Identification of <i>rho</i> as a substrate for botulinum toxin C<sub>3</sub>‐catalyzed ADP‐ribosylation

Quilliam, Lawrence A.; Lacal, Juan Carlos; Bokoch, Gary M.

doi:10.1016/0014-5793(89)81339-0

Cited by 39 publications

(12 citation statements)

References 29 publications

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“…In Fig. 3, the resulting autoradiogram revealed that C3 specifically labeled a species that migrated with an apparent molecular mass of approximately 25 kDa on a 11.25% polyacrylamide gel; this is consistent with previously published reports of the specificity for C3 catalyzed ribosylation of Rho (23,24,37). Lysates from cells cultured in the presence of C3 (lanes 2, 4, 6, 8, 10, and 12) showed a greatly reduced level of 32 P-labeled Rho indicating prior endogenous ADP-ribosylation.…”

Section: C3 Exoenzyme Inhibits Cell Proliferation-given a Role Forsupporting

confidence: 79%

ADP-ribosylation of the G Protein Rho Inhibits Integrin Regulation of Tumor Cell Growth

Udagawa

McIntyre

1996

Journal of Biological Chemistry

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Using a gastric derived tumor line, we investigated the involvement of ␤1 integrin and Rho in cell growth regulation in response to collagen. The addition of C3 exoenzyme from Clostridium botulinum to specifically ribosylate and inhibit the function of the rho gene products inhibited cellular proliferation in a dose-dependent fashion. C3 exoenzyme exhibited broad cytostatic activity toward a number of tumor lines and induced G 0 /G 1 accumulation, cyclin A inhibition, and pronounced alterations in cell morphology. Integrin-mediated adhesion to collagen led to the expression of the cyclin A gene whose expression could be blocked using anti-␤1 integrin monoclonal antibodies. Phospholipid levels were induced upon ␤1 integrin-mediated adhesion to collagen, and the phospholipid induction was inhibited by either antibodies to ␤1 integrin or pretreatment of cells with C3 exoenzyme. Significant reduction in phospholipid levels correlated with proliferation for a panel of tumor lines deprived of adhesion to substrate. These results implicate a novel role for integrins and Rho in the regulation of tumor growth in response to matrix.The integrins are a family of adhesion receptors that play a role in the interaction of cells with the extracellular matrix (1, 2). Cellular interactions with the extracellular matrix play a role in diverse processes such as differentiation (3, 4), lymphocyte activation (5), and tumor cell dissemination and metastasis (6). Collagen is a major component of the extracellular matrix that serves as a scaffold for cell binding but also plays a role in cell differentiation and growth (7).In vitro, interaction of cells with a collagen matrix can induce arachidonic acid production in HeLa cells (8) and glandular differentiation in a human colon carcinoma line (9). Clustering of the ␣2␤1 integrin, a receptor for collagen, induces stimulation of tyrosine phosphorylation and accumulation of GTP bound Ras in a human lymphoblastic cell line (10). The interaction of cells with the extracellular matrix can also regulate growth by creating a permissive effect for the action of mitogens (11, 12). Therefore, specific interaction between integrins and matrix components provide another level of growth regulation. A potential mediator of integrin signaling is the small GTP binding protein Rho. It is a member of the Ras family of GTPases that regulates the formation of actin stress fibers in response to growth factors (13). Integrins can also regulate the formation of actin stress fibers (14), which suggests a convergence of integrin and Rho signaling pathways.A role in mitogen signaling has been demonstrated for Rho using Val-14 mutations, analogous to Val-12 oncogenic mutations in Ras, which induced transformation of fibroblasts (15). Amplification of Rho by transfection resulted in cells that exhibited increased tumorigenicity, higher saturation density, and reduced serum dependence (16). Mutations in Dbl, a guanine exchange factor for Rho, also induced cellular transformation (17,18). Recent reports have demonst...

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Section: C3 Exoenzyme Inhibits Cell Proliferation-given a Role Forsupporting

confidence: 79%

ADP-ribosylation of the G Protein Rho Inhibits Integrin Regulation of Tumor Cell Growth

Udagawa

McIntyre

1996

Journal of Biological Chemistry

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“…How ADPribosylation inactivates Rho is not clear. ADP-ribosylation does not affect the interaction of Rho with its upstream regulators such as RhoGAP (29 kDa) (Paterson et al, 1990;Morii et al, 1991), GDP dissociation inhibitor, or stimulator proteins , nor does ADP-ribosylation alter its intrinsic or GAP-stimulated GTPase activities, or its intrinsic GDP/GTP exchange rate (Kikuchi et al, 1988;Quilliam et al, 1989;Paterson et al, 1990). It is therefore likely that ADP-ribosylation affects the interaction of Rho with its effector(s).…”

Section: Discussionmentioning

confidence: 99%

Physical association of the small GTPase Rho with a 68-kDa phosphatidylinositol 4-phosphate 5-kinase in Swiss 3T3 cells.

Ren

Bokoch

Traynor‐Kaplan

et al. 1996

MBoC

201

125

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Our previous work showed that post-translationally modified Rho in its GTP-bound state stimulated phosphatidylinositol 4-phosphate 5-kinase (PIP5K) activity in mouse fibroblast lysates. To investigate whether Rho physically interacts with PIP5K, we incubated immobilized Rho-GST with Swiss 3T3 cell lysates and tested for retained PIP5K activity. Rho-GST, but not Ras-GST or GST alone, bound significant PIP5K activity. The binding of PIP5K was independent of whether Rho was in a GTP-or GDP-bound state. An antibody against a 68-kDa human erythrocyte type I PIP5K recognized a single 68-kDa protein eluted from Rho-GST column. The Rho-associated PIP5K responded to phosphatidic acid differentially from the erythrocyte type I PIP5K, suggesting that it could be a distinct isoform not reported previously. Rho co-immunoprecipitated with the 68-kDa PIP5K from Swiss 3T3 lysates, demonstrating that endogenous Rho also interacts with PIP5K. ADP-ribosylation of Rho with C3 exoenzyme enhanced PIP5K binding by approximately eightfold, consistent with the ADP-ribosylated Rho functioning as a dominant negative inhibitor. These results demonstrate that Rho physically interacts with a 68-kDa PIP5K, although whether the association is direct or indirect is unknown. INTRODUCTIONThe small GTPase Rho plays a key role in the regulation of actin filament polymerization and cell growth (Hall, 1994;Olson et al., 1995). Like other GTPases, Rho proteins cycle between a GDP-bound inactive state and a GTP-bound active state. In the GTP-bound state, Rho activates downstream effector(s), leading to the assembly of actin stress fibers and focal adhesions. These effects can be blocked by ADP-ribosylation of endogenous Rho protein within the putative effectorbinding region (amino acids 32-42) with C3 exoenzyme from Clostridium botulinum (Paterson et al., 1990;Ridley and Hall, 1992). Rho proteins (193 amino acids)

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“…In contrast, the Rho-and Cdc42-activating toxin CNF1 (22, 40) had no effect on ACh release. C3 selectively ADP-ribosylates Rho isoforms at low concentrations (35,36), but also affects Rac at high doses (35,(37)(38)(39). C3 inhibited neurotransmission only when injected at micromolar concentrations into the presynaptic neuron.…”

Section: Discussionmentioning

confidence: 99%

“…Thus, ACh release from presynaptic neurons can be monitored by measuring the amplitude of the evoked transmembrane current in the postsynaptic neuron. To probe the role of Rho-related GTPases in neurotransmitter release, we used ToxB, which specifically glucosylates Rac, Cdc42, and Rho (34); LT, which glucosylates Rac, but has no effect on Rho and Cdc42 (25); and C3, which ADP-ribosylates RhoA, RhoB, RhoC, and Aplysia Rho (35,36) and, under certain circumstances, Rac (35,(37)(38)(39). Glucosylation or ADP-ribosylation in the effector domain of the various Rho, Rac, and Cdc42 isoforms disrupts their interaction with downstream effectors and thereby inactivates the intracellular pathways controlled by these GTPases.…”

Section: Effect Of Clostridial Toxins That Inactivate Rho-related Promentioning

confidence: 99%

A Rho-related GTPase Is Involved in Ca2+-dependent Neurotransmitter Exocytosis

Doussau¹,

Gasman²,

Humeau³

et al. 2000

Journal of Biological Chemistry

100

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Rho, Rac, and Cdc42 monomeric GTPases are well known regulators of the actin cytoskeleton and phosphoinositide metabolism and have been implicated in hormone secretion in endocrine cells. Here, we examine their possible implication in Ca 2؉ -dependent exocytosis of neurotransmitters. Using subcellular fractionation procedures, we found that RhoA, RhoB, Rac1, and Cdc42 are present in rat brain synaptosomes; however, only Rac1 was associated with highly purified synaptic vesicles. To determine the synaptic function of these GTPases, toxins that impair Rho-related proteins were microinjected into Aplysia neurons. We used lethal toxin from Clostridium sordellii, which inactivates Rac; toxin B from Clostridium difficile, which inactivates Rho, Rac, and Cdc42; and C3 exoenzyme from Clostridium botulinum and cytotoxic necrotizing factor 1 from Escherichia coli, which mainly affect Rho. Analysis of the toxin effects on evoked acetylcholine release revealed that a member of the Rho family, most likely Rac1, was implicated in the control of neurotransmitter release. Strikingly, blockage of acetylcholine release by lethal toxin and toxin B could be completely removed in <1 s by high frequency stimulation of nerve terminals. Further characterization of the inhibitory action produced by lethal toxin suggests that Rac1 protein regulates a late step in Ca 2؉ -dependent neuroexocytosis.

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Identification of rho as a substrate for botulinum toxin C₃‐catalyzed ADP‐ribosylation

Cited by 39 publications

References 29 publications

ADP-ribosylation of the G Protein Rho Inhibits Integrin Regulation of Tumor Cell Growth

ADP-ribosylation of the G Protein Rho Inhibits Integrin Regulation of Tumor Cell Growth

Physical association of the small GTPase Rho with a 68-kDa phosphatidylinositol 4-phosphate 5-kinase in Swiss 3T3 cells.

A Rho-related GTPase Is Involved in Ca2+-dependent Neurotransmitter Exocytosis

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Identification of rho as a substrate for botulinum toxin C3‐catalyzed ADP‐ribosylation

Cited by 39 publications

References 29 publications

ADP-ribosylation of the G Protein Rho Inhibits Integrin Regulation of Tumor Cell Growth

ADP-ribosylation of the G Protein Rho Inhibits Integrin Regulation of Tumor Cell Growth

Physical association of the small GTPase Rho with a 68-kDa phosphatidylinositol 4-phosphate 5-kinase in Swiss 3T3 cells.

A Rho-related GTPase Is Involved in Ca2+-dependent Neurotransmitter Exocytosis

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Identification of rho as a substrate for botulinum toxin C₃‐catalyzed ADP‐ribosylation