Rho P21 Research Articles

Regulation of Ca2+ sensitization by PKC and rho proteins in ovine cerebral arteries: effects of artery size and age.

G protein-regulated Ca2+ sensitivity of vascular contractile proteins plays an important role in cerebrovascular reactivity. The present study examines the intracellular mechanisms that govern G protein-regulated Ca2+ sensitivity in cerebral arteries of different size and age. We studied beta-escin-permeabilized segments of common carotid, basilar, and middle cerebral arteries from nonpregnant adult and near-term fetal sheep. Activation of protein kinase C (PKC) by (-)-indolactam V or a phorbol ester produced receptor-independent increases in Ca2+ sensitivity. Such increases were more marked in immature arteries and were inversely correlated with artery size in both mature and immature arteries. However, inhibitors of PKC did not significantly affect increases in Ca2+ sensitivity in responses to either serotonin (5-hydroxytryptamine, 5-HT) or guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS). Alternatively, deactivation of rho p21, a small G protein associated with Rho kinase, by exotoxin C3 fully prevented increases in Ca2+ sensitivity in responses to 5-HT or GTPgammaS in both adult and fetal arteries of all types. Neither inhibitors of PKC nor exotoxin C3 altered baseline Ca2+ sensitivity. We conclude that patterns of receptor- and/or G protein-mediated modulation of Ca2+ sensitivity are dependent on an intracellular pathway that involves activation of small G proteins and Rho kinase. In contrast, PKC has little, if any, role in agonist-induced Ca2+ sensitization under the present experimental conditions.

Cytotoxic necrotizing factor 1 from Escherichia coli: a toxin with a new intracellular activity for eukaryotic cells.

Certain pathogenic Escherichia coli strains elaborate a toxin, the cytotoxic necrotizing factor type 1 (CNF1). CNF1 covalently and specifically modifies the p21 Rho GTP-binding protein in mammalian cells by deamidation of the p21 Rho glutamine 63. CNF1 modification of Rho leads to permanent activation of the GTP-binding protein by blocking intrinsic and RhoGAP GTPase activities. Rho activation by CNF1 induces reorganization of the actin cytoskeleton into large stress fibers and the multiplication of focal contact points. Deamidation is a new catalytic activity described for an intracellularly acting toxin.

Neuromedin B receptor activation causes tyrosine phosphorylation of p125FAK by a phospholipase C independent mechanism which requires p21rho and integrity of the actin cytoskeleton.

Recent studies show that tyrosine phosphorylation by a number of neuropeptides may be an important intracellular pathway in mediating changes in cell function, particularly related to growth. Neuromedin B (NMB), a mammalian bombesin related peptide, functions through a distinct receptor, the neuromedin B receptor (NMB-R), of which little is known about its cellular basis of action. In the present study we explored the ability of NMB-R activation to cause tyrosine phosphorylation of focal adhesion kinase (p125(FAK)), an important substrate for tyrosine phosphorylation by other neuropeptides. NMB caused rapid increases in p125(FAK) phosphorylation which reached maximum at 2 min in both rat C6 glioblastoma cells which possess native NMB-Rs and rat neuromedin B receptor (rNMR-R) transfected BALB 3T3 cells. NMB had a half-maximal effect was at 0.4 nM and was 30-fold more potent than gastrin-releasing peptide (GRP). The stoichiometric relationships between increased p125(FAK) tyrosine phosphorylation and other cellular processes was similar in both C6 cells and rNMB-R transfected cells. TPA (1 microM) caused 45% and the calcium ionophore, A23187, 11% of maximal tyrosine phosphorylation of p125(FAK) seen with NMB. A23187 potentiated the effect of TPA. Pretreatment with the selective PKC inhibitor, GF109203X, inhibited TPA-induced p125(FAK) tyrosine phosphorylation, but it had no effect on the NMB stimulation. Pretreatment with thapsigargin completely inhibited NMB-stimulated increases in [Ca2+]i, but had no effect on NMB-stimulation of p125(FAK) phosphorylation either alone or with GF109203X. The tyrosine kinase inhibitor, tyrphostin A25, inhibited NMB-induced phosphorylation of p125(FAK) by 52%. However, tyrphostin A25 did not inhibit NMB-stimulated increases in [3H]inositol phosphates. Cytochalasin D, an agent which disrupts actin microfilaments, inhibited BN- and TPA-induced tyrosine phosphorylation of p125(FAK) completely. In contrast, colchicine, an agent which disrupts microtubules, had no effect. Pretreatment with Clostridium botulinum C3 exoenzyme which inactivates the small GTP-binding protein rho p21, also inhibited tyrosine phosphorylation of p125(FAK) by 55%. These results demonstrate that activation of NMB-R can cause rapid tyrosine phosphorylation of p125(FAK). NMB-induced tyrosine phosphorylation of p125(FAK) is independent of NMB-induced changes in [Ca2+]i or PKC. The integrity of the actin cytoskeleton but not of microtubules is necessary for NMB-stimulated phosphorylation of p125(FAK). The ras-related small GTP-binding protein rho p21 is at least partially involved in mediating NMB-induced tyrosine phosphorylation of p125(FAK). These results suggest that similar to some other neuropeptides, activation of this pathway may be an important mechanism in mediating cellular changes by this receptor such as growth.

Toxin-induced activation of the G protein p21 Rho by deamidation of glutamine.

Pathogenic Escherichia coli are responsible for a variety of diseases, including diarrhoea, haemolytic uraemic syndrome, kidney infection, septicaemia, pneumonia and meningitis. Toxins called cytotoxic necrotizing factors (CNFs) are among the virulence factors produced by uropathogenic (CNF1) or enteropathogenic (CNF2) E. coli strains that cause diseases in humans and animals, respectively. CNFs induce an increase in the content of actin stress fibres and focal contacts in cultured cells. Effects of CNFs on the actin cytoskeleton correlated with a decrease in the electrophoretic mobility of the GTP-binding protein Rho and indirect evidence indicates that CNF1 might constitutively activate Rho. Here we show that CNF1 catalyses the deamidation of a glutamine residue at position 63 of Rho, turning it into glutamic acid, which inhibits both intrinsic GTP hydrolysis and that stimulated by its GTPase-activating protein (GAP). Thus, this deamidation of glutamine 63 by CNF1 leads to the constitutive activation of Rho, and induces the reorganization of actin stress fibres. To our knowledge, CNF1 is the first example of a bacterial toxin acting by deamidation of a specific target protein.

Regulation of osteoclast function.

Regulation of osteoclast function.

The role of RhoA in tissue polarity and Frizzled signalling.

The tissue polarity genes of Drosophila are required for correct establishment of planar polarity in epidermal structures, which in the eye is shown in the mirror-image symmetric arrangement of ommatidia relative to the dorsoventral midline. Mutations in the genes frizzled (fz), dishevelled (dsh) and prickle-spiny-legs (pk-sple) result in the loss of this mirror-image symmetry. fz encodes a serpentine receptor-like transmembrane protein required for reception and transmission of a polarity signal. Little else is known of the signalling pathway(s) involved other than that Dsh acts downstream of Fz. We have identified mutations in the Drosophila homologue of RhoA p21 GTPase, and by analysis of their phenotype show that RhoA is required for the generation of tissue polarity. Genetic interactions indicate a role for RhoA in signalling mediated by Fz and Dsh, and furthermore suggest that JNK/SAPK-like kinases are involved. These data are consistent with a Fz/RhoA signalling cascade analogous to the yeast pheromone signalling pathway and that proposed for activation of the serum response factor (SRF) in vertebrate cells.

Analysis of the cellular functions of the small GTP-binding protein rho p21 with Clostridium botulinum C3 exoenzyme

rho p21 is a member of the ras superfamily of small GTPases. Clostridium botulinum C3 exoenzyme ADP-ribosylates rho p21 at the Asp41 residue located at an effector domain and inhibits its biological activity by interfering with the interaction with its downstream effectors. The amount of rho p21 in cells or tissues is determined by the in vitro ADP-ribosylation reaction with C3 exoenzyme and 32P NAD. The studies using C3 exoenzyme have revealed that rho p21 is involved in the regulation of stress fiber formation, cell adhesion, contractile ring formation during cytokinesis and serum response factor-mediated activation of immediate early genes. C3 exoenzyme is a valuable tool for elucidating the unidentified function of rho p21 because the exoenzyme specifically inhibits rho p21-mediated signal transduction pathways. A Glu173 substitution mutant of the C3 exoenzyme lacking ADP-ribosyltransferase activity is useful for a control experiment.

O-98 - Protein kinase N, a putative effector of the small GTPase rho p21. regulates transcription by serum response factor

O-98 - Protein kinase N, a putative effector of the small GTPase rho p21. regulates transcription by serum response factor

Involvement of rho p21 in Cyclic Strain-Induced Tyrosine Phosphorylation of Focal Adhesion Kinase (pp125FAK), Morphological Changes and Migration of Endothelial Cells

The molecular mechanisms by which endothelial cells sense and respond to physical forces remain to be elucidated. Recently we reported that cyclic strain-induced morphological change and migration of EC were regulated by the tyrosine phosphorylation of focal adhesion kinase (pp125FAK) and paxillin. The aim of the present study was to clarify the role of the small GTP-binding protein rho p21 in EC exposed to cyclic strain. Bovine aortic endothelial cells (EC) were subjected to 10% average strain at 60 cycle/min.Clostridium botulinumC3transferase (C3) was used as a specific inhibitor of rho p21. Preincubation of EC with C3inhibited ADP-ribosylation of rho (94%) and inhibited the morphological change, reorganization of actin filaments, and migration induced by cyclic strain. Moreover, C3inhibited the cyclic strain-induced tyrosine phosphorylation of pp125FAKand paxillin. These results demonstrate that rho downregulates the tyrosine phosphorylation of pp125FAKand paxillin and can modulate the morphological changes and migration induced by cyclic strain.

Rho-Mediated protein tyrosine phosphorylation in lysophosphatidic-acid-induced tumor-cell invasion.

Rat ascites hepatoma cell (MM1) invade a mesothelial cell monolayer in vitro in assay medium containing serum, but not in serum-free medium. Serum could be completely replaced by 1-oleoyl lysophosphatidic acid (LPA) in inducing invasion. LPA-induced invasion was inhibited by genistein, a tyrosine-kinase inhibitor. Protein tyrosine phosphorylation in response to LPA was thus analyzed in order to determine the molecular mechanism of invasion. LPA of invasion-inducible concentrations evoked a transient increase in tyrosine phosphorylation, mainly of 110- to 130-kDa proteins in MM1 cells but not in mesothelial cells. These concentrations of LPA were over 10 times higher (10 to 25 micron) than those necessary to produce a variety of biological actions, such as tyrosine phosphorylation in fibroblasts, neurite retraction and platelet aggregation. Protein tyrosine phosphorylation and invasion by MM1 cells induced by LPA are largely regulated by rho p21, because both were inhibited by Clostridium botulinum C3 exo-enzyme, which is known to specifically inactivate rho p21. Invasion of MCL by MM1 cells induced by serum and that by B16FE7 cells induced by LPA were inhibited by genistein or C3 as well. By immunoprecipitation, we detected p 125 focal adhesion kinase (FAK) as a major protein of 110- to 130-kDa tyrosine phosphorylated in response to LPA. Tyrosine phosphorylation of paxillin by LPA was also detected.

Regulation of vascular smooth muscle contraction. The roles of Ca2+, protein kinase C and myosin light chain phosphatase.

Vasoconstrictors bind to their receptors on the cell surface to active phospholipase C and Ca2+ channels, resulting in the mobilization of Ca2+ from intracellular are extracellular Ca2+ pools and protein kinase C activation. Vasoconstrictors are also thought to activate a distinct cellular mechanism for downregulating 20 kDa myosin light chain (MLC20) phosphatase activity, which involves Rho p21 and protein kinase C, resulting in an increase in the Ca2+ sensitivity of MLC20 phosphorylation. Protein kinase C also appears to activate a MLC20 phosphorylation-independent mechanism for contraction, contributing to the maintenance of agonist-induced contraction. On the other hand, vasorelaxants inhibit activation of phospholipase C and gating of Ca2+ channels, or stimulate Ca2+ extrusion across the plasma membrane, leading to a decrease in the [Ca2+]i. Vasorelaxants also appear to stimulate MLC20 phosphatase activity, resulting in a further reduction of contractile response. The modulatory mechanism for changing the Ca2+ sensitivity, together with the major regulatory mechanism for cellular Ca2+ metabolism, plays an important role in regulating vascular smooth muscle tone.

Regulation of exocytosis by the small GTP-binding protein rho in rat basophilic leukemia (RBL-2H3) cells

1. 1. We investigated the effect of Clostridium botulinum C3 ADP-ribosyltransferase upon β-hexosaminidase release induced by various stimuli from streptolysin-O (0.5-1 U/ml)-permeabilized rat basophilic leukemia (RBL-2H3) cells. 2. 2. The C3 transferase inhibited β-hexosaminidase release induced by Ca 2+ or by guanosine-5'-(3-thio-triphosphate) (GTPyS) plus Ca 2+. 3. 3. The C3 transferase also inhibited β-hexosaminidase release induced by stimulating high affinity IgE and m3 muscarinic acetylcholine receptors. 4. 4. The substrate for the C3 transferase was present in cytosol of RBL-2H3 cells, indicating the presence of rho p21. About 60% of the total cellular substrate protein remained within the cells permeabilized by 1 U/ml of streptolysin-O. 5. 5. The protein rho p21 appears to be regulated by several pathways and it may function as an integration point for exocytosis.

Synergistic Activation of Rat Brain Phospholipase D by ADP-ribosylation Factor and rhoA p21, and Its Inhibition by Clostridium botulinum C3 Exoenzyme

An activator of rat brain phospholipase D (PLD) that is distinct from the already identified PLD activator, ADP-ribosylation factor (ARF), was partially purified from bovine brain cytosol by a series of chromatographic steps. The partially purified preparation contained a 22-kDa substrate for Clostridium botulinum C3 exoenzyme ADP-ribosyltransferase, which strongly reacted with anti-rhoA p21 antibody, but not with anti-rac1 p21 or anti-cdc42Hs p21 antibody. Treatment of the partially purified PLD-activating factor with both C3 exoenzyme and NAD significantly inhibited the PLD-stimulating activity. These results suggest that rhoA p21 is, at least in part, responsible for the PLD-stimulating activity in the preparation. Recombinant isoprenylated rhoA p21 expressed in and purified from Sf9 cells activated rat brain PLD in a concentration- and GTP gamma S (guanosine 5'-O-(3-thiotriphosphate))-dependent manner. In contrast, recombinant non-isoprenylated rhoA p21 (fused to glutathione S-transferase) expressed in Escherichia coli failed to activate the PLD. This difference cannot be explained by a lower affinity of non-isoprenylated rhoA p21 for GTP gamma S, as the rates of [35S]GTP gamma S binding were very similar for both recombinant preparations and the GTP gamma S-bound form of non-isoprenylated rhoA p21 did not induce PLD activation. Interestingly, recombinant isoprenylated rhoA p21 and ARF synergistically activated rat brain PLD; a similar pattern was seen with the partially purified PLD-activating factor. The synergistic activation was inhibited by C3 exoenzyme-catalyzed ADP-ribosylation of recombinant isoprenylated rhoA p21 in a NAD-dependent manner. Inhibition correlated with the extent of ADP-ribosylation. These findings suggest that rhoA p21 regulates rat brain PLD in concert with ARF, and that isoprenylation of rhoA p21 is essential for PLD regulation in vitro.

Escherichia coli cytotoxic necrotizing factor 1: evidence for induction of actin assembly by constitutive activation of the p21 Rho GTPase.

Cytotoxic necrotizing factor type 1 (CNF1) induces in HEp-2 cells an increase in F-actin structures, which was detectable by fluorescence-activated cell sorter analysis 24 h after addition of this factor to the culture medium. Increase in F-actin was correlated with the augmentation of both the cell volume and the total cell actin content. Actin assembly-disassembly is controlled by small GTP-binding proteins of the Rho family, which have been reported recently to be modified by CNF1 treatment. Clostridium difficile toxin B and Clostridium botulinum exoenzyme C3, both known to act on the Rho GTPase, were used as biological tools to study the effect of CNF1 on this protein. CNF1 incubated before, during, or after exposure to the chimeric toxin C3B (which is the product of a genetic fusion between the DNA coding for C3 and the one coding for the B fragment of diphtheria toxin) protected HEp-2 cells from the disruption of F-actin structures caused by inactivation of the Rho GTPase through its ADP-ribosylation. On the other hand, C. difficile toxin B cytopathic effect was not observed upon preincubation of cells with CNF1. Toxins acting through a Rho-independent mechanism, such as cytochalasin D and Clostridium spiroforme iota-like toxin, could not be modified in their cellular activities by CNF1 treatment. All of our results suggest that CNF1 modifies the Rho molecule, thus probably protecting this GTPase from further bacterial toxin modification.

Two SH2 Domains of p120 Ras GTPase-activating Protein Bind Synergistically to Tyrosine Phosphorylated p190 Rho GTPase-activating Protein

p120 GTPase-activating protein (GAP) is a negative regulator of Ras that functions at a key relay point in signal transduction pathways that control cell proliferation. Among other proteins, p120 GAP associates with p190, a GAP for the Ras-related protein, Rho. To characterize the p120.p190 interaction further, we used bacterially expressed glutathione S-transferase fusion polypeptides to map the regions of p120 necessary for its interactions with p190. Our results show that both the N-terminal and the C-terminal SH2 domains of p120 are individually capable of binding p190 expressed in a baculovirus/insect cell system. Moreover, the two SH2 domains together on one polypeptide bind synergistically to p190, and this interaction is dependent on tyrosine phosphorylation of p190. In addition, mutation of the highly conserved Arg residues in the critical FLVR sequences of both SH2 domains of full-length p120 reduces binding to tyrosine-phosphorylated p190. The dependence on p190 phosphorylation for complex formation with p120 SH2 domains observed in vitro is consistent with analysis of the native p120.p190 complexes formed in vivo. These findings suggest that SH2-phosphotyrosine interaction is one mechanism by which the cell regulates p120.p190 association and thus may be a means for coordinating the Ras- and Rho-mediated signaling pathways.

The small gtp-binding protein, RHO p21, is involved in bone resorption by regulating cytoskeletal organization in osteoclasts

Rho protein (rho p21), a p21ras-related small guanine nucleotide binding protein, regulates cytoskeletal organization in a number of different types of cells. Evidence has indicated that Clostridium botulinum-derived ADP-ribosyltransferase (C3 exoenzyme) specifically ADP-ribosylates rho p21 at Asn41 and renders it functionally inactive. In this study, we examined the involvement of rho p21 in osteoclastic bone resorption using the C3 exoenzyme. When osteoclast-like multinucleated cells obtained from cocultures of mouse osteoblastic cells and bone marrow cells were placed on dentine slices, they formed ringed structures of podosomes containing F-actin (corresponding to the clear zone) within 8 hours. Many resorption pits were formed on dentine slices after culture for 24 hours. The C3 exoenzyme at 0.15-10 micrograms/ml added to the culture medium disrupted the ringed structure of podosomes in osteoclast-like cells in a dose-dependent manner. Correspondingly, pit formation by osteoclast-like cells on dentine slices was dose-dependently inhibited also by adding the C3 exoenzyme. Microinjection of the C3 exoenzyme into osteoclast-like cells placed on culture dishes completely disrupted the ringed podosome structure within 20 minutes. The amount of the rho p21 which was ADP-ribosylated by the C3 exoenzyme in vitro was much greater in purified osteoclast-like cells than in osteoblastic cells. Prior exposure of the purified osteoclast-like cell preparation to the C3 exoenzyme in vivo markedly decreased the amount of unribosylated rho p21. This indicated that the C3 exoenzyme incorporated into osteoclast-like cells effectively ADP-ribosylates rho p21 in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)

S27-4 - rho p21 and its signalling pathway; a potential drug target for cell adhesion and tumor metastasis

S27-4 - rho p21 and its signalling pathway; a potential drug target for cell adhesion and tumor metastasis

32] Inhibition of p21 Rho in intact cells by C3 diphtheria toxin chimera proteins

This chapter describes the realization of a fusion protein between exoenzyme C3 and diphtheria toxin (DT) fragment B and discusses the use of this molecule on cultured cells to inactivate the p21 Rho, thereby producing an alteration of the actin microfilament network. Clostridium botulinum ( C . botulinum ) C and D strains produce an ADP-ribosyltransferase called “exoenzyme C3,” which selectively modifies the p21 GTP-binding protein Rho on its asparagine-41. The inactivation of the Rho protein by ADP-ribosylation on introduction (by microinjection or forced pinocytosis) of exoenzyme C3 into the cytosol induces the loss of actin filaments in cultured cells. Thus, the use of exoenzyme C3 is of great help in understanding the functional role of the p21 Rho protein that belongs to the p21 Ras superfamily of small GTP-binding proteins. DT is synthesized by toxigenic Corynebacterium diphtheriae as a single-chain protein that is subsequently cleaved into two fragments linked by a disulfide bridge.

24] Purification and assay of kinases that interact with Rac/Cdc42

This chapter describes a method to detect a new family of Rho p21 interacting proteins. It also describes the use of the assay for expression screening and in the purification of an abundant Cdc42 and Rac1 target, which is the brain serine/threonine kinase p65-PAK. The GTPase-activating protein (GAP) overlay technique not only detects tissue levels of GAPs after sodium dodecyl sulfate (SDS) electrophoresis but also detects GTPase-inhibitory proteins. For these proteins to exert such an effect on the intrinsic GTPase activity of p21, they must remain bound (that is, they cannot act in a catalytic manner as do GAPs). After autoradiography of the nitrocellulose overlay filter, which holds the image of the nucleotide state of the test p21 within the original filter, some of these proteins can be observed as signals darker than the background. The process of detection of SDS–polyacrylamide-fractionated GTP-p21-binding proteins is described in the chapter.

Possible Involvement of a Small G-Protein Sensitive to Exoenzyme C3 of Clostridium botulinum in the Regulation of Myofilament Ca2+ Sensitivity in β-Escin Skinned Smooth Muscle of Guinea Pig Ileum

The effects of exoenzyme C3 of Clostridium botulinum on Ca(2+)- and drug-induced tension developments were investigated in beta-escin skinned smooth muscle of guinea pig ileum to test the involvement of a small G-protein in the regulation of myofilament Ca2+ sensitivity. C3 is known to ADP-ribosylate the rho p21 family of small G-proteins. Treatment with C3 (0.35 microgram/ml, for 30 min) shifted the pCa-tension curve rightward along the Ca2+ concentration axis, indicating a decrease in Ca2+ sensitivity of the contractile elements. The inhibitory effect of C3 was not preserved after treatment with GDP beta S (1 mM), an antagonist of GTP for the binding to G-proteins. Stimulation of muscarinic receptors with carbachol (CCh, 100 microM) shifted the pCa-tension curve leftward, indicating Ca2+ sensitization of tension development. The Ca(2+)-sensitizing effect of CCh was not observed after C3 treatment. When GTP gamma S (10 microM), an activator of G-proteins, was applied at a plateau of tension development produced by a moderate concentration of Ca2+, further increase in tension was elicited and the effect of GTP gamma S was inhibited by C3 treatment. The results suggest the possible involvement of a rho p21-like small G-protein in the regulation of Ca2+ sensitivity of smooth muscle myofilaments.