Family Of Small GTP-binding Proteins Research Articles

RGS-containing RhoGEFs: the missing link between transforming G proteins and Rho?

Many mitogens promote cell proliferation by acting on cell surface receptors that either possess an intrinsic protein-tyrosine kinase activity (Yarden et al., 1986) or interact with heterotrimeric GTPbinding proteins (G proteins). The latter are collectively known as G protein-coupled receptors (GPCRs) and comprise the largest group of cell surface molecules involved in signal transmission. With more than 1000 members, they are encoded by the biggest gene family in the human genome (Flower, 1999), and the importance and diversity of their physiological roles are directly supported by their link to a number of hereditary and acquired diseases (Spiegel, 1996; Stadel et al., 1997). Furthermore, these receptors and their ligands are the target of over 50% of all current therapeutic agents (Flower, 1999). GPCRs exhibit a common structural motif consisting of seven membrane-spanning regions (Dohlman et al., 1987), and can be activated by a diverse array of external stimuli, including growth factors, vasoactive polypeptides, chemoattractants, neurotransmitters, hormones, phospholipids, photons, odorants, and taste ligands. In general, agonist binding provokes rapid conformational changes in the transmembrane a helices, which result in the exposure of previously masked G protein binding sites in the intracellular loops (Altenbach et al., 1996; Bourne, 1997; Wess, 1997). This causes the exchange of GDP for GTP bound to the G protein a subunit, and GTP-bound a subunits or bg complexes then initiate intracellular signaling responses by acting on a variety of e€ector molecules. To date, sixteen distinct mammalian G protein a subunits have been identi®ed and divided into four families based on their sequence similarity: as, ai, aq, and a12 (Wilkie et al., 1992). The aq family of heterotrimeric G protein a subunits is coupled to phospholipase-C b to induce the hydrolysis of phosphatidylinositol bis phosphate, the consequent rise in the intracellular concentration of Ca, and the activation of protein kinase C, whereas the as and ai families are known to activate and inhibit adenylyl cyclase to increase and decrease the level of cyclic AMP, respectively. ai proteins also regulate ion channels, certain phospholipases and phosphodiesterases (Hamm, 1998). The targets for both members of the a12 family, a12 and a13, were unknown until recently. In addition, eleven-G protein g subunits and ®ve G protein b subunits have been cloned so far and they regulate certain ion channels, phospholipase-C, and phosphatidylinositol-3 kinases (Clapham and Neer, 1997). Through these G proteins subunits, GPCRs regulate a diverse array of physiological functions such as neurotransmission, chemoreception, photoreception, metabolism, growth, and di€erentiation. In addition to these physiological responses, there is also emerging evidence of the involvement of aberrant GPCR function in cellular transformation and oncogenesis (see Gutkind, 1998 for a recent review). In this regard, recent work suggests that the Rho family of small GTP-binding proteins plays a central role in the regulation of cell proliferation by GPCRs, and that the persistent activation of Rhomediated pathways by heterotrimeric G proteins can lead to cellular transformation. In this review, we will focus on recent e€orts addressing the molecular mechanisms by which GPCRs and G protein a subunits activate Rho, with emphasis on their contribution to the understanding on how this signal transducing system regulates normal and aberrant cell growth.

Regulation of Rac and Cdc42 Pathways by Gi during Lysophosphatidic Acid-induced Cell Spreading

The pertussis toxin-sensitive G protein, G(i), has been implicated in lysophosphatidic acid-induced cell mitogenesis and migration, but the mechanisms remain to be detailed. In the present study, we found that pertussis toxin blocks lysophosphatidic acid-induced cell spreading of NIH 3T3 fibroblasts on fibronectin. This prevention of cell spreading was eliminated by the expression of constitutively active mutants of Rho family small GTP-binding proteins, Rac and Cdc42, but not by Rho. In addition, activation of the endogenous forms was suppressed by pertussis toxin, indicating that G(i)-induced cell spreading is mediated through the Rac and Cdc42 pathway. Transfection of constitutively active mutants of G alpha(i) and G alpha(11) and G beta gamma subunits enhanced spreading of pertussis toxin-treated cells. G beta(1) with G gamma(12), a major G gamma form in fibroblasts, was more effective for increasing cell spreading than G beta(1)gamma(2) or G beta(1) plus G gamma(12)S2A, a mutant in which Ser-2, a phosphorylation site for protein kinase C, is replaced with alanine. In addition, a protein kinase C inhibitor diminished G beta(1)gamma(12)-induced cell spreading, suggesting a role for phosphorylation of the protein. These findings indicate that both G alpha(i) and G beta gamma stimulate Rac and Cdc42 pathways with lysophosphatidic acid-induced cell spreading on fibronectin.

Dynamic localization of rop GTPases to the tonoplast during vacuole development.

Vacuoles are essential pleomorphic organelles that undergo dynamic changes during cell growth and differentiation in plants. How developmental signals are linked to vacuole biogenesis and development is poorly understood. In this report, we show that a Rop GTPase is localized to developing vacuoles in pea (Pisum sativum cv Extra Early Alaska). Rop belongs to the RHO family of Ras-related small GTP-binding proteins that are key molecular switches in a wide variety of eukaryotic signal transduction pathways. Using indirect immunofluorescence and an anti-Rop antibody, we showed that Rop proteins accumulate to high levels in rapidly growing tapetal cells of pea anthers. In these cells, Rop is localized to an endomembrane system that exists as dynamic pleomorphic networks: a perinuclear fine network decorated with punctate dots, a network composed of small spheres and tubules, and interconnected chambers. Colocalization with a tonoplast annexin VCaB42 shows that these dynamic networks represent the tonoplast. Our results suggest that the dynamic Rop-containing tonoplast networks represent a unique stage of vacuole development. The specific localization of Rop to developing vacuoles supports a role for Rop in signal transduction that mediates vacuole development in plants.

Crystal structure and novel recognition motif of Rho ADP-ribosylating C3 exoenzyme from Clostridium botulinum: structural insights for recognition specificity and catalysis

Clostridium botulinum C3 exoenzyme inactivates the small GTP-binding protein family Rho by ADP-ribosylating asparagine 41, which depolymerizes the actin cytoskeleton. C3 thus represents a major family of the bacterial toxins that transfer the ADP-ribose moiety of NAD to specific amino acids in acceptor proteins to modify key biological activities in eukaryotic cells, including protein synthesis, differentiation, transformation, and intracellular signaling. The 1.7 Å resolution C3 exoenzyme structure establishes the conserved features of the core NAD-binding β-sandwich fold with other ADP-ribosylating toxins despite little sequence conservation. Importantly, the central core of the C3 exoenzyme structure is distinguished by the absence of an active site loop observed in many other ADP-ribosylating toxins. Unlike the ADP-ribosylating toxins that possess the active site loop near the central core, the C3 exoenzyme replaces the active site loop with an α-helix, α3. Moreover, structural and sequence similarities with the catalytic domain of vegetative insecticidal protein 2 (VIP2), an actin ADP-ribosyltransferase, unexpectedly implicates two adjacent, protruding turns, which join β5 and β6 of the toxin core fold, as a novel recognition specificity motif for this newly defined toxin family. Turn 1 evidently positions the solvent-exposed, aromatic side-chain of Phe209 to interact with the hydrophobic region of Rho adjacent to its GTP-binding site. Turn 2 evidently both places the Gln212 side-chain for hydrogen bonding to recognize Rho Asn41 for nucleophilic attack on the anomeric carbon of NAD ribose and holds the key Glu214 catalytic side-chain in the adjacent catalytic pocket. This proposed bipartite ADP-ribosylating toxin turn-turn (ARTT) motif places the VIP2 and C3 toxin classes into a single ARTT family characterized by analogous target protein recognition via turn 1 aromatic and turn 2 hydrogen-bonding side-chain moieties. Turn 2 centrally anchors the catalytic Glu214 within the ARTT motif, and furthermore distinguishes the C3 toxin class by a conserved turn 2 Gln and the VIP2 binary toxin class by a conserved turn 2 Glu for appropriate target side-chain hydrogen-bonding recognition. Taken together, these structural results provide a molecular basis for understanding the coupled activity and recognition specificity for C3 and for the newly defined ARTT toxin family, which acts in the depolymerization of the actin cytoskeleton. This β5 to β6 region of the toxin fold represents an experimentally testable and potentially general recognition motif region for other ADP-ribosylating toxins that have a similar β-structure framework.

Cytotoxic necrotizing factor type 1 of uropathogenic Escherichia coli kills cultured human uroepithelial 5637 cells by an apoptotic mechanism.

Pathogenic Escherichia coli associated with urinary tract infections (UTIs) in otherwise healthy individuals frequently produce cytotoxic necrotizing factor type 1 (CNF1), a member of the family of bacterial toxins that target the Rho family of small GTP-binding proteins. To gain insight into the function of CNF1 in the development of E. coli-mediated UTIs, we examined the effects of CNF1 intoxication on a panel of human cell lines derived from physiologically relevant sites (bladder, ureters, and kidneys). We identified one uroepithelial cell line that exhibited a distinctly different CNF1 intoxication phenotype from the prototypic one of multinucleation without cell death that is seen when HEp-2 or other epithelial cells are treated with CNF1. The 5637 bladder cell line detached from the growth surface within 72 h of CNF1 intoxication, a finding that suggested frank cytotoxicity. To determine the basis for the unexpected toxic effect of CNF1 on 5637 cells, we compared the degree of toxin binding, actin fiber formation, and Rho modification with those CNF1-induced events in HEp-2 cells. We found no apparent difference in the amount of CNF1 bound to 5637 cells and HEp-2 cells. Moreover, CNF1 modified Rho, in vivo and in vitro, in both cell types. In contrast, one of the classic responses to CNF1 in HEp-2 and other epithelial cell lines, the formation of actin stress fibers, was markedly absent in 5637 cells. Indeed, actin stress fiber induction by CNF1 did not occur in any of the other human bladder cell lines that we tested (J82, SV-HUC-1, or T24). Furthermore, the appearance of lamellipodia and filopodia in 5637 cells suggested that CNF1 activated the Cdc42 and Rac proteins. Finally, apoptosis was observed in CNF1-intoxicated 5637 cells. If our results with 5637 cells reflect the interaction of CNF1 with the transitional uroepithelium in the human bladder, then CNF1 may be involved in the exfoliative process that occurs in that organ after infection with uropathogenic E. coli.

Effect of lovastatin on small GTP binding proteins and on TGF-beta1 and fibronectin expression.

We have shown that lovastatin, an inhibitor of 3 hydroxy-3-methylglutary coenzyme A (HMG CoA) reductase, delays development and progression of diabetic nephropathy in streptozotocine-induced diabetic rats through suppression of glomerular transforming growth factor (TGF)-beta1 mRNA expression. We have also shown that lovastatin suppresses both control and high glucose (HG)-induced TGF-beta1 and fibronectin mRNA expression and protein synthesis by rat mesangial cell (RMC) and that this down-regulation by lovastatin is reversed by mevalonate. It was postulated that this down-regulation may be linked to signaling of small guanine triphosphate (GTP)-binding proteins and mediated by the limitation of isoprenoids such as farnesylpyrophosphate (FPP) and geranylgeranylpyrophosphate (GGPP) in RMC. To determine the isoprenoid and small GTP-binding proteins involved in TGF-beta1 and fibronectin expression. FPP or GGPP was added alone or in combination to RMC treated with lovastatin cultured under normal or high glucose condition. Suppression of TGF-beta1 and fibronectin expression by lovastatin was reversed effectively when GGPP was added alone. Partial reversal of lovastatin effect on fibronectin and TGF-beta1 expression was found when FPP was added alone. Adding both GGPP and FPP resulted in complete reversal of lovastatin effect on fibronectin but not TGF-beta1 suggesting that fibronectin and TGF-beta1 are regulated differently. Furthermore, luciferase activity of RMC cotransfected with fibronectin promoter reporter system and plasmid-expressing C3 exoenzyme (a specific inactivator of Rho family GTP binding proteins, pEFC3) was completely suppressed when compared with RMC cotransfected with empty vector, pEF. Because geranylgeranylation is usually involved in post-translational modification and membrane targeting of Rho family small GTP binding proteins, these data indicate that Rho family small GTP-binding proteins rather than Ras family small GTP binding proteins may play a key role in the TGF-beta1 and fibronectin expression in RMC.

Plant Rac Proteins Induce Superoxide Production in Mammalian Cells

The small GTP-binding protein family including Rac proteins represents a paradigm for signaling molecules shared by animal and plants. In mammalian cells, Rac induces the activation of NADPH oxidase leading to superoxide production. In plants, evidence suggests that resistance to pathogens depends on superoxide that is generated via NADPH oxidase-like enzymes. We have identified four closely related Rho/Rac genes from Zea mays that exhibit a high degree of homology to the human Rac. We hypothesized that these plant Rac proteins could function as their mammalian counterpart and activate an enzymatic complex that leads to superoxide production. Here, we show that like human Rac1, activated Zea mays Rac genes can induce superoxide production, when expressed in a mammalian system: NIH 3T3 cells. Our results suggest that in plants, Rac proteins can function as activators of oxidative burst and indicate the remarkable functional and structural conservation of Rho/Rac proteins between plant and animal kingdoms during evolution.

Cell Vacuolation Induced by the VacA Cytotoxin ofHelicobacter pylori Is Regulated by the Rac1 GTPase

Chronic gastric infection with the Gram-negative bacterium Helicobacter pylori is a major contributing factor in the development of duodenal ulcers and is believed to be a significant risk factor in the development of gastric tumors. The VacA cytotoxin of H. pylori is a 90-kDa secreted protein that forms trans-membrane ion channels. In epithelial cells, VacA activity is associated with the rapid formation of acidic vacuoles enriched for late endosomal and lysosomal markers. Rac1 is a member of the Rho family of small GTP-binding proteins that regulate reorganization of the actin cytoskeleton and intracellular signal transduction and are being shown increasingly to play a role in membrane trafficking events. In this study we report that: (i) green fluorescent-tagged Rac1 localizes around the perimeter of the vacuoles induced by VacA; (ii) expression of dominant negative Rac1 in epithelial cells inhibits vacuole formation; (iii) expression of constitutively active Rac1 potentiates the activity of VacA. Taken together, these data demonstrate a role for Rac1 in the regulation of VacA activity.

Altered focal adhesion regulation correlates with cardiomyopathy in mice expressing constitutively active rac1.

The ras family of small GTP-binding proteins exerts powerful effects upon cell structure and function. One member of this family, rac, induces actin cytoskeletal reorganization in nonmuscle cells and hypertrophic changes in cultured cardiomyocytes. To examine the effect of rac1 activation upon cardiac structure and function, transgenic mice were created that express constitutively activated rac1 specifically in the myocardium. Transgenic rac1 protein was expressed at levels comparable to endogenous rac levels, with activation of the rac1 signaling pathway resulting in two distinct cardiomyopathic phenotypes: a lethal dilated phenotype associated with neonatal activation of the transgene and a transient cardiac hypertrophy seen among juvenile mice that resolved with age. Neither phenotype showed myofibril disarray and hypertrophic hearts were hypercontractilein working heart analyses. The rac1 target p21-activated kinase translocated from a cytosolic to a cytoskeletal distribution, suggesting that rac1 activation was inducing focal adhesion reorganization. Corroborating results showed altered localizations of src in dilated cardiomyopathy and paxillin in both cardiomyopathic phenotypes. This study, the first examination of rac1-mediated cardiac effects in vivo, demonstrates that dilation and hypertrophy can share a common molecular origin and presents evidence that both timing and concurrent signaling from multiple pathways can influence cardiac remodeling.

Rem2, a new member of the Rem/Rad/Gem/Kir family of Ras-related GTPases

Here we report the molecular cloning and biochemical characterization of Rem2 (for Rem, Rad and Gem-related 2), a novel GTP-binding protein identified on the basis of its homology with the Rem, Rad, Gem and Kir (RGK) family of Ras-related small GTP-binding proteins. Rem2 mRNA was detected in rat brain and kidney, making it the first member of the RGK family to be expressed at relatively high levels in neuronal tissues. Recombinant Rem2 binds GTP saturably and exhibits a low intrinsic rate of GTP hydrolysis. Surprisingly, the guanine nucleotide dissociation constants for both Rem2 and Rem are significantly different than the majority of the Ras-related GTPases, displaying higher dissociation rates for GTP than GDP. Localization studies with green fluorescent protein (GFP)-tagged recombinant protein fusions indicate that Rem2 has a punctate, plasma membrane localization. Deletion of the C-terminal seven amino acid residues that are conserved in all RGK family members did not affect the cellular distribution of the GFP fusion protein, whereas a larger deletion, including much of the polybasic region of the Rem2 C-terminus, resulted in its redistribution to the cytosol. Thus Rem2 is a GTPase of the RGK family with distinctive biochemical properties and possessing a novel cellular localization signal, consistent with its having a unique role in cell physiology.

Multiple endocytic pathways of G protein-coupled receptors delineated by GIT1 sensitivity.

Recently, we identified a GTPase-activating protein for the ADP ribosylation factor family of small GTP-binding proteins that we call GIT1. This protein initially was identified as an interacting partner for the G protein-coupled receptor kinases, and its overexpression was found to affect signaling and internalization of the prototypical beta(2)-adrenergic receptor. Here, we report that GIT1 overexpression regulates internalization of numerous, but not all, G protein-coupled receptors. The specificity of the GIT1 effect is not related to the type of G protein to which a receptor couples, but rather to the endocytic route it uses. GIT1 only affects the function of G protein-coupled receptors that are internalized through the clathrin-coated pit pathway in a beta-arrestin- and dynamin-sensitive manner. Furthermore, the GIT1 effect is not limited to G protein-coupled receptors because overexpression of this protein also affects internalization of the epidermal growth factor receptor. However, constitutive agonist-independent internalization is not regulated by GIT1, because transferrin uptake is not affected by GIT1 overexpression. Thus, GIT1 is a protein involved in regulating the function of signaling receptors internalized through the clathrin pathway and can be used as a diagnostic tool for defining the endocytic pathway of a receptor.

Mapping the binding site for the GTP-binding protein Rac-1 on its inhibitor RhoGDI-1

Background: Members of the Rho family of small GTP-binding proteins, such as Rho, Rac and Cdc42, have a role in a wide range of cell responses. These proteins function as molecular switches by virtue of a conformational change between the GTP-bound (active) and GDP-bound (inactive) forms. In addition, most members of the Rho and Rac subfamilies cycle between the cytosol and membrane. The cytosolic guanine nucleotide dissociation inhibitors, RhoGDIs, regulate both the GDP/GTP exchange cycle and the membrane association/dissociation cycle. Results: We have used NMR spectroscopy and site-directed mutagenesis to identify the regions of human RhoGDI-1 that are involved in binding Rac-1. The results emphasise the importance of the flexible regions of both proteins in the interaction. At least one specific region (residues 46–57) of the flexible N-terminal domain of RhoGDI, which has a tendency to form an amphipathic helix in the free protein, makes a major contribution to the binding energy of the complex. In addition, the primary site of Rac-1 binding on the folded domain of RhoGDI involves the β4–β5 and β6–β7 loops, with a slight movement of the 3 10 helix accompanying the interaction. This binding site is on the same face of the protein as the binding site for the isoprenyl group of post-translationally modified Rac-1, but is distinct from this site. Conclusions: Isoprenylated Rac-1 appears to interact with three distinct sites on RhoGDI. The isoprenyl group attached to the C terminus of Rac-1 binds in a pocket in the folded domain of RhoGDI. This is distinct from the major site on this domain occupied by Rac-1 itself, which involves two loops at the opposite end to the isoprenyl-binding site. It is probable that the flexible C-terminal region of Rac-1 extends from the site at which Rac-1 contacts the folded domain of RhoGDI to allow the isoprenyl group to bind in the pocket at the other end of the RhoGDI molecule. Finally, the flexible N terminus of RhoGDI-1, and particularly residues 48–58, makes a specific interaction with Rac-1 which contributes substantially to the binding affinity.

Characterization of a small GTP-binding protein of the rab 5 family in Mesembryanthemum crystallinum with increased level of expression during early salt stress.

A cDNA encoding a member of the Ypt/Rab family of small GTP-binding proteins was cloned from the facultative CAM plant Mesembryanthemum crystallinum. Mcrab5b includes an open reading frame of 201 amino acids. The deduced amino acid sequence shows 91% similarity to LjRAB5b isolated from Lotus japonicus. The amino acid sequence of McRAB5b provides interesting features suggesting that McRAB5b and its homologue from Lotus japonicus represent a new subclass of Ypt/Rab proteins. The fact that proteins like McRAB5b and LjRAB5b were only found in plants and not in yeast or vertebrates suggests that they have plant-specific functions. The expression of Mcrab5b as investigated by northern blot hybridization and RT-PCR was stimulated under salt stress. After heterologous expression in Escherichia coli an antibody was raised against recombinant McRABSb protein. Western blot analysis revealed that McRAB5b was bound to membranes. It is present in a monomeric and a dimeric form in vitro and in vivo. In vitro only the monomeric protein exhibits a binding capacity for radiolabelled GTP, while the dimer is unable to do so, indicating that the activity may be regulated by monomer/dimer transition.

Linking Arf to Membrane Ruffles

In response to certain growth factors, cells reorganize their actin cytoskeleton, sometimes causing ruffling of their surface plasma membrane. Because the phospholipid phosphatidylinositol 4,5 bisphosphate [PI(4,5)P2] binds to certain actin-binding proteins, it has been implicated in signaling pathways that mediate this response. Although members of the Rho family of small GTP-binding proteins are thought to stimulate PI(4,5)P2 production in cells, Honda et al. report that another small GTP-binding protein, ARF6, may also regulate PI(4,5)P2 in cells by stimulating the α isoform of phosphatidylinositol 4-phosphate 5-kinase [PI(4)P5Kα]. When overexpressed in cells that were then stimulated with epidermal growth factor, ARF6 and PI(4)P5Kα colocalized to membrane ruffles. Phospholipase D2, which generates phosphatidic acid (PA), also localized to membrane ruffles. PA had a synergistic effect on the stimulation of PI(4)P5Kα by ARF6 in vitro. The authors suggest that an ARF6-dependent pathway regulates membrane ruffling and may work in parallel to regulatory pathways that depend on rho-related proteins. Honda, A., Nogami, M., Yokozeki, T., Yamazaki, M., Nakamura, H., Watanabe, H., Kawamoto, K., Nakayama, K., Morris, A.J., Frohman, M.A., and Kanaho, Y. (1999) Phosphatidylinositol 4-phosphate 5-kinase α is a downstream effector of the small G protein ARF6 in membrane ruffle formation. Cell 99 : 521-532. [Online Journal]

RA-GEF, a Novel Rap1A Guanine Nucleotide Exchange Factor Containing a Ras/Rap1A-associating Domain, Is Conserved between Nematode and Humans

A yeast two-hybrid screening for Ras-binding proteins in nematode Caenorhabditis elegans has identified a guanine nucleotide exchange factor (GEF) containing a Ras/Rap1A-associating (RA) domain, termed Ce-RA-GEF. Both Ce-RA-GEF and its human counterpart Hs-RA-GEF possessed a PSD-95/DlgA/ZO-1 (PDZ) domain and a Ras exchanger motif (REM) domain in addition to the RA and GEF domains. They also contained a region homologous to a cyclic nucleotide monophosphate-binding domain, which turned out to be incapable of binding cAMP or cGMP. Although the REM and GEF domains are conserved with other GEFs acting on Ras family small GTP-binding proteins, the RA and PDZ domains are unseen in any of them. Hs-RA-GEF exhibited not only a GTP-dependent binding activity to Rap1A at its RA domain but also an activity to stimulate GDP/GTP exchange of Rap1A both in vitro and in vivo at the segment containing its REM and GEF domains. However, it did not exhibit any binding or GEF activity toward Ras. On the other hand, Ce-RA-GEF associated with and stimulated GDP/GTP exchange of both Ras and Rap1A. These results indicate that Ce-RA-GEF and Hs-RA-GEF define a novel class of Rap1A GEF molecules, which are conserved through evolution.

An FH domain-containing Bnr1p is a multifunctional protein interacting with a variety of cytoskeletal proteins in Saccharomyces cerevisiae.

Proteins containing formin homology domains, FH1 and FH2, are involved in cytokinesis or establishment of cell polarity in a variety of organisms. Bni1p and Bnr1p are FH proteins and potential targets of the Rho family small GTP-binding proteins in S. cerevisiae. We have shown that Bnr1p is localized at the bud neck to interact with Hof1p, involved in cytokinesis. We report here that the overexpression of BNR1 causes a cytokinesis deficiency which is similar to the phenotypes of the septin mutants, including cdc3, cdc10, cdc11, and cdc12. The region required for the septin mutant phenotypes was mapped to Bnr1p (35-500), which coincided with the region required for the bud-neck localization. To further isolate a gene interacting with BNI1 or BNR1, a multicopy suppressor of the bni1 bnr1 mutant was isolated. This gene encoded Smy1p, a kinesin-related protein. Bnr1p, but not Bni1p, directly interacted with the C-terminal region of Smy1p. The Smy1p-interacting region of Bnr1p was mapped to a region containing the FH2 domain. Bnr1p also directly interacted with Bud6p, a novel actin-binding protein. Bnr1p is thus a multifunctional protein which interacts with the septin system, a microtubule-dependent motor protein, and the actin system, to regulate cytoskeletal functions in S. cerevisiae.

ADP-Ribosylation of Rho by C3 Ribosyltransferase Inhibits IL-2 Production and Sustained Calcium Influx in Activated T Cells

Activation of the T lymphocyte induces dramatic cytoskeletal changes, and there is increasing evidence that disruption of the cytoskeleton inhibits early and late events of T cell signal transduction. However, relatively little is known about the signaling molecules involved in activation-induced cytoskeletal rearrangement. The rho family of small GTP-binding proteins, which include rho, rac, and cdc42, regulates the cytoskeleton and coordinates various cellular functions via their many effector targets. In prior studies, the Clostridium botulinum toxin C3 exoenzyme has been used to ADP-ribosylate and inactivate rho. In this study, we demonstrate that treatment of T cells with C3 exoenzyme inhibits IL-2 transcription following ligation of the TCR. Inhibition of IL-2 expression correlated with loss of sustained increase in [Ca+2]i and mitogen activated protein kinase (MAPK/Erk) activity, but not with activation of the tyrosine kinase, lck. These findings are the first to show that ADP-ribosylation of rho by C3 ribosyltransferase (exoenzyme) inhibits IL-2 production due, in part, to the requirement for sustained calcium influx and MAPK activation after Ag receptor ligation.

Rho family small G proteins play critical roles in mechanical stress-induced hypertrophic responses in cardiac myocytes.

-Mechanical stress induces a variety of hypertrophic responses, such as activation of protein kinases, reprogramming of gene expression, and an increase in protein synthesis. In the present study, to elucidate how mechanical stress induces such events, we examined the role of Rho family small GTP-binding proteins (G proteins) in mechanical stress-induced cardiac hypertrophy. Treatment of neonatal rat cardiomyocytes with the C3 exoenzyme, which abrogates Rho functions, suppressed stretch-induced activation of extracellular signal-regulated protein kinases (ERKs). Overexpression of the Rho GDP dissociation inhibitor (Rho-GDI), dominant-negative mutants of RhoA (DNRhoA), or DNRac1 significantly inhibited stretch-induced activation of transfected ERK2. Overexpression of constitutively active mutants of RhoA slightly activated ERK2 in cardiac myocytes. Overexpression of C-terminal Src kinase, which inhibits functions of the Src family of tyrosine kinases, or overexpression of DNRas had no effect on stretch-induced activation of transfected ERK2. The promoter activity of skeletal alpha-actin and c-fos genes was increased by stretch, and these increases were completely inhibited by either cotransfection of Rho-GDI or pretreatment with C3 exoenzyme. Mechanical stretch increased phenylalanine incorporation into cardiac myocytes by approximately 1.5-fold compared with control, and this increase was also significantly suppressed by pretreatment with C3 exoenzyme. Overexpression of Rho-GDI or DNRhoA did not affect angiotensin II-induced activation of ERK. ERKs were activated by culture media conditioned by stretch of cardiomyocytes without any treatment, but not of cardiomyocytes with pretreatment by C3 exoenzyme. These results suggest that the Rho family of small G proteins plays critical roles in mechanical stress-induced hypertrophic responses.

Ras activity and cyclin D1 expression: An essential mechanism of mouse skin tumor development

ras is a family of small GTP-binding proteins that transduce signals from tyrosine-kinase receptors to the nucleus and thus play a role in the regulation of cell proliferation and differentiation. Several lines of evidence have shown that the cell-cycle machinery, specifically the circuit cyclin D1/cyclin-dependent kinase (cdk) 4 and 6-p16-pRb, lies downstream of ras. Point mutations that activate the ras protein and its downstream cascade have been observed in human and experimental tumors. In particular, ras mutations have been well characterized in the mouse skin two-stage carcinogenesis model, and a large body of literature has indicated that initiation with the genotoxic carcinogen 7,12-dimethylbenz[a]anthracene induces a specific point mutation in Ha-ras gene in this model. In the last few years, several studies have shown a correlation between ras activation and alterations in the expression of cyclin D1 as well as other cell cycle-regulated proteins, but the actual role of these alterations in tumor development had not been determined until a recent study provided genetic and biochemical evidence that cyclin D1 is a critical target of oncogenic ras in mouse skin carcinogenesis. Here we review these results, including the evidence that cyclin D1 has a role as a downstream mediator of ras activity during tumor development. We propose a model in which cyclin D1 has a unique growth-promoting role in tumor development but does not act as an oncogene independently of ras activity.

Cellular functions & pharmacological manipulations of the small GTPase Rho & Rho effectors

Rho is a member of the Ras-related family of small molecular weight GTP-binding proteins, and works as a molecular switch by shuttling between the GDP-bound inactive form and the GTP-bound active form. Cellular functions of Rho have been studied by two ways; one is to express or microinject constitutively active Rho mutants in cells to identify the active phenotype of Rho, and the other is to use botulinum C3 exoenzyme that specifically ADP-ribosylates and inactivates Rho in cells to find out phenotypes of Rho inactivation. These analyses have revealed that Rho is involved in cell to substratum adhesion and motility, cell contraction and cytokinesis through the reorganization of the actincytoskeleton and modulation of its activity. These actions of Rho are mediated by downstream Rho effectors. Several putative Rho effectors have been isolated on the basis of their selective binding to the GTP-bound form of Rho. Among them, the ROCK family of Rho-associated serine/threonine protein kinases inactivates cofilin and myosin phosphatase to induce stabilization of filamentous actin and increase in the actomyosin-based contractility. mDia binds profilin likely to promote actin polymerization. Thus, these effectors in combination are supposed to work in organization of various forms of the actin cytoskeleton. Furthermore, analyses using a ROCK specific inhibitor Y-27632 have suggested that the Rho-ROCK pathway works in contractions of vascular and bronchial smooth muscles under various pathological conditions and is involved in malignant cell transformation and tumor invasion and metastasis.