Table of contents

Rho GTPase-activating proteins (GAPs)

The Saccharomyces cerevisiae morphogenesis checkpoint delays mitosis in response to insults that impair actin organization and/or bud formation. The delay is due to accumulation of the inhibitory kinase Swe1p, which phosphorylates the cyclin-dependent kinase Cdc28p. Having screened through a panel of yeast mutants with defects in cell morphogenesis, it is reported that the polarity establishment protein Bem2p is required for the checkpoint response. Bem2p is a Rho-GTPase activating protein (GAP) previously shown to act on Rho1p; it also acts on Cdc42p, the GTPase primarily responsible for establishment of cell polarity in yeast. Whereas the morphogenesis role of Bem2p requires GAP activity, the checkpoint role of Bem2p does not. Instead, this function requires an N-terminal Bem2p domain. Thus, this single protein has a GAP-dependent role in promoting cell polarity and a GAP-independent role in responding to defects in cell polarity by enacting the checkpoint. Surprisingly, Swe1p accumulation occurs normally in bem2 mutant cells, but these cells are nevertheless unable to promote Cdc28p phosphorylation. Therefore, Bem2p defines a novel pathway in the morphogenesis checkpoint (Marquitz, 2002).

Small G proteins transduce signals from plasma-membrane receptors to control a wide range of cellular functions. These proteins are clustered into distinct families but all act as molecular switches, active in their GTP-bound form but inactive when GDP-bound. The Rho family of G proteins, which includes Cdc42Hs, activate effectors involved in the regulation of cytoskeleton formation, cell proliferation and the JNK signaling pathway. G proteins generally have a low intrinsic GTPase hydrolytic activity but there are family-specific groups of GTPase-activating proteins (GAPs) that enhance the rate of GTP hydrolysis by up to 100,000 times. The crystal structure of Cdc42Hs is reported, with the non-hydrolyzable GTP analog GMPPNP, in complex with the GAP domain of p50rhoGAP at 2.7A resolution. In the complex, Cdc42Hs interacts mainly through its switch I and II regions, with a shallow pocket on rhoGAP, which is lined with conserved residues. Arg 85 of rhoGAP interacts with the P-loop of Cdc42Hs, but from biochemical data and by analogy with the G-protein subunit G(i alpha1), it is proposed that the rhoGAP adopts a different conformation during the catalytic cycle that enables the rhoGAP to stabilize the transition state of the GTP-hydrolysis reaction (Rittinger, 1997).

Saccharomyces cerevisiae strain V918 was previously isolated in a search for thermosensitive autolytic mutants and found to bear a recessive mutation that causes the development of multinucleate swollen cells undergoing cell lysis. The BEM2 gene has been isolated by complementation of the phenotype of a V918 segregant. BEM2 encodes a Rho-GTPase-activating protein (GAP) that is thought to act as a modulator of the Rho1 small GTPase. The mutation causing the morphogenetic and autolytic phenotype in strain V918 and its segregants lies in the BEM2 gene, defining a new mutant allele, bem2-21. Mutants in the BEM2 gene display loss of cell polarity and depolarization of the actin cytoskeleton, causing a bud-emergence defect. Low resistance to sonication and to hydrolytic enzymes prove that the cell wall is less protective in bem2-21 mutants than in wild-type strains. Moreover, bem2-21 mutants are more sensitive than the wild-type to several antifungal drugs. Abnormally thick and wide septa develop and there exist thin areas in the cell wall, which probably account for cell lysis. The depolarization of actin in bem2-21 mutants does not preclude morphogenetic events such as cell elongation in homozygous diploid strains during nitrogen starvation in solid media, hyperpolarization of growth in a background bearing a mutated septin, or sporulation. Multinucleate cells from bem2-21 homozygous diploids undergo sporulation, giving rise to multispored asci ('polyads'), containing up to 36 spores. This phenomenon occurs only under osmotically stabilized conditions, suggesting that the integrity of the ascus wall is impaired in cells expressing the bem2-21 mutation. It is concluded that the function of the BEM2 gene product is essential for the maintenance of a functional cell wall (Cid, 1998).

PTPL1 is an intracellular protein-tyrosine phosphatase that contains five PDZ domains. A novel 150-kDa protein has been cloned, the four most C-terminal amino acid residues of this protein specifically interact with the fourth PDZ domain of PTPL1. The molecule contains a GTPase-activating protein (GAP) domain, a cysteine-rich, putative Zn2+- and diacylglycerol-binding domain, and a region of sequence homology to the product of the Caenorhabditis elegans gene ZK669.1a. The GAP domain is active on Rho, Rac, and Cdc42 in vitro but with a clear preference for Rho; the molecule is referred to as PTPL1-associated RhoGAP 1, or PARG1. Rho is inactivated by GAPs, and protein-tyrosine phosphorylation has been implicated in Rho signaling. Therefore, a complex between PTPL1 and PARG1 may function as a powerful negative regulator of Rho signaling, acting both on Rho itself and on tyrosine phosphorylated components in the Rho signal transduction pathway (Saras, 1997).

The ligation of available alpha6beta1 integrin in adherent LOX melanoma cells by laminin G peptides and integrin stimulatory antibodies, induces cell invasiveness. This occurs independent of the adhesion activity of integrins that are pre-bound to extracellular matrix This induced invasion involves an increase in tyrosine phosphorylation of a 190-kDa GTPase-activating protein for Rho family members (p190(RhoGAP); p190: see Drosophila RhoGAP) and membrane-protrusive activities at invadopodia. This tyrosine phosphorylation does not occur when the adherent cells are treated with non-activating antibody against beta1 integrin, control laminin peptides, or tyrosine kinase inhibitors genistein and herbimycin A. Although p190 and F-actin co-distribute in all cell cortex extensions, tyrosine-phosphorylated proteins, including p190, appear to associate with F-actin specifically in invadopodia. In addition, the localized matrix degradation and membrane-protrusive activities are blocked by treatment of LOX cells with tyrosine kinase inhibitors as well as microinjection of antibodies directed against p190, but not by non-perturbing antibodies or control buffers. It is suggested that activation of the alpha6beta1 integrin signaling regulates the tyrosine phosphorylation state of p190, which in turn connects downstream signaling pathways through Rho family GTPases to actin cytoskeleton in invadopodia, thus promoting membrane-protrusive and degradative activities necessary for cell invasion (Nakahara, 1998).

p120GAP forms distinct complexes with two phosphoproteins, p62 and p190. A cDNA (termed p190-B) has been cloned, encoding a protein with 51% amino acid identity to p190 (p190-A). The N-terminal portion of p190-B contains several motifs characteristic of a GTPase domain, while its C terminus contains a Rho GAP domain. A recombinant Rho GAP domain polypeptide shows GAP activity for RhoA, Rac1, and G25K/CDC42Hs. Immunoprecipitation and immunofluorescence studies demonstrates that p190-B protein is expressed in a variety of cells and localized diffusely in the cytoplasm and in fibrillar patterns that co-localize with the alpha 5 beta 1 integrin receptor for fibronectin. Adhesion of fibronectin-coated latex beads to cells results in recruitment of significant amounts of p190-B and Rho to the plasma membrane beneath the site of bead binding. In contrast, beads coated with polylysine or concanavalin A are unable to recruit either p190-B or Rho. Anti-beta 1 or anti-alpha 5 integrin antibody-coated beads are also able to recruit large amounts of p190-B and Rho. These results identify a novel second member of the p190 family and establish the existence of a novel transmembrane link between integrins and a new protein p190-B and Rho (Burbelo, 1995).

The integrin family of cell surface receptors mediates cell adhesion to components of the extracellular matrix (ECM). Integrin engagement with the ECM initiates signaling cascades that regulate the organization of the actin-cytoskeleton and changes in gene expression. The Rho subfamily of Ras-related low-molecular-weight GTP-binding proteins and several protein tyrosine kinases have been implicated in mediating various aspects of integrin-dependent alterations in cell homeostasis. Focal adhesion kinase (FAK or pp125FAK) is one of the tyrosine kinases predicted to be a critical component of integrin signaling. To elucidate the mechanisms by which FAK participates in integrin-mediated signaling, expression cloning has been used to identify cDNAs that encode potential FAK-binding proteins. A cDNA has been identified that encodes a new member of the GTPase-activating protein (GAP) family of GTPase regulators. This GAP, termed Graf (for GTPase regulator associated with FAK), binds to the C-terminal domain of FAK in an SH3 domain-dependent manner and preferentially stimulates the GTPase activity of the GTP-binding proteins RhoA and Cdc42. Subcellular localization studies using Graf-transfected chicken embryo cells indicate that Graf colocalizes with actin stress fibers, cortical actin structures, and focal adhesions. Graf mRNA is expressed in a variety of avian tissues and is particularly abundant in embryonic brain and liver. Graf represents the first example of a regulator of the Rho family of small GTP-binding proteins that exhibits binding to a protein tyrosine kinase. It is suggested that Graf may function to mediate cross talk between the tyrosine kinases such as FAK and the Rho family GTPases that control steps in integrin-initiated signaling events (Hildebrand, 1996).

The v-Src oncoprotein perturbs the dynamic regulation of the cellular cytoskeletal and adhesion network by a mechanism that is poorly understood. The effects of a temperature-dependent v-Src protein were examined on the regulation of p190 RhoGAP, a GTPase activating protein (GAP) that has been implicated in disruption of the organized actin cytoskeleton. Also addressed was the dependence of v-Src-induced stress fiber loss on inhibition of Rho activity. Activation of v-Src induces association of tyrosine phosphorylated p190 with p120(RasGAP) and stimulation of p120(RasGAP)-associated RhoGAP activity, although p120(RasGAP) itself is not a target for phosphorylation by v-Src in chicken embryo cells. These events require the catalytic activity of v-Src and are linked to loss of actin stress fibers during morphological transformation and not mitogenic signaling. Furthermore, these effects are rapidly reversible since switching off v-Src leads to dissociation of the p190/p120(RasGAP) complex, inactivation of p120(RasGAP)-associated RhoGAP activity and re-induction of actin stress fibers. In addition, transient transfection of Val14-RhoA, a constitutively active Rho protein that is insensitive to RhoGAPs, suppresses v-Src-induced stress fiber loss and cell transformation. Thus, an activated Src kinase requires the inactivation of Rho-mediated actin stress fiber assembly to induce its effects on actin disorganization. This work supports p190 as a strong candidate effector of v-Src-induced cytoskeletal disruption, most likely mediated by antagonism of the cellular function of Rho (Fincham, 1999).

Graf, the GTPase regulator associated with focal adhesion kinase has GAP activity for Rho A and Cdc42 in vitro. An attempt was made to determine whether Graf acts at the level of Cdc42, Rho, or both in vivo and whether Graf is a signal terminator or transducer for these proteins. Microinjection of Graf cDNA into subconfluent Swiss 3T3 cells (in the presence of serum) has marked effects on cell shape and actin localization. Graf expression causes clearing of stress fibers followed by formation of long actin based filopodial-like extensions. Similar phenotypes are observed following injection of the Rho-inhibitor C3 into these cells. The Graf response is dependent on GAP activity, since injection of Graf cDNA containing point mutations in the GAP domain (R236Q or N351V) that block enzymatic activity, does not confer this phenotype. Injection of Graf into Swiss 3T3 cells in which Rho has been down-regulated by serum starvation has no effect on cell morphology. Using this system, Graf is demonstrated to block sphingosine-1-phosphate (SPP) stimulated (Rho-mediated) stress fiber formation. Conversely, Graf expression does not inhibit bradykinin stimulated (Cdc42-mediated) filopodial extensions. These data indicate that Graf is a GAP for Rho in vivo. To further substantiate these results the effect of Graf over-expression on Rho-mediated neurite retraction in nerve growth factor (NGF)-differentiated PC12 cells was examined. In PC12 cells, which express relatively high levels of endogenous Graf, overexpression of Graf (but not Graf containing the R236Q mutation) enhances SPP-induced neurite retraction. These data indicate the possibility that Graf may be an effector for Rho in certain cell types (Taylor, 1999).

Rho GTPases direct actin rearrangements in response to a variety of extracellular signals. P190 RhoGAP (GTPase activating protein) is a potent Rho regulator that mediates integrin-dependent adhesion signaling in cultured cells. p190 RhoGAP is specifically expressed at high levels throughout the developing nervous system. Mice lacking functional p190 RhoGAP exhibit several defects in neural development that are reminiscent of those described in mice lacking certain mediators of neural cell adhesion. The defects reflect aberrant tissue morphogenesis and include abnormalities in forebrain hemisphere fusion, ventricle shape, optic cup formation, neural tube closure, and layering of the cerebral cortex. In cells of the neural tube floor plate of p190 RhoGAP mutant mice, polymerized actin accumulates excessively, suggesting a role for p190 RhoGAP in the regulation of Rho-mediated actin assembly within the neuroepithelium. Significantly, several of the observed tissue fusion defects seen in the mutant mice are also found in mice lacking MARCKS, the major substrate of protein kinase C (PKC), and p190 RhoGAP is found to be a PKC substrate in vivo. Upon either direct activation of PKC or in response to integrin engagement, p190 RhoGAP is rapidly translocated to regions of membrane ruffling, where it colocalizes with polymerized actin. Together, these results suggest that upon activation of neural adhesion molecules, the action of PKC and p190 RhoGAP leads to a modulation of Rho GTPase activity to direct several actin-dependent morphogenetic processes required for normal neural development (Brouns, 2000).

The Src tyrosine kinases have been implicated in several aspects of neural development and nervous system function; however, their relevant substrates in brain and their mechanism of action in neurons remain to be established clearly. The potent Rho regulatory protein p190 RhoGAP (GTPase-activating protein) has been identified as the principal Src substrate detected in the developing and mature nervous system. Mice lacking functional p190 RhoGAP exhibit defects in axon guidance and fasciculation. p190 RhoGAP is co-enriched with F-actin in the distal tips of axons, and overexpressing p190 RhoGAP in neuroblastoma cells promotes extensive neurite outgrowth, indicating that p190 RhoGAP may be an important regulator of Rho-mediated actin reorganization in neuronal growth cones. p190 RhoGAP transduces signals downstream of cell-surface adhesion molecules, and p190-RhoGAP-mediated neurite outgrowth is promoted by the extracellular matrix protein laminin. Together with the fact that mice lacking neural adhesion molecules or Src kinases also exhibit defects in axon outgrowth, guidance and fasciculation, these results suggest that p190 RhoGAP mediates a Src-dependent adhesion signal for neuritogenesis to the actin cytoskeleton through the Rho GTPase (Brouns, 2001).

It is proposed that the engagement of neural adhesion molecules results in Src activation and subsequent p190 RhoGAP phosphorylation in the developing brain. Such a signaling pathway most probably influences the various Rho-mediated actin reorganization events required for neural development, including the axonal guidance and fasciculation processes that are disrupted in the p190 RhoGAP mutant mice. Moreover, the defects in the morphogenesis of neuroepithelial tissues observed in p190 RhoGAP mutant mice may reflect disruption of the same pathways (Brouns, 2001).

Previous studies have shown that integrin/cell adhesion molecule-dependent signal transduction pathways are mediated by the Src kinases. In cultured neurons, activation of L1 or NCAM results in a rapid induction of the tyrosine phosphorylation of several proteins, and neurons lacking the Src or Fyn kinase are defective for neurite outgrowth on L1 or NCAM substrates, respectively. Src and Fyn, as well as L1, control axon fasciculation in the central nervous system. Experiments using fibroblasts lacking Src family members have shown that integrin engagement leads to inactivation of Rho through a mechanism requiring Src kinases, and that Rho inhibition correlates with Src-dependent tyrosine phosphorylation of p190 RhoGAP. These findings indicate that p190 RhoGAP mediates Src-dependent adhesion signaling to the actin cytoskeleton, and that cytoskeletal modulation almost certainly involves the Rho GTPase (Brouns, 2001 and references therein).

All of these data are consistent with a model for a Src/p190 RhoGAP adhesion pathway, which seeks to link the observed defects in the developing brain, the in vivo analysis of protein tyrosine phosphorylation, and the results of neuroblastoma cell-culture studies. Significantly, brain defects seen in Src/Fyn mutant mice resemble those seen in p190 RhoGAP mutant mice. Of particular interest are the axon guidance and fasciculation defects described in the olfactory system of Src/Fyn double-mutant mice at E11.5: the olfactory nerve is significantly defasciculated and axons 'wander' from the normal trajectory (Brouns, 2001 and references therein).

These fasciculation defects are strongly reminiscent of the defasciculation of cranial nerves detected at E10.5 in the p190 RhoGAP-/- mice described in this study, and the wandering behavior of olfactory axons reflects a similar disruption of axon guidance as that detected in the E16.5 cerebral cortex of p190 RhoGAP-/- mice. Notably, the Src/Fyn double-mutant mice, like the p190 RhoGAP-/- mice, die perinatally with axon guidance and fasciculation defects. However, additional functional redundancy provided by the tyrosine kinase Yes (which also appears to phosphorylate p190 RhoGAP in brain) and the p190-RhoGAP-related p190-B protein, together with the fact that there are likely to be additional Src family substrates in brain, make it difficult to relate the phenotypes among these various knockout animals in an unequivocal manner. Thus, definitive confirmation of the proposed relationships between Src kinase activity, p190 RhoGAP function and adhesion-mediated morphogenetic events await further genetic analysis (Brouns, 2001).

In addition to adhesion molecules, many different secreted growth factors, including epidermal growth factor, can promote tyrosine phosphorylation of p190 RhoGAP, suggesting that signals transduced by the numerous brain-expressed receptor tyrosine kinases may be mediated by p190 RhoGAP as well. Such receptors have also been implicated in a variety of morphogenetic events during neural development. In addition, mice lacking the Eph family receptor, EphB2 (Nuk), exhibit an anterior commissure guidance defect very similar to that seen in p190 RhoGAP mutant mice, suggesting that Eph ligands might signal through p190 RhoGAP. Significantly, EphB2 has been observed to associate with the binding partner of p190 RhoGAP -- p120 RasGAP. The fact that p190 RhoGAP is expressed widely throughout the nervous system at all stages of development, and is highly tyrosine phosphorylated in all regions of the brain, suggests that it is probably a common mediator of several diverse extracellular signals that affect many morphogenetic processes by precisely modulating Rho GTPase activity (Brouns, 2001).

Cell division is finely controlled by various molecules including small G proteins and kinases/phosphatases. Among these, Aurora B, RhoA, and the GAP MgcRacGAP have been implicated in cytokinesis, but their underlying mechanisms of action have remained unclear. MgcRacGAP is shown to colocalize with Aurora B and RhoA, but not Rac1/Cdc42, at the midbody. Aurora B phosphorylates MgcRacGAP on serine residues and this modification induces latent GAP activity toward RhoA in vitro. Expression of a kinase-defective mutant of Aurora B disrupts cytokinesis and inhibits phosphorylation of MgcRacGAP at Ser387, but not its localization to the midbody. Overexpression of a phosphorylation-deficient MgcRacGAP-S387A mutant, but not phosphorylation-mimic MgcRacGAP-S387D mutant, arrests cytokinesis at a late stage and induces polyploidy. Together, these findings indicate that during cytokinesis, MgcRacGAP, a GAP for Rac/Cdc42, is functionally converted to a RhoGAP through phosphorylation by Aurora B (Minoshima, 2003).

Guanine nucleotide dissociation inhibitors (GDIs), proteins that associate with GTPases to maintain the existing nucleotide-bound state

Members of the Rho subfamily of GTP-binding proteins contain a region of amino acid sequence (residues 122-134) that is absent from other Ras-like proteins and is termed the Rho insert region. To address the functional role of this domain, a Cdc42Hs/Ras chimera has been constructed in which loop 8 from Ha-Ras has been substituted for the region in Cdc42Hs that contains the 13-amino acid insert region. The insert region of Cdc42Hs is not essential for its interactions with various target/effector molecules or for interactions with the guanine nucleotide exchange factor, Dbl, or the Cdc42 GTPase-activating protein (GAP). However, the regulation of GDP dissociation and GTP hydrolysis on Cdc42Hs by the Rho GDP-dissociation inhibitor (GDI) is extremely sensitive to changes in the insert region, such that a Cdc42Hs/Ha-Ras chimera that lacks this insert is no longer susceptible to a GDI-induced inhibition of GDP dissociation and GTP hydrolysis. The insensitivity to GDI activity is not due to the inability of the GDI molecule to bind to the Cdc42Hs/Ha-Ras chimera; in fact, the GDI is fully capable of stimulating the release of this chimera from membranes (Wu, 1997).

Rho GTPases are synthesized as cytosolic proteins but have the capacity to associate with membranes by virtue of a series of posttranslational modifications of a COOH-terminal CAAX (prenylation, AAX tripeptide proteolysis, and carboxyl methylation) motif. Unlike Ras proteins, prenylated Rho proteins can be sequestered in the cytosol by their interaction with RhoGDI (guanine nucleotide dissociation inhibitor), a class of proteins shown to inhibit the release of GDP from Rho. Rho GTPases can cycle on and off membranes conferred by interaction with RhoGDI; this interaction is integral to Rho biological activity. Consistent with this extra variable, the subcellular localization of Rho family proteins has proven to be more complex than that of Ras proteins, perhaps reflecting the more varied functions of Rho proteins. Rho proteins have been localized in cytosol, plasma membranes (PMs), including cholesterol-rich microdomains, subplasmalemmal actin mesh, Golgi, endosomes, multivesicular bodies, and nuclei. Moreover, upon activation, Rho proteins have been shown to translocate from cytosol to membranes (Michaelson, 2001 and references therein).

Rho and Arf family small GTPases are well-known regulators of cellular actin dynamics. ARAP3, a member of the ARAP family of dual GTPase activating proteins (GAPs) for Arf and Rho family GTPases has been identified in a screen for PtdIns(3,4,5)P3 binding proteins. PtdIns(3,4,5)P3 is the lipid product of class I phosphoinositide 3OH-kinases (PI3Ks) and is a signaling molecule used by growth factor receptors and integrins in the regulation of cell dynamics. A Rho GAP, ARAP3 prefers RhoA as a substrate, and it can be activated in vitro by the direct binding of Rap proteins to a neighboring Ras binding domain (RBD). This activation by Rap is GTP dependent and specific for Rap versus other Ras family members. No evidence was found for direct regulation of ARAP3's Rho GAP activity by PtdIns(3,4,5)P3 in vitro, but PI3K activity is required for activation by Rap in a cellular context, suggesting that PtdIns(3,4,5)P3-dependent translocation of ARAP3 to the plasma membrane may be required for further activation by Rap. These results indicate that ARAP3 is a Rap-effector that plays an important role in mediating PI3K-dependent crosstalk between Ras, Rho, and Arf family small GTPases (Krugmann, 2004).

Subcellular localization of Rho

Determinants of membrane targeting of Rho proteins were investigated in live cells with green fluorescent fusion proteins expressed with or without Rho-guanine nucleotide dissociation inhibitor (GDI)alpha. The hypervariable region determines to which membrane compartment each protein is targeted. Targeting is regulated by binding to RhoGDIalpha in the case of RhoA, Rac1, Rac2, and Cdc42hs but not RhoB or TC10. Although RhoB localizes to the plasma membrane, Golgi, and motile peri-Golgi vesicles, TC10 localizes to PMs and endosomes. Inhibition of palmitoylation mislocalizes H-Ras, RhoB, and TC10 to the endoplasmic reticulum. Although overexpressed Cdc42hs and Rac2 are observed predominantly on endomembrane, Rac1 is predominantly at the PM. RhoA is cytosolic even when expressed at levels in vast excess of RhoGDIalpha. Oncogenic Dbl stimulates translocation of green fluorescent protein (GFP)-Rac1, GFP-Cdc42hs, and GFP-RhoA to lamellipodia. RhoGDI binding to GFP-Cdc42hs is not affected by substituting farnesylation for geranylgeranylation. A palmitoylation site inserted into RhoA blocks RhoGDIalpha binding. Mutations that render RhoA, Cdc42hs, or Rac1, either constitutively active or dominant negative abrogate binding to RhoGDIalpha and redirect expression to both PMs and internal membranes. Thus, despite the common essential feature of the CAAX (prenylation, AAX tripeptide proteolysis, and carboxyl methylation) motif, the subcellular localizations of Rho GTPases, like their functions, are diverse and dynamic (Michaelson, 2001).

The small GTP-binding protein, Rho1/RhoA plays a central role in cytokinetic actomyosin ring (CAR) assembly and cytokinesis. Concentration of Rho proteins at the division site is a general feature of cytokinesis, yet the mechanisms for recruiting Rho to the division site for cytokinesis remain poorly understood. Budding yeast utilizes two mechanisms to concentrate Rho1 at the division site. During anaphase, the primary mechanism for recruiting Rho1 is binding to its guanine nucleotide exchange factors (GEFs). GEF-dependent recruitment requires that Rho1 has the ability to pass through its GDP or unliganded state prior to being GTP-loaded. This model was tested by generating viable yeast lacking all identifiable Rho1 GEFs. Later, during septation and abscission, a second GEF-independent mechanism contributes to Rho1 bud neck targeting. This GEF-independent mechanism requires the Rho1 polybasic sequence that binds to acidic phospholipids, including phosphatidylinositol 4,5-bisphosphate (PIP2). This latter mechanism is functionally important because Rho1 activation or increased cellular levels of PIP2 promote cytokinesis in the absence of a contractile ring. These findings comprehensively define the targeting mechanisms of Rho1 essential for cytokinesis in yeast, and are likely to be relevant to cytokinesis in other organisms (Yoshida, 2009).

PIP2 concentrates at the division site in many organisms. The molecular function of PIP2 in cytokinesis is poorly understood, but is likely to be complex. PIP2 is not required for CAR assembly, but is essential for completion of cytokinesis after furrow ingression. PIP2 regulates both endocytosis and exocytosis and both processes are required for executing the late stages of cytokinesis. Interestingly, a recent study showed that a lipid-binding protein Anillin has a role in maintaining RhoA at the division site during late stage cytokinesis in human cells. Based on the current results, it is proposed that one way that PIP2 promotes cytokinesis is to facilitate the local accumulation of Rho GTPases and their GEFs (Yoshida, 2009).

These findings also have implications for the regional organization of the plasma membrane. Small GTPases that localize to the plasma membrane either contain multiple sites of lipid modification or have a single site of lipid modification and a polybasic sequence. Functional differences between these classes of GTPases are only just being defined. However, the K-Ras polybasic sequence appears to be important for its segregation into plasma membrane nanoclusters that are distinct from clusters containing H-Ras. This study has demonstrated that the C-terminal PBS domain of Rho1 is necessary and sufficient for targeting to a specific plasma membrane region: the membrane microdomain formed by split septin rings (Yoshida, 2009).

Signaling upstream of Rho

Lysophosphatidic acid (LPA) utilizes a G-protein-coupled receptor to activate the small GTP-binding protein Rho and to induce rapid remodeling of the actin cytoskeleton. The signal transduction from LPA receptors to Rho activation has been studied. Analysis of the G-protein-coupling pattern of LPA receptors (by labeling activated G-proteins with [alpha-32P]GTP azidoanilide) reveals interaction with proteins of the Gq, Gi, and G12 subfamilies. In COS-7 cells, expression of GTPase-deficient mutants of Galpha12 and Galpha13 trigger Rho activation as measured by increased Rho-GTP levels. In Swiss 3T3 cells, incubation with LPA or microinjection of constitutively active mutants of Galpha12 and Galpha13 induce formation of actin stress fibers and assembly of focal adhesions in a Rho-dependent manner. Interestingly, the LPA-dependent cytoskeletal reorganization is suppressed by microinjected antibodies directed against Galpha13, whereas Galpha12-specific antibodies show no inhibition. The tyrosine kinase inhibitor tyrphostin A 25 and the epidermal growth factor (EGF) receptor-specific tyrphostin AG 1478 completely block actin stress fiber formation caused by LPA or activated Galpha13, but not the effects of activated Galpha12. Expression of the dominant negative EGF receptor mutant EGFR-CD533 also markedly prevents the LPA- and Galpha13-induced actin polymerization. Coexpression of EGFR-CD533 and activated Galpha13 in COS-7 cells result in decreased Rho-GTP levels, when compared with expression of activated Galpha13 alone. These data indicate that in Swiss 3T3 cells, G13 (but not G12) is involved in the LPA-induced activation of Rho. These results suggest an involvement of the EGF receptor in this pathway (Gohla, 1998).

Netrin-1 (see Drosophila Netrins) is known to function as a chemoattractant for several classes of developing axons and as a chemorepellent for other classes of axons, apparently dependent on the receptor type expressed by responsive cells. In culture, growth cones of embryonic Xenopus spinal neurons exhibit chemoattractive turning toward the source of netrin-1 but show chemorepulsive responses in the presence of a competitive analog of cAMP or an inhibitor of protein kinase A (see Drosophila PKA). Both attractive and repulsive responses are abolished by depleting extracellular calcium and by adding a blocking antibody against the netrin-1 receptor Deleted in Colorectal Cancer. Thus, nerve growth cones may respond to the same guidance cue with opposite turning behavior, dependent on other coincident signals that set the level of cytosolic cAMP (Ming, 1997).

Previous studies have shown that BDNF-induced turning of growth cones also exhibits either attraction or repulsion, depending on differences in cyclic-AMP-dependent activity in neurons. The currient studies suggest that Ca2+ signaling (acting downstream from the BDNF receptor known as TrkB) lies upstream from the cAMP-dependent step in the cascade of events, since attractive turning induced by a forskolin gradient is not affected by removal of extracellular CA2+ (Song, 1997). A netrin-induced Ca2+ influx may trigger a rise in cAMP through activation of Ca2+-dependent adenylate cyclases, thus creating a cAMP gradient within the growth cone, a condition known to result in an attractive response of Xenopus growth cones. It is possible that a gradient of cytosolic Ca2+ induced by netrin-1 is responsible for triggering the repulsive response of the growth cone, but the effect is normally overridden by the attractive response due to a cAMP gradient generated by the Ca2+ gradient. Inhibition of cAMP-dependent processes using competitive cAMP analogs may thus unmask the repulsive action of the cytosolic Ca2+ gradient. In principle, cAMP and the cAMP-dependent protein kinase pathway could regulate either the receptors for different diffusible guidance cues or the activity of the downstream effector molecules activated by these receptors. The cAMP-dependent protein kinase may thereby act as a gating mechanism, being differentially permissive for a receptor-induced signaling cascade, depending on the cascade's functional status. One group of potential downstream targets of PKA is small GTP-binding proteins of the rho family, e.g., rhoA, rac1 and cdc42, which are known to mediate morphological changes by regulating the actin cytoskeleton and to play a role in growth cone turning. It is known that PKA can phosphorylate rhoA, leading to the translocation of membrane-associated rhoA to the cytoplasm, thus providing an additional mechanism for its inactivation. The observation that lowering PKA activity converts netrin-1-induced attraction into repulsion suggests the intriguing possibility that activation of an UNC-5-like protein may down-regulate PKA. UNC-5 appears to be either a receptor or a component of a receptor complex, involved in netrin-mediated repulsion. For example, UNC-5 may inhibit adenylate cyclase activity or stimulate phosphodiesterase, which lowers the cAMP level and consequently PKA activity (Ming, 1997).

A transient transfection system was used to identify regulators and effectors for Tec and Bmx, members of the Tec non-receptor tyrosine kinase family. Tec and Bmx are found to activate serum response factor (SRF), in synergy with constitutively active alpha subunits of the G12 family of GTP-binding proteins, in transiently transfected NIH 3T3 cells. The SRF activation is sensitive to C3, suggesting the involvement of Rho. The kinase and Tec homology (TH) domains of the kinases are required for SRF activation. In addition, kinase-deficient mutants of Bmx are able to inhibit Galpha13- and Galpha12-induced SRF activation, and to suppress thrombin-induced SRF activation in cells lacking Galphaq/11, where thrombin's effect is mediated by G12/13 proteins. Expression of Galpha12 and Galpha13 stimulates the autophosphorylation and transphosphorylation activities of Tec. Thus, the evidence indicates that Tec kinases are involved in Galpha12/13-induced, Rho-mediated activation of SRF. Furthermore, Src, which has been previously shown to activate kinase activities of Tec kinases, activates SRF predominantly in Rho-independent pathways in 3T3 cells, as shown by the fact that C3 does not block Src-mediated SRF activation. However, the Rho-dependent pathway becomes significant when Tec is overexpressed. Thus, Tec/Bmx non-receptor tyrosine kinases are involved in regulation of Rho and serum response factor by Galpha12/13 (Mao, 1998a).

Proteins of the Ras superfamily, Ras, Rac, Rho, and Cdc42, control the remodelling of the cortical actin cytoskeleton following growth factor stimulation. A major regulator of Ras, Ras-GAP, contains several structural motifs, including an SH3 domain and two SH2 domains, and there is evidence that they harbor a signaling function. A monoclonal antibody to the SH3 domain of Ras-GAP blocks Ras signaling in Xenopus oocytes. Microinjection of this antibody into Swiss 3T3 cells prevents the formation of actin stress fibers stimulated by growth factors or activated Ras, but not membrane ruffling. This inhibition is bypassed by coinjection of activated Rho, suggesting that the Ras-GAP SH3 domain is necessary for endogenous Rho activation. In agreement, the antibody blocks lysophosphatidic acid-induced neurite retraction in differentiated PC12 cells. Microinjection of full-length Ras-GAP triggers stress fiber polymerization in fibroblasts in an SH3-dependent manner, strongly suggesting an effector function, in addition to its role as a Ras downregulator. These results support the idea that Ras-GAP connects the Ras and Rho pathways and, therefore, regulates the actin cytoskeleton through a mechanism that probably does not involve p190 Rho-GAP (Leblanc, 1998).

Integrin-mediated adhesion is a critical regulator of cell migration. Integrin-mediated adhesion to high fibronectin concentrations induces a stop signal for cell migration by inhibiting cell polarization and protrusion. On fibronectin, the stop signal is generated through alpha5beta1 integrin-mediated signaling to the Rho family of GTPases. Specifically, Cdc42 and Rac1 activation exhibit a biphasic dependence on fibronectin concentration that parallels optimum cell polarization and protrusion. In contrast, RhoA activity increases with increasing substratum concentration. Cross talk between Cdc42 and Rac1 is required for substratum-stimulated protrusion, whereas RhoA activity is inhibitory. Cdc42 activity is inhibited by Rac1 activation, suggesting that Rac1 activity may down-regulate Cdc42 activity and promote the formation of stabilized rather than transient protrusion. Furthermore, expression of RhoA down-regulates Cdc42 and Rac1 activity, providing a mechanism whereby RhoA may inhibit cell polarization and protrusion. These findings implicate adhesion-dependent signaling as a mechanism to stop cell migration by regulating cell polarity and protrusion via the Rho family of GTPases (Cox, 2001).

FRT thyroid epithelial cells synthesize fibronectin and organize a network of fibronectin fibrils at the basal surface of the cells. Fibronectin fibril formation is enhanced by the overexpression of the ubiquitous beta1A integrin and is inhibited by the expression of the dominant-negative beta1B subunit. The hypothesis was tested that RhoA activity might mediate the integrin-dependent fibronectin fibrillogenesis and might counteract beta1B integrin inhibitory effect. FRT-beta1A cells were transfected with a vector carrying a dominant negative form of RhoA (RhoAN19) or treated with the C3 transferase exoenzyme. Both treatments inhibit fibronectin assembly and causes loss of actin microfilaments and adhesion plaques. FRT-beta1B cells were also transfected with the constitutively activated form of RhoA (RhoAV14) or treated with the E. coli cytotoxic necrotizing factor 1, which directly activates RhoA. Either treatment restores microfilament and adhesion plaque assembly and promotes fibronectin fibril organization. A great increase in fibronectin fibril assembly was also obtained by treatment of FRT-beta1B cells with TGF-beta. These data indicate that RhoA is required to promote fibronectin matrix assembly in FRT cells and that the activation of the signal transduction pathway downstream of RhoA can overcome the inhibitory effect of beta1B integrin (Cali, 1999).

The ephrins, ligands of Eph receptor tyrosine kinases, have been shown to act as repulsive guidance molecules and to induce collapse of neuronal growth cones. Ephrin-A5 collapse is mediated by activation of the small GTPase Rho and its downstream effector Rho kinase. In ephrin-A5-treated retinal ganglion cell cultures, Rho is activated and Rac is downregulated. Pretreatment of ganglion cell axons with C3-transferase, a specific inhibitor of the Rho GTPase, or with Y-27632, a specific inhibitor of the Rho kinase, strongly reduces the collapse rate of retinal growth cones. These results suggest that activation of Rho and its downstream effector Rho kinase are important elements of the ephrin-A5 signal transduction pathway. Currently not much is known as to how ligand-induced activation of EphA receptor tyrosine kinases regulates the Rho and the Cdc42/Rac pathways. EphA receptors might interact via autophosphorylated juxtamembrane tyrosine residues, with RasGAP (Ras GTPase-activating protein), which is constitutively associated with RhoGAP. RhoGAP is a negative regulator of Rho, and the strong activation of Rho by fc-ephrin-A5 in these experiments would require inactivation of RhoGAP activity. It remains to be shown if additional elements (p62 dok) of the RasGAP-RhoGAP complex are responsible for such an inhibition (Wahl, 2000).

Remodeling of filamentous actin into distinct arrangements is precisely controlled by members of the Rho family of small GTPases. A well characterized member of this family is RhoA, whose activation results in reorganization of the cytoskeleton into thick actin stress fibers terminating in integrin-rich focal adhesions. Regulation of RhoA is required to maintain adhesion in stationary cells, but is also critical for cell spreading and migration. Despite its biological importance, the signaling events leading to RhoA activation are not fully understood. Several independent studies have implicated tyrosine phosphorylation as a critical event upstream of RhoA. Consistent with this, studies have demonstrated the existence of a protein tyrosine phosphatase (PTPase), sensitive to the dipeptide aldehyde calpeptin, acting upstream of RhoA. The SH2 (Src homology region 2)-containing PTPase Shp-2 has been identified as a calpeptin-sensitive PTPase; calpeptin interferes with the catalytic activity of Shp-2 in vitro and with Shp-2 signaling in vivo. Perturbation of Shp-2 activity by a variety of genetic manipulations results in raised levels of active RhoA. Together, these studies identify Shp-2 as a PTPase acting upstream of RhoA to regulate its activity and contribute to the coordinated control of cell movement (Schoenwaelder, 2000).

Ligation of several growth factor receptors results in a loss of stress fibers and focal adhesions, indicative of decreased RhoA activity. One mechanism contributing to this effect is suggested by findings here that Shp-2 negatively regulates RhoA activity. A model is invisioned wherein recruitment of Shp-2 by growth factor receptors brings this PTPase into close proximity with a critical RhoA GEF. Dephosphorylation of the GEF by Shp-2 downregulates its activity, resulting in decreased RhoA-GTP levels, in turn leading to a loss of stress fibers and focal adhesions, and effecting the motile state of the cell (Schoenwaelder, 2000).

Wnt signaling via the Frizzled (Fz) receptor controls cell polarity and movement during development, but the molecular nature of Wnt/Fz polarity signal transduction remains poorly defined. In human cells and during Xenopus embryogenesis, Wnt/Fz signaling activates the small GTPase Rho, a key regulator of cytoskeleton architecture. Wnt/Fz activation of Rho requires the cytoplasmic protein Dishevelled (Dvl) and a novel Formin homology protein Daam1 (see Drosophila DAAM). Daam1 binds to both Dvl and Rho, and mediates Wnt-induced Dvl-Rho complex formation. Inhibition or depletion of Daam1 prevents Wnt/Fz activation of Rho and of Xenopus gastrulation, but not of ß-catenin signaling. This study illustrates a molecular pathway from Wnt/Fz signaling to Rho activation in cell polarity signal transduction (Habas, 2001).

Because Dvl2 PDZ domain is required for Fz/Dvl signaling to Rho proteins associated with the PDZ domain, interacting proteins were sought using the yeast two-hybrid technique. The widely expressed human Daam1 protein contains 1078 amino acids, and belongs to the family of Formin homology (FH) proteins that have been implicated in cell polarity from yeast to human. Formin is the product of the limb deformity locus and is required for limb morphogenesis in mice. Daam1 shares 22% to 30% identity with, and thus is distantly related to, several known mammalian FH proteins. Like other FH proteins, Daam1 contains a central proline-rich FH1 domain and a more carboxyl FH2 domain, and represents a novel subfamily that includes a closely related Daam2, Xenopus and zebrafish Daam, and a Drosophila ortholog, dDaam. The Daam subfamily exhibits extensive similarity both within and outside the FH1 and FH2 domains, including the amino and carboxyl terminal regions. Since several FH proteins bind to Rho, Rac, or Cdc42, Daam1 may also bind Rho GTPases (Habas, 2001).

The Daam1 amino terminus binds to Rho-GDP or Rho-GTP, suggesting a role for Daam1 as a scaffolding protein to recruit Rho-GDP (via the amino terminus) and a Rho-GEF (via the C-Daam1 portion), thereby enhancing Rho-GTP formation. The Daam1 amino terminus binds Rho-GTP with apparently higher affinity, raising an intriguing possibility of positive feedback control, a theme common in cell polarization. Polarity establishment relies on signal amplifications that interpret a small difference in a polarity signal field into a polarized cellular response. DFz1 (Frizzled) exhibits a polarized localization that depends on Dsh function, suggesting a positive feedback loop. Rho-GTP binding to the Daam1 amino terminus may stabilize Daam1 in its activated state, or recruit/activate additional Daam1, thereby promoting an amplification of Rho activation. Such a feedback loop would resemble one in pheromone-induced polarity in yeast. The mating pheromone, via its serpentine receptor and the trimeric G protein, recruits and activates a GEF specific for Cdc42. Activated Cdc42, in turn, is required for the GEF localization, thereby leading to further and polarized Cdc42 activation. The possibility that Daam1 may function primarily in such a feedback control cannot be ruled out. In this scenario, Wnt/Fz signaling initiates Rho activation without Daam1, and the activated Rho together with Dvl recruits/activates Daam1 to amplify Rho activation. In any event, Daam1 function is essential for Rho activation triggered by Wnt/Fz signaling (Habas, 2001).

Daam1 is distantly related to several distinct mammalian FH proteins, such as FRL (30% identity), FHOS (27%), mDia1 (28%), and mDia2 (22%), whose functions in GTPase signaling remain to be fully understood. FRL and FHOS bind specifically to Rac in a nucleotide-independent manner, and an activated FHOS is antagonized by Rac and Rac mutants, leading to the suggestion that FRL and FHOS are scaffolding proteins linking Rac to other proteins. Members of the mDia subfamily of FH proteins (see Drosophila Diaphanous) bind to Rho-GTP (and Rho-GDP in some cases), and are proposed to be Rho targets. However, since actin fiber induction by the activated mDia can be blocked by inhibition of Rho in some instances, and the activated mDia can cause RhoA activation, the relationship between mDia and Rho, and between FH proteins and Rho GTPases in general, may be complex and needs further investigation (Habas, 2001).

Vertebrate gastrulation involves polarization and intercalation of dorsal mesodermal cells along the mediolateral axis (convergence), resulting in the elongation of the anterioposterior axis (extension). This morphogenetic process is governed by Wnt-11 PCP signaling. In Xenopus gastrula, endogenous Rho activation is detected mainly in dorsal tissue, and is abolished when Wnt-11/Fz/Xdsh signaling or Daam1 function is inhibited. Conversely, ectopic Wnt-11/Fz/Xdsh signaling or C-Daam1 activates RhoA on the ventral side. Thus, Wnt-11/Fz signaling, via Xdsh and Daam1, is necessary and sufficient for RhoA activation during gastrulation, consistent with the previous finding that interference of Rho function inhibits gastrulation. In an explant assay, inhibition or depletion of Daam1 perturbs morphogenetic movements, whereas C-Daam1 restores the movements even when Wnt-11/Fz or Xdsh is inhibited. Daam1 thus functions downstream of Wnt-11/Fz/Xdsh in governing gastrulation. Finally, inhibition or depletion of Daam1 in the embryo blocks gastrulation and phenocopies the morphogenetic defects caused by inhibition of Wnt-11, Fz, or Xdsh signaling (Habas, 2001).

A molecular pathway for the Wnt/Fz activation of Rho is suggested, which is referred to as the Wnt/Rho pathway to distinguish it molecularly from Wnt/ß-catenin and Wnt/Ca2+ pathways. A Wnt signal activates a Fz receptor, which translocates Dsh to the plasma membrane and promotes Dsh-Daam1-RhoA complex formation and RhoA activation, likely via the recruitment of a Rho-GEF by the Daam1 scaffolding protein. Activated RhoA generates polarized cytoskeleton remodeling via the ROCK kinase, and perhaps also induces changes in gene expression. The zebrafish knypek gene product, a glypican, facilitates Wnt signal reception, whereas LRP5/6, which is the Fz coreceptor for Wnt/ß-catenin signaling, participates in neither PCP signaling nor RhoA activation. Whether and how other PCP gene products function in the Wnt/Rho pathway or in parallel pathways remains to be elucidated (Habas, 2001).

Binding partners for the Cdc42 effector CIP4 were identified by the yeast two-hybrid system, as well as by testing potential CIP4-binding proteins in coimmunoprecipitation experiments. One of the CIP4-binding proteins, DAAM1, was characterised in more detail. DAAM1 is a ubiquitously expressed member of the mammalian diaphanous-related formins, which include proteins such as mDia1 and mDia2. DAAM1 binds to the SH3 domain of CIP4 in vivo. Ectopically expressed DAAM1 localizes in dotted pattern at the dorsal side of transfected cells and the protein accumulates in the proximity to the microtubule organizing center. Moreover, ectopic expression of DAAM1 induces a marked alteration of the cell morphology, seen as rounding up of the cells, the formation of branched protrusions as well as a reduction of stress-fibres in the transfected cells. Coimmunoprecipitation experiments demonstrated that DAAM1 bind to RhoA and Cdc42 in a GTP-dependent manner. Moreover, DAAM1 interacts and collaborate with the non-receptor tyrosine kinase Src in the formation of branched protrusions. Taken together, these data indicate that DAAM1 communicates with Rho GTPases, CIP4 and Src in the regulation of the signalling pathways that co-ordinate the dynamics of the actin filament system (Aspenstrom, 2006).

Eph tyrosine kinase receptors and their membrane-bound ligands, ephrins, are presumed to regulate cell-cell interactions. The major consequence of bidirectional activation of Eph receptors and ephrin ligands is cell repulsion. In this study, Xenopus Dishevelled (Xdsh) is found to form a complex with Eph receptors and ephrin-B ligands and mediate the cell repulsion induced by Eph and ephrin. In vitro re-aggregation assays with Xenopus animal cap explants revealed that co-expression of a dominant-negative mutant of Xdsh affects the sorting of cells expressing EphB2 and those expressing ephrin-B1. Co-expression of Xdsh induces the activation of RhoA and Rho kinase in the EphB2-overexpressing cells and in the cells expressing EphB2-stimulated ephrin-B1. Therefore, Xdsh mediates both forward and reverse signaling of EphB2 and ephrin-B1, leading to the activation of RhoA and its effector protein Rho kinase. The inhibition of RhoA activity in animal caps significantly prevents the EphB2- and ephrin-B1-mediated cell sorting. It is proposed that Xdsh, which is expressed in various tissues, is involved in EphB and ephrin-B signaling related to regulation of cell repulsion via modification of RhoA activity (Tanaka, 2003).

Plexins are functional receptors for Semaphorin axon guidance cues. Previous studies have established that some Plexins directly bind RACGTP and RHO. Recent work in C. elegans has shown that semaphorin 1 (smp-1 and smp-2) and plexin 1 (plx-1) are required to prevent anterior displacement of the ray 1 cells in the male tail. plx-1 is shown genetically to be part of the same functional pathway as smp-1 and smp-2 for male ray positioning. RAC GTPase genes mig-2 and ced-10 probably function redundantly, whereas unc-73, which encodes a GEF for both of these GTPases, is required cell autonomously for preventing anterior displacement of ray 1 cells. RNAi analysis indicates that rho-1-encoded RHO GTPase, plus let-502 and K08B12.5-encoded RHO-kinases, are also required to prevent anterior displacement of ray 1 cells, suggesting that different kinds of RHO-family GTPases act similarly in ray 1 positioning. At low doses of wild-type mig-2 and ced-10, the Semaphorin 1 proteins no longer act through PLX-1 to prevent anterior displacements of ray 1, but have the opposite effect, acting through PLX-1 to mediate anterior displacements of ray 1. These results suggest that Plexin 1 senses levels of distinct RHO and RAC GTPases. At normal levels of RHO and RAC, Semaphorin 1 proteins and PLX-1 prevent a forward displacement of ray 1 cells, whereas at low levels of cycling RAC, Semaphorin 1 proteins and PLX-1 actively mediate their anterior displacement. Endogenously and ectopically expressed SMP-1 and SMP-2 suggest that the hook, a major source of Semaphorin 1 proteins in the male tail, normally attracts PLX-1-expressing ray 1 cells (Dalpé, 2004).

Several components of noncanonical Wnt signaling pathways are involved in the control of convergence and extension (CE) movements during zebrafish and Xenopus gastrulation. However, the complexity of these pathways and the wide patterns of expression and activity displayed by some of their components immediately suggest additional morphogenetic roles beyond the control of CE. The key modular intracellular mediator Dishevelled, through a specific activation of RhoA GTPase, controls the process of convergence of endoderm and organ precursors toward the embryonic midline in the zebrafish embryo. Three Wnt noncanonical ligands wnt4a, silberblick/wnt11, and wnt11-related regulate this process by acting in a largely redundant way. The same ligands are also required, nonredundantly, to control specific aspects of CE that involve interaction of Dishevelled with mediators different from that of RhoA GTPase. Overall, these results uncover a late, previously unexpected role of noncanonical Wnt signaling in the control of midline assembly of organ precursors during vertebrate embryo development (Matsui, 2005).

HEF1, p130Cas (see CAS/CSE1 segregation protein), and Efs/Sin define the Cas family of proteins. In interphase cells, Cas proteins predominantly localize to focal adhesions. During initial integrin engagement, induced by cell attachment to the extracellular matrix, Cas proteins are phosphorylated by focal adhesion kinase (FAK) and subsequently targeted by Src family kinases. The result of Cas-FAK-Src interactions is extensive tyrosine phosphorylation of Cas proteins, nucleating formation of complexes with adaptor proteins including CrkII, C3G, and DOCK180, providing both prosurvival and promotility signaling. HEF1 undergoes a striking relocalization to the spindle at mitosis, but a function for HEF1 in mitotic signaling has not been demonstrated. Overexpression of HEF1 leads to failure of cells to progress through cytokinesis, while depletion of HEF1 by siRNA leads to defects earlier in M-phase before cleavage furrow formation. These defects can be explained mechanistically by the determination that HEF1 regulates the activation cycle of RhoA. Inactivation of RhoA has long been known to be required for cytokinesis, while it has recently been determined that activation of RhoA at the entry to M-phase is required for cellular rounding. Increased HEF1 sustains RhoA activation, whereas depleted HEF1 reduces RhoA activation. Further, it study demonstrates that chemical inhibition of RhoA is sufficient to reverse HEF1-dependent cellular arrest at cytokinesis. Finally, HEF1 has been demonstrated to associate with the RhoA-GTP exchange factor ECT2, an ortholog of the Drosophila cytokinetic regulator Pebble, providing a direct means for HEF1 control of RhoA. It is concluded that HEF1 is a novel component of the cell division control machinery, and that HEF1 activity impacts division as well as cell attachment signaling events (Dadke, 2006).

When presented with a gradient of chemoattractant, many eukaryotic cells respond with polarized accumulation of the phospholipid PtdIns(3,4,5)P3. This lipid asymmetry is one of the earliest readouts of polarity in neutrophils, Dictyostelium and fibroblasts. However, the mechanisms that regulate PtdInsP3 polarization are not well understood. Using a cationic lipid shuttling system, exogenous PtdInsP3 was delivered to neutrophils. Exogenous PtdInsP3 elicits accumulation of endogenous PtdInsP3 in a positive feedback loop that requires endogenous phosphatidylinositol-3-OH kinases (PI3Ks) and Rho family GTPases. This feedback loop is important for establishing PtdInsP3 polarity in response to both chemoattractant and to exogenous PtdInsP3; it may function through a self-organizing pattern formation system. Emergent properties of positive and negative regulatory links between PtdInsP3 and Rho family GTPases may constitute a broadly conserved module for the establishment of cell polarity during eukaryotic chemotaxis (Weiner, 2003).

Lymphocyte recruitment is regulated by signaling modules based on the activity of Rho and Rap small guanosine triphosphatases that control integrin activation by chemokines. This study shows that Janus kinase (JAK) protein tyrosine kinases control chemokine-induced Lymphocyte function-associated antigen 1 (LFA-1) and Integrin alpha4beta1 (VLA-4) mediated adhesion as well as human T lymphocyte homing to secondary lymphoid organs. JAK2 and JAK3 isoforms, but not JAK1, mediate CXCL12-induced LFA-1 triggering to a high affinity state. Signal transduction analysis showed that chemokine-induced activation of the Rho module of LFA-1 affinity triggering is dependent on JAK activity, with VAV1 mediating Rho activation by JAKs in a Galphai-independent manner. Furthermore, activation of Rap1A by chemokines is also dependent on JAK2 and JAK3 activity. Importantly, activation of Rap1A by JAKs is mediated by RhoA and PLD1, thus establishing Rap1A as a downstream effector of the Rho module. Thus, JAK tyrosine kinases control integrin activation and dependent lymphocyte trafficking by bridging chemokine receptors to the concurrent and hierarchical activation of the Rho and Rap modules of integrin activation (Montresor, 2013).

Anillin regulates cell-cell junction integrity by organizing junctional accumulation of Rho-GTP and actomyosin

Anillin is a scaffolding protein that organizes and stabilizes actomyosin contractile rings and was previously thought to function primarily in cytokinesis. Using Xenopus laevis embryos as a model system to examine Anillin's role in the intact vertebrate epithelium, this study found that a population of Anillin surprisingly localizes to epithelial cell-cell junctions throughout the cell cycle, whereas it was previously thought to be nuclear during interphase. Furthermore, Anillin was shown to play a critical role in regulating cell-cell junction integrity. Both tight junctions and adherens junctions are disrupted when Anillin is knocked down, leading to altered cell shape and increased intercellular spaces. Anillin interacts with Rho, F-actin, and myosin II, all of which regulate cell-cell junction structure and function. When Anillin is knocked down, active Rho (Rho-guanosine triphosphate [GTP]), F-actin, and myosin II are misregulated at junctions. Indeed, increased dynamic 'flares' of Rho-GTP are observed at cell-cell junctions, whereas overall junctional F-actin and myosin II accumulation is reduced when Anillin is depleted. It is proposed that Anillin is required for proper Rho-GTP distribution at cell-cell junctions and for maintenance of a robust apical actomyosin belt, which is required for cell-cell junction integrity. These results reveal a novel role for Anillin in regulating epithelial cell-cell junctions (Reyes, 2014).

Rac regulation of RhoA in cell motility

The precise spatio-temporal dynamics of protein activity are often critical in determining cell behaviour, yet for most proteins they remain poorly understood; it remains difficult to manipulate protein activity at precise times and places within living cells. Protein activity has been controlled by light, through protein derivatization with photocleavable moieties or using photoreactive small-molecule ligands. However, this requires use of toxic ultraviolet wavelengths, activation is irreversible, and/or cell loading is accomplished via disruption of the cell membrane (for example, through microinjection). This study reports the development of a new approach to produce genetically encoded photoactivatable derivatives of Rac1, a key GTPase regulating actin cytoskeletal dynamics in metazoan cells. Rac1 mutants were fused to the photoreactive LOV (light oxygen voltage) domain from phototropin, sterically blocking Rac1 interactions until irradiation unwound a helix linking LOV to Rac1. Photoactivatable Rac1 (PA-Rac1) could be reversibly and repeatedly activated using 458- or 473-nm light to generate precisely localized cell protrusions and ruffling. Localized Rac activation or inactivation was sufficient to produce cell motility and control the direction of cell movement. Myosin was involved in Rac control of directionality but not in Rac-induced protrusion, whereas PAK was required for Rac-induced protrusion. PA-Rac1 was used to elucidate Rac regulation of RhoA in cell motility. Rac and Rho coordinate cytoskeletal behaviours with seconds and submicrometre precision. Their mutual regulation remains controversial, with data indicating that Rac inhibits and/or activates Rho. Rac was shown to inhibit RhoA in mouse embryonic fibroblasts, with inhibition modulated at protrusions and ruffles. A PA-Rac crystal structure and modelling revealed LOV-Rac interactions that will facilitate extension of this photoactivation approach to other proteins (Wu, 2009).

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).

Table of contents

Rho1: Biological Overview | Regulation | Protein Interactions | Developmental Biology | Effects of Mutation | References

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