Dominant Negative Mutants Of Cdc42 Research Articles

Activin B-activated Cdc42 signaling plays a key role in regulating adipose-derived mesenchymal stem cells-mediated skin wound healing

BackgroundIn our previous study, activin B in combination with ADSCs enhances skin wound healing. However, the underlying molecular mechanisms are not well studied. Cdc42 is recognized to play a critical role in the regulation of stem cells.MethodsPull-down assay was performed to investigate the activity of Cdc42. The dominant-negative mutant of Cdc42 (Cdc42N17) was used to explore the role of Cdc42 in activin B-induced ADSCs migration, proliferation, and secretion in vitro. Cdc42N17-transfected ADSCs were injected into a full-thickness excisional wound model to explore their efficiency in wound healing in vivo. The wound healing efficacy was evaluated by the wound closure rates and histological examination. The neovascularization and wound contraction were detected by immunohistochemistry staining of CD31 and α-SMA. Finally, the underlying mechanisms were explored by RNA sequencing.ResultsCdc42N17 inhibited ADSCs migration, proliferation, and secretion induced by activin B. Furthermore, Cdc42N17-transfected ADSCs inhibited the wound closure rate and suppressed the expression of CD31 and α-SMA induced by activin B in vivo. The RNA sequencing showed that the differentially expressed genes in Cdc42N17-transfected ADSCs versus ADSCs were associated with cell migration, proliferation, and adhesion. Further study revealed that the Cdc42-Erk-Srf pathway was required for activin B-induced proliferation in ADSCs.ConclusionsOur study indicates that Cdc42 plays a crucial role in ADSCs-mediated skin wound healing induced by activin B.Graphical

Cdc42 Facilitates Axonogenesis by Enhancing Microtubule Stabilization in Primary Hippocampal Neurons.

The establishment of polarity is an essential process in early neuronal development. Cdc42, a GTPase of the Rho family, is a key regulator of cytoskeletal dynamics and neuronal polarity. However, the mechanisms underlying the action of cdc42 in regulating axonogenesis have not been elucidated. Here, we expressed wild-type cdc42, a constitutively active cdc42 mutant (cdc42F28L) and a dominant negative cdc42 mutant (cdc42N17), respectively, in the primary hippocampal neurons to alter the activity of cdc42. We found that cdc42 activities were paralleled with the capacities to promote axonogenesis in the cultured neurons. Cdc42 also enhanced microtubule stability in the cultured neurons. Pharmacologically stabilizing microtubules significantly abrogated the defective axonogenesis induced by cdc42 inhibition. Moreover, cdc42 promoted the dephosphorylation of collapsing response mediator protein-2 (CRMP-2) at Thr514 by increasing GSK-3β phosphorylation at Ser9 in the cultured neurons. These findings suggest that cdc42 may facilitate axonogenesis by promoting microtubule stabilization in rat primary hippocampal neurons.

Cdc42 is involved in NC1 peptide-regulated BTB dynamics through actin and microtubule cytoskeletal reorganization.

Noncollagenous domain 1 (NC1)-peptide is a biologically active peptide derived from the C-terminal region of collagen α3(IV) chain, a structural constituent protein at the basement membrane in the rat testis, likely via proteolytic cleavage of matrix metalloproteinase 9. Studies have shown that this NC1 peptide regulates testis function by inducing Sertoli cell blood-testis barrier (BTB) remodeling and is also capable of inducing elongate spermatid exfoliation through its disruptive effects on the organization of actin- and microtubule (MT)-based cytoskeletons at these cell adhesion sites. However, the underlying molecular mechanism remains unknown. NC1 peptide was found to exert its biologic effects through an activation of small GTPase cell division control protein 42 homolog (Cdc42) because cooverexpression of the dominant negative mutant of Cdc42 [namely, Cdc42-T17N (via a single mutation of amino acid residue 17 from the N terminus from Thr to Asn by site-directed mutagenesis, making it constitutively inactive)] and NC1 peptide was able to block the NC1 peptide-induced Sertoli cell tight junction-permeability barrier disruption. Their cooverexpression also blocked the NC1 peptide-induced misdistribution of BTB-associated proteins at the cell-cell interface and also disruptive cytoskeletal organization of F-actin and MTs through changes in spatial expression of the corresponding actin and MT regulatory proteins. Interestingly, NC1 peptide was also found to induce an up-regulation of phosphorylated (p)-ribosomal protein S6 (rpS6) (namely, p-rpS6-S235/S236) and a concomitant down-regulation of p-Akt1/2 (namely, p-Akt1-S473 and p-Akt2-S474), but these changes could not be blocked by overexpression of Cdc42-T17N. More importantly, NC1 peptide-induced Cdc42 activation was effectively blocked by treatment of Sertoli cell epithelium with a p-Akt1/2 activator SC79, which is also capable of blocking NC1 peptide-induced down-regulation of p-Akt1-S473 and p-Akt2/S474, but not p-rpS6-S235/S236 up-regulation. In summary, these findings illustrate that Cdc42 is working downstream of the mammalian target of rapamycin complex 1/rpS6/Akt1/2 signaling pathway to support NC1 peptide-mediated effects on Sertoli cell function in the testis using the rat as an animal model.-Su, W., Cheng, C. Y. Cdc42 is involved in NC1 peptide-regulated BTB dynamics through actin and microtubule cytoskeletal reorganization.

Matrix rigidity regulates spatiotemporal dynamics of Cdc42 activity and vacuole formation kinetics of endothelial colony forming cells

Recent evidence has shown that endothelial colony forming cells (ECFCs) may serve as a cell therapy for improving blood vessel formation in subjects with vascular injury, largely due to their robust vasculogenic potential. The Rho family GTPase Cdc42 is known to play a primary role in this vasculogenesis process, but little is known about how extracellular matrix (ECM) rigidity affects Cdc42 activity during the process. In this study, we addressed two questions: Does matrix rigidity affect Cdc42 activity in ECFC undergoing early vacuole formation? How is the spatiotemporal activation of Cdc42 related to ECFC vacuole formation? A fluorescence resonance energy transfer (FRET)-based Cdc42 biosensor was used to examine the effects of the rigidity of three-dimensional (3D) collagen matrices on spatiotemporal activity of Cdc42 in ECFCs. Collagen matrix stiffness was modulated by varying the collagen concentration and therefore fibril density. The results showed that soft (150Pa) matrices induced an increased level of Cdc42 activity compared to stiff (1kPa) matrices. Time-course imaging and colocalization analysis of Cdc42 activity and vacuole formation revealed that Cdc42 activity was colocalized to the periphery of cytoplasmic vacuoles. Moreover, soft matrices generated faster and larger vacuoles than stiff matrices. The matrix-driven vacuole formation was enhanced by a constitutively active Cdc42 mutant, but significantly inhibited by a dominant-negative Cdc42 mutant. Collectively, the results suggest that matrix rigidity is a strong regulator of Cdc42 activity and vacuole formation kinetics, and that enhanced activity of Cdc42 is an important step in early vacuole formation in ECFCs.

HnRNP Q Regulates Cdc42-Mediated Neuronal Morphogenesis

The RNA-binding protein hnRNP Q has been implicated in neuronal mRNA metabolism. Here, we show that knockdown of hnRNP Q increased neurite complexity in cultured rat cortical neurons and induced filopodium formation in mouse neuroblastoma cells. Reexpression of hnRNP Q1 in hnRNP Q-depleted cells abrogated the morphological changes of neurites, indicating a specific role for hnRNP Q1 in neuronal morphogenesis. A search for mRNA targets of hnRNP Q1 identified functionally coherent sets of mRNAs encoding factors involved in cellular signaling or cytoskeletal regulation and determined its preferred binding sequences. We demonstrated that hnRNP Q1 bound to a set of identified mRNAs encoding the components of the actin nucleation-promoting Cdc42/N-WASP/Arp2/3 complex and was in part colocalized with Cdc42 mRNA in granules. Using subcellular fractionation and immunofluorescence, we showed that knockdown of hnRNP Q reduced the level of some of those mRNAs in neurites and redistributed their encoded proteins from neurite tips to soma to different extents. Overexpression of dominant negative mutants of Cdc42 or N-WASP compromised hnRNP Q depletion-induced neurite complexity. Together, our results suggest that hnRNP Q1 may participate in localization of mRNAs encoding Cdc42 signaling factors in neurites, and thereby may regulate actin dynamics and control neuronal morphogenesis.

Collybistin splice variants differentially interact with gephyrin and Cdc42 to regulate gephyrin clustering at GABAergic synapses

Collybistin (CB) is a guanine-nucleotide-exchange factor (GEF) selectively activating Cdc42. CB mutations cause X-linked mental retardation due to defective clustering of gephyrin, a postsynaptic protein associated with both glycine and GABA(A) receptors. Using a combination of biochemistry and cell biology we provide novel insights into the roles of the CB2 splice variants, CB2(SH3+) and CB2(SH3-), and their substrate, Cdc42, in regulating gephyrin clustering at GABAergic synapses. Transfection of Myc-tagged CB2(SH3+) and CB2(SH3-) into cultured neurons revealed strong, but distinct, effects promoting postsynaptic gephyrin clustering, denoting mechanistic differences in their function. In addition, overexpression of constitutively active or dominant-negative Cdc42 mutants identified a new function of Cdc42 in regulating the shape and size of postsynaptic gephyrin clusters. Using biochemical assays and native brain tissue, we identify a direct interaction between gephyrin and Cdc42, independent of its activation state. Finally, our data show that CB2(SH3-), but not CB2(SH3+), can form a ternary complex with gephyrin and Cdc42, providing a biochemical substrate for the distinct contribution of these CB isoforms in gephyrin clustering at GABAergic postsynaptic sites. Taken together, our results identify CB and Cdc42 as major regulators of GABAergic postsynaptic densities.

Polarized migration of lymphatic endothelial cells is critically dependent on podoplanin regulation of Cdc42

We have shown previously that T1α/podoplanin is required for capillary tube formation by human lung microvascular lymphatic endothelial cells (HMVEC-LLy) and that cells with decreased podoplanin expression fail to properly activate the small GTPase RhoA shortly after the beginning of the lymphangiogenic process. The objective of this study was to determine whether podoplanin regulates HMVEC-LLy migration and whether this regulation is via modulation of small GTPase activation. In analysis of scratch wound assays, we found that small interfering RNA (siRNA) depletion of podoplanin expression in HMVEC-LLy inhibits VEGF-induced microtubule-organizing center (MTOC) and Golgi polarization and causes a dramatic reduction in directional migration compared with control siRNA-transfected cells. In addition, a striking redistribution of cortical actin to fiber networks across the cell body is observed in these cells, and, remarkably, it returns to control levels if the cells are cotransfected with a dominant-negative mutant of Cdc42. Moreover, cotransfection of a dominant-negative construct of Cdc42 into podoplanin knockdown HMVEC-LLy completely abrogated the effect of podoplanin deficiency, rescuing MTOC and Golgi polarization and cell migration to control level. Importantly, expression of constitutively active Cdc42 construct, like podoplanin knockdown, decreased RhoA-GTP level in HMVEC-LLy, demonstrating cross talk between both GTPases. Taken together, the results indicate that polarized migration of lymphatic endothelial cells in response to VEGF is mediated via a pathway of podoplanin regulation of small GTPase activities, in particular Cdc42.

Integrin β4 Regulates Migratory Behavior of Keratinocytes by Determining Laminin-332 Organization

Whether alpha6beta4 integrin regulates migration remains controversial. beta4 integrin-deficient (JEB) keratinocytes display aberrant migration in that they move in circles, a behavior that mirrors the circular arrays of laminin (LM)-332 in their matrix. In contrast, wild-type keratinocytes and JEB keratinocytes, induced to express beta4 integrin, assemble laminin-332 in linear tracks over which they migrate. Moreover, laminin-332-dependent migration of JEB keratinocytes along linear tracks is restored when cells are plated on wild-type keratinocyte matrix, whereas wild-type keratinocytes show rotation over circular arrays of laminn-332 in JEB keratinocyte matrix. The activities of Rac1 and the actin cytoskeleton-severing protein cofilin are low in JEB keratinocytes compared with wild-type cells but are rescued following expression of wild-type beta4 integrin in JEB cells. Additionally, in wild-type keratinocytes Rac1 is complexed with alpha6beta4 integrin. Moreover, Rac1 or cofilin inactivation induces wild-type keratinocytes to move in circles over rings of laminin-332 in their matrix. Together these data indicate that laminin-332 matrix organization is determined by the alpha6beta4 integrin/actin cytoskeleton via Rac1/cofilin signaling. Furthermore, our results imply that the organizational state of laminin-332 is a key determinant of the motility behavior of keratinocytes, an essential element of skin wound healing and the successful invasion of epidermal-derived tumor cells.

The Role of SPECs, Small Cdc42-binding Proteins, in F-actin Accumulation at the Immunological Synapse

SPEC1 and SPEC2 are structurally similar Cdc42-binding proteins of 79 and 84 amino acid residues, respectively. We investigated the role of SPEC2 in T cell function due to its high mRNA expression in lymphocytes. Western blot analysis revealed abundant SPEC2 protein in lymphocytes, which in glutathione S-transferase-capture experiments specifically interacted with only GTP-bound Cdc42. Immunofluorescence experiments revealed that the SPEC2 protein was diffusely localized in the cytoplasm and at the cell membrane in unstimulated Jurkat T cells and Raji B cells. Recruitment of SPEC2 within Jurkat T cells to the antigen-presenting cell interface occurred following incubation with staphylococcal enterotoxin E superantigen-loaded B cells and colocalized there with F-actin and Cdc42. T cell receptor (TCR) activation studies using anti-CD3 antibody-coated polystyrene beads showed that SPEC2 was recruited to the site of bead contact, which was not observed with anti-major histocompatibility complex antibody-coated beads. Accumulation of SPEC2 following TCR engagement occurred as early as 5 min, before obvious F-actin accumulation. Biochemical studies with Jurkat T cells demonstrated that N-terminal cysteine residues in SPEC2 were palmitoylated. Overexpression studies of the related SPEC1 showed that it also was recruited to the activated TCR. Mutational analysis revealed that localization of SPEC1 to the TCR required two N-terminal cysteine residues. Furthermore, a SPEC1 Cdc42 Rac-interacting binding mutant, containing an intact N terminus but defective in Cdc42 binding, completely blocked F-actin accumulation at the activated TCR. Taken together these results suggest that SPECs may play important roles in Cdc42-mediated F-actin accumulation at the immunological synapse.

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.

Functional Characterization of Gαo Signaling through G Protein-Regulated Inducer of Neurite Outgrowth 1

G protein-regulated inducer of neurite outgrowth 1 (GRIN1) was initially identified as a binding protein for guanosine 5'-3-O-(thio)triphosphate-bound Galphaz. GRIN1 is specifically expressed in brain and interacts selectively with activated alpha subunits of the Gi subfamily. GRIN1 colocalizes with Galphao at the growth cone of neuronal cells and promotes neurite extension in Neuro2a cells when coexpressed with constitutively active mutant GalphaoQ205L. These results suggest that GRIN1 functions as a downstream target for Galphao. However, GRIN1 does not contain domains that are homologous to known signaling motifs. To understand the mechanisms of Galphao-GRIN1 pathway, we analyzed functional domains of GRIN1 that are involved in binding with Galphao or with its targeting to the plasma membrane. Using pull-down assays with glutathione S-transferase-fused GRIN1 deletion mutants, Galphao binding regions were localized to amino acid residues 716 to 746 and 797 to 827 of GRIN1. The Galphao binding region of GRIN1 did not demonstrate GTPase accelerating activity for Galphao. GRIN1 localized in the cell periphery in Neuro2a cells, and two cysteine residues at C-terminal region of GRIN1 (Cys818 and Cys819) were shown to be critical for its membrane targeting. Coexpression of GRIN1 with GalphaoQ205L or GRIN1Delta(717-827), which lacks Galphao binding region, promoted microspike formation in Swiss 3T3 cells or neurite extension in Neuro2a cells. The dominant-negative mutant of Cdc42 blocked these morphological changes. Coexpression of GRIN1 and GalphaoQ205L stimulated the formation of GTP-bound Cdc42 in Swiss 3T3 cells. These results suggest that the binding of activated Galphao to GRIN1 induces activation of Cdc42, which leads to morphological changes in neuronal cells.

BNIP-2 induces cell elongation and membrane protrusions by interacting with Cdc42 via a unique Cdc42-binding motif within its BNIP-2 and Cdc42GAP homology domain

The Cdc42 small GTPase regulates cytoskeletal reorganization and cell morphological changes that result in cellular extensions, migration, or cytokinesis. We previously showed that BNIP-2 interacted with Cdc42 and its cognate inactivator, p50RhoGAP/Cdc42GAP via its BNIP-2 and Cdc42GAP homology (BCH) domain, but its cellular and physiological roles still remain unclear. We report here that following transient expression of BNIP-2 in various cells, the expressed protein was located in irregular spots throughout the cytoplasm and concentrated at the leading edge of cellular extensions. The induced cell elongation and membrane protrusions required an intact BCH domain and were variously inhibited by coexpression of dominant negative mutants of Cdc42 (completely inhibited), Rac1 (partially inhibited), and RhoA (least inhibited). Presence of the Cdc42/Rac1 interactive binding (CRIB) motif alone as the dominant negative mutant of p21-activated kinase also inhibited the BNIP-2 effect. Bioinformatic analyses together with progressive deletional mutagenesis and binding studies revealed that a distal part of the BNIP-2 BCH domain contained a sequence with low homology to CRIB motif. However, in contrary to most effectors, BNIP-2 binding to Cdc42 was mediated exclusively via the unique sequence motif 285VPMEYVGI 292. Cells expressing the BNIP-2 mutants devoid of this motif or/and the 34-amino acids immediately upstream to this sequence failed to elicit cell elongation and membrane protrusions despite that the protein still remained in the cytoplasm and interacted with Cdc42GAP. Evidence is presented where BNIP-2 in vivo induces cell dynamics by recruiting Cdc42 via its BCH domain, thus providing a novel mechanism for regulating Cdc42 signaling pathway.

Cdc42/Rac1 participates in the control of telomerase activity in human nasopharyngeal cancer cells

Telomerase, a specialized ribonucleoprotein reverse transcriptase that directs the synthesis of telomeric DNA, is repressed in normal human somatic cells, but is activated in most cancers. Little is known concerning how telomerase activity is activated and maintained in cancer cells. We have previously shown that protein kinase C-ζ (PKCζ) controls telomerase activity in nasopharyngeal cancer (NPC) cells. Since PKCζ activity is known to be modulated by Cdc42/Rac1, we investigated the effects of inhibiting Cdc42 and Rac1 on the telomerase activity of NPC-076 cells. Treatment of NPC cells with antisense oligonucleotides against Cdc42 or Rac1 produced an inhibition of telomerase activity. Similarly, transient expression of dominant-negative mutants of Cdc42 or Rac1, but not the wild-type Cdc42 or Rac1, also produced an inhibition of telomerase activity in NPC cells. This inhibition of telomerase activity is not associated with a transcriptional down-regulation of hTERT, the key regulator of telomerase. We suggest that Cdc42/Rac1 participates in the posttranscriptional control of telomerase activity in NPC cells.

Cdc42 and the actin-related protein/neural Wiskott-Aldrich syndrome protein network mediate cellular invasion by Cryptosporidium parvum.

Cryptosporidium parvum invasion of epithelial cells involves host cell membrane alterations which require a remodeling of the host cell actin cytoskeleton. In addition, an actin plaque, possibly associated with the dense-band region, forms within the host cytoplasm at the host-parasite interface. Here we show that Cdc42 and RhoA, but not Rac1, members of the Rho family of GTPases, are recruited to the host-parasite interface in an in vitro model of human biliary cryptosporidiosis. Interestingly, activation of Cdc42, but not RhoA, was detected in the infected cells. Neural Wiskott-Aldrich syndrome protein (N-WASP) and p34-Arc, actin-regulating downstream effectors of Cdc42, were also recruited to the host-parasite interface. Whereas cellular expression of a constitutively active mutant of Cdc42 promoted C. parvum invasion, overexpression of a dominant negative mutant of Cdc42, or depletion of Cdc42 mRNA by short interfering RNA-mediated gene silencing, inhibited C. parvum invasion. Expression of the WA fragment of N-WASP to block associated actin polymerization also inhibited C. parvum invasion. Moreover, inhibition of host cell Cdc42 activation by dominant negative mutation inhibited C. parvum-associated actin remodeling, membrane protrusion, and dense-band formation. In contrast, treatment of cells with a Rho inhibitor, exoenzyme C3, or cellular overexpression of dominant negative mutants of RhoA and Rac1 had no effect on C. parvum invasion. These data suggest that C. parvum invasion of target epithelia results from the organism's ability to activate a host cell Cdc42 GTPase signaling pathway to induce host cell actin remodeling at the attachment site.

Concerted Regulation of Cell Dynamics by BNIP-2 and Cdc42GAP Homology/Sec14p-like, Proline-rich, and GTPase-activating Protein Domains of a Novel Rho GTPase-activating Protein, BPGAP1

RhoA, Cdc42, and Rac1 are small GTPases that regulate cytoskeletal reorganization leading to changes in cell morphology and cell motility. Their signaling pathways are activated by guanine nucleotide exchange factors and inactivated by GTPase-activating proteins (GAPs). We have identified a novel RhoGAP, BPGAP1 (for BNIP-2 and Cdc42GAP Homology (BCH) domain-containing, Proline-rich and Cdc42GAP-like protein subtype-1), that is ubiquitously expressed and shares 54% sequence identity to Cdc42GAP/p50RhoGAP. BP-GAP1 selectively enhanced RhoA GTPase activity in vivo although it also interacted strongly with Cdc42 and Rac1. "Pull-down" and co-immunoprecipitation studies indicated that it formed homophilic or heterophilic complexes with other BCH domain-containing proteins. Fluorescence studies of epitope-tagged BPGAP1 revealed that it induced pseudopodia and increased migration of MCF7 cells. Formation of pseudopodia required its BCH and GAP domains but not the proline-rich region, and was differentially inhibited by coexpression of the constitutively active mutant of RhoA, or dominant negative mutants of Cdc42 and Rac1. However, the mutant without the proline-rich region failed to confer any increase in cell migration despite the induction of pseudopodia. Our findings provide evidence that cell morphology changes and migration are coordinated via multiple domains in BPGAP1 and present a novel mode of regulation for cell dynamics by a RhoGAP protein.

PTP mu-dependent growth cone rearrangement is regulated by Cdc42.

PTP mu is expressed in the developing nervous system and promotes growth and guidance of chick retinal ganglion cells. Using a newly developed growth cone rearrangement assay, we examined whether the small G-proteins were involved in PTP mu-dependent signaling. The stimulation of retinal cultures with purified PTP mu resulted in a striking morphological change in the growth cone, which becomes dominated by filopodia within 5 min of addition. This rearrangement in response to PTP mu stimulation was mediated by homophilic binding. We perturbed GTPase signaling using Toxin B, which inhibits Cdc42, Rac, and Rho, as well as the toxin Exoenzyme C3 that inhibits Rho. The PTP mu-induced growth cone rearrangement was blocked by Toxin B, but not by Exoenzyme C3. This result suggests that either Cdc42 or Rac are required but not Rho. To determine which GTPase was involved in PTP mu signaling, we utilized dominant-negative mutants of Cdc42 and Rac. Dominant-negative Cdc42 blocked PTP mu-induced rearrangement, while wild-type Cdc42 and dominant-negative Rac did not. Together, these results suggest a molecular signaling cascade beginning with PTP mu homophilic binding at the plasma membrane and the activation of Cdc42, which acts on the actin cytoskeleton to result in rearrangement of the growth cone.

Cdc42 plays a critical role in assembly of sarcomere units in series of cardiac myocytes

Cardiomyocyte hypertrophy is observed in various cardiovascular diseases and causes heart failure. We here examined the role of small GTP-binding proteins of Rho family in phenylephrine (PE)-or leukocyte inhibitory factor (LIF)-induced hypertrophic morphogenesis of cultured neonatal rat cardiomyocytes. Both LIF and PE increased cell size of cardiomyocytes. LIF induced an increase in the length/width ratio of cardiomyocytes, while PE did not change the ratio. Adenoviral gene transfer of constitutively active mutants of Cdc42 increased the length/width ratio of cardiomyocytes and dominant negative mutants of Cdc42 conversely inhibited LIF-induced cell-elongation, while mutants of RhoA and Rac1 did not affect the length/width ratio of cardiomyocytes. These results suggest that Cdc42, but not RhoA and Rac1, is involved in LIF-induced sarcomere assembly in series in cardiomyocytes.

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.

Cdc42 and Rac1 are necessary for autotaxin-induced tumor cell motility in A2058 melanoma cells

Autotaxin (ATX) is a strong motogen that can increase invasiveness and angiogenesis. In the present study, we investigated the signal transduction mechanism of ATX-induced tumor cell motility. Unlike N19RhoA expressing cells, the cells expressing N17Cdc42 or N17Rac1 showed reduced motility against ATX. ATX activated Cdc42 and Rac1 and increased complex formation between these small G proteins and p21-activated kinase (PAK). Furthermore, ATX phosphorylated focal adhesion kinase (FAK) that was not shown in cells expressing dominant negative mutants of Cdc42 or Rac1. Collectively, these data strongly indicate that Cdc42 and Rac1 are essential for ATX-induced tumor cell motility in A2058 melanoma cells, and that PAK and FAK might be also involved in the process.

Assembly of Epithelial Tight Junctions Is Negatively Regulated by Par6

Epithelial cells display apical-basal polarity, and the apical surface is segregated from the basolateral membranes by a barrier called the tight junction (TJ). TJs are constructed from transmembrane proteins that form cell-cell contacts—claudins, occludin, and junctional adhesion molecule (JAM)—plus peripheral proteins such as ZO-1 [1, 2]. The Par proteins (partitioning-defective) Par3 and Par6, plus atypical protein kinase C (aPKC) function in the formation or maintenance of TJs [3–5] and more generally in metazoan cell polarity establishment [6, 7]. Par6 contains a PDZ domain and a partial CRIB (Cdc42/Rac interactive binding) domain and binds the small GTPase Cdc42. Here, we show that Par6 inhibits TJ assembly in MDCK II epithelial cells after their disruption by Ca2+ depletion but does not inhibit adherens junction (AJ) formation. Transepithelial resistance and paracellular diffusion assays confirmed that assembly of functional TJs is delayed by Par6 overexpression. Strikingly, the isolated, N-terminal fragment of PKCζ, which binds Par6, also inhibits TJ assembly. Activated Cdc42 can disrupt TJs [3–5], but neither a dominant-negative Cdc42 mutant nor the CRIB domain of γPAK (p21-activated kinase), which inhibits Cdc42 function, observably inhibit TJ formation. These results suggest that Cdc42 and Par6 negatively regulate TJ assembly in mammalian epithelial cells.