Comparison of morphological changes induced by ras superfamily small GTPases

Signaling proteins from the same family can have markedly different roles in a given cellular context. Expression of one hundred constitutively active human small GTPases is found to induce cell morphologies that fall into nine distinct classes. An algorithm is developed for pairs of classes that predicted amino acid positions that can be exchanged to create mutants with switched functionality. The algorithm was validated by creating switch-of-function mutants for Rac1, CDC42, H-Ras, RalA, Rap2B, and R-Ras3. Contrary to expectations, the relevant residues are mostly outside known interaction surfaces and are structurally far apart from one another. This study shows that specificity in protein families can be explored by combining genome-wide experimental functional classification with the creation of switch-of-function mutants (Heo, 2003).

Fifty six of the expressed small GTPase constructs triggered no significant morphology changes, while 44 others induced marked morphology changes. The induced morphologies were clearly distinguishable from one another and fell into only nine distinct classes. The Rho family members Rho6, Rho7, RhoE, and ARHE induced a marked cell rounding. Cells transfected with CDC42, CDC42h, TC10, and TCL constructs showed extensions of thin processes that have been termed filopodia, while cells transfected with Rac1, Rac2, Rac3, and RhoG constructs extended lamellipodia that consisted of mostly circular membrane sheets. Transfection of RhoA, RhoB, and RhoC constructs induced polymerized actin bundles or stress fibers that reached across the cell. Only RhoD and RhoH did not show a significant morphology change (Heo, 2003).

Arf family small GTPases induced two types of morphologies. Several members of the Arl family induced a shrunken morphology, while Arf6 had one of the most distinct morphologies with multiple characteristics that include broader cell arms, local membrane spreading, filopodia extensions as well as actin polymerization throughout the cell body and along the cell periphery. Within the shrunken morphology class, Arl 1, Arl 2, and Arl 3 could be considered as a subclass with less pronounced shrinkage and occasional induction of short filopodia type processes that have been termed microspikes in other studies (Heo, 2003).

Cells transfected with Ras family small GTPases also show two distinguishable morphology classes. The oncogenic H-, K-, and N-Ras induce a marked polarized morphology with membrane ruffles and strong actin staining at a polar end of the cells, while cells transfected with most of the remaining members show cell spreading combined with hairlike filopodia formation with pronounced polymerized actin boutons at their ends. The spreading of these cells has a resemblance to eyelashes and looks markedly different from the morphology of lamellipodia induced by Rac or RhoG or the polarized morphology induced by Ras (Heo, 2003).

Finally, several of the Rab family members also have a strong effect on cell morphology. Rab4B, Rab13, Rab22A, Rab23, and Rab35 induce a local spread morphology characterized by local lamellipodia extensions and occasional filopodia induction. Rab8 and Rab8B have the most dramatic effect on cell morphology of all constructs tested and, like Arf6, fall into the multiple morphology class characterized by large branched structures with local lamellipodia and filopodia (Heo, 2003).

In conclusion, this study shows that the structural fold of Ras superfamily small GTPases can induce nine different morphology classes. Furthermore, the residues have been discovered that define the filopodia, lamellipodia, polar, and eyelash morphologies and it was unexpectedly found that the locations of the switch-of-function sites are mostly outside the known effector interaction surfaces and are far apart from each other. These engineered small GTPases with a changed functional selectivity will be useful as tools in pull-down assays to identify the function-specific binding partners as perturbation constructs to investigate crosstalk between signaling processes and for testing whether particular cell functions are physiologically relevant by creating mutant model organisms. Finally, this study introduced an algorithm and a genome-based experimental classification strategy that can be employed to classify the functional space of protein families and to understand the structural basis of functional specificity (Heo, 2003).

Cdc42 in yeast

Signaling molecules such as Cdc42 and mitogen-activated protein kinases (MAPKs) can function in multiple pathways in the same cell. Proposed here is one mechanism by which such factors may be directed to function in a particular pathway, such that a specific response is elicited. Using genomic approaches, a new component of the Cdc42- and MAPK-dependent signaling pathway has been identified that regulates filamentous growth (FG) in yeast. This factor, called Msb2, is a FG-pathway-specific factor that promotes differential activation of the MAPK for the FG pathway, Kss1. Msb2 is localized to polarized sites on the cell surface and interacts with Cdc42 and with the osmosensor for the high osmolarity glycerol response (HOG) pathway, Sho1. Msb2 is glycosylated and is a member of the mucin family, proteins that in mammalian cells promote disease resistance and contribute to metastasis in cancer cells. Remarkably, loss of the mucin domain of Msb2 causes hyperactivity of the FG pathway, demonstrating an inhibitory role for mucin domains in MAPK pathway activation. Taken together, these data suggest that Msb2 is a signaling mucin that interacts with general components, such as Cdc42 and Sho1, to promote their function in the FG pathway (Cullen, 2004).

Msb2 is a member of the mucin family of proteins, which are glycosylated cell-surface adhesion proteins. In mammalian cells, mucins act as barriers to pathogen infection and are key factors in metastasis in a variety of human cancers. In addition, two membrane-spanning mucins in humans, MUC1 and MUC4, function as signaling molecules. Like Msb2, MUC1 and MUC4 are cell-surface integral-membrane proteins whose cytoplasmic tails interact with signaling molecules at the head of a cascade. MUC1 is a docking protein for ß-catenin, and tyrosine phosphorylation of the cytoplasmic domain of MUC1 activates a MAPK pathway, the Grb2-Sos-Ras-MEK-ERK2 pathway. MUC4 binds to the tyrosine kinase ErbB2/HER2/Neu, to trigger phosphorylation of ErbB2 and potentiate signaling through the ErbB2/ErbB3 heterodimeric receptor complex (Cullen, 2004 and references therein).

Several findings from this study may be extrapolated to signaling mucins in general. First, as for Msb2, the mucin domains of MUC1 and MUC4 (and others) may have inhibitory roles. Hence, mutation of mucin domains may cause pathway activation and contribute to cancer progression in mammalian cells. The sequence similarity of mucin tandem repeats makes them highly susceptible to recombination-mediated deletion, as can occur for Msb2. Moreover, if, as for Msb2, the hyperactivity is dominant, then inappropriate pathway activation in mucin-deleted receptors may be prevalent among human cancers. The duality of mucin function in signaling pathway function should be a consideration in studies of adhesion-dependent developmental responses in normal mammalian cells and for appropriate drug design in human tumors. Indeed, MUC1 has been reported to have positive and negative roles in signaling. A second consequence of this study comes from the finding that Msb2 interacts with Cdc42 to redirect cell polarity. This finding suggests a role for signaling mucins in regulating polarized growth. Intriguingly, both MUC1 and MUC4 are localized to the apical surfaces of epithelial cells. Elucidation of the roles of signaling mucins in signaling pathway activation and polarized growth will help define the mechanisms by which these molecules induce metastasis in human cancers (Cullen, 2004 and references therein).

Cellular polarization is often a response to distinct extracellular or intracellular cues, such as nutrient gradients or cortical landmarks. However, in the absence of such cues, some cells can still select a polarization axis at random. Positive feedback loops promoting localized activation of the GTPase Cdc42p are central to this process in budding yeast. This study explores spontaneous polarization during bud site selection in mutant yeast cells that lack functional landmarks. These cells do not select a single random polarization axis, but continuously change this axis during the G1 phase of the cell cycle. This is reflected in traveling waves of activated Cdc42p which randomly explore the cell periphery. Integrated computational and in vivo analyses of these waves reveal a negative feedback loop that competes with the aforementioned positive feedback loops to regulate Cdc42p activity and confer dynamic responsiveness on the robust initiation of cell polarization (Ozubuda, 2005).

The formation of Cdc42p activity waves can be modeled in terms of a feedback control of activators and inhibitors of Cdc42p. Interactions between scaffold protein Bem1p, the GEF Cdc24p, and Cdc42p are suggested to form an actin-independent, positive feedback loop that enhances the recruitment of activators to the polar cap. It is hypothesized that this positive feedback loop accounts for the initial symmetry breaking. A second, actin-mediated, positive feedback could be mediated by the actin-based delivery of secretory vesicles containing, for example, Cdc42p or Cdc24p. Indeed, sometimes faint Gic2p1-208-GFP-labeled dots (Gic2p is a Cdc42-interacting protein) are observed moving toward the polar cap (Ozubuda, 2005).

Positive feedback loops allow an initially homogenous system to polarize in a random static direction. Because there is a limited number of activated Cdc42p molecules distributed along the membrane, a uniform distribution will still exhibit small local concentration deviations about the average. Due to the positive feedback regulation, local concentration maximums will grow at the expense of the surrounding areas in the membrane. This mechanism can explain the initial symmetry breaking in, for example, latrunculin-treated cells, but cannot explain the traveling Cdc42p activity waves (Ozubuda, 2005).

It is therefore proposed that in addition to the positive regulation, a negative feedback loop must exist. Because waves are not observed in latrunculin-treated cells, it is likely that the negative feedback loop is mediated by the actin cytoskeleton. A potential molecular implementation of the negative loop might be the delivery of vesicles containing GAPs along the actin cables. Alternatively, recent experiments show that actin-dependent endocytosis might disperse polarizing factors. Because actin patch components (associated with endocytosis) can be recruited by Cdc42p-GTP, this indicates that Cdc42p could stimulate dispersal of polarized factors in an actin-dependent manner, effectively establishing a negative feedback loop. The negative feedback loop provides the system with the potential to exhibit traveling waves. To observe traveling waves, it is important that the negative feedback regulation operates at a slower rate than the positive regulation. It is proposed that the actin-independent positive feedback reacts faster to a change in Cdc42p activation state than the regulation mediated by the actin cytoskeleton. This is a reasonable assumption because nucleation and polymerization of new actin cables followed by vesicle transport might be a slower process than the formation of the Bem1p scaffolding complex that relies on relatively fast protein-protein interactions and rapid diffusion. It is proposed that the waves observed in rsr1delta cells, lacking the Ras GTPase Rsr1p (Bud1p) are a result of a competition between a fast, actin-independent, positive feedback regulation and a slow and therefore delayed, negative feedback regulation. Because the inactivation always lags behind the spreading activation, this induces a wave motion. This implies that in rsr1delta cells, the actin-mediated negative feedback dominates the actin-mediated positive feedback. Wandering of the polar cap was not observed in earlier experiments in which the position of the polar cap was monitored by using a GFP fusion of wild-type Cdc42p or a constitutively activated version of Cdc42p. The strains used in these experiments carried a functional RSR1 gene and expressed higher levels of activated Cdc42p compared to wild-type. This elevated Cdc42p activity leads to a stronger actin-mediated positive feedback, which might explain the absence of Cdc42p activity waves in these strains (Ozubuda, 2005).

It has been concluded that Cdc42 is essential for chemotaxis in higher eukaryotes such as Dictyostelium and neutrophils. In these cells, interlocked positive and negative feedback loops have been identified among Cdc42, its regulators, and the actin network. Similar arguments that explain the Cdc42p activity waves in budding yeast might apply to the rotating pseudopod waves observed in Dictyostelium or the minCDE oscillatory waves in Escherichia coli. Mechanisms responsible for symmetry breaking are inherently coupled to mechanisms that enable restructuring of patterns. A design of competing feedback regulation loops combines efficient polarization with the ability to dynamically respond to varying intracellular or environmental conditions (Ozubuda, 2005).

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

An important problem in polarized morphogenesis is how polarized transport of membrane vesicles is spatiotemporally regulated. This study reports that a local change in the transbilayer phospholipid distribution of the plasma membrane regulates the axis of polarized growth. Type 4 P-type ATPases Lem3p-Dnf1p and -Dnf2p are putative heteromeric phospholipid flippases in budding yeast that are localized to polarized sites on the plasma membrane. The lem3Δ mutant exhibits prolonged apical growth due to a defect in the switch to isotropic bud growth. In lem3Δ cells, the small GTPase Cdc42p remains polarized at the bud tip where phosphatidylethanolamine remains exposed on the outer leaflet. Intriguingly, phosphatidylethanolamine and phosphatidylserine stimulate GTPase-activating protein (GAP) activity of Rga1p and Rga2p toward Cdc42p, whereas PI(4,5)P2 inhibits it. It is proposed that a redistribution of phospholipids to the inner leaflet of the plasma membrane triggers the dispersal of Cdc42p from the apical growth site, through activation of GAPs (Saito, 2007).

Polarized membrane growth, which occurs by membrane transport and insertion to a specific area of the cell surface, plays an essential role in cell morphogenesis, but it is largely unknown how it is spatiotemporally regulated. The budding yeast Saccharomyces cerevisiae undergoes changes in polarized membrane growth that are dependent on rearrangements of the actin cytoskeleton. During the early phase of budding, the bud grows apically, and later switches to an isotropic phase, resulting in an ellipsoidal bud shape. Cells that are delayed in the switching step form an elongated bud. During cytokinesis, cells repolarize the growth site to the mother-daughter neck of the cell division site (bud neck). The small GTPase Cdc42p plays a key role in directing these events by polarizing regulators of the actin cytoskeleton to growth sites. The timing of the growth switch is regulated in a cell cycle-dependent manner: activation of the cyclin-dependent kinase Cdc28p by Clb cyclins at an early G2 phase triggers the rearrangement of the actin cytoskeleton for the apical-isotropic growth switch and entry into mitosis; inactivation of Clb/Cdc28p at the end of mitosis leads to the repolarization of the actin cytoskeleton to the bud neck. However, it is not known how the reorganization of the actin cytoskeleton is spatially regulated downstream of Clb/Cdc28p (Saito, 2007).

Various cell types display an asymmetric distribution of phospholipids across the plasma membrane. This lipid asymmetry is generated and maintained by ATP-driven lipid transporters or translocases that vary in their lipid specificity. A subfamily (classified as 'flippase') of the type 4 P-type ATPases has been implicated in the translocation of phospholipids from the external to the cytosolic leaflet. Dnf1p and Dnf2p, members of this subfamily in the budding yeast, are hypothesized to translocate phospholipids from the outer to the inner leaflet of the plasma membrane. Lem3p, a member of the Cdc50p family, is a potential noncatalytic subunit of both Dnf1p and Dnf2p, and is essential for the exit of Dnf1p and Dnf2p from the endoplasmic reticulum. These proteins are primarily localized to the plasma membrane of polarized growth sites, such as emerging buds, small buds, and the bud neck of dividing cells, but their functions remain to be elucidated. In this study, it is proposed that the transbilayer phospholipid redistribution by Lem3p-Dnf1p and -Dnf2p (Lem3p-Dnf1/2p) at the apical growth site changes the mode of membrane growth by triggering downregulation of Cdc42p (Saito, 2007).

Dual modes of cdc42 recycling fine-tune polarized morphogenesis

In budding yeast, the highly conserved small GTPase Cdc42 localizes to the cortex at a cell pole and orchestrates the trafficking and deposition of cell surface materials required for building a bud or mating projection (shmoo). Using a combination of quantitative imaging and mathematical modeling, this study elucidated mechanisms of dynamic recycling of Cdc42 that balance diffusion. Rdi1, a guanine nucleotide dissociation inhibitor (GDI), mediates a fast recycling pathway, while actin patch-mediated endocytosis accounts for a slower one. These recycling mechanisms are restricted to the same region of the nascent bud, as both are coupled to the Cdc42 GTPase cycle. It was found that a single dynamic parameter, the rate of internalization inside the window of polarized delivery, is tuned to give rise to distinct shapes of Cdc42 distributions that correlate with distinct morphogenetic fates, such as the formation of a round bud or a pointed shmoo (Slaughter, 2009).

Modeling vesicle traffic reveals unexpected consequences for Cdc42p-mediated polarity establishment

Polarization in yeast has been proposed to involve a positive feedback loop whereby the polarity regulator Cdc42p orients actin cables, which deliver vesicles carrying Cdc42p to the polarization site. Previous mathematical models treating Cdc42p traffic as a membrane-free flux suggested that directed traffic would polarize Cdc42p, but it remained unclear whether Cdc42p would become polarized without the membrane-free simplifying assumption. This study presents mathematical models that explicitly consider stochastic vesicle traffic via exocytosis and endocytosis, providing several new insights. The findings suggest that endocytic cargo influences the timing of vesicle internalization in yeast. Moreover, the models provide quantitative support for the view that integral membrane cargo proteins would become polarized by directed vesicle traffic given the experimentally determined rates of vesicle traffic and diffusion. However, such traffic cannot effectively polarize the more rapidly diffusing Cdc42p in the model without making additional assumptions that seem implausible and lack experimental support. These findings suggest that actin-directed vesicle traffic would perturb, rather than reinforce, polarization in yeast (Layton, 2011).

The simulations suggest that in contrast to bulk cargo, an integral membrane cargo protein that diffuses slowly and is actively concentrated into both exocytic and endocytic vesicles would become effectively polarized. The key parameters in these models (vesicle dimensions and trafficking frequencies, degree of cargo trapping into endocytic patches, and diffusion constant) are all constrained within a factor of ~2 by experimental data, so the ability of the models to reproduce the experimentally observed v-SNARE distribution provides strong quantitative support for the prevailing hypothesis that v-SNARE polarization is due to polarized vesicle traffic. In budded cells, v-SNAREs are concentrated in the bud and largely absent from the mother plasma membrane. One possible basis for this is a diffusion barrier at the mother-bud neck. Indeed, the septin ring, which localizes to the neck, is thought to create such a barrier. Recent studies suggested that the septin ring might also promote endocytic patch formation. The effect of septin-biased endocytosis on the distribution of cargo in the plasma membrane was modeled and it was found that when vesicle delivery to a central window is coupled with a sufficient septin bias of endocytosis around the window, recycling cargo proteins are restricted to the vicinity of the window. This effect provides an alternative explanation for why v-SNAREs and similar cargo proteins are restricted to the bud. Moreover, like septin-restricted diffusion, it would also keep such proteins focused near the polarization site in unbudded cells (Layton, 2011).

Structure of Cdc42

The RhoGDI proteins serve as key multifunctional regulators of Rho family GTP-binding proteins. The 2.6 Å X-ray crystallographic structure of the Cdc42/RhoGDI complex reveals two important sites of interaction between GDI and Cdc42. (1) The amino-terminal regulatory arm of the GDI binds to the switch I and II domains of Cdc42 leading to the inhibition of both GDP dissociation and GTP hydrolysis. (2) The geranylgeranyl moiety of Cdc42 inserts into a hydrophobic pocket within the immunoglobulin-like domain of the GDI molecule leading to membrane release. The structural data demonstrate how GDIs serve as negative regulators of small GTP-binding proteins and how the isoprenoid moiety is utilized in this critical regulatory interaction (Hoffman, 2000).

Dbl-related oncoproteins are guanine nucleotide exchange factors (GEFs) specific for Rho guanosine triphosphatases (GTPases) and invariably possess tandem Dbl (DH) and pleckstrin homology (PH) domains. While it is known that the DH domain is the principal catalytic subunit, recent biochemical data indicate that for some Dbl-family proteins, such as Dbs and Trio, PH domains may cooperate with their associated DH domains in promoting guanine nucleotide exchange of Rho GTPases. In order to gain an understanding of the involvement of these PH domains in guanine nucleotide exchange, the crystal structure of a DH/PH fragment from Dbs in complex with Cdc42 has been determined. The complex features the PH domain in a unique conformation distinct from the PH domains in the related structures of Sos1 and Tiam1.Rac1. Consequently, the Dbs PH domain participates with the DH domain in binding Cdc42, primarily through a set of interactions involving switch 2 of the GTPase. Comparative sequence analysis suggests that a subset of Dbl-family proteins will utilize their PH domains similarly to Dbs (Rossman, 2002).

Cdc42 interaction with a GEF

The Rho family of small GTP-binding proteins plays important roles in the regulation of actin cytoskeleton organization and cell growth. Activation of these GTPases involves the replacement of bound GDP with GTP, a process catalyzed by the Dbl-like guanine-nucleotide exchange factors, all of which seem to share a putative catalytic motif termed the Dbl homology (DH) domain, followed by a pleckstrin homology (PH) domain. The role has been examined of a Dbl-like molecule, the faciogenital dysplasia gene product (FGD1), which when mutated in its Dbl homology domain, cosegregates with the developmental disease Aarskog-Scott syndrome. A polypeptide of FGD1 encompassing the DH and PH domains can bind specifically to the Rho family GTPase Cdc42Hs and stimulate the GDP-GTP exchange of the isoprenylated form of Cdc42Hs. Microinjection of this FGD1 polypeptide into Swiss 3T3 fibroblast cells induces the formation of peripheral actin microspikes, similar to that observed when cells are injected with a constitutively active form of Cdc42Hs. This effect of FGD1 on actin organization is readily inhibited by coinjection of a dominant-negative mutant of Cdc42Hs. Examination of NIH 3T3 cells expressing the FGD1 fragment revealed that similar to cells expressing Dbl, two independent elements downstream of Cdc42Hs, (the Jun NH2-terminal kinase and the p70 S6 kinase) became activated. Hence, these results indicate that FGD1, through its DH and PH domains, acts as a Cdc42Hs-specific guanine-nucleotide exchange factor and suggest that the Cdc42Hs GTPase may have a role in mammalian development (Zheng, 1996).

Cdc42 interaction with guanine nucleotide exchange factor RhoGDI

Cdc42, a Rho-related small GTP binding protein, plays pivotal roles in actin cytoskeletal organization, Golgi vesicular trafficking, receptor endocytosis, and cell cycle progression. However, the target/effectors mediating these cellular activities and, in particular, those responsible for Cdc42-mediated cell growth regulation and transformation are still being determined. This study examines how the regulatory protein RhoGDI influences the cellular responses elicited by activated Cdc42. X-ray crystallographic analysis of the Cdc42-RhoGDI complex suggests that arginine 66 of Cdc42 is essential for its interaction with RhoGDI. Mutation of either arginine 66 or arginine 68 within the Switch II domain of Cdc42 completely abolishes the binding of Cdc42 to RhoGDI without affecting the binding of other known regulators or target/effectors of this GTP binding protein. Introduction of the RhoGDI binding-defective mutation R66A within a constitutively active Cdc42(F28L) background is accompanied by changes in cell shape and an accumulation of Cdc42 in the Golgi. The most striking change was that unlike Cdc42(F28L), which is able to induce the transformation of NIH 3T3 fibroblasts as assayed by their growth in low serum or their ability to form colonies in soft-agar, the Cdc42(F28L,R66A) mutant is transformation-defective. Likewise, the introduction of RhoGDI siRNA into Cdc42(F28L)-transfected cells inhibits their transformation. Taken together, these results indicate that despite being a negative regulator of Cdc42 activation and GTP hydrolysis, RhoGDI plays an essential role in Cdc42-mediated cellular transformation (Lin, 2003).

Control of local Rho GTPase crosstalk by Abr

The Rho GTPases-Rho, Rac, and Cdc42-regulate the dynamics of F-actin (filamentous actin) and myosin-2 with considerable subcellular precision. Consistent with this ability, active Rho and Cdc42 occupy mutually exclusive zones during single-cell wound repair and asymmetric cytokinesis, suggesting the existence of mechanisms for local crosstalk, but how local Rho GTPase crosstalk is controlled is unknown. Using a candidate screen approach for Rho GTPase activators (guanine nucleotide exchange factors; GEFs) and Rho GTPase inactivators (GTPase-activating proteins; GAPs), Abr, a protein with both GEF and GAP activity, was found to regulate Rho and Cdc42 during single-cell wound repair. Abr is targeted to the Rho activity zone via active Rho. Within the Rho zone, Abr promotes local Rho activation via its GEF domain and controls local crosstalk via its GAP domain, which limits Cdc42 activity within the Rho zone. Depletion of Abr attenuates Rho activity and wound repair. Abr is the first identified Rho GTPase regulator of single-cell wound healing. Its novel mode of targeting by interaction with active Rho allows Abr to rapidly amplify local increases in Rho activity using its GEF domain while its ability to inactivate Cdc42 using its GAP domain results in sharp segregation of the Rho and Cdc42 zones. Similar mechanisms of local Rho GTPase activation and segregation enforcement may be employed in other processes that exhibit local Rho GTPase crosstalk (Vaughan, 2011).

Nudel binds Cdc42GAP to modulate Cdc42 activity at the leading edge of migrating cells

Cdc42GAP promotes inactivation of Cdc42, a small GTPase whose activation at the leading edge by guanine nucleotide exchange factors is critical for cell migration. How Cdc42GAP is regulated to ensure proper levels of active Cdc42 is poorly understood. Nudel, a cytoplasmic dynein regulator, competes with Cdc42 for binding Cdc42GAP. Consequently, Nudel can inhibit Cdc42GAP-mediated inactivation of Cdc42 in a dose-dependent manner. Both Nudel and Cdc42GAP exhibit leading-edge localization in migrating cells. The localization of Nudel requires its phosphorylation by Erk1/2. Depleting Nudel by RNAi or overexpression of a nonphosphorylatable mutant abolishes Cdc42 activation and cell migration. The data thus uncover Nudel as a regulator of Cdc42 during cell migration. Nudel facilitates cell migration by sequestering Cdc42GAP at the leading edge to stabilize active Cdc42 in response to extracellular stimuli. Excess active Cdc42 may in turn control its own activity by recruiting Cdc42GAP from Nudel (Shen, 2008).

Cdc42- and IRSp53-dependent contractile filopodia tether presumptive lens and retina to coordinate epithelial invagination

The vertebrate lens provides an excellent model with which to study the mechanisms required for epithelial invagination. In the mouse, the lens forms from the head surface ectoderm. A domain of ectoderm first thickens to form the lens placode and then invaginates to form the lens pit. The epithelium of the lens placode remains in close apposition to the epithelium of the presumptive retina as these structures undergo a coordinated invagination. This study shows that F-actin-rich basal filopodia that link adjacent presumptive lens and retinal epithelia function as physical tethers that coordinate invagination. The filopodia, most of which originate in the presumptive lens, form at E9.5 when presumptive lens and retinal epithelia first come into close contact, and have retracted by E11.5 when invagination is complete. At E10.5 - the lens pit stage - there is approximately one filopodium per epithelial cell. Formation of filopodia is dependent on the Rho family GTPase Cdc42 and the Cdc42 effector IRSp53 (Baiap2). Loss of filopodia results in reduced lens pit invagination. Pharmacological manipulation of the actin-myosin contraction pathway showed that the filopodia can respond rapidly in length to change inter-epithelial distance. These data suggest that the lens-retina inter-epithelial filopodia are a fine-tuning mechanism to assist in lens pit invagination by transmitting the forces between presumptive lens and retina. Although invagination of the archenteron in sea urchins and dorsal closure in Drosophila are known to be partly dependent on filopodia, this mechanism of morphogenesis has not previously been identified in vertebrates (Chauhan, 2009).

Intracellular localization of Cdc42

Cdc42Hs may play a role in cell morphogenesis by acting on targets in the Golgi that direct polarized growth at the plasma membrane. Immunocytochemical and fractionation approaches have been used to provide a description of the localization of the mammalian Cdc42 protein (designated Cdc42Hs) in vivo. A specific anti-peptide antibody was generated against the C-terminal region of Cdc42Hs. Using affinity-purified preparations of this antibody in indirect immunofluorescence experiments, Cdc42Hs was found to be localized to the Golgi apparatus. Similar to the well-characterized non-clathrin coat proteins ADP-ribosylation factor (ARF) and beta-COP, the perinuclear clustering of Cdc42Hs is rapidly dispersed upon exposure of the cells to the drug brefeldin A, suggesting that it too may play a role in the processes of intracellular lipid and protein transport. Employing cell lines possessing inducible forms of ARF, a tight coupling of the nucleotide-bound state of ARF and the subcellular localization of Cdc42Hs is demonstrated. Specifically, the expression of wild-type ARF has no effect on the brefeldin A sensitivity of Cdc42Hs while, as is the case for ARF and beta-COP, expression of a GTPase-deficient form of ARF [ARF(Q71L)] renders these Golgi-localized proteins resistant to brefeldin A treatment. Moreover, the induced expression of a mutant form of ARF with a low affinity for nucleotide results in constitutive redistribution of Cdc42Hs in the absence of brefeldin A treatment. Thus, Mammalian cdc42 is a brefeldin A-sensitive component of the Golgi apparatus (Erickson, 1996).

Signaling upstream of Cdc42

E-cadherin is a transmembrane protein that mediates Ca(2+)-dependent cell-cell adhesion. Cdc42, a member of the Rho family of small GTPases, participates in cytoskeletal rearrangement and cell cycle progression. Recent evidence reveals that members of the Rho family modulate E-cadherin function. To further examine the role of Cdc42 in E-cadherin-mediated cell-cell adhesion, an assay was developed for active Cdc42 using the GTPase-binding domain of the Wiskott-Aldrich syndrome protein. Initiation of E-cadherin-mediated cell-cell attachment significantly increases in a time-dependent manner the amount of active Cdc42 in MCF-7 epithelial cell lysates. By contrast, Cdc42 activity is not increased under identical conditions in MCF-7 cells incubated with anti-E-cadherin antibodies nor in MDA-MB-231 (E-cadherin negative) epithelial cells. By fusing the Wiskott-Aldrich syndrome protein/GTPase-binding domain to a green fluorescent protein, activation of endogenous Cdc42 by E-cadherin was demonstrated in live cells. These data indicate that E-cadherin activates Cdc42, demonstrating bi-directional interactions between the Rho- and E-cadherin signaling pathways (Kim, 2000).

IQGAP1 contains a number of protein recognition motifs through which it binds to targets. Several in vitro studies have documented that IQGAP1 interacts directly with calmodulin, actin, E-cadherin, beta-catenin, and the small GTPases Cdc42 and Rac. Nevertheless, direct demonstration of in vivo function of mammalian IQGAP1 is limited. Using a novel assay to evaluate in vivo function of IQGAP1, it has been shown that microinjection of IQGAP1 into early Xenopus embryos generates superficial ectoderm lesions at late blastula stages. This activity was retained by the mutated variants of IQGAP1 in which the calponin homology domain or the WW domain was deleted. By contrast, deletion of the IQ (IQGAP1-DeltaIQ), Ras-GAP-related (IQGAP1-DeltaGRD), or C-terminal (IQGAP1-DeltaC) domains abrogates the effect of IQGAP1 on the embryos. None of the latter mutants bind Cdc42, suggesting that the binding of Cdc42 by IQGAP1 is critical for its function. Moreover, overexpression of IQGAP1, but not IQGAP1-DeltaGRD, significantly increases the amount of active Cdc42 in embryonic cells. Co-injection of wild type IQGAP1 with dominant negative Cdc42, but not the dominant negative forms of Rac or Rho, blocks the effect of IQGAP1 on embryonic ectoderm. Together these data indicate that the activity of IQGAP1 in embryonic ectoderm requires Cdc42 function (Sokol, 2001).

The Ras-GAP related protein IQGAP1 binds several proteins, including actin, calmodulin, E-cadherin and the Rho family GTPase Cdc42. To gain insight into its in vivo function, IQGAP1 was overexpressed in mammalian cells. Transfection of IQGAP1 significantly increased the levels of active, GTP-bound Cdc42, resulting in the formation of peripheral actin microspikes. By contrast, transfection of an IQGAP1 mutant lacking part of the GAP-related domain (IQGAP1deltaGRD) substantially decreased the amount of GTP-bound Cdc42 in cell lysates. Consistent with these findings, IQGAP1DeltaGRD blocks Cdc42 function in cells that stably overexpress constitutively active Cdc42 and abrogate the effect of bradykinin on Cdc42. In cells transfected with IQGAP1deltaGRD, bradykinin is unable to activate Cdc42, translocate Cdc42 to the membrane fraction, or induce filopodia production. IQGAP1deltaGRD transfection alters cellular morphology, producing small, round cells that closely resemble Cdc42-/- cells. Some insight into the mechanism was provided by in vitro analysis, which revealed that IQGAP1deltaGRD increases the intrinsic GTPase activity of Cdc42, thereby increasing the amount of inactive, GDP-bound Cdc42. These data imply that IQGAP1 has a crucial role in transducing Cdc42 signaling to the cytoskeleton (Swart-Mataraza, 2002).

Rho family members are key regulators of the actin cytoskeleton, and control transcriptional targets through the activation of the JNK/SAPK pathway. Evidence for the role of Rho GTPases downstream of frizzled first arose from the analysis of Drosophila mutants affecting the establishment of planar cell polarity (PCP) of epithelia in developing eyes, dorsal thorax, and wings. Genetic dissection of the PCP pathway has shown that the Rho GTPase RhoA and Rac are activated in a pathway involving Frizzled and Dishevelled. A growing amount of data support the idea that a vertebrate equivalent of the PCP pathway activated downstream of Wnt-11/Xfz7 controls at least some of the cellular behaviors involved during mediolateral intercalation of the cells in the dorsal marginal zone (DMZ). Wnt-11/Xfz7 signaling plays a major role in the regulation of convergent extension movements affecting the DMZ of gastrulating Xenopus embryos. In order to provide data concerning the molecular targets of Wnt-11/Xfz7 signals, the regulation of the Rho GTPase Cdc42 by Wnt-11 was analyzed. In animal cap ectoderm, Cdc42 activity increases as a response to Wnt-11 expression. This increase is inhibited by pertussis toxin, or sequestration of free Gßgamma subunits by exogenous Galphai2 or Galphat. Activation of Cdc42 is also produced by the expression of bovine Gß1 and Ggamma2. This process is abolished by a PKC inhibitor, while phorbol esther treatment of ectodermal explants activates Cdc42 in a PKC-dependent way, implicating PKC downstream of Gßgamma. In activin-treated animal caps and in the embryo, interference with Gßgamma signaling rescues morphogenetic movements inhibited by Wnt-11 hyperactivation, thus phenocopying the dominant negative version of Cdc42 (N17Cdc42). Conversely, expression of Gß1gamma2 blocks animal cap elongation. This effect is reversed by N17Cdc42. Together, these results strongly argue for a role of Gßgamma signaling in the regulation of Cdc42 activity downstream of Wnt-11/Xfz7 in mesodermal cells undergoing convergent extension. This idea is further supported by the observation that expression of Galphat in the DMZ causes severe gastrulation defects (Penzo-Mendez, 2003).

Efficient chemotaxis requires directional sensing and cell polarization. A signaling mechanism is described involving Gβγ, PAK-associated guanine nucleotide exchange factor (PIXα), Cdc42, and p21-activated kinase (PAK) 1. This pathway is utilized by chemoattractants to regulate directional sensing and directional migration of myeloid cells. The results suggest that Gβγ binds PAK1 and, via PAK-associated PIXα, activates Cdc42, which in turn activates PAK1. Thus, in this pathway, PAK1 is not only an effector for Cdc42, but it also functions as a scaffold protein required for Cdc42 activation. This Gβγ-PAK1/PIXα/Cdc42 pathway is essential for the localization of F-actin formation to the leading edge, the exclusion of PTEN from the leading edge, directional sensing, and the persistent directional migration of chemotactic leukocytes. Although ligand-induced production of PIP3 is not required for activation of this pathway, PIP3 appears to localize the activation of Cdc42 by the pathway (Li, 2003).

Chemoattractants play a central role in regulation of inflammatory reactions by attracting and activating leukocytes at sites of injury and inflammation. Most chemoattractants bind to serpentine cell surface receptors that activate the Gi family of G proteins to elicit a range of responses in leukocytes that includes chemotaxis. Chemotaxis is an intriguing biological process in which cells interpret gradients of chemoattractants to move toward higher concentrations of chemoattractants. During chemotaxis, chemoattractants elicit a number of changes in cells. These include morphological changes characterized by cell elongation and formation of lamellae at the leading edge. There are also biochemical changes characterized by the polarized distribution of some intracellular proteins. Many of these processes depend on cytoskeleton reorganization. The Rho GTPase family of small G proteins has been demonstrated to play a key role in mediating extracellular stimulus-induced cytoskeleton reorganization, often by stimulating the formation of various polymerized actin structures (Li, 2003).

Cdc42 has been shown to interact with and/or activate proteins known to be involved in cytoskeleton reorganization, including the PAK kinases and Wiscott Aldrich syndrome protein (WASP). However, the precise mechanisms by which Cdc42 regulates cytoskeleton reorganization are not clear. There are three highly homologous PAK isoforms that contain a C-terminal kinase domain, a PIX binding domain, several proline-rich domains, and an autoregulatory segment. The autoregulatory segment contains a CRIB (Cdc42/Rac interaction/binding) domain, dimerization sequences, an inhibitory switch domain, and a kinase inhibition sequence. The PIX domain binds with a very high affinity to PIX guanine nucleotide exchange factors (GEFs), consisting of PIXα and PIXβ (also called Cool1 and Cool2, respectively). PAK and PIX proteins are readily coimmunoprecipitated from unstimulated cells, suggesting that these two proteins are normally associated. PIXα has been shown to stimulate nucleotide exchange activity for Cdc42 and Rac in vivo and in vitro (Li, 2003 and references therein).

PAK1 protein forms a homodimer. The binding of GTP bound Cdc42 or Rac dissociates the PAK dimer, leading to PAK activation. In addition, translocation of PAK proteins to membranes, such as that translocation mediated by binding to Nck, moderately activates PAK kinase activity. One effect of PAK1 activation is the regulation of actin cytoskeletal organization and dynamics, which may underlie PAK's role in regulating cell migration and axon guidance (Li, 2003).

Chemoattractants have been shown to activate Rac, Cdc42, and PAK kinases in leukocytes. The recent discovery of P-Rex1 [a novel Rac activator serving as a Gβγ and phosphatidylinositol (3,4,5) triphosphate (PIP3)-dependent GEF for Rac], suggests that chemoattractants may regulate Rac via P-Rex1; however, the mechanisms by which chemoattractants regulate Cdc42 remain elusive. In this report, a signaling mechanism has been identified by which Gβγ directly interacts with PAK1 and activates Cdc42 through PAK1-associated PIXα. Activated Cdc42 in turn activates PAK1. Utilizing short interference RNA (siRNA)-mediated knockdown and mouse gene-targeting approaches, it has been demonstrated that chemoattractants use this pathway to specifically activate Cdc42, but not Rac, in myeloid cells and reveal an essential role for this signaling pathway in establishing cell directionality during chemotaxis (Li, 2003).

Cell polarization and migration in response to chemokines is essential for proper development of the immune system and activation of immune responses. Recent studies of chemokine signaling have revealed a critical role for PI3-Kinase, which is required for polarized membrane association of pleckstrin homology (PH) domain-containing proteins and activation of Rho family GTPases that are essential for cell polarization and actin reorganization. Additional data argue that tyrosine kinases are also important for chemokine-induced Rac activation. However, how and which kinases participate in these pathways remain unclear. The Tec kinases Itk and Rlk play an important role in chemokine signaling in T lymphocytes. Chemokine stimulation induces transient membrane association of Itk and phosphorylation of both Itk and Rlk, and purified T cells from Rlk-/-Itk-/- mice exhibits defective migration to multiple chemokines in vitro and decreased homing to lymph nodes upon transfer to wt mice. SDF-1alpha is a chemokine that acts via the broadly expressed receptor CXCR4. Expression of a dominant-negative Itk impairs SDF-1alpha-induced migration, cell polarization, and activation of Rac and Cdc42. Thus, Tec kinases are critical components of signaling pathways required for actin polarization downstream from both antigen and chemokine receptors in T cells (Takesono, 2004).

To determine whether chemokine stimulation activates Tec kinases, the effects were examined of SDF-1alpha on phosphorylation of endogenous Itk, the major Tec kinase expressed in the Jurkat T cell line. In this cell line, Itk is constitutively localized at the plasma membrane due to a deficiency of the lipid phosphatase PTEN. Exposure of Jurkat cells to SDF-1alpha leads to a rapid increase in Itk tyrosine phosphorylation. Consistent with the known role of Lck in activation of Itk and previous reports that Lck is important for chemokine-induced migration of Jurkat cells, inhibition of Src family kinases with PP2 prevents Itk phosphorylation. Furthermore, examination of a transfected murine Rlk construct revealed that SDF-1alpha also induces tyrosine phosphorylation of Rlk. Thus, chemokines appear to stimulate phosphorylation of both Itk and Rlk (Takesono, 2004).

Itk has been shown to be required for TCR-induced actin polarization and the activation of Cdc42 and its downstream effector WASP, a critical regulator of the actin cytoskeleton that contributes to lymphocyte chemotaxis. To determine whether similar defects are observed downstream of chemokine receptor signaling, polarization was examined of Jurkat cells that were bound to beads coated with either fibronectin or fibronectin and SDF-1alpha. In the absence of SDF-1alpha or in the presence of SDF-1alpha at 0°C, cells bound the fibronectin-coated beads yet retained a spherical morphology. Stimulation of wt Jurkat cells or Jurkat cells expressing GFP with beads coated with fibronectin and SDF-1? at 37°C led to rapid changes in cell morphology, with increased F-actin accumulation associated with engulfment of the beads. However, cells expressing ItkKD-GFP failed to elongate and exhibited polarized actin accumulation upon SDF-1alpha stimulation. Moreover, ItkKD-GFP cells also showed decreased actin polymerization in response to SDF-1alpha as assessed by flow cytometry of fluorescently labeled phalloidin binding (Takesono, 2004).

To determine whether Itk also regulates chemokine-induced activation of Rho family GTPases, a GST-PAK-CRIB pull-down assay was used to isolate active, GTP bound Cdc42 and Rac. Stimulation of GFP-expressing Jurkat cells with SDF-1alpha led to the rapid activation of both Rac and Cdc42. However, Jurkat cells expressing ItkKD-GFP showed impaired SDF-1alpha-induced Rac and Cdc42 activation. In contrast, activation of Erk in response to SDF-1alpha was only minimally affected in the ItkKD-GFP cells, suggesting that some chemokine-mediated signaling pathways are intact in these cells (Takesono, 2004).

Coordination of Rho GTPase activities during cell protrusion

The GTPases Rac1, RhoA and Cdc42 act together to control cytoskeleton dynamics. Recent biosensor studies have shown that all three GTPases are activated at the front of migrating cells, and biochemical evidence suggests that they may regulate one another: Cdc42 can activate Rac1, and Rac1 and RhoA are mutually inhibitory. However, their spatiotemporal coordination, at the seconds and single-micrometre dimensions typical of individual protrusion events, remains unknown. This paper examine GTPase coordination in mouse embryonic fibroblasts both through simultaneous visualization of two GTPase biosensors and using a 'computational multiplexing' approach capable of defining the relationships between multiple protein activities visualized in separate experiments. It was found that RhoA is activated at the cell edge synchronous with edge advancement, whereas Cdc42 and Rac1 are activated 2 micro-m behind the edge with a delay of 40 s. This indicates that Rac1 and RhoA operate antagonistically through spatial separation and precise timing, and that RhoA has a role in the initial events of protrusion, whereas Rac1 and Cdc42 activate pathways implicated in reinforcement and stabilization of newly expanded protrusions (Machacek, 2009).

PTEN-mediated apical segregation of phosphoinositides controls epithelial morphogenesis through Cdc42

Formation of the apical surface and lumen is a fundamental, yet poorly understood, step in epithelial organ development. PTEN localizes to the apical plasma membrane during epithelial morphogenesis to mediate the enrichment of PtdIns(4,5)P2 at this domain during cyst development in three-dimensional culture. Ectopic PtdIns(4,5)P2 at the basolateral surface causes apical proteins to relocalize to the basolateral (BL) surface. Annexin 2 (Anx2) binds PtdIns(4,5)P2 and is recruited to the apical surface. Anx2 binds Cdc42, recruiting it to the apical surface. Cdc42 recruits aPKC to the apical surface. Loss of function of PTEN, Anx2, Cdc42, or aPKC prevents normal development of the apical surface and lumen. It is concluded that the mechanism of PTEN, PtdIns(4,5)P2, Anx2, Cdc42, and aPKC controls apical plasma membrane and lumen formation (Martin-Belmonte, 2007).

Formation of the apical surface and lumen is a central problem in understanding how epithelial tissues arrange themselves into tubes and other hollow structures, such as cysts. This study has uncovered a molecular mechanism of AP surface and lumen formation. PTEN is needed for segregation of PtdIns(4,5)p2 to the apical plasma membrane (PM) and PtdIns(3,4,5)p3 to the BL PM. Apical PtdIns(4,5)p2 recruits Anx2, which in turn recruits Cdc42 to the apical PM, causing the organization of the sub-apical actin cytoskeleton and formation of the apical surface and lumen. Cdc42 binds and localizes the Par6/aPKC complex to the apical PM to promote establishment of polarity (Martin-Belmonte, 2007).

PtdIns(4,5)p2 is thus a key determinant of the apical surface. Similarly, PtdIns(3,4,5)p3 is a key determinant of the BL surface (Gassama-Diagne, 2006). Together, PtdIns(4,5)p2 and PtdIns(3,4,5)p3 play complementary roles in epithelial polarity. More generally, phosphoinositides have emerged as general determinants of membrane identity. An advantage of epithelia is that exogenous phosphoinositides can be inserted into limited ectopic locations. These gain-of-function experiments provide a direct test of the role of the lipid in specifying domain identity (Martin-Belmonte, 2007).

To exert its effects, PtdIns(4,5)p2 interacts with Anx2, which clusters this lipid with high affinity and specificity. Ectopic PtdIns(4,5)p2 in the BL surface recruits Anx2 to the BL PM. Although loss of Anx2 by RNAi prevents lumen formation, RNAi of Anx2 did not produce as strong a phenotype as expression of the DN Anx2CM or RNAi of PTEN or Cdc42. One explanation could be the existence of >20 annexin family members, which might have redundancy with each other. Indeed, Anx2 knockout (KO) mice are viable. Alternatively, even low levels of Anx2 might suffice to exert its function (Martin-Belmonte, 2007).

Cdc42 interacts with Anx2 in a GTP-dependent manner. Cdc42 is activated during cystogenesis. Most activated Cdc42 relocalizes from cell-cell contacts to the apical pole as lumens form. RNAi of Cdc42 caused malformation of the central lumen in cysts but did not affect polarization of MDCK cells in 2D monolayers. This effect of Cdc42 depletion in cysts highlights the importance of using 3D models in analysis of lumen formation. Interestingly, an intracellular accumulation of Anx2 was seen in the cells with reduced Cdc42, suggesting a potential positive feedback loop whereby Cdc42 and Anx2 each promote the localization of the other at the lumen of mature cysts. Anx2 might work by recruiting Cdc42 or a GEF for Cdc42, and this GEF may in turn activate Cdc42 at this location. In contrast, active Cdc42 might promote the exocytosis of Anx2 and other apical proteins (Martin-Belmonte, 2007).

Formation of the apical surface and lumen has been suggested to be mediated by exocytosis of large intracellular vacuoles, termed vacuolar apical compartment (VAC). Formation of endothelial lumens occurs by vacuolar exocytosis. Cdc42 is needed for the exocytosis of secretory vesicles from neuroendocrine cells, apparently via rearrangement of the actin cytoskeleton, and this may be analogous to the fusion of VACs or smaller vesicles with the apical surface. Accumulation of apparent VACs was seen when Cdc42 was depleted. Similarly, DN Cdc42 blocks capillary lumen formation. Perhaps during normal MDCK cyst lumen formation smaller vesicles are rapidly exocytosed to form the lumen. Inhibition of this by Cdc42 depletion may cause the accumulation of larger, more easily detected VACs. Indeed, this may be the defect underlying the phenotypes observed with loss of function of PTEN, Anx2, Cdc42, or aPKC. Cdc42 is also needed for exit of apical and BL proteins from the trans-Golgi network (TGN), so Cdc42 may act at multiple levels in the formation of the apical surface (Martin-Belmonte, 2007).

Localized active Cdc42 may promote formation of the apical surface and lumen by additional mechanisms. Active Cdc42 binds to Par6, a member of the Par3/Par6/aPKC complex that regulates TJ and polarity formation. In Drosophila, Par6/aPKC functions at the apical PM independently of Par3, which is associated with the junctional complex. Indeed, Par6/aPKC localizes at the apical PM of MDCK cysts independently of Par3, and the disruption of aPKC function inhibits normal lumen formation. Mutation of zebrafish aPKCλ causes defects in lumen formation in the intestine. These data suggest the existence of two distinct Par complexes for the establishment of epithelial polarity: a complex of Par6/aPKC localized to the apical PM and involved in the formation of this domain; and a complex that also includes Par3, localized at the TJs and required for their formation (Martin-Belmonte, 2007).

It has been reported that inhibition of Rac1 or β1-integrin in cysts leads to inversion of polarity orientation and abnormal organization of laminin. Rac1, β1-integrin, and laminin may be part of a pathway that determines orientation of the axis of polarity, while PTEN, PtdIns(4,5)p2, Anx2, Cdc42, and Par6/aPKC are part of a pathway that controls formation of the apical surface and lumen. The Rac1/β1-integrin/laminin pathway might be upstream and/or parallel to the PTEN/PtdIns(4,5)p2/Anx2/Cdc42 pathway, and it determines the location of the apical surface. Because activation of Rac1 at the primordial adhesions of epithelial cells controls the association and activation of the Par3/Par6/aPKC complex to induce TJ biogenesis and cell polarity, one potential connection between these pathways might be the targeting of PTEN to the apical domain through its interaction with TJs. PTEN localizes to the adherens junctions in fly epithelium through its interaction with Bazooka/Par3. This study shows that PTEN is needed for apical PM and lumen formation during cyst development and that Par3 localizes specifically to the TJs in MDCK cyst. This observation is consistent with previous studies showing that the expression of DN Par-3 cells disrupted MDCK cyst morphogenesis, causing the lack of a central lumen (Martin-Belmonte, 2007).

PAKs are effectors of Cdc42

The GTPases Rac and Cdc42Hs (human Cdc42) control diverse cellular functions. In addition to being mediators of intracellular signaling cascades, they have important roles in cell morphogenesis and mitogenesis. A novel PAK-related kinase, PAK4 (see Drosophila PAK kinase, has been identified as a new effector molecule for Cdc42Hs. PAK4 interacts only with the activated form of Cdc42Hs through its GTPase-binding domain (GBD). Co-expression of PAK4 and the constitutively active Cdc42HsV12 causes the redistribution of PAK4 to the brefeldin A-sensitive compartment of the Golgi membrane and the subsequent induction of filopodia and actin polymerization. Importantly, the reorganization of the actin cytoskeleton is dependent on PAK4 kinase activity and on its interaction with Cdc42Hs. Thus, unlike other members of the PAK family, PAK4 provides a novel link between Cdc42Hs and the actin cytoskeleton. The cellular locations of PAK4 and Cdc42Hs suggest a role for the Golgi in cell morphogenesis (Abo, 1998).

Coordination of the different cytoskeleton networks in the cell is of central importance for morphogenesis, organelle transport, and motility. The Rho family proteins are well characterized for their effects on the actin cytoskeleton, but increasing evidence indicates that they may also control microtubule (MT) dynamics. A novel Cdc42/Rac effector, X-p21-activated kinase (PAK)5, colocalizes and binds to both the actin and MT networks and its subcellular localization is regulated during cell cycle progression. In transfected cells, X-PAK5 promotes the formation of stabilized MTs that are associated in bundles and interferes with MTs dynamics, slowing both the elongation and shrinkage rates and inducing long paused periods. X-PAK5 subcellular localization is regulated tightly, since coexpression with active Rac or Cdc42 induces its shuttling to actin-rich structures. Thus, X-PAK5 is a novel MT-associated protein that may communicate between the actin and MT networks during cellular responses to environmental conditions (Cau, 2001).

PAK4 differs from other members of the PAK family in sequence and in many of its functions. An important function of this kinase is to mediate the induction of filopodia in response to Cdc42. PAK4 also regulates the activity of the protein kinase LIM kinase 1 (LIMK1). PAK4 interacts specifically with LIMK1 in binding assays. Immune complex kinase assays reveal that both wild-type and constitutively active PAK4 phosphorylates LIMK1 even more strongly than PAK1, and activated PAK4 stimulates LIMK1's ability to phosphorylate cofilin. Immunofluorescence experiments reveal that PAK4 and LIMK1 cooperate to induce cytoskeletal changes in C2C12 cells. Furthermore, dominant negative LIMK1 and a mutant cofilin inhibit the specific cytoskeletal and cell shape changes that are induced in response to a constitutively activated PAK4 mutant (Dan, 2001).

p21-activated protein kinases (PAKs) are involved in signal transduction processes initiating a variety of biological responses. They become activated by interaction with Rho-type small GTP-binding proteins Rac and Cdc42 in the GTP-bound conformation, thereby relieving the inhibition of the regulatory domain (RD) on the catalytic domain (CD). Proteolytic digestion of PAK produces a heterodimeric RD-CD complex consisting of a regulatory fragment (residues 57 to 200) and a catalytic fragment (residues 201 to 491) that is active in the absence of Cdc42. Cdc42-GppNHp binds with low affinity [K(d) 0.6 microM] to intact kinase, whereas the affinity to the isolated regulatory fragment is much higher [K(d) 18 nM], suggesting that the difference in binding energy is used for the conformational change leading to activation. The full-length kinase, the isolated RD, and surprisingly also their complexes with Cdc42 behave as dimers on a gel filtration column. Cdc42-GppNHp interaction with the RD-CD complex is also of low affinity and does not dissociate the RD from the CD. After autophosphorylation of the kinase domain, Cdc42 binds with high (14 nM) affinity and dissociates the RD-CD complex. Assuming that the RD-CD complex mimics the interaction in native PAK, this indicates that the small G protein may not simply release the RD from the CD. It acts in a more subtle allosteric control mechanism to induce autophosphorylation, which in turn induces the release of the RD and thus full activation (Buchwald, 2001).

The PAK family of protein kinases has recently attracted considerable attention as an effector of the Rho family of small G proteins and as an upstream regulator of MAPK signaling pathways during cellular events such as re-arrangement of the cytoskeleton and apoptosis. A novel human PAK family kinase has been cloned that has been designated as PAK5. PAK5 contains a CDC42/Rac1 interactive binding (CRIB) motif at the N-terminus and a Ste20-like kinase domain at the C-terminus. PAK5 is structurally most related to PAK4 and PAK6 to make up the PAK-II subfamily. PAK5 preferentially binds to CDC42 in the presence of GTP and the CRIB motif is essential for this interaction. PAK5 is a functional protein kinase but unlike PAK-I family kinases (PAK1, 2, and 3), the kinase activity of PAK5 does not seem to require the binding of CDC42. Overexpression of PAK5 activates the JNK kinase pathway but not p38 or ERK pathways. PAK5 transcript is predominantly expressed in brain as revealed by Northern blot and in situ hybridization. The expression pattern of PAK5 is distinct from that of PAK4 and PAK6, suggesting a functional division among PAK-II subfamily kinases based on differential tissue distribution (Pandey, 2002).

The Rho GTPases play distinctive roles in cytoskeletal reorganization associated with growth and differentiation. The Cdc42/Rac-binding p21-activated kinase (PAK) and Rho-binding kinase (ROK) act as morphological effectors for these GTPases. Two related novel brain kinases have been isolated whose p21-binding domains resemble that of PAK, whereas the kinase domains resemble that of myotonic dystrophy kinase-related ROK. These approximately 190-kDa myotonic dystrophy kinase-related Cdc42-binding kinases (MRCKs) preferentially phosphorylate nonmuscle myosin light chain at serine 19, which is known to be crucial for activating actin-myosin contractility. The p21-binding domain binds GTP-Cdc42 but not GDP-Cdc42. The multidomain structure includes a cysteine-rich motif resembling that of protein kinase C and n-chimaerin and a putative pleckstrin homology domain. MRCK alpha and Cdc42V12 colocalize, particularly at the cell periphery in transfected HeLa cells. Microinjection of plasmid encoding MRCK alpha results in actin and myosin reorganization. Expression of kinase-dead MRCK alpha blocks Cdc42V12-dependent formation of focal complexes and peripheral microspikes. This was not due to possible sequestration of the p21, since a kinase-dead MRCK alpha mutant, defective in Cdc42 binding, is an equally effective blocker. Coinjection of MRCK alpha plasmid with Cdc42 plasmid, at concentrations where Cdc42 plasmid by itself elicits no effect, leads to the formation of the peripheral structures associated with a Cdc42-induced morphological phenotype. These Cdc42-type effects are not promoted upon coinjection with plasmids of kinase-dead or Cdc42-binding-deficient MRCK alpha mutants. These results suggest that MRCK alpha may act as a downstream effector of Cdc42 in cytoskeletal reorganization (Leung, 1998).

Diaphanous-related formin is an effector for Cdc42

Mammalian Diaphanous-related formins (Drfs) act as Rho small GTPase effectors during growth factor-induced cytoskeletal remodeling and cell division. While both p140 mDia1 (herein called Drf1) and p134 mDia2 (Drf3) have been shown to bind in vitro to activated RhoA-C, and Drf3 has also been shown to bind to Cdc42, little is known about the cellular function of these GTPase effector pairs. Thus, the murine Drf genes have been targeted to address their various contributions to small GTPase signaling in cytoskeletal remodeling and development. Drf1 +/+, +/-, and -/- cell lines were derived from embryonic stem cells. While some Drf1 +/- lines have fewer actin stress fibers, several Drf1 +/- and -/- cells were more motile and had more abundant lamella and filopodia. Because the apparent 'gain-of-function' corresponds with elevated levels of Drf3 protein expression, it was hypothesized that the effects on the actin cytoskeleton are due to Cdc42 utilization of Drf3 as an effector. In this study, it was found that inactive Drf3 variants and microinjected Drf3 antibodies interfer with Cdc42-induced filopodia. In addition, Drf3 contains a previously unidentified CRIB-like motif within its GTPase binding domain (GBD). By fluorescent resonance energy transfer (FRET) analysis, it has been demonstrated that this motif is required for Cdc42 binding and Drf3 recruitment to the leading edge and, surprisingly, to the microtubule organizing center (MTOC) of migrating fibroblasts. These observations extend the role of the mammalian Drfs in cell signaling and demonstrate that Cdc42 not only activates Drf3, but guides the effector to sites at the cell cortex where it remodels the actin cytoskeleton (Peng, 2003).

WASP interaction with Cdc42

Cdc42 is a small GTPase of the Rho family which regulates the formation of actin filaments to generate filopodia. Although there are several proteins such as PAK, ACK (see Drosophila Ack) and WASP (Wiskott-Aldrich syndrome protein) that bind Cdc42 directly, none of these can account for the filopodium formation induced by Cdc42. Before it can induce filopodium formation, Cdc42 must bind a WASP-related protein, N-WASP, that is richest in neural tissues but is expressed ubiquitously. N-WASP induces extremely long actin microspikes only when co-expressed with active Cdc42, whereas WASP, which is expressed in hematopoietic cells, does not, despite the structural similarities between WASP and N-WASP. In a cell-free system, addition of active Cdc42 significantly stimulates the actin-depolymerizing activity of N-WASP, creating free barbed ends from which actin polymerization can then take place. This activation seems to be caused by exposure of N-WASP's actin-depolymerizing region induced by Cdc42 binding (Miki, 1998).

Although small GTP-binding proteins of the Rho family have been implicated in signaling to the actin cytoskeleton, the exact nature of the linkage has remained obscure. A mechanism is described that links one Rho family member, Cdc42, to actin polymerization. N-WASP (see Drosophila WASp), a ubiquitously expressed Cdc42-interacting protein, is required for Cdc42-stimulated actin polymerization in Xenopus egg extracts. The C terminus of N-WASP binds to the Arp2/3 complex and dramatically stimulates its ability to nucleate actin polymerization. Although full-length N-WASP is less effective, its activity can be greatly enhanced by Cdc42 and phosphatidylinositol (4,5) bisphosphate. Therefore, N-WASP and the Arp2/3 complex comprise a core mechanism that directly connects signal transduction pathways to the stimulation of actin polymerization (Rohatgi, 1999).

WASP was identified as the gene product whose mutation causes the human hereditary disease Wiskott-Aldrich syndrome. WASP contains many functional domains and has been shown to induce the formation of clusters of actin filaments in a manner dependent on Cdc42. However, there has been no report investigating what domain(s) is (are) important for the function. A detailed analysis on the domain-function relationship of WASP has been carried out, with the following results: (1) the C-terminal verprolin-cofilin-acidic domain is essential for the regulation of actin cytoskeleton; (2) the clustering of WASP itself is distinct from actin clustering. The partial protein containing the region from the N-terminal pleckstrin homology domain to the basic residue-rich region also clusters especially around the nucleus as wild type WASP without inducing actin clustering. (3) Quite unexpectedly, a WASP mutant deficient in binding to Cdc42 still induces actin cluster formation, indicating that direct interaction between Cdc42 and WASP is not required for the regulation of actin cytoskeleton. This result may explain why no Wiskott-Aldrich syndrome patients have been identified with a missense mutation in the Cdc42-binding site (Kato, 1999).

Native WASp was purified from bovine thymus and its ability to stimulate actin nucleation by Arp2/3 complex was studied. WASp alone is inactive in the presence or absence of 0.5 microM GTP-Cdc42. Phosphatidylinositol 4,5 bisphosphate [PIP(2)] micelles allow WASp to activate actin nucleation by Arp2/3 complex, and this is further enhanced twofold by GTP-Cdc42. Filaments nucleated by Arp2/3 complex and WASp in the presence of PIP(2) and Cdc42 concentrate around lipid micelles and vesicles, providing that Cdc42 is GTP-bound and prenylated. Thus, the high concentration of WASp in neutrophils (9 microM) is dependent on interactions with both acidic lipids and GTP-Cdc42 to activate actin nucleation by Arp2/3 complex. The results also suggest that membrane binding increases the local concentrations of Cdc42 and WASp, favoring their interaction (Higgs, 2000).

The Rho-family GTPase, Cdc42, can regulate the actin cytoskeleton through activation of Wiskott-Aldrich syndrome protein (WASP) family members. Activation relieves an autoinhibitory contact between the GTPase-binding domain and the carboxy-terminal region of WASP proteins. The autoinhibited structure of the GTPase-binding domain of WASP, which can be induced by the C-terminal region or by organic co-solvents, is reported. In the autoinhibited complex, intramolecular interactions with the GTPase-binding domain occlude residues of the C terminus that regulate the Arp2/3 actin-nucleating complex. Binding of Cdc42 to the GTPase-binding domain causes a dramatic conformational change, resulting in disruption of the hydrophobic core and release of the C terminus, enabling its interaction with the actin regulatory machinery. These data show that 'intrinsically unstructured' peptides such as the GTPase-binding domain of WASP can be induced into distinct structural and functional states depending on context (A. S. Kim, 2000).

Wiskott-Aldrich syndrome protein (WASP) activation at the site of T cell-APC interaction is a two-step process, with recruitment dependent on the proline-rich domain and activation dependent on binding of Cdc42-GTP to the GTPase binding domain. WASP recruitment occurs through binding to the C-terminal Src homology 3 domain of Nck. In contrast, WASP activation requires Vav-1. In Vav-1-deficient T cells, WASP recruitment proceeds normally, but localized activation of Cdc42 and WASP is disrupted. The recruitment and activation of WASP are coordinated by tyrosine-phosphorylated Src homology 2 domain-containing leukocyte protein of 76 kDa, which functions as a scaffold, bringing Nck and WASP into proximity with Vav-1 and Cdc42-GTP. Taken together, these findings reconstruct the signaling pathway leading from TCR ligation to localized WASP activation (Zeng, 2003).

An important signaling pathway to the actin cytoskeleton links the Rho family GTPase Cdc42 to the actin-nucleating Arp2/3 complex through N-WASP. Nevertheless, these previously identified components are not sufficient to mediate Cdc42-induced actin polymerization in a physiological context. In this paper, the biochemical purification of Toca-1 (transducer of Cdc42-dependent actin assembly) as an essential component of the Cdc42 pathway is described. Toca-1 binds both N-WASP and Cdc42 and is a member of the evolutionarily conserved PCH protein family. Toca-1 promotes actin nucleation by activating the N-WASP-WIP/CR16 complex, the predominant form of N-WASP in cells. Thus, the cooperative actions of two distinct Cdc42 effectors, the N-WASP-WIP complex and Toca-1, are required for Cdc42-induced actin assembly. These findings represent a significantly revised view of Cdc42-signaling and shed light on the pathogenesis of Wiskott-Aldrich syndrome (Ho, 2004).

Sequence analysis reveals that Toca-1 is structurally related to proteins of the PCH (pombe Cdc15 h) family; these proteins have been implicated recently in a wide variety of actin-dependent processes, including cytokinesis, membrane trafficking, and cellular morphogenesis. This protein family is conserved throughout eukaryotic evolution and includes human formin binding protein 17 (FBP17), human Cdc42-interacting protein 4 (CIP4), human syndapins, D. melanogaster Cip4, C. elegans CE27939, S. cerevisiae Bzz1p, and S. pombe Cdc15. Members of this protein family are defined by a common domain structure that includes a FER/CIP4 homology (FCH) domain at the N terminus and one or two Src homology 3 (SH3) domains at the C terminus. The FCH domain is found in a large number of proteins involved in signal transduction, but its function is largely unknown. In addition, many PCH proteins are also predicted to contain coiled-coil domains. In the case of Toca-1, FBP17, CIP4, and D. melanogaster Cip4, one of these coiled-coil regions has homology to a domain called HR1 (protein kinase C-related kinase homology region 1), which was originally identified as a Rho-interactive module in several RhoA binding proteins. The functional conservation of Toca-1 across species is highlighted by the finding that Toca-1 homologs from X. tropicalis and D. melanogaster can complement the MCAP2B activity in an in vitro assay system (Ho, 2004).

The following view of the Cdc42 signaling pathway is proposed. Formation of PIP2 on membranes (such as a vesicle surface) leads to the recruitment and activation of Cdc42. Prenylated Cdc42 inserts into the membrane and forms high avidity sites that recruit Toca-1 and the N-WASP-WIP complex. Activation of N-WASP then could proceed through one of two paths: both Cdc42 and Toca-1 could cooperate to activate the N-WASP-WIP complex, or Toca-1 could function indirectly by relieving the inhibition of N-WASP by WIP (see the Drosophila homolog Verprolin 1). Toca-1 is ideally positioned to be an important regulatory node for the Cdc42 pathway. The function of Toca-1 suggests a specific mechanism by which PCH family proteins can influence actin nucleation in a wide variety of cellular processes such as vesicle motility and cytokinesis. Important future questions include the precise biochemical mechanism by which the N-WASP-WIP complex is activated by Toca-1 and Cdc42, as well as investigation into the regulation of Toca-1 itself by other signals (Ho, 2004).

Cdc42 and the cell cycle

During cytokinesis in animal cells, an equatorial actomyosin-based contractile ring divides the cell into two daughter cells. The position of the contractile ring is specified by a signal that emanates from the mitotic spindle. This signal has not been identified and it is not understood how the components of the contractile ring assemble. It is also unclear how the ring constricts or how new plasma membrane inserts specifically behind the leading edge of the constricting furrow. The Rho family of small GTPases regulate polarized changes in cell growth and cell shape by affecting the formation of actin structures beneath the plasma membrane, but their role in cytokinesis is unclear. The function of two Rho family members has been studied during the early cell divisions of Xenopus embryos by injecting modified forms of Rho and Cdc42. Both inhibition and constitutive activation of either GTPase blocks cytokinesis. Furrow specification occurs normally, but ingression of the furrow is inhibited. Newly inserted cleavage membranes appear aberrantly on the outer surface of the embryo. Rho localizes to the cortex and regulates the levels of cortical F-actin. These results show that Rho regulates the assembly of actin filaments in the cortex during cytokinesis, that local activation of Rho is important for proper constriction of the contractile furrow, and that Cdc42 plays a role in furrow ingression. Moreover, these observations reveal that furrow ingression and membrane insertion are not strictly linked. Neither Rho nor Cdc42 appear to be required for establishment of the cell-division plane (Drechsel, 1997).

During vertebrate egg maturation, cytokinesis initiates after one pole of the bipolar metaphase I spindle attaches to the oocyte cortex, resulting in the formation of a polar body and the mature egg. It is not known what signal couples the spindle pole positioning to polar body formation. This question was approached by drawing an analogy to mitotic exit in budding yeast; asymmetric spindle attachment to the appropriate cortical region is the common regulatory cue. In budding yeast, the small G protein Cdc42 plays an important role in mitotic exit following the spindle pole attachment. This study shows that inhibition of Cdc42 activation blocks polar body formation. The oocytes initiate anaphase but fail to properly form and direct a contractile ring. Endogenous Cdc42 is activated at the spindle pole-cortical contact site immediately prior to polar body formation. The cortical Cdc42 activity zone, which directly overlays the spindle pole, is circumscribed by a cortical RhoA activity zone; the latter defines the cytokinetic contractile furrow. As the RhoA ring contracts during cytokinesis, the Cdc42 zone expands, maintaining its complementary relationship with the RhoA ring. Cdc42 signaling may thus be an evolutionarily conserved mechanism that couples spindle positioning to asymmetric cytokinesis (Ma, 2006).

Polar body emission during oocyte maturation requires cyclin B degradation, RhoA contractile ring, and Cdc42-mediated membrane protrusion

Vertebrate oocyte maturation is an extreme form of asymmetric cell division, producing a mature egg alongside a diminutive polar body. Critical to this process is the attachment of one spindle pole to the oocyte cortex prior to anaphase. This study reports that asymmetric spindle pole attachment and anaphase initiation are required for localized cortical activation of Cdc42, which in turn defines the surface of the impending polar body. The Cdc42 activity zone overlaps with dynamic F-actin and is circumscribed by a RhoA-based actomyosin contractile ring. During cytokinesis, constriction of the RhoA contractile ring is accompanied by Cdc42-mediated membrane outpocketing such that one spindle pole and one set of chromosomes are pulled into the Cdc42 enclosure. Unexpectedly, the guanine nucleotide exchange factor Ect2, which is necessary for contractile ring formation, does not colocalize with active RhoA. Polar body emission thus requires a classical RhoA contractile ring and Cdc42-mediated membrane protrusion (Zhang, 2008).

Cdc42 activation is first observed at the spindle-cortex contact site shortly (within a few minutes) following anaphase initiation, suggesting that activation of Cdc42 might be temporarily and mechanistically coupled to anaphase. Attempts were made to inhibit anaphase without altering spindle assembly or the perpendicular spindle attachment to the oocyte cortex. The role of cyclin B degradation and the resultant loss of Cdk1 activity during amphibian meiosis is controversial. While it has been reported that stabilization of Cdk1 activity via prevention of cyclin B destruction does not prevent polar body formation, it has also been reported that experimental stabilization of Cdk1 by other means does block polar body emission and that this blockade can be relieved by Cdk1 inhibition. The role of cyclin B degradation on polar body emission was reinvestigated. A truncated form of cyclin B1 (ΔN cyclin B1 was employed that lacks the destruction box required for APC-targeted degradation. Injection of ΔN cyclin B1 mRNA efficiently eliminated the transient inactivation of MPF seen in control oocytes. Oocytes injected with ΔN cyclin B1 underwent progesterone-induced GVBD indistinguishably from control oocytes. However, oocytes injected with ΔN cyclin B1 mRNA failed to emit the first polar body. Similarly, injection of methylubiquitin effectively inhibited cyclin B degradation and inhibited first polar body emission (Zhang, 2008).

Time-lapse imaging (or 4D imaging) experiments were carried to determine the effect of ΔN cyclin B1 on Cdc42 activation in live oocytes, using eGFP-wGBD, a GFP fusion protein containing the GTPase-binding domain of WASP (wGBD) that binds only active (GTP-bound) Cdc42. Control oocytes exhibited Cdc42 activation approximately 2 hr after GVBD, emitted the first polar body, and then arrested in metaphase II indefinitely. In contrast, ΔN cyclin B1-injected oocytes failed to activate Cdc42, and the spindle remained intact and attached to the cortex for an extended period of time. Similarly, methylubiquitin-injected oocytes exhibited intact metaphase I spindles that remained asymmetrically attached to the cortex, with no Cdc42 activation (Zhang, 2008).

Given the controversy regarding whether the transient degradation of cyclin B is required for homolog separation in Xenopus oocytes, chromosome dynamics were observed in live oocytes injected with ΔN cyclin B1 mRNA. Fluorescent antibodies against Xenopus Aurora B were used to track endogenous Aurora B, a chromosome passenger kinase. Fluorescent anti-Aur B faithfully tracked chromosomes through the metaphase I (00:10) to metaphase II (00:52) transition. In addition, fluorescent anti-Aur B also clearly marked the central spindle or spindle midzone (MZ) at anaphase/telophase (arrows, 00:20–00:24), and the midbody following polar body emission. These data are consistent with previous conclusions based on staining of fixed oocytes of other species. It is noteworthy that in contrast to mitosis in which Aurora B is completely transferred to the central spindle at anaphase, in the oocytes Aurora B persisted with the segregated chromosome homologs. Presumably, Aurora B signal persisted at anaphase I due to the cohesion of sister centromeres, which only resolve at anaphase II upon fertilization (Zhang, 2008).

Having established the utility of fluorescent anti-Aur B in tracking chromosomes in live oocytes, the fate of chromosomes was followed in oocytes injected with ΔN cyclin B1. Metaphase I spindles remained asymmetrically positioned against the animal pole cortex for an extended period of time, with no homolog separation. Similarly, oocytes injected with methylubiquitin did not separate chromosome homologs. These data clearly indicate that cyclin B degradation is required for anaphase initiation and for Cdc42 activation (Zhang, 2008).

In vivo imaging reveals a role for Cdc42 in spindle positioning and planar orientation of cell divisions during vertebrate neural tube closure

Specialization of the cell-division process is a common feature of developing embryos, but most studies on vertebrate cell division have focused on cells dividing in culture. This study used in vivo four-dimensional confocal microscopy to explore the role of Cdc42 in governing cell division in the developing neural epithelium of Xenopus laevis. Cdc42 was found to be crucial for stable positioning of the metaphase spindle in these cells, but was not required for spindle positioning in epidermal epithelial cells. Divisions in the Xenopus neural plate are planar oriented, and rotations of mitotic spindles are essential for establishing this orientation. When Cdc42 is disrupted, spindles over-rotate and the final orientation of divisions is changed. Finally, the planar orientation of cell divisions in this tissue seems to be independent of planar cell polarity (PCP) signaling and does not require normal neural morphogenesis. These data provide new insights into the coordination of cell division and morphogenesis in epithelial cell sheets and reveal novel, cell-type-specific roles for Cdc42 in spindle positioning and spindle orientation (Kieserman, 2009).

Alteration of Cdc42 function has a dramatic effect on the extent of mitotic-spindle rotation in the Xenopus neural plate. Whereas control cells have a limited range of rotation, with very few spindles rotating more than 120°, cells with altered Cdc42 function had spindles that rotated in excess of 200°. These data suggest a model for the orientation of cell divisions in the neural plate. Spindle positioning in general involves interactions between astral microtubules and the cell cortex, and it is proposed that spindle rotations are initiated essentially at random, and that astral microtubules identify 'catch points' on the cell cortex that stop rotation prior to anaphase and in accordance with the long axis of a cell. One such catch point that is necessary for stopping spindle rotation so that the final division angle is aligned with the cellular long axis is Cdc42-dependent. In this model, if the Cdc42-dependent catch point is disrupted, spindles rotate excessively, and eventually are able to stop by responding to other, Cdc42-independent mechanisms. This general mechanism, by which multiple inputs 'compete' to control spindle orientation, has been demonstrated in Drosophila neuroblasts (Kieserman, 2009 and references therein).

S6K functions downstream of Cdc42

The 70 kDa ribosomol S6 kinase (pp70S6k) plays an important role in the progression of cells through G1 phase of the cell cycle. However, little is known of the signaling molecules that mediate its activation. Rho family G proteins regulate pp70S6k activity in vivo. Activated alleles of Cdc42 and Rac1, but not RhoA, stimulate pp70S6k activity in multiple cell types. Activation requires an intact effector domain and isoprenylation of Cdc42 and Rac1. Coexpression of Dbl, an exchange factor for Cdc42, also activates pp70S6k. Growth factor-induced activation of pp70S6k is abrogated by dominant negative alleles of Cdc42 and Rac1. In addition, Cdc42 and Rac1 form GTP-dependent complex with the catalytically inactive form of pp70S6k in vitro and in vivo, suggesting a mechanism by which these G proteins activate pp70S6k (Chou, 1996).

The mammalian target of rapamycin (mTOR) regulates cell growth and proliferation via the downstream targets ribosomal S6 kinase 1 (S6K1) and eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1). Phosphatidic acid (PA) has been identified as a mediator of mitogenic activation of mTOR signaling. This study tests the hypotheses that phospholipase D 1 (PLD1) is an upstream regulator of mTOR and that the previously reported S6K1 activation by Cdc42 is mediated by PLD1. Overexpression of wild-type PLD1 is found to increase S6K1 activity in serum-stimulated cells, whereas a catalytically inactive PLD1 exerts a dominant-negative effect on S6K1. More importantly, eliminating endogenous PLD1 by RNAi leads to drastic inhibition of serum-stimulated S6K1 activation and 4E-BP1 hyperphosphorylation in both HEK293 and COS-7 cells. Knockdown of PLD1 also results in reduced cell size, suggesting a critical role for PLD1 in cell growth control. Using a rapamycin-resistant S6K1 mutant, Cdc42's action has been demonstrated to be through the mTOR pathway. When Cdc42 is mutated in a region specifically required for PLD1 activation, its ability to activate S6K1 in the presence of serum is hindered. However, when exogenous PA is used as a stimulus, the PLD1-inactive Cdc42 mutant behaves similarly to the wild-type protein. These observations reveal the involvement of PLD1 in mTOR signaling and cell size control, and provide a molecular mechanism for Cdc42 activation of S6K1. A new cascade is proposed to connect mitogenic signals to mTOR through Cdc42, PLD1, and PA (Fang, 2003).

Cdc42 and endocytosis

Dendritic cells (DCs) developmentally regulate antigen uptake by controlling their endocytic capacity. Immature DCs actively internalize antigen. However, mature DCs are poorly endocytic, functioning instead to present antigens to T cells. Endocytic downregulation reflects a decrease in endocytic activity controlled by Rho family GTPases, especially Cdc42. Blocking Cdc42 function by Toxin B treatment or injection of dominant-negative inhibitors of Cdc42 abrogates endocytosis in immature DCs. In mature DCs, injection of constitutively active Cdc42 or microbial delivery of a Cdc42 nucleotide exchange factor reactivates endocytosis. DCs regulate endogenous levels of Cdc42-GTP with activated Cdc42 detectable only in immature cells. It is concluded that DCs developmentally regulate endocytosis at least in part by controlling levels of activated Cdc42 (Garrett, 2000).

Cdc42 associates with gammaCop

The Ras-related GTP-binding protein Cdc42 is implicated in a variety of biological activities including the establishment of cell polarity in yeast, the regulation of cell morphology, motility and cell-cycle progression in mammalian cells and the induction of malignant transformation. A Cdc42 mutant (Cdc42F28L) has been identified that binds GTP in the absence of a guanine nucleotide exchange factor, but still hydrolyses GTP with a turnover number identical to that for wild-type Cdc42. Expression of this mutant in NIH 3T3 fibroblasts causes cellular transformation, mimicking many of the characteristics of cells transformed by the Dbl oncoprotein, a known guanine nucleotide exchange factor for Cdc42. New Cdc42 targets have been sought in an effort to understand how Cdc42 mediates cellular transformation. The gamma-subunit of the coatomer complex (gammaCOP) has been identified as a specific binding partner for activated Cdc42. The binding of Cdc42 to gammaCOP is essential for a transforming signal distinct from those elicited by Ras (Wu, 2000).

Cdc42 and cell polarity

A novel effector of Rac and Cdc42, hPar-6, has been identified that is the human homolog of a cell-polarity determinant in C. elegans. hPar-6 contains a PDZ domain and a Cdc42/Rac interactive binding (CRIB) motif, and interacts with Rac1 and Cdc42 in a GTP-dependent manner. hPar-6 also binds directly to an atypical protein kinase C isoform, PKC, and forms a stable ternary complex with either Rac1 or Cdc42 and PKC. This association results in stimulation of PKC kinase activity. Moreover, hPar-6 potentiates cell transformation by Rac1/Cdc42 and its interaction with Rac1/Cdc42 is essential for this effect. Cell transformation by hPar-6 involves a PKC-dependent pathway distinct from the pathway mediated by Raf (Qui, 2000).

Many direct targets of Rac1 and Cdc42 have been identified, but none has been shown to have a direct role in cell transformation by Rac1 and Cdc42. hPar-6 is a novel effector of Rac1 and Cdc42 that promotes PKCzeta-dependent transformation by both GTPases. Although it has been suggested that PAK1 may also contribute to transformation by Rac1 in Rat1 fibroblasts, PAK1 does not enhance transformation by activated Raf or activated Rac1 in NIH-3T3 cells, and studies using effector domain mutants indicate that interaction of PAK1 with Rac1 does not correlate with cell-cycle progression or transformation. Thus, hPar-6 appears to be the first effector shown to directly mediate transformation by Rac1 and Cdc42. The identification of PKCzeta as a downstream effector of hPar-6 represents the first elucidation of a signaling pathway linking Rac1/Cdc42 to cell transformation. A model is presented depicting two separate pathways downstream of Ras that lead to cell polarity and growth control: these pathways can contribute to cell transformation. One pathway is comprised of Rac/Cdc42, hPar-6 and PKCzeta, and the other is mediated by Raf, MEK and MAP kinase (Qui, 2000).

The mechanism by which hPar-6 regulates the kinase activity of PKCzeta is currently under investigation. Subcellular targeting by interaction with specific proteins provides an attractive mechanism for PKC isozyme-specific regulation. It is possible that hPar-6 and PKCzeta are translocated by Rac1 or Cdc42 to the membrane, where PKCzeta could interact with an activator. One candidate activator is the phosphatidylinositol 3-kinase (PI3-kinase) target PDK1, since PDK1 and PKCzeta associate in vivo via their catalytic domains, and both PI3-kinase and PDK1 stimulate PKCzeta activity. Consistent with this model, it has been demonstrated that PI3-kinase can act as a link between Ras and Rac in transformation and that membrane-targeted PKCzeta is constitutively active. The observation that hPar-6 alone exhibits little, if any, transforming activity is also consistent with the membrane-targeting model. It should also be noted that although overexpression of hPar-6 alone (i.e., in the absence of Rac1[G12V]) is sufficient to activate PKCzeta kinase activity, overexpression of hPar-6 and PKCzeta only marginally promotes focus formation, suggesting that activated Rac1 is necessary to target PKCzeta to substrates involved in transformation. However, the possibility that Rac1 activates some other pathway that is also necessary for transformation cannot be ruled out. In addition to being activated by hPar-6, PKCzeta might in turn phosphorylate hPar-6. In this regard, it should be noted that there is a putative PKCzeta-phosphorylation site in mammalian Par-6 (Qui, 2000).

The mechanism underlying transformation by hPar-6 and PKCzeta is not yet clear. Stimulation of cell proliferation and inhibition of apoptosis are, however, important characteristics of cell transformation. In this regard, it has been shown that Rac1 and Cdc42 induce cyclin D1 transcription and accumulation, phosphorylation and inactivation of the tumor suppressor protein Rb, and activation of the transcription factor E2F. Inactivation of Rb may be necessary for Rac1/Cdc42 stimulation of cell proliferation, and it is possible that hPar-6 and PKCzeta have a role in this pathway. In addition, Ras, Rac1, Cdc42 and PKCzeta are all able to activate the transcription factor NF-kappaB. NF-kappaB activation is associated with mitogenesis, anti-apoptotic activity and cell transformation. Thus, the hPar-6-PKCzeta pathway might mediate NF-kappaB activation, and thereby contribute to cell transformation by Rac1 and Ras. Another possibility is that the hPar-6-PKCzeta pathway may mediate growth control by Rac1/Cdc42 by inducing downregulation of the pro-apoptotic protein Par-4 (prostate apoptosis response-4; unrelated to the C. elegans Par gene product). Par-4 interacts with PKCzeta and overexpression of PKCzeta downregulates Par-4, an event that appears important for Ras transformation and tumor progression. Thus, cyclin D1, Rb, E2F, NF-kappaB and Par-4 all warrant further investigation as possible downstream targets of the hPar-6-PKCzeta pathway (Qui, 2000).

Polarity is a fundamental feature of all eukaryotic cells. Rac, Cdc42, Par-6 and atypical PKCs appear to be conserved in diverse metazoans, including Drosophila, C. elegans, Xenopus, mouse and humans. The CRIB motif of Par-6 is also conserved, suggesting that it interacts with Rac and/or Cdc42 in these different species. In C. elegans, inhibition of Cdc42 function by RNA-mediated gene interference (RNAi) produces defects in cell polarity similar to those observed in par and pkc-3 mutants, while in mammalian cells, Par-6 is localized to tight junctions, together with atypical PKC and ASIP, the mammalian homolog of Par-3. Moreover in C. elegans, Par-6 interacts with Par-3, and in Drosophila the Par-3 homolog has an important role in the asymmetric cleavage of epithelial cells and neuroblasts. Taken together, these observations suggest that Rac or Cdc42, Par-6, atypical PKC, and perhaps Par-3, constitute a conserved pathway that regulates cell polarity. As hPar-6 and PKCzeta mediate cell transformation by Rac1 and Cdc42, there may be a link between cell-polarity signaling and growth control: aberrant cell-polarity signaling could lead to oncogenic transformation. In the light of the important roles of Rac1/Cdc42 in Ras-induced transformation, hPar-6 and PKCzeta could represent potential targets for anti-cancer therapeutics (Qui, 2000).

Cellular asymmetry is critical for the development of multicellular organisms. Homologs of proteins necessary for asymmetric cell division in Caenorhabditis elegans associate with each other in mammalian cells and tissues. mPAR-3 and mPAR-6 exhibit similar expression patterns and subcellular distributions in the CNS and associate through their PDZ (PSD-95/Dlg/ZO-1) domains. mPAR-6 binds to Cdc42/Rac1 GTPases, and mPAR-3 and mPAR-6 bind independently to atypical protein kinase C aPKC) isoforms. In vitro, mPAR-3 acts as a substrate and an inhibitor of aPKC. It is concluded that mPAR-3 and mPAR-6 have a scaffolding function, coordinating the activities of several signaling proteins that are implicated in mammalian cell polarity (Lin, 2000).

Generation of asymmetry in the one-cell embryo of C. elegans establishes the anterior-posterior axis (A-P), and is necessary for the proper identity of early blastomeres. Conserved PAR proteins are asymmetrically distributed and are required for the generation of this early asymmetry. The small G protein Cdc42 is a key regulator of polarity in other systems, and recently it has been shown to interact with the mammalian homolog of PAR-6. The function of Cdc42 in C. elegans had not yet been investigated, however. C. elegans cdc-42 plays an essential role in the polarity of the one-cell embryo and the proper localization of PAR proteins. Inhibition of cdc-42 using RNA interference results in embryos with a phenotype that is nearly identical to par-3, par-6, and pkc-3 mutants, and asymmetric localization of these and other PAR proteins is lost. CDC-42 physically interacts with PAR-6 in a yeast two-hybrid system, consistent with data on the interaction of human homologs. It is concluded that CDC-42 acts in concert with the PAR proteins to control the polarity of the C. elegans embryo, and the interaction of CDC-42 and the PAR-3/PAR-6/PKC-3 complex has been evolutionarily conserved as a functional unit (Gotta, 2001).

Phenotypic and two-hybrid data suggest that CDC-42 might activate the PAR-3/PAR-6/PKC-3 complex through interaction with PAR-6. CDC-42 appears to be necessary for the activity of the complex as well as for its correct localization. It is also possible that the PAR-3/PAR-6/PKC-3 complex has a role in activating CDC-42, since its initial anterior localization seems CDC-42 independent. One way that CDC-42 and the PAR-3/PAR-6/PKC-3 complex might direct polarity is through the regulation of the actin cytoskeleton. In par-3 mutants and in cdc-42(RNAi) embryos, enrichment of actin at the anterior of early embryos is lost. Further, cosuppression of par-2 and cdc-42(RNAi) mutant phenotypes suggests that CDC-42 and PAR-2 have counterbalancing, antagonistic activities. Because PAR-2 has a RING finger, a motif that has been proposed to be involved in ubiquitin-mediated protein degradation, CDC-42 might normally activate a protein that is a target of PAR-2. Future biochemical and in vivo studies should help to reveal the nature of these interactions and identify downstream targets (Gotta, 2001 and references therein).

Cdc42 is a small GTPase that is required for cell polarity establishment in eukaryotes as diverse as budding yeast and mammals. Par6 is also implicated in metazoan cell polarity establishment and asymmetric cell divisions. Cdc42.GTP interacts with proteins that contain a conserved sequence called a CRIB motif. Uniquely, Par6 possesses a semi-CRIB motif that is not sufficient for binding to Cdc42. An adjacent PDZ domain is also necessary and is required for biological effects of Par6. The crystal structure of a complex between Cdc42 and the Par6 GTPase-binding domain is reported in this study. The semi-CRIB motif forms a beta-strand that inserts between the four strands of Cdc42 and the three strands of the PDZ domain to form a continuous eight-stranded sheet. Cdc42 induces a conformational change in Par6, detectable by fluorescence resonance energy transfer spectroscopy. Nuclear magnetic resonance studies indicate that the semi-CRIB motif of Par6 is at least partially structured by the PDZ domain. The structure highlights a novel role for a PDZ domain as a structural scaffold (Garrard, 2003).

C. elegans embryonic polarity requires the asymmetrically distributed proteins PAR-3, PAR-6 and PKC-3. The rho family GTPase CDC-42 regulates the activities of these proteins in mammals, flies and worms. To clarify its mode of action in C. elegans, the interaction between PAR-6 and CDC-42 was disrupted in vivo, and also the distribution of GFP-tagged CDC-42 was determined in the early embryo. Mutant PAR-6 proteins unable to interact with CDC-42 accumulate asymmetrically, at a reduced level, but this asymmetry is not maintained during the first division. Constitutively active GFP::CDC-42 becomes enriched in the anterior during the first cell cycle in a domain that overlaps with PAR-6. The asymmetry is dependent on PAR-2, PAR-5 and PAR-6. Furthermore, it was found that overexpression of constitutively active GFP::CDC-42 increased the size of the anterior domain. It is concluded that the CDC-42 interaction with PAR-6 is not required for the initial establishment of asymmetry but is required for maximal cortical accumulation of PAR-6 and to maintain its asymmetry (Aceto, 2006).

In C. elegans one-cell embryos, polarity is conventionally defined along the anteroposterior axis by the segregation of partitioning-defective (PAR) proteins into anterior (PAR-3, PAR-6) and posterior (PAR-1, PAR-2) cortical domains. The establishment of PAR asymmetry is coupled with acto-myosin cytoskeleton rearrangements. The small GTPases RHO-1 and CDC-42 are key players in cytoskeletal remodeling and cell polarity in a number of different systems. This study investigated he roles of these two GTPases and the RhoGEF ECT-2 in polarity establishment in C. elegans embryos. CDC-42 is shown to be required to remove PAR-2 from the cortex at the end of meiosis and to localize PAR-6 to the cortex. By contrast, RHO-1 activity is required to facilitate the segregation of CDC-42 and PAR-6 to the anterior. Loss of RHO-1 activity causes defects in the early organization of the myosin cytoskeleton but does not inhibit segregation of myosin to the anterior. It is therefore proposed that RHO-1 couples the polarization of the acto-myosin cytoskeleton with the proper segregation of CDC-42, which, in turn, localizes PAR-6 to the anterior cortex (Schonegg, 2006).

CDC-42 orients cell migration during epithelial intercalation in the Caenorhabditis elegans epidermis.

Cell intercalation is a highly directed cell rearrangement that is essential for animal morphogenesis. As such, intercalation requires orchestration of cell polarity across the plane of the tissue. CDC-42 is a Rho family GTPase with key functions in cell polarity, yet its role during epithelial intercalation has not been established because its roles early in embryogenesis have historically made it difficult to study. To circumvent these early requirements, this study used tissue-specific and conditional loss-of-function approaches to identify a role for CDC-42 during intercalation of the Caenorhabditis elegans dorsal embryonic epidermis . CDC-42 activity is enriched in the medial tips of intercalating cells, which extend as cells migrate past one another. Moreover, CDC-42 is involved in both the efficient formation and orientation of cell tips during cell rearrangement. Using conditional loss-of-function it was shown that the PAR complex (see Drosophila PAR complex) functions in tip formation and orientation. Additionally, the sole C. elegans Eph receptor, VAB-1 (see Drosophila Eph), was found to function during this process in an Ephrin-independent manner. Using epistasis analysis, it was shown that vab-1 lies in the same genetic pathway as cdc-42 and is responsible for polarizing CDC-42 activity to the medial tip. Together, these data establish a previously uncharacterized role for polarized CDC-42, in conjunction with PAR-6, PAR-3 and an Eph receptor, during epithelial intercalation.

Spontaneous cell polarization through actomyosin-based delivery of the Cdc42 GTPase

Cell polarization can occur in the absence of any spatial cues. To investigate the mechanism of spontaneous cell polarization, an assay was used in yeast where expression of an activated form of Cdc42, a Rho-type guanosine triphosphatase (GTPase) required for cell polarization, could generate cell polarity without any recourse to a preestablished physical cue. The polar distribution of Cdc42 in this assay required targeted secretion directed by the actin cytoskeleton. A mathematical simulation showed that a stable polarity axis could be generated through a positive feedback loop in which a stochastic increase in the local concentration of activated Cdc42 on the plasma membrane enhanced the probability of actin polymerization and increased the probability of further Cdc42 accumulation to that site (Wedlich-Soldner, 2003).

Robust cell polarity is a dynamic state established by coupling transport and GTPase signaling

Yeast cells can initiate bud formation at the G1/S transition in a cue-independent manner. This study investigated the dynamic nature of the polar cap and the regulation of the GTPase Cdc42 in the establishment of cell polarity. Using analysis of fluorescence recovery after photobleaching, it was found that Cdc42 exchanged rapidly between the polar caps and cytosol and that this rapid exchange required its GTPase cycle. A previously proposed positive feedback loop involving actomyosin-based transport of the Cdc42 GTPase is required for the generation of robust cell polarity during bud formation in yeast. Inhibition of actin-based transport results in unstable Cdc42 polar caps. Unstable polarity was also observed in mutants lacking Bem1, a protein implicated in a feedback loop for Cdc42 activation through a signaling pathway. When Bem1 and actin are both inhibited, polarization completely fails. These results suggest that cell polarity is established through coupling of transport and signaling pathways and maintained actively by balance of flux (Wedlich-Soldner, 2004).

Scaffold-mediated symmetry breaking by Cdc42p

Cell polarization generally occurs along a single well-defined axis that is frequently determined by environmental cues such as chemoattractant gradients or cell-cell contacts, but polarization can also occur spontaneously in the apparent absence of such cues, through a process called symmetry breaking. In Saccharomyces cerevisiae, cells are born with positional landmarks that mark the poles of the cell and guide subsequent polarization and bud emergence to those sites, but cells lacking such landmarks polarize towards a random cortical site and proliferate normally. The landmarks employ a Ras-family GTPase, Rsr1p, to communicate with the conserved Rho-family GTPase Cdc42p, which is itself polarized and essential for cytoskeletal polarization. Yeast Cdc42p is effectively polarized to a single random cortical site even in the combined absence of landmarks, microtubules and microfilaments. Among a panel of Cdc42p effectors and interacting proteins, it was found that the scaffold protein Bem1p is uniquely required for this symmetry-breaking behaviour. Moreover, polarization is dependent on GTP hydrolysis by Cdc42p, suggesting that assembly of a polarization site involves cycling of Cdc42p between GTP- and GDP-bound forms, rather than functioning as a simple on/off switch (Irazoqui, 2003).

Cdc42 regulates the Par-6 PDZ domain through an allosteric CRIB-PDZ transition

Regulation of protein interaction domains is required for cellular signaling dynamics. This study shows that the PDZ protein interaction domain from the cell polarity protein Par-6 is regulated by the Rho GTPase Cdc42. Cdc42 binds to a CRIB domain adjacent to the PDZ domain, increasing the affinity of the Par-6 PDZ for its carboxy-terminal ligand by approximately 13-fold. Par-6 PDZ regulation is required for function; mutational disruption of Cdc42-Par-6 PDZ coupling leads to inactivation of Par-6 in polarized MDCK epithelial cells. Structural analysis reveals that the free PDZ domain has several deviations from the canonical PDZ conformation that account for its low ligand affinity. Regulation results from a Cdc42-induced conformational transition in the CRIB-PDZ module that causes the PDZ to assume a canonical, high-affinity PDZ conformation. The coupled CRIB and PDZ architecture of Par-6 reveals how simple binding domains can be combined to yield complex regulation (Peterson, 2004).

This study describes a set of Cdc42-dependent and -independent interactions for the Par-6 CRIB-PDZ module involving the known ligands Par-3 and Pals1/Sdt and a carboxy-terminal ligand identified in a peptide library screen. The functional relevance of Cdc42 regulation of carboxy-terminal ligand binding by Par-6 was established in polarized epithelial cells. The intrinsic low affinity of the Par-6 PDZ domain was found to arise from structural deviations from the canonical PDZ conformation, both in the peptide binding pocket and in regions that contact Cdc42. Binding of Cdc42 causes an allosteric transition in the CRIB-PDZ, which leads to a typical PDZ conformation that binds C-terminal ligand with high affinity. A simple thermodynamic cycle model indicates that binding of Cdc42 or carboxy-terminal peptide should induce the Par-6 PDZ allosteric transition, which was verified by X-ray crystallography (Peterson, 2006).

Although protein interaction domains must be regulated to yield the complex signaling dynamics observed in cells, little is known about the mechanisms underlying this regulation. PDZ domains are very common in metazoans and are also found in many bacteria and yeast. Individual PDZ domains have been shown to bind to carboxy-terminal ligands with affinities ranging from high nanomolar to low micromolar. Like most isolated domains, PDZ domains typically bind their ligands in a constitutive manner. The Par-6 PDZ differs in this respect, with low intrinsic affinity for ligand that can be increased into the low micromolar range upon Cdc42 binding. This may be a common regulatory mechanism used by protein interaction domains. However, as ligand screens are often performed with isolated domains, ligands regulated in this manner are likely to be missed. As with the Par-6 PDZ domain, ligand binding may only be observed with the proper set of intra- and/or intermolecular ligands (Peterson, 2006).

The different affinities of free and Cdc42-bound Par-6 for carboxy-terminal ligand result from an allosteric transition in the PDZ domain. Analysis of multiple sequence alignments and double mutant cycles has suggested that energetic pathways within PDZ domains may support allostery. In this study, a pathway of physical connectivity was identified that runs between the two PDZ helices. This pathway is consistent with the conformational changes that occur in the Par-6 PDZ upon Cdc42 binding as it connects the Cdc42 and carboxy-terminal ligand binding sites. The energetic coupling in PDZ domains, along with the Par-6 PDZ results discussed in this study, suggest that allostery may be a general feature of the PDZ domain family (Peterson, 2006).

Allostery appears to be a common mechanism used by Cdc42 effectors to translate binding into changes in activity. Rho GTPases, including Cdc42, are found in many different cellular systems and, as such, utilize a diverse array of effectors. Over 60 targets have been identified for the prototypical Rho GTPases, Rho, Rac, and Cdc42. Besides Par-6, Cdc42 also regulates several widely varying classes of proteins, including kinases (p21 activated kinase, PAK) and actin regulatory molecules (Wiskott-Aldrich syndrome protein, WASP). Although each of these effectors has an entirely different domain structure, each contains a CRIB motif for binding to Cdc42 (Peterson, 2006).

Recent studies have begun to uncover the molecular mechanism by which binding to the CRIB motif in PAK and WASP is translated into activity modulation. In these proteins, the CRIB is part of a larger module that regulates the protein's kinase or Arp2/3 activating domains, respectively. In both cases, residues carboxy-terminal to the CRIB motif form an intramolecular interaction that inactivates the protein. Cdc42 binding to the CRIB leads to a conformational transition in the regulatory module that is incompatible with the intramolecular interaction leading to activation (Peterson, 2006).

The mechanism of Par-6 regulation by Cdc42 shows that the CRIB is a versatile motif that can be coupled to diverse domains to regulate effector function. Rather than the specialized regulatory module found in PAK and N-WASP, the Par-6 CRIB is coupled to a PDZ protein interaction domain. However, in all cases, the CRIB motif transmits binding information to the adjacent domain through allosteric changes. While binding of Cdc42 to PAK and N-WASP leads to a decrease in affinity of the adjacent domain for their intramolecular ligands, Cdc42 binding to Par-6 leads to an increase in affinity for an intermolecular PDZ ligand (Peterson, 2006).

Many diverse systems require Cdc42 and Par-6 activity for cell polarity. The molecular mechanism by which polarity is controlled by these signaling molecules is poorly understood. One function of Cdc42 in cell polarity appears to be regulation of aPKC kinase activity through binding to Par-6. Although aPKC has a conserved carboxy-terminal sequence (-SLEDCV-COOH) with similarities to the Par-6 PDZ ligand identified it this study, it does not bind to the Par-6 PDZ domain. As such, it is expected that Cdc42 regulates formation of an unidentified Par-6 complex (Peterson, 2006).

The Cdc42-Par-6 interaction may also play a role in localization of Par-6, aPKC, and their ligands as inactivation of Cdc42 leads to symmetrical localization of these proteins. Cooperative binding of Cdc42 and Par-6 PDZ ligands would then allow for correct spatial and temporal activation. This may be an essential feature of Par-6 regulation as localization is a defining feature of cell polarization. Future work will be directed at further exploring the role of Cdc42-Par-6 PDZ ligand coupling in cell polarity (Peterson, 2006).

A Rich1/Amot complex regulates the Cdc42 GTPase and apical-polarity proteins in epithelial cells

Using functional and proteomic screens of proteins that regulate the Cdc42 GTPase, a network of protein interactions have been identified that center around the Cdc42 RhoGAP Rich1 and organize apical polarity in MDCK epithelial cells. Rich1 binds the scaffolding protein angiomotin (Amot) and is thereby targeted to a protein complex at tight junctions (TJs) containing the PDZ-domain proteins Pals1, Patj, and Par-3. Regulation of Cdc42 by Rich1 is necessary for maintenance of TJs, and Rich1 is therefore an important mediator of this polarity complex. Furthermore, the coiled-coil domain of Amot, with which it binds Rich1, is necessary for localization to apical membranes and is required for Amot to relocalize Pals1 and Par-3 to internal puncta. It is proposed that Rich1 and Amot maintain TJ integrity by the coordinate regulation of Cdc42 and by linking specific components of the TJ to intracellular protein trafficking (Wells, 2006).

Cdc42 and the actin cytoskeleton in yeast

The Saccharomyces cerevisiae BNI1 gene product (Bni1p) is a member of the formin family of proteins, which participate in cell polarization, cytokinesis, and vertebrate limb formation. During mating pheromone response, bni1 mutants show defects both in polarized morphogenesis and in reorganization of the underlying actin cytoskeleton. In two-hybrid experiments, Bni1p forms complexes with the activated form of the Rho-related guanosine triphosphatase Cdc42p, with actin, and with two actin-associated proteins, profilin and Bud6p (Aip3p). Both Bni1p and Bud6p (like Cdc42p and actin) localize to the tips of mating projections. Bni1p may function as a Cdc42p target that links the pheromone response pathway to the actin cytoskeleton (Evangelista, 1997).

Cdc42 and convergent extension in Xenopus

Rho GTPases are molecular switches that regulate many essential cellular processes, including actin dynamics, cell adhesion, cell-cycle progression, and transcription. The Xenopus homolog of Rho GTPase Cdc42 has been isolated and its potential role during gastrulation movements in early Xenopus embryos has been examined. XCdc42 is expressed in tissues undergoing extensive morphogenetic changes, such as the deep layers of involuting mesoderm and posterior neuroectoderm during gastrulation, and somitic mesoderm at neurula stages. Overexpression of either wild-type (WT) or dominant-negative (DN) XCdc42 interferes with convergent extension movements in intact embryos, activin-stimulated animal caps, and dorsal marginal zone explants. These effects occur without affecting mesodermal specification. Overexpression of WT or DN XCdc42 leads to the decrease and increase of cell adhesiveness of blastomeres, respectively, as demonstrated by the cell adhesion assay. In addition, when overexpressed, PKC-alpha, XWnt-5a, and Mfz-3 inhibit activin-induced convergent extension in animal cap explants. This inhibition can be rescued by coexpression of DN XCdc42, implying that XCdc42 acts downstream of the Wnt/Ca2+ signaling pathway involving PKC activation. XCdc42 also lies downstream of XWnt-5a in the regulation of Ca2+-dependent cell adhesion. Taken together, these results suggest that XCdc42 plays a role in the regulation of convergent extension movements during gastrulation through the protein kinase C-mediated Wnt/Ca2+ pathway (Choi, 2002.

Mesenchymal-epithelial transition during somitic segmentation is regulated by differential roles of Cdc42 and Rac1

Mesenchymal-epithelial transitions (MET) are crucial for vertebrate organogenesis. The roles of Rho family GTPases in such processes during actual development remain largely unknown. By electroporating genes into chick presomitic mesenchymal cells, it was demonstrated that Cdc42 and Rac1 play important and different roles in the MET that generates the vertebrate somites. Presomitic mesenchymal cells, which normally contribute to both the epithelial and mesenchymal populations of the somite, are hyperepithelialized when Cdc42 signaling is blocked. Conversely, cells taking up genes that elevate Cdc42 levels remain mesenchymal. Thus, Cdc42 activity levels appear critical for the binary decision that defines the epithelial and mesenchymal somitic compartments. Proper levels of Rac1 are necessary for somitic epithelialization, since cells with activated or inhibited Rac1 fail to undergo correct epithelialization. Furthermore, Rac1 appears to be required for Paraxis to act as an epithelialization-promoting transcription factor during somitogenesis (Nakaya, 2004).

A complementary pattern of phenotypes was obtained by different levels of Cdc42 activity: enhanced epithelialization and mesenchymal maintenance by inhibition and activation of Cdc42, respectively. Thus, during normal somitogenesis, different levels of Cdc42 activity appear to be critical for the binary determination during MET: Cdc42 activity needs to be low for cell epithelialization, whereas cells require high activity to maintain their mesenchymal state. Cdc42 has been reported, mainly by experiments in vitro, to assemble with several associated molecules, such as Par6, aPKC (atypical protein kinase C), and Par3 in polarizing cells. The experimental system developed in this study can be used to clarify the roles of these members in establishing epithelial structures during vertebrate morphogenesis. Recently, another member of Cdc42 subfamily, TC10, was reported to bind to N-WASP, although it remains unclear whether the binding is only to the CRIB domain. Which member among Cdc42 subfamily plays a role during somitogenesis awaits further analysis (Nakaya, 2004).

Another important finding in this study highlights differential roles between Cdc42 and Rac1 in the somitic MET. Unlike the case for Cdc42, however, overactivation and inhibition of Rac1 did not show a complementary phenotype between each other; in both cases the electroporated cells were primarily localized in the mesenchymal area, with some cells remaining in the epithelial territory. It is likely that the Rac1 activity needs to be maintained at an appropriate level to accomplish the correct MET during somitogenesis or, alternatively, that switching between the negative forms and active forms of Rac1 is important. The importance of proper Rac1 activity levels was corroborated by several lines of evidence: Rac1-activated cells that remained in the epithelial territory are not 'normal epithelial cells,' since they display aberrant accumulation of N-cadherin without polarized distribution of ZO-1. Similarly, Rac1-activated cells and Rac1-inactivated cells residing in the mesenchymal compartment also exhibit aberrantly upregulated N-cadherin and poorly organized actin polymerization, respectively. Thus, cells with an inappropriate level of Rac1 activity are neither 'normal mesenchyme' nor 'normal epithelium,' regardless of the position they occupy within a forming somite (Nakaya, 2004).

LIM-kinase functions downstream of Cdc42

The rapid turnover of actin filaments and the tertiary meshwork formation are regulated by a variety of actin-binding proteins. Protein phosphorylation of cofilin, an actin-binding protein that depolymerizes actin filaments, suppresses its function. Thus, cofilin is a terminal effector of signaling cascades that evokes actin cytoskeletal rearrangement. When wild-type LIMK2 (see Drosophila LIM-kinase1) and kinase-dead LIMK2 (LIMK2/KD) are respectively expressed in cells, LIMK2, but not LIMK2/KD, phosphorylates cofilin and induces formation of stress fibers and focal complexes. LIMK2 activity toward cofilin phosphorylation is stimulated by coexpression of activated Rho and Cdc42, but not Rac. Importantly, expression of activated Rho and Cdc42, respectively, induces stress fibers and filopodia, whereas both Rho-induced stress fibers and Cdc42-induce filopodia are abrogated by the coexpression of LIMK2/KD. In contrast, the coexpression of LIMK2/KD with the activated Rac does not affect Rac-induced lamellipodia formation. These results indicate that LIMK2 plays a crucial role both in Rho- and Cdc42-induced actin cytoskeletal reorganization, at least in part by inhibiting the functions of cofilin. Together with recent findings that LIMK1 participates in Rac-induced lamellipodia formation, LIMK1 and LIMK2 function under control of distinct Rho subfamily GTPases and are essential regulators in the Rho subfamilies-induced actin cytoskeletal reorganization (Sumi, 1999).

Cdc42 in neurons

The multidomain shank/ProSAP/SSTRIP proteins are major scaffold proteins in glutamatergic synapses in the mammalian brain; expression of shank1/SSTRIP in hippocampal neurons induces morphological changes in dendritic spines, suggesting that shank1 is involved in synapse formation and activity-dependent changes of synaptic structure. Using part of the proline-rich region of shank1 in a yeast two hybrid screen, the insulin receptor substrate IRSp53 has been identified as an interaction partner. Overlay assays verified a strong interaction between a proline-rich sequence (residues 911-940) in shank1 and the SH3 domain of IRSp53. When coexpressed in HEK cells, shank1 colocalizes with IRSp53 in intracellular structures, preventing targeting of IRSp53 to filopodia that are induced by IRSp53 expression in the absence of shank1. IRSp53 also binds to the activated form of the small G-protein cdc42. Interestingly, IRSp53 coprecipitates with shank1 from transfected HEK cells in a small G-protein-regulated manner. Thus, IRSp53 constitutes a cdc42-regulated ligand for shank1 that may provide a molecular basis for small G-protein mediated effects on the structure of the postsynaptic complex (Soltau, 2002).

Cdc42 and proliferation

The Rho family small GTPase Cdc42 is critical for diverse cellular functions including the regulation of actin organization, cell polarity, intracellular membrane trafficking, transcription, cell cycle progression and cell transformation. Like other members of the Rho family, Cdc42 cycles between the GTP-bound, active state, and the inactive, GDP-bound state under tight regulation, and it is believed that the GTP bound form of Cdc42 represents the active signaling module in eliciting effector activation and cellular responses. The constitutively active mutant, V12, derived from the analogous mutations found in oncogenic Ras that are GTPase-defective, and a 'fast-cycling' self-activating mutant, F28, of Cdc42, have been widely in use to study the cellular effects of Cdc42. The constitutively active V12 mutant of Cdc42, when stably expressed in cells, behaves in a dominant negative fashion in inhibiting cell proliferation while the F28 mutant is growth stimulatory. The V12 mutant fails to transform NIH3T3 cells while F28 potently stimulates anchorage-independent growth. The growth inhibitory effect of the V12 mutant correlates with activation of JNK2 and suppression of the cyclin D1 and NF-kappaB expressions that are instead upregulated by the F28 mutant. Furthermore, the V12 mutant suppresses, whereas the F28 mutant potentiates, or has no effect on, a wide variety of oncogene-induced cell transformation, including that by the Dbl family GEFs Dbl, Vav and Lbc and the oncogenic Ras, ErbB-2, PDGF B or Raf. These results raise the possibility that over-saturation or constitutive activation of Cdc42 signal may negatively impact on cell proliferation and that both the activation and deactivation steps, or the complete GTPase cycle, of Cdc42 is required for proper function (Vanni, 2005).

Serotonin-induced regulation of the actin network for learning-related synaptic growth requires Cdc42, N-WASP, and PAK in Aplysia sensory neurons

Application of Clostridium difficile toxin B, an inhibitor of the Rho family of GTPases, at the Aplysia sensory to motor neuron synapse blocks long-term facilitation (LTF) and the associated growth of new sensory neuron varicosities induced by repeated pulses of serotonin (5-HT). cDNAs encoding Aplysia Rho, Rac, and Cdc42 have been isolated and it has been found that Rho and Rac had no effect but that overexpression in sensory neurons of a dominant-negative mutant of ApCdc42 or the CRIB domains of its downstream effectors PAK and N-WASP selectively reduces the long-term changes in synaptic strength and structure. FRET analysis indicates that 5-HT activates ApCdc42 in a subset of varicosities contacting the postsynaptic motor neuron and that this activation is dependent on the PI3K and PLC signaling pathways. The 5-HT-induced activation of ApCdc42 initiates reorganization of the presynaptic actin network leading to the outgrowth of filopodia, some of which are morphological precursors for the learning-related formation of new sensory neuron varicosities (Udo, 2005).

Actin is enriched in both the pre- and postsynaptic compartments of most neurons. Although the activity-dependent modulation of actin dynamics at postsynaptic spines has been well documented, the extent and role of actin regulation in presynaptic terminals is not well understood. During development, reorganization of actin in growth cones has been shown to play an important role in axonal pathfinding. However, in mature neurons, it has been suggested that the presynaptic actin network functions more as an intracellular scaffold rather than as a propulsive force, that contributes directly to the type of frank structural remodeling reported for postsynaptic dendritic spines. In Aplysia, repeated applications of 5-HT (which lead to LTF) induce a slower and more persistent alteration in the dynamics of the presynaptic actin network, leading to the growth of new sensory neuron synapses. The data indicate that Cdc42 is one of the key molecular regulators of this learning-related modulation of presynaptic actin organization (Udo, 2005).

The family of Rho GTPases has been shown to play an important role in neuronal development, for example, the establishment of polarity, axon guidance, and the growth and maintenance of dendritic spines. ApCdc42 is involved in long-term synaptic plasticity in Aplysia, suggesting that Cdc42 may also have a role in learning and memory storage in the mature nervous system. It was surprising that Rac, which is functionally related to Cdc42 and known to regulate spine morphology and memory consolidation in mice, does not significantly contribute to LTF and the associated structural changes. By contrast, Rho tends to oppose the effects of Cdc42 on long-term synaptic plasticity, which is consistent with the ways in which these two proteins regulate actin dynamics (Udo, 2005).

In Aplysia, the activation of ApCdc42 in sensory neurons leads to the outgrowth of filopodia from presynaptic varicosities. Interestingly, 5-HT stimulation by itself naturally induces filopodia; this induction is dependent on the activation of ApCdc42. Filopodia have been proposed to be morphological precursors of dendritic spines in the mammalian central nervous system, and this process may be regulated by neuronal activity. The 5-HT-induced activation of Cdc42 in Aplysia triggers not only the formation of filopodia but also the molecular maturation of neurotransmitter release sites. A major synaptic vesicle protein, synaptophysin, accumulates at the tips of 5-HT-induced filopodia, some of which then give rise to new varicosities. These observations support the following ideas: (1) filopodia represent one type of morphological precursor for the growth of new presynaptic varicosities during learning-related synaptic plasticity, and (2) the formation of filopodia and initial assembly of the presynaptic compartment can be induced by the activation of Cdc42 (Udo, 2005).

5-HT is a modulatory neurotransmitter released from facilitating interneurons that make synaptic contacts onto sensory neurons. Most of the 5-HT receptors are known to be G protein coupled, and G proteins such as Gα12 and Gα13 have been shown to link to Rho. However, the signaling pathways for Rac/Cdc42 appear to be different from those for Rho, and the molecular cascade between G protein-coupled receptors and Cdc42 is not well understood. The current results suggest that 5-HT activates ApCdc42 through pathways involving PLC and PI3 kinase. PLC produces two independent second messengers (diacylglycerol and inositol triphosphate) to initiate a variety of cellular functions. It has also been shown that some isoforms of PLC are able to directly bind to Rac/Cdc42 and to enhance its activity. Since an increase in the internal concentration of calcium is known to stimulate actin polymerization, PLC may send multiple signals to regulate actin-related structures. Like PLC, PI3 kinase also plays a key role in regulating cell growth and survival. PI3 kinase is thought to interact directly with Cdc42 and stimulate its activity. Although PI3 kinase is usually activated by receptor tyrosine kinases such as Trk receptors, recent evidence suggests that some PI3 kinase isoforms (such as type 1B) can be upregulated by their interaction with Gβγ. Thus, it is possible that PI3 kinase may be activated by 5-HT receptors as well as Trk-like receptors and that PLC and PI3K may send signals to ApCdc42 via independent pathways (Udo, 2005).

The synapse-specific nature of the 5-HT-induced activation of ApCdc42 suggests the possibility of a coordinated interaction between the presynaptic sensory neuron and the postsynaptic motor neuron. This could be mediated by a variety of different cell adhesion or trans-synaptic signaling molecules. For example, ephexin and IQGAP, which bind to the EphA receptor and cadherin, respectively, are known to modulate the activity of Cdc42. The identification of such molecules in Aplysia should provide additional molecular insights into the upstream signaling pathways that activate ApCdc42 (Udo, 2005).

The differential activation of ApCdc42 at a spatially distinct subset of presynaptic sensory neuron varicosities is consistent with previous studies, which have shown that the initial segment and cell body of the postsynaptic motor neuron is a preferred site for new sensory neuron varicosity formation induced by 5-HT. It is proposed that 5-HT receptors coupled to G proteins (Gαq and Gβγ) activate the PLC and PI3 kinase pathways, which in turn upregulate ApCdc42 at specific presynaptic varicosities. The selective activation of ApCdc42 leads to the formation of actin-based filopodia by activating downstream effector proteins such as N-WASP and to a lesser extent PAK. Presynaptic components, including synaptic vesicles, appear to be recruited to the tips of specific filopodia, possibly through actin-myosin-dependent transport, which then become transformed into new functional sensory neuron varicosities. Thus, 5-HT-induced regulation of the Cdc42 signaling pathways and the consequent reorganization of the presynaptic actin network appear to be a part of the initial molecular cascade required for the growth of new sensory neuron synapses associated with long-term memory (Udo, 2005).

Cdc42-mediated tubulogenesis controls cell specification

Understanding how cells polarize and coordinate tubulogenesis during organ formation is a central question in biology. Tubulogenesis often coincides with cell-lineage specification during organ development. Hence, an elementary question is whether these two processes are independently controlled, or whether proper cell specification depends on formation of tubes. To address these fundamental questions, the functional role of Cdc42 in pancreatic tubulogenesis was studied. Evidence is presented that Cdc42 is essential for tube formation, specifically for initiating microlumen formation and later for maintaining apical cell polarity. Finally, it was shown that Cdc42 controls cell specification non-cell-autonomously by providing the correct microenvironment for proper control of cell-fate choices of multipotent progenitors (Kesavan, 2009).

Signals that control tubulogenesis also control cell specification. For example, signaling pathways acting as chemoattractants during epithelial branching, e.g., Bnl/Fgf signaling, also determine whether a Drosophila tracheal epithelial cell becomes a tip or stalk cell. Correspondingly, maintenance of a tip cell phenotype is regulated by FGF signaling in the mammary gland (Kesavan, 2009).

Detailed comparison between the complete and mosaic ablation of Cdc42 and the inhibition of aPKC activity demonstrates that pancreatic cell-fate specification requires tubes to ensure that multipotent Pdx1+ pancreatic progenitors are confined to distinct microenvironments. Pancreatic progenitors in the periphery of the branching epithelium are continuously exposed to basal lamina components, e.g., laminins, and mesenchymal cells, whereas the more centrally localized progenitors primarily interact with one another (until E13.5). It is only later when tubes are beginning to form (E13.5 to E15.5) that the latter cells become exposed to basement membrane components and mesenchymal cells. Hence, the consequence of ablating Cdc42 in all pancreatic progenitors is that virtually all progenitors become exposed to the same environment as the peripheral progenitors. Consistently, the progenitors maintain expression of Ptf1a and differentiate into acinar cells, the normal fate of peripheral 'tip cell' progenitors. As a consequence endocrine and duct cell differentiation become compromised. Interestingly, blocking aPKC activity also resulted in reduced endocrine cell specification, but acinar specification was unaffected. Limited invasion by mesenchymal cells and ECM (laminin) may explain the lack of effect on acinar differentiation, whereas failure to generate continuous tubes most likely explains the endocrine phenotype. Importantly, in vitro explant experiments demonstrated that Cdc42 does not affect endocrine lineages directly as they were as capable as WT epithelium of differentiating into endocrine cells. In conclusion, these results provide an explanation for how a direct role of Cdc42 in tissue architecture secondarily specifies microenvironments permissive for specification of multipotent Pdx1+ pancreatic progenitors (Kesavan, 2009).

Finally, current attempts to develop protocols for generating insulin-producing beta cells from various stem/progenitor cell sources are based on how pancreatic beta cells normally develop in vivo. So far, it has been unclear if and how the tissue architecture/microenvironment influences cell specification during beta cell development. Consequently most in vitro differentiation protocols do not take these issues into consideration. The data demonstrate that pancreatic cell-fate specification requires tubes. Tubulogenesis ensures confinement of multipotent Pdx1+ pancreatic progenitors to distinct microenvironments, which is a prerequisite for appropriate cell-fate determination. This emphasizes the significance of understanding the underlying molecular principles for how distinct microenvironments along the 'tip-trunk' axis control pancreatic cell-fate choices in vivo. Filling in these gaps will most likely provide new strategies for developing robust and efficient protocols to generate beta cells from stem/progenitor cells in vitro (Kesavan, 2009).

Cdc42: Biological Overview | Regulation | Developmental Biology | Effects of Mutation and Ectopic Expression | References

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