino acid sequence determinants mediating recruitment of Bem3 to its physiological sites of action and those important for Bem3 function are not clear. Here, we show that Bem3's localization is guided by two distinct targeting regions-the PX-PH-domain-containing TD1 and the coiled-coil-containing TD2. TD2 localization is largely mediated by its interaction with the polarisome component Epo1 via heterotypic coiled-coil interaction. This finding reveals a novel role for the polarisome in linking Bem3 to its functional target, Cdc42. We also show that the coiled-coil domain of Bem3 interacts homotypically and this interaction is important for the regulation of Cdc42 by Bem3. Moreover, we show that overexpression of a longer version of the TD2 domain disrupts septin-ring assembly in a RhoGAP-independent manner, suggesting that TD2 may be capable of interacting with proteins implicated in septin-ring assembly. Furthermore, we show that the longer version of TD2 interacts with Kss1, a MAPK involved in filamentous growth. Kss1 is reported to localize mainly in the nucleus. We find that Kss1 also localizes to the sites of polarized growth and Bem3 interacts with Kss1 at the septin-ring assembly site. Our study provides new insights in Bem3's localization and function.

Complex Haploinsufficiency-Based Genetic Analysis of the NDR/Lats Kinase Cbk1 Provides Insight into Its Multiple Functions in Candida albicans.

Although the analysis of genetic interactions and networks is a powerful approach to understanding biology, it has not been applied widely to the pathogenic yeast Candida albicans Here, we describe the use of both screening and directed genetic interaction studies based on complex haploinsufficiency to probe the function of the R: egulation of A: ce2 and M: orphogenesis (RAM) pathway in C. albicans A library of 5200 Tn7-mutagenized derivatives of a parental strain heterozygous at CBK1, the key kinase in the RAM pathway, was screened for alterations in serum-induced filamentation. Following confirmation of phenotypes and identification of insertion sites by sequencing, a set of 36 unique double heterozygous strains showing complex haploinsufficiency was obtained. In addition to a large set of genes regulated by the RAM transcription factor Ace2, genes related to cell wall biosynthesis, cell cycle, polarity, oxidative stress, and nitrogen utilization were identified. Follow-up analysis led to the first demonstration that the RAM pathway is required for oxidative stress tolerance in a manner related to the two-component-regulated kinase Chk1 and revealed a potential direct connection between the RAM pathway and the essential Mps1 spindle pole-related kinase. In addition, genetic interactions with CDC42-related genes MSB1, a putative scaffold protein, and RGD3, a putative Rho GTPase-activating protein (GAP) were identified. We also provide evidence that Rgd3 is a GAP for Cdc42 and show that its localization and phosphorylation are dependent on Cbk1.