Timing it right: Precise ON/OFF switches for Rho1 and Cdc42 GTPases in cytokinesis

In many eukaryotes, cytokinesis requires an actomyosin contractile ring that is crucial for cell constriction and new membrane organization. Two studies in this issue (Onishi et al. 2013. J. Cell Biol. http://dx.doi.org.10.1083/jcb.201302001 and Atkins et al. 2013. J. Cell Biol. http://dx.doi.org.10.1083/jcb.201301090) establish that precise activation and/or inactivation of Rho1 and Cdc42 GTPases is important for the correct order and successful completion of events downstream of actomyosin ring constriction in budding yeast.Cytokinesis is the terminal step in the cell cycle through which one cell physically divides into two daughters (Schroeder, 1990; Balasubramanian et al., 2004; Green et al., 2012). In many eukaryotes, ranging from yeast to human, cytokinesis depends on a division apparatus (known by several names, such as actomyosin ring, contractile ring, and cleavage furrow), which is composed of >100 proteins, including filamentous actin, the motor protein myosin II, IQGAP, and F-BAR domain–containing proteins (Oliferenko et al., 2009; Pollard and Wu, 2010). The cytokinetic–actomyosin ring generates constrictive force as well as guides the assembly of new membranes and the cell wall (in yeasts and fungi). Finally, through the process of abscission, the remaining cytoplasmic connections are resolved to liberate the two daughters (Neto et al., 2011; Green et al., 2012). How the later steps of cytokinesis (such as membrane/cell wall assembly and abscission) are coordinated with the earlier steps of cytokinesis (such as actomyosin ring maturation and contraction) remains poorly understood. Two papers in this issue of The Journal of Cell Biology (Atkins et al.; Oishi et al.) significantly clarify the molecular controls that coordinate the terminal steps of cytokinesis. From these studies, a picture emerges of exquisite and previously unappreciated temporal regulation of Rho1/A and Cdc42 family GTPases (Fig. 1) that is important for successful completion of cytokinesis in the budding yeast Saccharomyces cerevisiae.Open in a separate windowFigure 1.The highs and lows of Rho1 and Cdc42 during the cell cycle. Activity profiles of Rho1 and Cdc42 through the budding yeast cell cycle and the proposed functions for activation and inactivation of Rho1 and Cdc42. P1, P2, and T1 refer to peak 1, peak 2, and trough 1, respectively, of the activity of the GTPases described. PS and SS refer to primary septum and secondary septum, respectively. MEN refers to the mitotic exit network signaling module.The Rho superfamily of GTPases, which comprise Cdc42, Rho, and Rac (Ridley, 1995; Tapon and Hall, 1997), regulate actin cytoskeletal remodeling and function during cell polarization and cytokinesis. In yeast and animal cells, Rho1/A (Rho1 in yeast and RhoA in animals) plays important roles in major aspects of actomyosin ring function (Tolliday et al., 2002; Piekny et al., 2005; Yoshida et al., 2006; Fededa and Gerlich, 2012). In its GTP-bound active form, Rho1/A binds to the actin filament nucleator formin to regulate actin polymerization at the division site. In animals, it also binds to the Rho-associated protein kinase (ROCK) through which it regulates myosin II contractility. Although Rho1/A has a clear role in cytokinesis, whether regulation of Cdc42, another key member of the Rho superfamily, is important for cytokinesis is unknown.Onishi et al. (2013) further examine the role of Rho1 in cytokinesis in budding yeast. In this organism, the division septum is assembled in two stages, each thought to be indispensable. A primary septum, largely composed of chitin, is first assembled concomitant with actomyosin ring constriction. Subsequently, a secondary septum, largely composed of 1,3-β-glucan, is assembled on both sides of the primary septum (Bi and Park, 2012). Using electron microscopy, Onishi et al. (2013) found that even in wild-type cells, small gaps in the primary septum were masked by additional growth of the secondary septum. Furthermore, they found that the secondary septum, in addition to forming part of the cell wall of the daughter cells, itself might participate in cytokinetic abscission. Because the secondary septum was able to bypass partial loss of the primary septum, Onishi et al. (2013) searched for mechanisms regulating secondary septum assembly through high dosage genetic suppressor analysis with mutants strongly defective in primary septum synthesis. Remarkably, the authors found that up-regulation of Rho1 GTPase or down-regulation of Cdc42 GTPase activities led to secondary septum assembly and viability, even in cells devoid of Chs2, the enzyme involved in primary septum synthesis. These and previous studies (Tolliday et al., 2002; Yoshida et al., 2006) led to two conclusions: (1) Rho1 activation was key to actomyosin ring assembly and (2) Rho1 activation was essential for secondary septum synthesis and abscission. Through the use of temporally regulated expression of a constitutively active version of Rho1, Onishi et al. (2013) found that these two high activity states of Rho1 had to be interrupted by a phase in which Rho1 was maintained in an inactive state. The presence of a trough of Rho1 activity explains why secondary septum assembly occurs only after actomyosin ring constriction and primary septum assembly, despite the localization of Rho1-GTP effector Fks1 (enzyme that synthesizes 1,3-β-glucan in the secondary septum) before actomyosin ring constriction.How is Rho1 temporarily inactivated, during actomyosin ring constriction and primary septum formation, to facilitate progression of cytokinesis? Onishi et al. (2013) reasoned that the SH3 and transglutaminase (TGc) domain protein Cyk3 (Korinek et al., 2000), a component of the actomyosin ring, might be part of this mechanism because both septa formed simultaneously in cyk3 mutants (possibly as a result of premature Fks1 activation in the absence of a mechanism maintaining Rho1 in its inactive GDP-bound form). Onishi et al. (2013) found that the TGc domain of Cyk3, which lacks enzymatic activity, physically interacted preferentially with GDP-bound Rho1. This biochemical interaction could also be observed in fluorescence-based protein interaction assays, leading them to conclude that the TGc domain of Cyk3 functioned akin to a GDP dissociation inhibitor for Rho1. Thus, it appears that the two peaks and one trough of Rho1 activity are all important for proper cytokinesis.Although Onishi et al. (2013) found that down-regulation of Cdc42 promoted secondary septum assembly and cytokinesis, their study was focused on Rho1. The complementary study by Atkins et al. (2013) sheds light on how Cdc42 inhibition is regulated and how such an inhibition might regulate cytokinesis. These authors measured the fraction of active GTP-bound Cdc42 during the cell cycle using the Cdc42-GTP reporter CRIB (Burbelo et al., 1995). Interestingly, they found that Cdc42 was active in two peaks: in anaphase and during cell polarization at G1/S. These two phases of peak Cdc42 activity were interrupted by a period of trough during cytokinesis, when Cdc42 was predominantly GDP bound. Because expression of an activated form of Cdc42 was toxic to cells partially defective in actomyosin ring function and cytokinesis, Atkins et al. (2013) concluded that active Cdc42 interfered with cytokinesis.How is Cdc42 inactivated in a temporally precise manner, and what downstream cytokinetic events depend on Cdc42 inactivation? Through a variety of genetic and biochemical experiments, Atkins et al. (2013) found that the Cdc5 protein kinase (related to Polo kinase in animals) was important for the inactivation of Cdc42 via phosphorylation of Bem2 and Bem3, which are GTPase-activating proteins for Cdc42 (Bi and Park, 2012). Consistently, Atkins et al. (2013) found that bem2 mutants (in which Cdc42 is inappropriately active) were defective in cell separation, suggesting a role for Bem2 (and likely Bem3) in aspects of actomyosin ring function or septum assembly. Through protein localization experiments, Atkins et al. (2013) found that Iqg1 (Epp and Chant, 1997; Osman and Cerione, 1998; Shannon and Li, 1999), a protein essential for actomyosin ring assembly and septum formation, and Inn1 (Sanchez-Diaz et al., 2008; Nishihama et al., 2009), a protein that links the plasma membrane to the actomyosin ring and participates in primary septum assembly, failed to properly localize in bem2 mutants. Conversely, increasing the level of Iqg1 rescued the cytokinesis defect of bem2 mutants, establishing that Iqg1 was a key effector affected by increased Cdc42 activity. Interestingly, Iqg1 localization and the cell separation defect were rectified in double mutants defective in bem2 and the Cdc42 effector kinase Ste20, suggesting that the down-regulation of a known canonical Cdc42 response pathway was key to proper cytokinesis. Thus, it appears that inactivation of Cdc42 is essential for the localization of proteins important for actomyosin ring constriction and secondary septum assembly to the division site.Where do these studies leave us, and what are the open questions that emerge? An important question that follows is what is the precise temporal correlation between the activities of Rho1 and Cdc42 and is the temporary inactivation of Rho1 and Cdc42 activities necessary to avoid cross talk between these GTPase signaling pathways? Simultaneous analysis of Rho1 and Cdc42 activity and function in the same cell populations should begin to address this question. A second important question is precisely how does inhibition of Cdc42 lead to Iqg1 and Inn1 localization and what are the targets of Rho1 (other than Fks1) that participate in secondary septum formation during its second activity peak? The studies of Onishi et al. (2013) and Atkins et al. (2013) are remarkable in their breadth and depth, in that they have together shed detailed mechanistic insight into the physiological roles of proteins that are evolutionarily highly conserved. Whether similar mechanisms operate in other organisms can now be investigated.     

Pathogenicity island-dependent activation of Rho GTPases Rac1 and Cdc42 in Helicobacter pylori infection

Helicobacter pylori has been identified as the major aetiological agent in the development of chronic gastritis and duodenal ulcer, and it plays a role in the development of gastric carcinoma. Attachment of H. pylori to gastric epithelial cells leads to nuclear and cytoskeletal responses in host cells. Here, we show that Rho GTPases Rac1 and Cdc42 were activated during infection of gastric epithelial cells with either the wild-type H. pylori or the mutant strain cagA. In contrast, no activation of Rho GTPases was observed when H. pylori mutant strains (virB7 and PAI) were used that lack functional type IV secretion apparatus. We demonstrated that H. pylori-induced activation of Rac1 and Cdc42 led to the activation of p21-activated kinase 1 (PAK1) mediating nuclear responses, whereas the mutant strain PAI had no effect on PAK1 activity. Activation of Rac1, Cdc42 and PAK1 represented a very early event in colonization of gastric epithelial cells by H. pylori. Rac1 and Cdc42 were recruited to the sites of bacterial attachment and are therefore probably involved in the regulation of local and overall cytoskeleton rearrangement in host cells. Finally, actin rearrangement and epithelial cell motility in H. pylori infection depended on the presence of a functional type IV secretion system encoded by the cag pathogenicity island (PAI).     

Distinct Roles for Rho Versus Rac/Cdc42 GTPases Downstream of Vav2 in Regulating Mammary Epithelial Acinar Architecture

Non-malignant mammary epithelial cells (MECs) undergo acinar morphogenesis in three-dimensional Matrigel culture, a trait that is lost upon oncogenic transformation. Rho GTPases are thought to play important roles in regulating epithelial cell-cell junctions, but their contributions to acinar morphogenesis remain unclear. Here we report that the activity of Rho GTPases is down-regulated in non-malignant MECs in three-dimensional culture with particular suppression of Rac1 and Cdc42. Inducible expression of a constitutively active form of Vav2, a Rho GTPase guanine nucleotide exchange factor activated by receptor tyrosine kinases, in three-dimensional MEC culture activated Rac1 and Cdc42; Vav2 induction from early stages of culture impaired acinar morphogenesis, and induction in preformed acini disrupted the pre-established acinar architecture and led to cellular outgrowths. Knockdown studies demonstrated that Rac1 and Cdc42 mediate the constitutively active Vav2 phenotype, whereas in contrast, RhoA knockdown intensified the Vav2-induced disruption of acini, leading to more aggressive cell outgrowth and branching morphogenesis. These results indicate that RhoA plays an antagonistic role to Rac1/Cdc42 in the control of mammary epithelial acinar morphogenesis.     

Stimulation of phospholipase C-beta2 by the Rho GTPases Cdc42Hs and Rac1.

Neutrophils contain a soluble guanine-nucleotidebinding protein, made up of two components with molecular masses of 23 and 26 kDa, that mediates stimulation of phospholipase C-beta2 (PLCbeta2). We have identified the two components of the stimulatory heterodimer by amino acid sequencing as a Rho GTPase and the Rho guanine nucleotide dissociation inhibitor LyGDI. Using recombinant Rho GTPases and LyGDI, we demonstrate that PLCbeta2 is stimulated by guanosine 5'-O-(3-thiotriphosphate) (GTP[S])-activated Cdc42HsxLyGDI, but not by RhoAxLyGDI. Stimulation of PLCbeta2, which was also observed for GTP[S]-activated recombinant Rac1, was independent of LyGDI, but required C-terminal processing of Cdc42Hs/Rac1. Cdc42Hs/Rac1 also stimulated PLCbeta2 in a system made up of purified recombinant proteins, suggesting that this function is mediated by direct protein-protein interaction. The Cdc42Hs mutants F37A and Y40C failed to stimulate PLCbeta2, indicating that the Cdc42Hs effector site is involved in this interaction. The results identify PLCbeta2 as a novel effector of the Rho GTPases Cdc42Hs and Rac1, and as the first mammalian effector directly regulated by both heterotrimeric and low-molecular-mass GTP-binding proteins.     

The GIT-PIX protein complex: a hub to ARF and Rac/Cdc42 GTPases

We recently described that the tumor suppressor factor Scribble anchors the PIX exchange factor for Rac/Cdc42 and the ARF-GAP GIT proteins at the plasma membrane. Because it has been postulated that the GIT-PIX proteins dimerize and tightly self-assemble to form a high molecular weight complex, this nexus may be capable of linking together important signalling molecules to control cytosqueleton polymerization and membrane dynamics. To date, most studies that have tempted to unravel the function of these proteins have found their implication in a great variety of cellular functions (receptor recycling, endo-exocytosis, cell migration, synapse formation...) but have mostly neglected to consider the multimeric organization of this hub. There is no doubt that our comprehension of physiopathological disorders such as cancers will be improved when the nature of the complex pathways integrated by the GIT-PIX nodule will be understood.     

Specific contributions of the small GTPases Rho, Rac, and Cdc42 to Dbl transformation.

Dbl is a representative prototype of a growing family of oncogene products that contain the Dbl homology/pleckstrin homology elements in their primary structures and are associated with a variety of neoplastic pathologies. Members of the Dbl family have been shown to function as physiological activators (guanine nucleotide exchange factors) of the Rho-like small GTPases. Although the expression of GTPase-defective versions of Rho proteins has been shown to induce a transformed phenotype under different conditions, their transformation capacity has been typically weak and incomplete relative to that exhibited by dbl-like oncogenes. Moreover, in some cases (e.g. NIH3T3 fibroblasts), expression of GTPase-defective Cdc42 results in growth inhibition. Thus, in attempting to reconstitute dbl-induced transformation of NIH3T3 fibroblasts, we have generated spontaneously activated ("fast-cycling") mutants of Cdc42, Rac1, and RhoA that mimic the functional effects of activation by the Dbl oncoprotein. When stably expressed in NIH3T3 cells, all three mutants caused the loss of serum dependence and showed increased saturation density. Furthermore, all three stable cell lines were tumorigenic when injected into nude mice. Our data demonstrate that all three Dbl targets need to be activated to promote the full complement of Dbl effects. More importantly, activation of each of these GTP-binding proteins contributes to a different and distinct facet of cellular transformation.     

Role of the small Rho GTPases Rac1 and Cdc42 in host cell invasion of Campylobacter jejuni

Host cell invasion of the food-borne pathogen Campylobacter jejuni is one of the primary reasons of tissue damage in humans but molecular mechanisms are widely unclear. Here, we show that C. jejuni triggers membrane ruffling in the eukaryotic cell followed by invasion in a very specific manner first with its tip followed by the flagellar end. To pinpoint important signalling events involved in the C. jejuni invasion process, we examined the role of small Rho family GTPases. Using specific GTPase-modifying toxins, inhibitors and GTPase expression constructs we show that Rac1 and Cdc42, but not RhoA, are involved in C. jejuni invasion. In agreement with these observations, we found that internalization of C. jejuni is accompanied by a time-dependent activation of both Rac1 and Cdc42. Finally, we show that the activation of these GTPases involves different host cell kinases and the bacterial fibronectin-binding protein CadF. Thus, CadF is a bifunctional protein which triggers bacterial binding to host cells as well as signalling leading to GTPase activation. Collectively, our results suggest that C. jejuni invade host target cells by a unique mechanism and the activation of the Rho GTPase members Rac1 and Cdc42 plays a crucial role in this entry process.     

GTPases of the Rho subfamily are required for Brucella abortus internalization in nonprofessional phagocytes: direct activation of Cdc42

Members of the genus Brucella are intracellular alpha-Proteobacteria responsible for brucellosis, a chronic disease of humans and animals. Little is known about Brucella virulence mechanisms, but the abilities of these bacteria to invade and to survive within cells are decisive factors for causing disease. Transmission electron and fluorescence microscopy of infected nonprofessional phagocytic HeLa cells revealed minor membrane changes accompanied by discrete recruitment of F-actin at the site of Brucella abortus entry. Cell uptake of B. abortus was negatively affected to various degrees by actin, actin-myosin, and microtubule chemical inhibitors. Modulators of MAPKs and protein-tyrosine kinases hampered Brucella cell internalization. Inactivation of Rho small GTPases using clostridial toxins TcdB-10463, TcdB-1470, TcsL-1522, and TcdA significantly reduced the uptake of B. abortus by HeLa cells. In contrast, cytotoxic necrotizing factor from Escherichia coli, known to activate Rho, Rac, and Cdc42 small GTPases, increased the internalization of both virulent and non-virulent B. abortus. Expression of dominant-positive Rho, Rac, and Cdc42 forms in HeLa cells promoted the uptake of B. abortus, whereas expression of dominant-negative forms of these GTPases in HeLa cells hampered Brucella uptake. Cdc42 was activated upon cell contact by virulent B. abortus, but not by a noninvasive isogenic strain, as proven by affinity precipitation of active Rho, Rac, and Cdc42. The polyphasic approach used to discern the molecular events leading to Brucella internalization provides new alternatives for exploring the complexity of the signals required by intracellular pathogens for cell invasion.     

Peroxisomal protein import and ERAD: variations on a common theme

Despite their distinct biological functions, there is a surprising similarity between the composition of the machinery that imports proteins into peroxisomes and the machinery that degrades endoplasmic reticulum (ER)-associated proteins. The basis of this similarity lies in the fact that both machineries make use of the same basic mechanistic principle: the tagging of a substrate by monoubiquitylation or polyubiquitylation and its subsequent recognition and ATP-dependent removal from a membrane by ATPases of the ATPases associated with diverse cellular activities (AAA) family of proteins. We propose that the ER-associated protein degradation (ERAD)-like removal of the peroxisomal import receptor is mechanically coupled to protein translocation into the organelle, giving rise to a new concept of export-driven import.     

The sevenless signaling pathway: variations of a common theme

Many developmental processes are regulated by intercellular signaling mechanisms that employ the activation of receptor tyrosine kinases. One model system that has been particular useful in determining the role of receptor tyrosine kinase-mediated signaling processes in cell fate determination is the developing Drosophila eye. The specification of the R7 photoreceptor cell in each ommatidium of the developing Drosophila eye is dependent on activation of the Sevenless receptor tyrosine kinase. This review will focus on the genetic and biochemical approaches that have identified signaling molecules acting downstream of the Sevenless receptor tyrosine kinase which ultimately trigger differentiation of the R7 photoreceptor cell.     

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

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

Galanin stimulates neurite outgrowth from sensory neurons by inhibition of Cdc42 and Rho GTPases and activation of cofilin

We and others have previously shown that the neuropeptide galanin modulates neurite outgrowth from adult sensory neurons via activation of the second galanin receptor; however, the intracellular signalling pathways that mediate this neuritogenic effect have yet to be elucidated. Here, we demonstrate that galanin decreases the activation state in adult sensory neurons and PC12 cells of Rho and Cdc42 GTPases, both known regulators of filopodial and growth cone motility. Consistent with this, activated levels of Rho and Cdc42 levels are increased in the dorsal root ganglion of adult galanin knockout animals compared with wildtype controls. Furthermore, galanin markedly increases the activation state of cofilin, a downstream effector of many of the small GTPases, in the cell bodies and growth cones of sensory neurons and in PC12 cells. We also demonstrate a reduction in the activation of cofilin, and alteration in growth cone motility, in cultured galanin knockout neurons compared with wildtype controls. These data provide the first evidence that galanin regulates the Rho family of GTPases and cofilin to stimulate growth cone dynamics and neurite outgrowth in sensory neurons. These findings have important therapeutic implications for the treatment of peripheral sensory neuropathies.

     

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.     

Desmoglein 3 acting as an upstream regulator of Rho GTPases, Rac-1/Cdc42 in the regulation of actin organisation and dynamics

Desmoglein 3 (Dsg3), a member of the desmoglein sub-family, serves as an adhesion molecule in desmosomes. Our previous study showed that overexpression of human Dsg3 in several epithelial lines induces formation of membrane protrusions, a phenotype suggestive of Rho GTPase activation. Here we examined the interaction between Dsg3 and actin in detail and showed that endogenous Dsg3 colocalises and interacts with actin, particularly the junctional actin in a Rac1-dependent manner. Ablation of Rac1 activity by dominant negative Rac1 mutant (N17Rac1) or the Rac1 specific inhibitor (NSC23766) directly disrupts the interaction between Dsg3 and actin. Assembly of the junctional actin at the cell borders is accompanied with enhanced levels of Dsg3, while inhibition of Dsg3 by RNAi results in profound changes in the organisation of actin cytoskeleton. In accordance, overexpression of Dsg3 results in a remarkable increase of Rac1 and Cdc42 activities and to a lesser extent, RhoA. The enhancements in Rho GTPases are accompanied by the pronounced actin-based membrane structures such as lamellipodia and filopodia, enhanced rate of actin turnover and cell polarisation. Together, our results reveal an important novel function for Dsg3 in promoting actin dynamics through regulating Rac1 and Cdc42 activation in epithelial cells.     

The TRE17 oncogene encodes a component of a novel effector pathway for Rho GTPases Cdc42 and Rac1 and stimulates actin remodeling

The Rho family GTPases Cdc42 and Rac1 play fundamental roles in transformation and actin remodeling. Here, we demonstrate that the TRE17 oncogene encodes a component of a novel effector pathway for these GTPases. TRE17 coprecipitated specifically with the active forms of Cdc42 and Rac1 in vivo. Furthermore, the subcellular localization of TRE17 was dramatically regulated by these GTPases and mitogens. Under serum-starved conditions, TRE17 localized predominantly to filamentous structures within the cell. Epidermal growth factor (EGF) induced relocalization of TRE17 to the plasma membrane in a Cdc42-/Rac1-dependent manner. Coexpression of activated alleles of Cdc42 or Rac1 also caused complete redistribution of TRE17 to the plasma membrane, where it partially colocalized with the GTPases in filopodia and ruffles, respectively. Membrane recruitment of TRE17 by EGF or the GTPases was dependent on actin polymerization. Finally, we found that a C-terminal truncation mutant of TRE17 induced the accumulation of cortical actin, mimicking the effects of activated Cdc42. Together, these results identify TRE17 as part of a novel effector complex for Cdc42 and Rac1, potentially contributing to their effects on actin remodeling. The present study provides insights into the regulation and cellular function of this previously uncharacterized oncogene.     

A role for the small molecular weight GTPases, Rho and Cdc42, in muscarinic receptor signaling to focal adhesion kinase

An enhanced tyrosine phosphorylation of focal adhesion kinase (FAK) is elicited during neuronal growth cone remodeling and requires the maintenance of agonist-sensitive pools of phosphatidylinositol 4,5-bisphosphate (PIP2). Rho family GTPases are putative regulators of both PIP2 synthesis and growth cone remodeling, including neurite outgrowth elicited by muscarinic cholinergic receptor (mAChR) stimulation. In this study, we investigated the interrelationships among Rho family GTPases, PIP2 synthesis, and mAChR signaling to FAK in SH-SY5Y neuroblastoma cells. Preincubation with Clostridium difficile toxin B (Tox B), an inhibitor of Rho, Rac, and Cdc42, attenuated mAChR-stimulated FAK and paxillin tyrosine phosphorylation and lysophosphatidic acid (LPA)-induced FAK phosphorylation to a similar extent (75% decreases at 200 pg/ml Tox B) but did not affect mitogen-activated protein kinase activation elicited by either phorbol ester or an mAChR agonist. In contrast, preincubation with selective inhibitors of either Rho (C3 exoenzyme) or Rho kinase (HA-1 077) resulted in 80-90% reductions in LPA-induced FAK phosphorylation but only 40-50% decreases in mAChR-stimulated phosphorylation. Moreover, mAChR-mediated FAK phosphorylation was significantly attenuated in cells scrape-loaded with dominant-negative N17Cdc42 but not N17Rac1. Tox B had little or no effect on agonist-sensitive pools of PIP2 but inhibited mAChR-driven actin cytoskeletal remodeling. The results suggest that the Rho family GTPases, Rho and Cdc42, link mAChR stimulation to increases in FAK phosphorylation independently of effects on PIP2 synthesis.