Hepatocyte polarity establishment and apical lumen formation are organized by Par3, Cdc42, and aPKC in conjunction with Lgl.

Hepatocytes differ from columnar epithelial cells by their multipolar organization, which follows the initial formation of central lumen-sharing clusters of polarized cells as observed during liver development and regeneration. The molecular mechanism for hepatocyte polarity establishment, however, has been comparatively less studied than those for other epithelial cell types. Here, we show that the tight junction protein Par3 organizes hepatocyte polarization via cooperating with the small GTPase Cdc42 to target atypical protein kinase C (aPKC) to a cortical site near the center of cell-cell contacts. In 3D Matrigel culture of human hepatocytic HepG2 cells, which mimics a process of liver development and regeneration, depletion of Par3, Cdc42, or aPKC results in an impaired establishment of apicobasolateral polarity and a loss of subsequent apical lumen formation. The aPKC activity is also required for bile canalicular (apical) elongation in mouse primary hepatocytes. The lateral membrane-associated proteins Lgl1 and Lgl2, major substrates of aPKC, seem to be dispensable for hepatocyte polarity establishment because Lgl-depleted HepG2 cells are able to form a single apical lumen in 3D culture. On the other hand, Lgl depletion leads to lateral invasion of aPKC, and overexpression of Lgl1 or Lgl2 prevents apical lumen formation, indicating that they maintain proper lateral integrity. Thus, hepatocyte polarity establishment and apical lumen formation are organized by Par3, Cdc42, and aPKC; Par3 cooperates with Cdc42 to recruit aPKC, which plays a crucial role in apical membrane development and regulation of the lateral maintainer Lgl.

SKA1 regulates actin cytoskeleton remodelling via activating Cdc42 and influences the migration of pancreatic ductal adenocarcinoma cells.

OBJECTIVES: Spindle and kinetochore-associated protein 1(SKA1), originally identified as a protein essential for proper chromosome segregation, has been recently linked to multiple malignancies. This study aimed to explore the biological, clinical role and molecular mechanism of SKA1 in pancreatic carcinogenesis. MATERIALS AND METHODS: SKA1 expression was detected in 145 pancreatic ductal adenocarcinoma (PDAC) specimens by immunohistochemistry. Biological behaviour assays were used to determine the role of SKA1 in PDAC progression in vitro and in vivo. Using isobaric tags for relative and absolute quantitation (iTRAQ), SKA1's downstream proteins were examined. Moreover, cytochalasin B and ZCL278 were used to explore the changes of SKA1-induced signalling and cell morphology, with further confirmation by immunoblotting and immunofluorescence assays. RESULTS: Increased SKA1 expression was significantly correlated with tumour size and cellular differentiation degree in PDAC tissues. Furthermore, elevated levels of SKA1 reflected shorter overall survival (P = .019). As for biological behaviour, SKA1 acted as a tumour promotor in PDAC, overexpression of SKA1 facilitates cell proliferation, migration and invasion in vitro and in vivo. Mechanistically, we demonstrated that SKA1 enhanced pancreatic cancer aggressiveness by inhibiting G2/M arrest and regulating actin cytoskeleton organization via activating Cdc42. CONCLUSIONS: This study revealed novel roles for SKA1 as an important regulator of actin cytoskeleton organization and an oncogene in PDAC cells, which may provide insights into developing novel therapeutics.

miR-29a/b/c function as invasion suppressors for gliomas by targeting CDC42 and predict the prognosis of patients.

BACKGROUND: The lethality and poor outcome of high-grade gliomas result from the tumour relentless invasion. miR-29a/b/c downexpressions contribute to several human tumourigenesis. However, their relevance to prognosis and invasion in gliomas remains unclear. METHODS: Relationships of miR-29a/b/c and CDC42 expressions to grade and survival-time in 147 human gliomas were analysed by in situ hybridisation and immunohistochemistry. Dual-luciferase reporter assay was used to identify CDC42 as a target of miR-29a/b/c. Underlining mechanisms by which miR-29a/b/c inhibited glioma cell migration and invasion were studied by in vitro and in vivo assays. RESULTS: miR-29a/b/c expressions were inversely correlated with glioma grades, but positively correlated with patients' survival. Two distinct subgroups of grade I-IV glioma patients with different prognoses were identified according to miR-29a/b/c expressions. miR-29a/b/c overexpressions suppressed glioma cell migration and invasion through targeting CDC42 and subsequently decreasing phosphorylated PAK1/2/3, LIMK1/2 and cofilin, the pivotal downstream effectors of CDC42. Moreover, CDC42 expression was positively correlated with glioma grades, but inversely correlated with miR-29a/b/c expressions and patients' survival. In glioblastoma cell lines, CDC42-knockdown could mimic the anti-tumour effects of miR-29a/b/c. CONCLUSIONS: miR-29a/b/c are important tumour suppressors and novel prognostic biomarkers of gliomas, and miR-29a/b/c and CDC42 are potential therapeutic candidates for malignant gliomas.

Probing Cdc42 Polarization Dynamics in Budding Yeast Using a Biosensor.

Cdc42 is a small guanosine triphosphatase (GTPase) that plays a central role in polarity development in diverse cell types. Since the activity of Cdc42 is dynamically controlled in time and space, it is required to develop a biosensor to monitor its activation in vivo. In this chapter, we describe the construction and usage of a simple and robust biosensor for monitoring active Cdc42 in budding yeast. This affinity-based biosensor uses a red fluorescent protein fused to a Cdc42- and Rac-interactive binding motif from one of the Cdc42 effector proteins. Because it binds specifically to the GTP-bound Cdc42, this biosensor can be used to monitor Cdc42 activation in vivo. This or similar biosensors can be widely used for studying GTPase signaling in other cell types because of the conserved CRIB motif present among GTPase targets.

Aerobic exercise regulates Rho/cofilin pathways to rescue synaptic loss in aged rats.

PURPOSE: The role of exercise to prevent or reverse aging-induced cognitive decline has been widely reported. This neuroprotection is associated with changes in the synaptic structure plasticity. However, the mechanisms of exercise-induced synaptic plasticity in the aging brain are still unclear. Thus, the aim of the present study is to investigate the aging-related alterations of Rho-GTPase and the modulatory influences of exercise training. METHODS: Young and old rats were used in this study. Old rats were subjected to different schedules of aerobic exercise (12 m/min, 60 min/d, 3d/w or 5d/w) or kept sedentary for 12 w. After 12 w of aerobic exercise, the synapse density in the cortex and hippocampus was detected with immunofluorescent staining using synaptophysin as a marker. The total protein levels of RhoA, Rac1, Cdc42 and cofilin in the cortex and hippocampus were detected with Western Blot. The activities of RhoA, Rac1 and Cdc42 were determined using a pull down assay. RESULTS: We found that synapse loss occurred in aging rats. However, the change of expression and activity of RhoA, Rac1 and Cdc42 was different in the cortex and hippocampus. In the cortex, the expression and activity of Rac1 and Cdc42 was greatly increased with aging, whereas there were no changes in the expression and activity of RhoA. In the hippocampus, the expression and activity of Rac1 and Cdc42 was greatly decreased and there were no changes in the expression and activity of RhoA. As a major downstream substrate of the Rho GTPase family, the increased expression of cofilin was only observed in the cortex. High frequency exercise ameliorated all aging-related changes in the cortex and hippocampus. CONCLUSIONS: These data suggest that aerobic exercise reverses synapse loss in the cortex and hippocampus in aging rats, which might be related to the regulation of Rho GTPases.

FLIM FRET Visualization of Cdc42 Activation by Netrin-1 in Embryonic Spinal Commissural Neuron Growth Cones.

Netrin-1 is an essential extracellular chemoattractant that signals through its receptor DCC to guide commissural axon extension in the embryonic spinal cord. DCC directs the organization of F-actin in growth cones by activating an intracellular protein complex that includes the Rho GTPase Cdc42, a critical regulator of cell polarity and directional migration. To address the spatial distribution of signaling events downstream of netrin-1, we expressed the FRET biosensor Raichu-Cdc42 in cultured embryonic rat spinal commissural neurons. Using FLIM-FRET imaging we detected rapid activation of Cdc42 in neuronal growth cones following application of netrin-1. Investigating the signaling mechanisms that control Cdc42 activation by netrin-1, we demonstrate that netrin-1 rapidly enriches DCC at the leading edge of commissural neuron growth cones and that netrin-1 induced activation of Cdc42 in the growth cone is blocked by inhibiting src family kinase signaling. These findings reveal the activation of Cdc42 in embryonic spinal commissural axon growth cones and support the conclusion that src family kinase activation downstream of DCC is required for Cdc42 activation by netrin-1.

Ratiometric Imaging Using a Single Dye Enables Simultaneous Visualization of Rac1 and Cdc42 Activation.

Biosensors that report endogenous protein activity in vivo can be based on environment-sensing fluorescent dyes. The dyes can be attached to reagents that bind selectively to a specific conformation of the targeted protein, such that binding leads to a fluorescence change. Dyes that are sufficiently bright for use at low, nonperturbing intracellular concentrations typically undergo changes in intensity rather than the shifts in excitation or emission maxima that would enable precise quantitation through ratiometric imaging. We report here mero199, an environment-sensing dye that undergoes a 33 nm solvent-dependent shift in excitation. The dye was used to generate a ratiometric biosensor of Cdc42 (CRIB199) without the need for additional fluorophores. CRIB199 was used in the same cell with a FRET sensor of Rac1 activation to simultaneously observe Cdc42 and Rac1 activity in cellular protrusions, indicating that Rac1 but not Cdc42 activity was reduced during tail retraction, and specific protrusions had reduced Cdc42 activity. A novel program (EdgeProps) used to correlate localized activation with cell edge dynamics indicated that Rac1 was specifically reduced during retraction.

GTPases Rho distribution in intraepithelial and invasive neoplasias of the uterine cervix.

PURPOSE OF INVESTIGATION: To evaluate the distribution of GTPases RhoA, RhoB, and Cdc42 in cervical intraepithelial neoplasias (CIN) and invasive neoplasias of the uterine cervix. MATERIALS AND METHODS: samples of neoplastic lesions of the uterine cervix of 44 patients were classified in: CIN I (n = 10), CIN II (n = 10), CIN III (n = 09), and invasive carcinoma (n = 15). Antibodies anti-RhoA, anti-RhoB, and anti-Cdc42 were used and staining was classified as: negative, mild, moderate, and intense positive. RESULTS: When compared with dysplastic cells, superficial cells showed: higher expression of RhoB in CIN I (p = 0.0018), and lower expression of Cdc42 in CIN I (p = 0.0225). The authors observed higher expression of RhoA (p = 0.0002) and RhoB (p = 0.0046) in CIN dysplastic cells when compared with invasive carcinoma cells. CONCLUSIONS: GTPases Rho may be involved with the regulation of biological processes, important to the progression of cervical neoplasias. Probably, RhoA is important for maintenance of cell differentiation and RhoB protects cells from malignant cervical neoplasia.

A new genetically encoded single-chain biosensor for Cdc42 based on FRET, useful for live-cell imaging.

Cdc42 is critical in a myriad of cellular morphogenic processes, requiring precisely regulated activation dynamics to affect specific cellular events. To facilitate direct observations of Cdc42 activation in live cells, we developed and validated a new biosensor of Cdc42 activation. The biosensor is genetically encoded, of single-chain design and capable of correctly localizing to membrane compartments as well as interacting with its upstream regulators including the guanine nucleotide dissociation inhibitor. We characterized this new biosensor in motile mouse embryonic fibroblasts and observed robust activation dynamics at leading edge protrusions, similar to those previously observed for endogenous Cdc42 using the organic dye-based biosensor system. We then extended our validations and observations of Cdc42 activity to macrophages, and show that this new biosensor is able to detect differential activation patterns during phagocytosis and cytokine stimulation. Furthermore, we observe for the first time, a highly transient and localized activation of Cdc42 during podosome formation in macrophages, which was previously hypothesized but never directly visualized.

Remarkable reductions of PAKs in the brain tissues of scrapie-infected rodent possibly linked closely with neuron loss.

Prion diseases are irreversible progressive neurodegenerative diseases characterized in the brain by PrP(Sc) deposits, neuronal degeneration, gliosis and by cognitive, behavioral and physical impairments, leading to severe incapacity and inevitable death. Proteins of the p21-activated kinase (PAK) family are noted for roles in gene transcription, cytoskeletal dynamics, cell cycle progression and survival signaling. In the present study, we aimed to identify the potential roles of PAKs during prion infection, utilizing the brains of scrapie agent-infected hamsters. Western blots and immunohistochemical assays showed that brain levels of PAK3 and PAK1, as well as their upstream activator Rac/cdc42 and downstream substrate Raf1, were remarkably reduced at terminal stage. Double-stained immunofluorescent assay demonstrated that PAK3 was expressed mainly in neurons. Dynamic analyses of the brain samples collected at the different time points during the incubation period illustrated successive decreases of PAK3, PAK1 and Raf1, especially phosphor Raf1, which correlated well with neuron loss. Rac/cdc42 in the brain tissues increased at early stage and reached to the top at mid-late stage, but diminished at final stage. Unlike the alteration of PAKs in vivo, PAK3 and PAK1, as well as Rac/cdc42 and Raf1 in the prion-infected cell line SMB-S15 remained unchanged compared with those of its normal cell line SMB-PS. Our data here indicate that the functions of PAKs and their associated signaling pathways are seriously affected in the brains of prion disease, which appear to associate closely with the extensive neuron loss.

Environment-sensing merocyanine dyes for live cell imaging applications.

Fluorescent biosensors based on environmentally sensitive dyes enable visualization and quantification of endogenous protein activation within living cells. Merocyanine dyes are especially useful for live cell imaging applications, as they are extraordinarily bright, have long wavelengths of excitation and emission, and can exhibit readily detectable fluorescence changes in response to environment. We sought to systematically examine the effects of structural features on key photophysical properties, including dye brightness, environmental responsiveness, and photostability, through the synthesis of a library of 25 merocyanine dyes, derived from combinatorial reaction of 5 donor and 5 acceptor heterocycles. Four of these dyes showed optimal properties for specific imaging applications and were subsequently prepared with reactive side chains and enhanced aqueous solubility using a one-pot synthetic method. The new dyes were then applied within a biosensor design for Cdc42 activation, where dye mero60 showed a remarkable 1470% increase in fluorescence intensity on binding activated Cdc42 in vitro. The dye-based biosensors were used to report activation of endogenous Cdc42 in living cells.

Overexpression of small GTPases directly correlates with expression of δ-catenin and their coexpression predicts a poor clinical outcome in nonsmall cell lung cancer.

δ-catenin can affect cytoskeletal assembly, and promote cell migration by regulating the activity of small GTPases. While many malignancies have been shown to be positive for δ-catenin, it is still unclear whether δ-catenin and small GTPases are coexpressed in tumor cells, and so is the relationship between their coexpression and prognosis in the tumor patients. In this study, immunohistochemistry was performed to examine expressive levels of δ-catenin, cdc42, and Rac1 in 135 cases of nonsmall cell lung cancer (NSCLC), including 60 cases with follow-up records. Thirty samples of paired lung cancer tissues and adjacent normal lung tissues were collected to analyze mRNA and protein expression of δ-catenin and small GTPases. The effects of δ-catenin on small GTPases expression and invasive ability of lung cancer cells were also evaluated. Compared with normal lung tissues, both mRNA and protein levels of δ-catenin and small GTPases were increased in lung cancer tissues (P < 0.05), and the expression of small GTPases directly correlated with that of δ-catenin (P < 0.001). In addition, δ-catenin and small GTPases tended to be coexpressed in lung adenocarcinoma, advanced stages, and primary tumors with lymph node metastasis (all P < 0.05). The patients with coexpression of δ-catenin and small GTPases had a shorter survival time than those without coexpression (P < 0.05). Furthermore, δ-catenin overexpression could enhance invasive ability of lung cancer cells by upregulating protein and transcriptional level of small GTPases. Therefore, δ-catenin likely upregulates the activity of small GTPases at transcriptional level, and their coexpression may predict a poor clinical outcome in NSCLC patients.

The association between CDC42 and caveolin-1 is involved in the regulation of capacitation and acrosome reaction of guinea pig and mouse sperm.

In the mammalian sperm, the acrosome reaction (AR) is considered to be a regulated secretion that is an essential requirement for physiological fertilization. The AR is the all-or-nothing secretion system that allows for multiple membrane fusion events. It is a Ca(2)(+)-regulated exocytosis reaction that has also been shown to be regulated by several signaling pathways. CDC42 has a central role in the regulated exocytosis through the activation of SNARE proteins and actin polymerization. Furthermore, the lipid raft protein caveolin-1 (CAV1) functions as a scaffold and guanine nucleotide dissociation inhibitor protein for CDC42, which is inactivated when associated with CAV1. CDC42 and other RHO proteins have been shown to localize in the acrosome region of mammalian sperm; however, their relationship with the AR is unknown. Here, we present the first evidence that CDC42 and CAV1 could be involved in the regulation of capacitation and the AR. Our findings show that CDC42 is activated early during capacitation, reaching an activation maximum after 20 min of capacitation. Spontaneous and progesterone-induced ARs were inhibited when sperm were capacitated in presence of secramine A, a specific CDC42 inhibitor. CAV1 and CDC42 were co-immunoprecipitated from the membranes of noncapacitated sperm; this association was reduced in capacitated sperm, and our data suggest that the phosphorylation (Tyr14) of CAV1 by c-Src is involved in such reductions. We suggest that CDC42 activation is favored by the disruption of the CAV1-CDC42 interaction, allowing for its participation in the regulation of capacitation and the AR.

Interleukin-1α, -6, and -8 decrease Cdc42 activity resulting in loss of articular chondrocyte phenotype.

Small GTPase proteins mediate changes in cellular morphology and other cellular functions. The aim of this study was to examine signaling of the small GTPase Cdc42 by stimulating chondrocytes grown in monolayer with long- (96 h) or short- (2 and 30 min) term exposure to interleukin-1α (IL-1α), IL-6, or IL-8. Quantitative PCR was used to determine changes in collagen type IIB (COL2A1), aggrecan (AGG), and matrix metalloproteinase-13 (MMP-13) gene expression after prolonged cytokine exposure. Effects of short-term treatment with IL-α, IL-6, or IL-8 on endogenous GTP-bound Cdc42 levels were assessed using an affinity assay, and on actin filament organization using confocal microscopy. Cytokine treatments significantly decreased COL2A1 and AGG expression and increased MMP-13 expression. Short exposure to IL-1α, IL-6, or IL-8 decreased endogenous GTP-Cdc42 and increased stress fibers, which were reversed with cytochalasin D treatment. These results show that IL-mediated Cdc42 signaling modifies chondrocyte phenotype and morphology. This may lend insight into the altered chondrocyte phenotype in catabolic conditions such as osteoarthritis.

Methylmercury exposure downregulates the expression of Racl and leads to neuritic degeneration and ultimately apoptosis in cerebrocortical neurons.

Methylmercury (MeHg) has been recognized as a neurotoxicant targeted on the central nervous system including cerebellum and cerebral cortex. Some molecular targets of MeHg have been identified using cerebellar neuronal cells, but little is known in the cerebrocortical neuronal cells. In this study, the molecular mechanism underlying MeHg-induced cell death in cerebrocortical neurons was investigated using a primary culture of embryonic rat cortical neuronal cells. The cultured cells exhibited apoptosis 3 days after exposure to 100 nM MeHg, suggesting the involvement of caspase-dependent apoptotic pathways. We demonstrated for the first time that neuritic degeneration precedes MeHg-induced apoptotic death in neurons exposed to 100 nM MeHg. Immunocytochemical and ELISA analyses for neurite-specific proteins namely, tau and MAP2, showed that injury to tau-positive axons was first induced followed by damage to the dendrites and cellular bodies. To further investigate the factors responsible for neuronal death, we investigated the expression levels of Rho-family proteins (Rac1, Cdc42, and RhoA), which regulate neuritic functions and apoptosis in neurons. Western blot analysis demonstrated that MeHg downregulated the expression levels of Rac1 and Cdc42 but did not affect RhoA. The exposure concentration and time course studies confirmed that Rac1 is targeted during an early stage of MeHg-induced cytotoxicity. The results indicate that neuritic degeneration, in particular axonal degeneration triggered by the downregulation of Rac1 expression, contributes to MeHg-induced apoptotic cell death in cultured cerebrocortical neurons.

Cdc42 is highly expressed in colorectal adenocarcinoma and downregulates ID4 through an epigenetic mechanism.

Cdc42, a member of Rho GTPases family, is involved in the regulation of several cellular functions, such as rearrangement of actin cytoskeleton, membrane trafficking, cell-cycle progression, and transcriptional regulation. Aberrant expression or activity of Cdc42 has been reported in several tumours. Here, the specific role of Cdc42 in development and progression of colorectal cancer was analyzed through microarrays technology. A comparative analysis of Cdc42 overexpressing cells versus cells with decreased Cdc42 levels through siRNA revealed that Cdc42 overexpression down-regulated the potential tumour suppressor gene ID4. Results were validated by quantitative RT-PCR and the methylation status of the specific promoter, analyzed. Methylation-specific PCR and bisulfite sequencing PCR analysis revealed that Cdc42 induced the methylation of the CpG island of the ID4 promoter. Colorectal adenocarcinoma samples were compared with the corresponding adjacent normal tissue of the same patient in order to determine specific gene expression levels. The downregulation of ID4 by Cdc42 was also found of relevance in colorectal adenocarcinoma biopsies. Cdc42 was found to be overexpressed with high incidence (60%) in colorectal cancer samples, and this expression was associated with silencing of ID4 with statistical significance (p<0.05). Cdc42 may have a role in the development of colon cancer. Furthermore, inhibition of Cdc42 activity may have a direct impact in the management of colorectal cancer.

Syndecan-1 interaction with the LG4/5 domain in laminin-332 is essential for keratinocyte migration.

Laminin 5/laminin 332 (LN332) is an adhesion substrate for epithelial cells. After secretion of LN332, a regulated cleavage occurs at the carboxy-terminus of its alpha3 subunit, which releases a tandem of two globular modules named LG4/5. We show that the presence of the LG4/5 domain in precursor LN332 decreases its integrin-mediated cell adhesion properties in comparison with mature LN332. Whereas cell adhesion to the recombinant LG4/5 fragment relies solely on the heparan sulfate proteoglycan (HSPG) receptor syndecan-1, we reveal that both syndecan-1 and the alpha3beta1 integrin bind to precursor LN332. We further demonstrate that syndecan-1 mediated cell adhesion to the LG4/5 fragment and pre-LN332 allows the formation of fascin-containing protrusions, depending on the GTPases Rac and Cdc42 activation. Reducing syndecan-1 expression in normal keratinocytes prevents cell protrusions on pre-LN332 with subsequent failure of the peripheral localization of the alpha3beta1 integrin. We finally show that cell migration on pre-LN332 requires syndecan-1. Therefore, the LG4/5 domain in precursor LN332 appears to trigger intracellular signaling events, which participate in keratinocyte motility.

The Par polarity complex regulates Rap1- and chemokine-induced T cell polarization.

Cell polarization is required for virtually all functions of T cells, including transendothelial migration in response to chemokines. However, the molecular pathways that establish T cell polarity are poorly understood. We show that the activation of the partitioning defective (Par) polarity complex is a key event during Rap1- and chemokine-induced T cell polarization. Intracellular localization and activation of the Par complex are initiated by Rap1 and require Cdc42 activity. The Rac activator Tiam1 associates with both Rap1 and components of the Par complex, and thereby may function to connect the Par polarity complex to Rap1 and to regulate the Rac-mediated actin remodelling required for T cell polarization. Consistent with these findings, Tiam1-deficient T cells are impaired in Rap1- and chemokine-induced polarization and chemotaxis. Our studies implicate Tiam1 and the Par polarity complex in polarization of T cells, and provide a mechanism by which chemokines and Rap1 regulate T cell polarization and chemotaxis.

Endothelin 1 stimulates beta1Pix-dependent activation of Cdc42 through the G(salpha) pathway.

Beta1Pix (PAK-interacting exchange factor) is a recently identified guanine nucleotide exchange factor (GEF) for the Rho family small G protein Cdc42/Rac. On stimulation with extracellular signals, GEFs induce the exchange of guanosine diphosphate to guanosine triphosphate, resulting in the activation of the small guanosine 5C-triphosphatases. This activation enables the signal to propagate to downstream effectors. Herein, we show that G(salpha) stimulation by cholera toxin increased Cdc42 activation by endothelin-1 (ET-1), whereas pertussis toxin had no effect. H-89, a protein kinase A (PKA) inhibitor, strongly inhibited Cdc42 activation by ET-1. Moreover, the overexpression of beta1Pix enhanced ET-1-induced Cdc42 activation. The essential role of beta1Pix in ET-1-induced Cdc42 activation was evidenced by the blocking of Cdc42 activation in cells expressing beta1Pix mutant lacking the ability to bind PAK (beta1Pix SH3m[W43K]) or mutant lacking GEF activity (beta1PixdeltaDH). The overexpression of mutant lacking the pleckstrin homology domain beta1PixdeltaPH, which is unable to bind phospholipids, had no effect on Cdc42 activation. These results demonstrate that beta1Pix, along with PKA, plays a crucial role in the regulation of Cdc42 activation by ET-1.