RhoGap function in yeast

The nonessential RGD1 gene encodes a Rho-GTPase activating protein for the Rho3 and Rho4 proteins in Saccharomyces cerevisiae. RGD1 acts somewhere upstream of the PKC pathway. Given that Rgd1p has been shown to have a Rho-GAP activity toward Rho3p and Rho4p, the defect of PKC pathway activation at late exponential phase observed in the rgd1 mutants might be mediated by the small GTPases Rho3p and Rho4p. Previous studies have revealed genetic interactions between RGD1 and the SLG1 and MID2 genes, encoding two putative sensors for cell integrity signaling, and VRP1 encoding an actin and myosin interacting protein involved in polarized growth. To better understand the role of Rgd1p, multicopy suppressor genes of the cell lethality of the double mutant rgd1Delta mid2Delta, RHO1 and RHO2 encoding two small GTPases, MKK1 encoding one of the MAP-kinase kinases in the protein kinase C (PKC) pathway, and MTL1, a MID2-homolog were each shown to suppress the rgd1Delta defects strengthening the functional links between RGD1 and the cell integrity pathway. Study of the transcriptional activity of Rlm1p, which is under the control of Mpk1p, the last kinase of the PKC pathway, and follow-up of the PST1 transcription, which is positively regulated by Rlm1p, indicate that the lack of RGD1 function diminishes the PKC pathway activity. It is hypothesized that rgd1Delta inactivation, at least through the hyperactivation of the small GTPases Rho3p and Rho4p, alters the secretory pathway and/or the actin cytoskeleton and decreases activity of the PKC pathway (de Bettignies, 2001).

In Saccharomyces cerevisiae, the Rho family of GTPases is thought to have a central role in the polarized growth process. The main functions assigned to these GTPases involve bud formation and cell surface growth, which might occur through the involvement of the actin cytoskeleton and the secretory pathway. Genetic and functional analyses have allowed the identification of five Rho members in yeast: Cdc42 and Rho1 to Rho4. These small GTPases function as binary switches, which are turned on and off by binding to GTP or GDP, respectively. The GTP-bound form interacts with its specific target and performs its cell functions. Small GTPases are regulated by GAPs (GTPase-activating proteins), GEFs (GDP-GTP exchange factors), and a GDP dissociation inhibitor (de Bettignies, 2001).

During the sequencing of the genome of S. cerevisiae, a new gene encoding a protein with a Rho-GAP homology domain was identified. This protein, called Rgd1p (for related GAP domain), was shown in vitro to be a GTPase activating protein for the Rho3 and Rho4 proteins. Thus, in activating the hydrolysis of GTP, Rgd1p negatively regulates the action of these two Rho proteins. Rho3p and Rho4p play a role in bud formation and have some partially overlapping functions. Deletion of RHO4 does not affect cell growth, whereas deletion of RHO3 causes a severe growth delay and a decrease in cell viability. Overexpression of RHO4 suppresses the growth defect in rho3 cells. Depletion of both RHO3 and RHO4 gene products results in lysis of cells with a small bud, which can be prevented by the presence of osmotic stabilizer in the medium. In this latter condition, Rho3p- and Rho4p-depleted cells lose cell polarity as revealed by chitin delocalization and by random distribution of actin patches (de Bettignies, 2001).

Genetic interactions occur between RGD1 and the SLG1 and MID2 genes. SLG1 has also been designated HCS77 and WSC1, but for simplicity this gene is referred to here as SLG1. Slg1p and Mid2p are both plasma membrane proteins with partial overlapping functions. They act upstream of the protein kinase C (PKC) pathway and are thought to monitor the state of the cell surface and relay the information to Pkc1p. Protein kinase C is mostly regulated by the small GTPase Rho1p in vivo. Pkc1p activates a mitogen-activated protein (MAP) kinase cascade, named the PKC pathway, consisting of Bck1p, Mkk1p/Mkk2p, and the MAP kinase Mpk1p. Activation of this pathway is particularly important in response to various external stresses, including high temperature, low osmolarity, and cell wall disruption, as well as being important during mating. The protein Slg1 is linked to the PKC pathway by the finding that this MAP kinase cascade is activated by heat stress via Slg1p. A direct interaction of Slg1p with Rom2p, one of the Rho1p-GEFs, has been recently reported and this interaction is responsible for the activation of the PKC pathway through Rho1p (de Bettignies, 2001).

The loss of RGD1 function amplifies the phenotype due to the SLG1 deletion and the small-budded double-mutant cells die because of defects in cell wall structure and lysis upon bud growth. In parallel, the inactivation of MID2, the other putative sensor for cell integrity signaling in S. cerevisiae, exacerbates the specific phenotype of the rgd1Delta mutant with an increase in dead cells at late exponential phase in minimal medium. Taken together, these results suggest that Rgd1p has a regulatory role in connection with both the PKC pathway and the actin cytoskeleton organization in S. cerevisiae (de Bettignies, 2001).

To further elucidate the function of RGD1, multicopy suppressors of the viability defect of the rgd1Delta mutation were isolated in minimal medium. Phenotypic and genetic analysis has allowed the identification of several multicopy as well as monocopy suppressors of rgd1Delta: the RHO1 and RHO2 genes encoding two GTPases involved in actin cytoskeleton organization, the MID2-homolog MTL1, and the MKK1 gene coding for one of the MAP-kinase kinases of the PKC pathway. Considering the suppressor effect of additional PKC pathway components, it has been shown that activation of the PKC pathway prevents lethality of rgd1Delta cells. Analysis of the transcriptional activity of Rlm1p, one of the targets of the last kinase in the PKC pathway, and study of the PST1 transcription, which is positively regulated by Rlm1p, shows that the rgd1Delta mutation decreases the activity of this MAP-kinase pathway in minimal medium at late exponential phase. This decrease in PKC pathway activity is at least partly responsible for the rgd1Delta cell viability loss under particular growth or physiological conditions (de Bettignies, 2001).

RhoGAP in C. elegans

Embryonic morphogenesis involves the coordinate behaviour of multiple cells and requires the accurate balance of forces acting within different cells through the application of appropriate brakes and throttles. In C. elegans, embryonic elongation is driven by Rho-binding kinase (ROCK) and actomyosin contraction in the epidermis. This study identified an evolutionary conserved, actin microfilament-associated RhoGAP (RGA-2) that behaves as a negative regulator of LET-502/ROCK. The small GTPase RHO-1 is the preferred target of RGA-2 in vitro, and acts between RGA-2 and LET-502 in vivo. Two observations show that RGA-2 acts in dorsal and ventral epidermal cells to moderate actomyosin tension during the first half of elongation: (1) time-lapse microscopy shows that loss of RGA-2 induces localised circumferentially oriented pulling on junctional complexes in dorsal and ventral epidermal cells; (2) specific expression of RGA-2 in dorsal/ventral, but not lateral, cells rescues the embryonic lethality of rga-2 mutants. It is proposed that actomyosin-generated tension must be moderated in two out of the three sets of epidermal cells surrounding the C. elegans embryo to achieve morphogenesis (Diogon, 2007).

C. elegans embryos establish cortical domains of PAR proteins of reproducible size before asymmetric cell division. The ways in which the size of these domains is set remain unknown. This study identified the GTPase-activating proteins (GAPs) RGA-3 and RGA-4, which regulate the activity of the small GTPase RHO-1. rga-3/4(RNAi) embryos have a hypercontractile cortex, and the initial relative size of their anterior and posterior PAR domains is altered. Thus, RHO-1 activity appears to control the level of cortical contractility and concomitantly the size of cortical domains. These data support the idea that in C. elegans embryos the initial size of the PAR domains is set by regulating the contractile activity of the acto-myosin cytoskeleton through the activity of RHO-1. RGA-3/4 have functions different from CYK-4, the other known GAP required for the first cell division, showing that different GAPs cooperate to control the activity of the acto-myosin cytoskeleton in the first cell division of C. elegans embryos (Schonegg, 2007).

Two redundant GTPase activating proteins (GAPs) -- RGA-3 and RGA-4 -- regulate Rho GTPase function at the plasma membrane in early C. elegans embryos. Knockdown of both RhoGAPs resulted in extensive membrane ruffling, furrowing and pronounced pseudo-cleavages. In addition, the non-muscle myosin NMY-2 and RHO-1 accumulated on the cortex at sites of ruffling. RGA-3 and RGA-4 are GAPs for RHO-1, but most probably not for CDC-42, because only RHO-1 is epistatic to the two GAPs, and the GAPs have no obvious influence on CDC-42 function. Furthermore, knockdown of either the RHO-1 effector, LET-502, or the exchange factor for RHO-1, ECT-2, alleviates the membrane-ruffling phenotype caused by simultaneous knockdown of both RGA-3 and RGA-4 [rga-3/4 (RNAi)]. GFP::PAR-6 and GFP::PAR-2 are localized at the anterior and posterior part of the early C. elegans embryo, respectively showing that rga-3/4 (RNAi) does not interfere with polarity establishment. Most importantly, upon simultaneous knockdown of RGA-3, RGA-4 and the third RhoGAP present in the early embryo, CYK-4, NMY-2 spread over the entire cortex and GFP::PAR-2 localization at the posterior cortex is greatly diminished. These results indicate that the functions of CYK-4 are temporally and spatially distinct from RGA-3 and RGA-4 (RGA-3/4). RGA-3/4 and CYK-4 also play different roles in controlling LET-502 activation in the germ line, because rga-3/4 (RNAi), but not cyk-4 (RNAi), aggravate the let-502(sb106) phenotype. It is proposed that RGA-3/4 and CYK-4 control with which effector molecules RHO-1 interacts at particular sites at the cortex in the zygote and in the germ line (Schmutz, 2007).

Identification and domain structure of mammalian p190 RhoGap

In mitogenically stimulated and tyrosine kinase-transformed cells, a substantial fraction of the ras GTPase-activating protein (GAP) forms a complex with a protein termed p190. Several cDNAs encoding the p190 protein have been cloned. Analysis of the predicted protein sequence reveals three distinct domains with homology to previously described sequences. An N-terminal domain of p190 contains sequence motifs that are found in all of the known GTPases. At the C-terminus of the protein is a domain that contains sequences very similar to those found in the breakpoint cluster region gene product, n-chimerin, and rho GAP, all of which have been shown to possess intrinsic GAP activity on small GTPases. Finally, a 778 aa segment in the middle of p190 is nearly identical in sequence to a recently described transcriptional repressor (Settleman, 1992).

p190 is a GTPase-activating protein (GAP) for the Rho family of GTPases. The GAP domain of p190 is at the C terminus of the protein. At its N terminus, p190 contains a GTP binding domain of unknown significance. A mutation (Ser36 --> Asn) has been introduced into this domain of p190 that decreased its ability to bind guanine nucleotide when expressed as a hemagglutinin (HA)-tagged protein in COS cells. In vitro, both the wild type and S36N mutant HA-p190 proteins showed similar GAP activities toward RhoA, but when expressed in NIH 3T3 fibroblasts only wild type p190 appeared able to function as a RhoGAP. Wild type HA-p190 induces a phenotype of rounded cells with long, beaded extensions similar to those seen when Rho function is disrupted by ADP-ribosylation. HA-p190(S36N), although expressed at a similar level to the wild type protein, has no discernible effect on the cells. The beaded extension phenotype induced by wild type HA-p190 requires GAP function. A GAP-defective mutant, p190(R1283A), has no effect on cell morphology. Moreover, the beaded extension phenotype can be suppressed by co-expression of a gain-of-function Rho mutant, RhoA(G14V), or Rac mutant, Rac1(G12V). Activation of the Jun kinase (JNK) via muscarinic receptors is inhibited by wild type HA-p190, but JNK activity is enhanced by the S36N mutant. Co-expression of HA-p190 with a fragment containing only the mutated GTP binding domain partially inhibits the beaded extension phenotype, suggesting that it may sequester a factor required for p190 function. Taken together these data demonstrate that within the cell, the Rho/Rac GAP activity of p190 can be regulated by the N-terminal GTP binding domain (Tatsis, 1998).

Coordination of Rho and Rac GTPase function via p190B RhoGAP

The Rac GTPase regulates Rho signaling in a broad range of physiological settings and in oncogenic transformation. This study reports a novel mechanism by which crosstalk between Rac and Rho GTPases is achieved. Activated Rac1 binds directly to p190B Rho GTPase-activating protein (RhoGAP), a major modulator of Rho signaling. p190B colocalizes with constitutively active Rac1 in membrane ruffles. Moreover, activated Rac1 is sufficient to recruit p190B into a detergent-insoluble membrane fraction, a process that is accompanied by a decrease in GTP-bound RhoA from membranes. p190B is recruited to the plasma membrane in response to integrin engagement. Collagen type I, a potent inducer of Rac1-dependent cell motility in HeLa cells, counteracts cytoskeletal collapse resulting from overexpression of wild-type p190B, but not that resulting from overexpression of a p190B mutant specifically lacking the Rac1-binding sequence. Furthermore, this p190B mutant exhibits dramatically enhanced RhoGAP activity, consistent with a model whereby binding of Rac1 relieves autoinhibition of p190B RhoGAP function. Collectively, these observations establish that activated Rac1, through direct interaction with p190B, modulates subcellular RhoGAP localization and activity, thereby providing a novel mechanism for Rac control of Rho signaling in a broad range of physiological processes (Bustos, 2008).

p190 RhoGap acts downstream of Integrins and Src

The critical pathways through which protein-tyrosine kinases induce cellular proliferation and malignant transformation are not well defined. Since microinjection of antibodies against p21ras can block the biological effects of both normal and oncogenic tyrosine kinases, it is likely that they require functional p21ras to transmit their mitogenic signals. No biochemical link has been established, however, between tyrosine kinases and p21ras. A non-catalytic domain of cytoplasmic tyrosine kinases, SH2, has been identified that regulates the activity and specificity of the kinase domain. The presence of two adjacent SH2 domains in the p21ras GTPase-activating protein (GAP) indicates that GAP might interact directly with tyrosine kinases. GAP, and two co-precipitating proteins of relative molecular masses 62,000 and 190,000 (p62 and p190) are phosphorylated on tyrosine in cells that have been transformed by cytoplasmic and receptor-like tyrosine kinases. The phosphorylation of these polypeptides correlates with transformation in cells expressing inducible forms of the v-src or v-fps encoded tyrosine kinases. Furthermore, GAP, p62 and p190 are also rapidly phosphorylated on tyrosine in fibroblasts stimulated with epidermal growth factor. These results suggest a mechanism by which tyrosine kinases might modify p21ras function, and implicate GAP and its associated proteins as targets of both oncoproteins and normal growth factor receptors with tyrosine kinase activity. These data support the idea that SH2 sequences direct the interactions of cytoplasmic proteins involved in signal transduction (Ellis, 1990).

Analysis of C3H10T1/2 murine fibroblasts overexpressing wild type and dominant negative variants of c-Src has demonstrated a requirement for c-Src in EGF-induced mitogenesis. Correlating with the ability of c-Src variants to potentiate or inhibit EGF-dependent DNA synthesis is the phosphotyrosine content of multiple cellular proteins, including p190-RhoGAP, a protein thought to regulate growth factor-induced actin cytoskeleton remodeling by modulating the activity of the small GTP binding protein, Rho. Because the in vivo phosphotyrosine content of p190 varies with the level of active c-Src and not with EGF treatment, p190 is considered to be a preferred substrate of c-Src. To determine whether tyrosyl phosphorylation of p190 (by c-Src) could influence EGF-dependent actin remodeling, conventional and confocal immunofluorescence microscopy was used to examine the intracellular distribution of p190, actin, and p120RasGAP in EGF-stimulated or unstimulated 10T1/2 Neo control cells and cells that stably overexpress wild-type (K+) or kinase-defective (K-) c-Src. In all cell lines, EGF induces a rapid and transient condensation of p190 and RasGAP into cytoplasmic, arclike structures. However, in K+ cells the rate of appearance and number of cells exhibiting arcs is increased when compared with control cells. Conversely, K- cells exhibit delayed arc formation and a reduction in the number of cells forming arcs. EGF-induced actin stress fiber disassembly and reassembly occurs with the same kinetics and frequency as does p190 and RasGAP rearrangements in all three cell lines. These results, together with the documented Rho-GAP activity intrinsic to p190 and the ability of Rho to modulate actin stress fiber formation, suggest that c-Src regulates EGF-dependent actin cytoskeleton reorganization through phosphorylation of p190 (Chang, 1995).

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).

p190 RhoGAP is a 190-kDa protein that stably associates with p120 RasGAP and regulates actin dynamics through members of the Rho family of small GTPases. Previous studies have indicated a direct relationship between levels of p190 tyrosine phosphorylation, the extent and kinetics of epidermal growth factor (EGF)-induced actin rearrangements, and EGF-induced cell cycle progression, suggesting that p190 links Ras-mediated mitogenic signaling with signaling through the actin cytoskeleton. Determining which tyrosine residues in p190 are phosphorylated, what factors regulate phosphorylation of these sites, and what effect tyrosine phosphorylation has on p190 function is key to understanding the role(s) that p190 may play in these processes. To begin investigating these questions, biochemical approaches have been used to characterize the number and relative levels of in vivo-phosphorylated tyrosine residues on endogenous p190 from C3H10T1/2 murine fibroblasts. Only two tryptic phosphopeptides containing phosphotyrosine (p-Tyr), a major site, identified as Y1105, and a minor, unidentified site, were detected. Phosphorylation of Y1105, but not the minor site, is modulated in vivo to a greater extent by overexpression of c-Src than by the EGF receptor and is efficiently catalyzed by c-Src in vitro, indicating that Y1105 is a selective and preferential target of c-Src both in vitro and in vivo. In vitro and in vivo coprecipitation analysis using glutathione S-transferase (GST) fusion proteins containing wild-type and Y1105F variants of the p190 middle domain, variants of full-length p190 ectopically expressed in COS-7 cells, and endogenous p190 and p120 in C3H10T1/2 cells, reveals that p190 can bind to p120 in the presence and absence of p190 tyrosine phosphorylation. p-Tyr-independent complexes comprise 10% to 20% of the complexes formed in the presence of p-Tyr. Mutation of Y1105 from Tyr to Phe results in complete loss of p-Tyr-dependent complex formation, indicating that p-Y1105 is the sole p-Tyr residue mediating binding to p120. These studies describe a specific mechanism by which c-Src can regulate p190-p120 association and also document a significant role for p-Tyr-independent means of p190-p120 binding (Roof, 1998).

The interaction of cells with their substrate triggers cascades of signal transduction that result in profound changes in cell morphology. The nature of these signals and how they are integrated to orchestrate changes in cell shape are beginning to be elucidated. In particular, adhesive interactions between cells and their substrate, mediated by cell-surface integrins and extracellular matrix (ECM) proteins, appear to result in massive rearrangement of the cell cytoskeleton via the small G-protein, Rho. In mouse fibroblasts, the interaction between cells and their substrate results in the rapid recruitment to the cytoskeleton of RasGAP (p120RasGAP), its associated protein of 190 kilodaltons, the GTPase activating protein for RhoA (p190RhoGAP) and the focal adhesion kinase (p125FAK). Similar results were obtained when cells were plated on ECM proteins, such as fibronectin, suggesting that the phenomenon is integrin mediated. These studies suggest that in fibroblasts, cell-substrate interaction triggered by integrin engagement results in the recruitment to the cytoskeleton of signaling molecules such as p120RasGAP, p190RhoGAP and p125FAK and may be involved in the formation of membrane cytoskeleton-associated signaling complexes that are important in cytoarchitectural reorganization (Sharma, 1998).

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) 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).

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).

p190 RhoGAP is a multi-domain protein that is thought to regulate actin cytoskeleton dynamics. It can be phosphorylated both in vitro and in vivo at multiple sites by the Src tyrosine kinase and one or more of these sites is postulated to modulate p190 function. One of the regions which is multiply phosphorylated by Src in vitro is the N-terminal GTP binding domain. Using a partially purified, bacterially expressed recombinant protein that includes the GTP binding domain (residues 1-389), it has been shown that GTP binds to this fragment in a specific and saturable manner that is both time- and dose-dependent and that tyrosine phosphorylation of this fragment by c-Src results in a loss of GTP binding activity. These findings suggest that tyrosine phosphorylation of the p190 N-terminal domain can alter its ability to bind GTP (Roof, 2000).

Engagement of beta2 integrins on human neutrophils induces activation of RhoA, as indicated by the increased ratio of GTP:GTP + GDP recovered on RhoA and translocation of RhoA to a membrane fraction. The clustering of beta2 integrins also induces a time-dependent increase in GDP bound to RhoA, which correlates with beta2 integrin-induced activation of p190RHOGAP: The activation of p190RhoGAP is completely blocked by PP1, a selective inhibitor of Src family tyrosine kinases. However, clustering of beta2 integrins does not increase the basal tyrosine phosphorylation of p190RhoGAP, nor does it affect the amount of p120RasGAP bound to p190RHOGAP: Instead, the beta2 integrin-induced activation of p190RhoGAP is accompanied by increased tyrosine phosphorylation of a p190RhoGAP-associated protein, p120RasGAP, and accumulation of both p120RasGAP and p190RhoGAP in a membrane fraction. PP1 blocks the beta2 integrin-induced phosphorylation of p120RasGAP, as well as the translocation of p190RhoGAP and p120RasGAP, but it does not affect the accumulation of RhoA in the membrane fraction. PP1 also increases the GTP:GTP + GDP ratio recovered on RhoA immunoprecipitated from beta2 integrin-stimulated cells. Thus, in neutrophils, beta2 integrin-induced activation of p190RhoGAP requires a signal from a Src family tyrosine kinase, but it does not occur via the signaling pathway responsible for activation of RHOA (Dib, 2001).

p190 RhoGAP is a tyrosine phosphorylated protein that contains an N-terminal GTP binding domain, a middle domain (MD) that mediates interaction with p120 RasGAP and a C-terminal GTPase-activating protein (GAP) domain that is specific for the Rho family of small GTPases. Evidence is accumulating to suggest that p190 participates in actin cytoskeleton rearrangements that occur following transformation by v-Src or stimulation by growth factors, and that tyrosine phosphorylation of p190 by Src influences these processes. The current study was performed to establish whether p190RhoGAP directly participates in epidermal growth factor-induced actin stress fiber disassembly and how c-Src is involved in this process. The results support a model in which the p190 MD negatively regulates the activity of the GAP domain and that c-Src phosphorylation of Y1105 is necessary, but insufficient on its own, for actin stress fiber disassembly (Haskell, 2001).

RhoGap regulates cell fate

Mature adipocytes and myocytes are derived from a common mesenchymal precursor. While IGF-1 promotes the differentiation of both cell types, the signaling pathways that specify the distinct cell fates are largely unknown. The Rho GTPase and its regulator, p190-B RhoGAP, are components of a critical switch in the adipogenesis-myogenesis 'decision.' Cells derived from embryos lacking p190-B RhoGAP exhibit excessive Rho activity, are defective for adipogenesis, but undergo myogenesis in response to IGF-1 exposure. In vitro, activation of Rho-kinase by Rho inhibits adipogenesis and is required for myogenesis. The activation state of Rho following IGF-1 signaling is determined by the tyrosine-phosphorylation status of p190-B RhoGAP and its resulting subcellular relocalization. Moreover, adjusting Rho activity is sufficient to alter the differentiation program of adipocyte and myocyte precursors. Together, these results identify the Rho GTPase as an essential modulator of IGF-1 signals that direct the adipogenesis-myogenesis cell fate decision (Sordella, 2003).

p190 RhoGap and the determination of cell size and animal size

Rho GTPases regulate several aspects of tissue morphogenesis during animal development. Mice lacking the Rho-inhibitory protein, p190-B RhoGAP, are 30% reduced in size and exhibit developmental defects strikingly similar to those seen in mice lacking the CREB transcription factor. In p190-B RhoGAP-deficient mice, CREB phosphorylation is substantially reduced in embryonic tissues. Embryo-derived cells contain abnormally high levels of active Rho protein, are reduced in size, and exhibit defects in CREB activation upon exposure to insulin or IGF-1. The cell size defect is rescued by expression of constitutively activated CREB, and in wild-type cells, expression of activated Rho or dominant-negative CREB results in reduced cell size. Together, these results suggest that activity of the Rho GTPase modulates a signal from insulin/IGFs to CREB that determines cell size and animal size during embryogenesis (Sordella, 2002).

These observations indicate that activity of the Rho GTPase and the CREB transcription factor are important determinates of cell and animal size during embryonic development, thereby defining a novel biological function for both of these widely expressed regulatory proteins. In fibroblasts derived from mice lacking p190-B RhoGAP, it appears that Rho is performing a largely cell-autonomous role that can influence responsiveness to insulin/IGFs through activation of the Rho target ROK. However, the fact that mutant fibroblasts are not completely defective in insulin/IGF-1 responsiveness suggests that at least some aspect of insulin-promoted growth is Rho insensitive. Moreover, the observation that mice lacking p190-B RhoGAP appear to be uniformly reduced in size, while phospho-CREB levels are not substantially reduced in every tissue, raises the possibility that there is additional complexity involved in determining organism size (Sordella, 2002).

It is possible, for example, that modulation of CREB is a more significant determinant of cell size at earlier stages of development in some tissues. In addition, there may be both cell-autonomous and -nonautonomous mechanisms involved in vivo. Indeed, PI-3 kinase, which appears to play a role in the pathway proposed here, has been previously implicated in cell size regulation, and has been reported to have both cell-autonomous and -nonautonomous functions in C. elegans. In addition, while IRS plays a well-documented cell-autonomous role in regulating cell size in Drosophila, studies in mice reveal an additional cell-nonautonomous role in controlling animal size. It is also worth noting that both insulin and IGF-1 gene expression have been found to be CREB responsive, raising the possibility that impaired CREB activity can also contribute in a cell-nonautonomous manner to growth regulation by affecting levels of secreted and circulating insulin/IGF. Such findings point to a complex regulatory system for the control of cell, organ, and animal size that involves both cell-autonomous and -nonautonomous mechanisms that may require an overlapping set of proteins. In fact, there may be additional compensatory mechanisms that serve to uniformly adjust the size of organs to accommodate changes that may be limited to a subset of organs in the mutant animals (Sordella, 2002).

p190 RhoGap functions in neurons

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).

During development of the central nervous system, oligodendrocyte progenitor cells differentiate into mature myelinating cells. The molecular signals that promote this process, however, are not well defined. One molecule that has been implicated in oligodendrocyte differentiation is the Src family kinase Fyn. In order to probe the function of Fyn in this system, a yeast two hybrid screen was performed. Using Fyn as bait, p190 RhoGAP was isolated in the screen of an oligodendrocyte cDNA library. Coimmunoprecipitation and in vitro binding assays have verified that p190 RhoGAP binds to the Fyn SH2 domain. Phosphorylation of p190 requires active Fyn tyrosine kinase and is increased threefold upon differentiation of primary oligodendrocytes. Moreover, complex formation between p190 and p120 RasGAP occurs in differentiated oligodendrocytes. p190 RhoGAP activity is known to regulate the RhoGDP:RhoGTP ratio. Indeed, expression of dominant negative Rho in primary oligodendrocytes causes a hyperextension of processes. Conversely, constitutively activated Rho causes reduced process formation. These findings define a pathway in which Fyn activity regulates the phosphorylation of p190, leading to an increase in RhoGAP activity with a subsequent increase in RhoGDP, which in turn, regulates the morphological changes that accompany oligodendrocyte differentiation (Wolf, 2001).

In rats, fear conditioning, which is known to alter synaptic efficacy in lateral amygdala (LA), was used to study molecular mechanisms underlying long-term memory. Following fear conditioning, the tyrosine phosphorylated protein p190 RhoGAP becomes associated with GRB2 in LA significantly more in conditioned than in controls. RasGAP and Shc were also found to associate with GRB2 in LA significantly more in the conditioned animals. Inhibition of the p190 RhoGAP-downstream kinase ROCK in LA during fear conditioning, by using the ROCK inhibitor Y-27632, which has been shown to be cell permeable, nontoxic, and highly specific for ROCK, impairs long- but not short-term memory. Thus, the p190 RhoGAP/ROCK pathway, which regulates the morphology of dendrites and axons during neural development, plays a central role, through a GRB2-mediated molecular complex, in fear memory formation in the lateral amygdala (Lamprecht, 2002).

Abl family kinases, which include the mammalian Abl and Arg (Abl-related gene) kinases, regulate neuronal morphogenesis in developing metazoa. Activation of Abl kinase activity directs changes in actin-dependent processes such as membrane ruffling, filopodial protrusion, and cell motility. However, the mechanisms by which increased Abl or Arg kinase activity promote cytoskeletal rearrangements are unclear. Evidence is provided that the Rho inhibitor p190RhoGAP (GTPase-activating protein) is an Arg substrate in the postnatal mouse brain. p190RhoGAP has reduced phosphotyrosine content in postnatal arg-/- mouse brain extracts relative to wild-type extracts. In addition, the adhesion-dependent stimulation of p190RhoGAP phosphorylation observed in wild-type cells is not observed in arg-/- fibroblasts and neurons. Arg can phosphorylate p190RhoGAP in vitro and in vivo on tyrosine (Y) 1105. Arg can stimulate p190RhoGAP to inhibit Rho and Arg-mediated phosphorylation is required for this stimulation. Phosphorylation by Arg also promotes p190RhoGAP's association with p120RasGAP and stimulates p190RhoGAP's ability to induce neuritogenesis in neuroblastoma cells. These results demonstrate that p190RhoGAP is an Arg substrate in the developing brain and suggest that Arg mediates the adhesion-dependent regulation of neuronal morphogenesis in the postnatal brain by phosphorylating p190RhoGAP (Hernández, 2004).

The majority of excitatory synaptic transmission in the brain occurs at dendritic spines, which are actin-rich protrusions on the dendrites. The asymmetric nature of these structures suggests that proteins regulating cell polarity might be involved in their formation. Indeed, the polarity protein PAR-3 is required for normal spine morphogenesis. However, this function is independent of association with atypical protein kinase C (aPKC) and PAR-6. This study shows that PAR-6 together with aPKC plays a distinct but essential role in spine morphogenesis. Knockdown of PAR-6 inhibits spine morphogenesis, whereas overexpression of PAR-6 increases spine density, and these effects are mediated by aPKC. Using a FRET biosensor, it was further shown that p190 RhoGAP and RhoA act downstream of the PAR-6/aPKC complex. These results define a role for PAR-6 and aPKC in dendritic spine biogenesis and maintenance, and reveal an unexpected link between the PAR-6/aPKC complex and RhoA activity (H. Zhang, 2008).

RhoGAP: Biological Overview | Regulation | References

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