The rho gene product expressed in E. Coli is a substrate of botulinum ADP-ribosyltransferase C3

Aktories, Klaus; Braun, U.; Rösener, Sigrid; Just, Ingo; Hall, Alan

doi:10.1016/s0006-291x(89)80199-8

Cited by 248 publications

(133 citation statements)

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“…In almost all studies, Clostridium botulinum C3 ADP-ribosyltransferase (C3) has been employed to inhibit the activation of RhoA. However, C3 is not a RhoA-specific inhibitor, since it also modifies at least RhoB and RhoC [Aktories et al, 1989;Maehama et al, 1994]. Blocking RhoA function with dominant negative RhoA may require high levels of expression because most of the endogenous RhoA is inactive, and, therefore, the relatively weak promoter SV40 of the N19-RhoA construct used in this study may not be able to induce sufficient expression.…”

Section: Discussionmentioning

confidence: 99%

The role of RhoA in the regulation of cell morphology and motility

Tkach¹,

Bock²,

Berezin³

2005

Cell Motil. Cytoskeleton

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Members of the Rho family of small GTPases are key regulators of the actin cytoskeleton, particularly in relation to the cell shape changes and the adhesion dynamic that drive cell migration. Here, we report the effect of activation or inhibition of the function of RhoA on cell motility and morphology. Both in the presence and the absence of serum, expression of constitutively active RhoA dramatically inhibited L929 fibroblasts' cell motility, and induced a rounding of the cells and a decrease in the number of processes per cell. In contrast, expression of a dominant negative mutant of RhoA had no effect on cell motility or morphology in steady-state conditions with or without serum in the medium. Inhibition of p160ROCK, a kinase effector of RhoA, only partially inhibited cell migration. Conversely, when cells were submitted to a period of serum deprivation followed by addition of serum, inhibition of endogenous RhoA by expression of the dominant negative mutant of RhoA impeded cell motility after serum stimulation. Thus, RhoA activity is required for stimulation of cell locomotion by serum factors. It was also observed that the addition of serum factors to quiescent L929 and NR6wtEGFR fibroblasts resulted in a delayed motility response of several hours compared to the immediately induced morphological changes, indicating the absence of a previously assumed direct correlation between changes in cell motility and cell morphology in response to serum addition. The motility response of L929 and NR6wtEGFR fibroblasts to serum stimulation required protein synthesis. Cell Motil.

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Section: Discussionmentioning

confidence: 99%

The role of RhoA in the regulation of cell morphology and motility

Tkach¹,

Bock²,

Berezin³

2005

Cell Motil. Cytoskeleton

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“…Interference with the rhodependent pathway leads to a diminished tyrosine phosphorylation of paxillin and p 125 focal adhesion kinase (FAK), two proteins closely connected to actin and focal adhesions (13,14). Clostridium botulinum C3 exoenzyme ADP-ribosylates and inactivates rho A, B, and C proteins (12,15). The ADPribosylation of rho inhibits several cell functions, including the formation of stress fibers and focal adhesions.…”

Section: Introductionmentioning

confidence: 99%

Bombesin-induced gastrin release from canine G cells is stimulated by Ca2+ but not by protein kinase C, and is enhanced by disruption of rho/cytoskeletal pathways.

Seensalu¹,

Avedian²,

Song³

et al. 1997

J. Clin. Invest.

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“…In the presence of GDP (10 #M), inhibition by illumination of ADP-ribose incorporation into the recombinant rho A protein by C3 was reduced. Not only the normal rho A protein but also its Val-14 mutant, exhibiting reduced GTPase activity [6], can be ADP-ribosylated by C3 [12]. As observed for normal rho A protein (see Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Therefore, we used these membranes to study whether the light regulation of C3 substrates observed in crude ROS membranes is due to a direct interaction of rhodopsin with the small G proteins. Since rho proteins (A, B and C) are substrates of C3 [4,[9][10][11][12][13], we used recombinant human rho A protein expressed in E. coli for the reconstitution with urea-treated ROS membranes. As is shown herein, ADP-ribosylation of rho A protein by C3 is regulated by light when studied in this reconstitution assay in a manner similar to endogenous C3 substrates.…”

Section: Discussionmentioning

confidence: 99%

“…Recently, several proteins have been described, apparently specifically regulating rho protein functions, such as a GTPase-activating protein (GAP) [6], a GDP dissociation inhibitor (GDI) [7] and a GDP dissociation stimulator (GDS) [8]. In addition, all three rho proteins can be ADP-ribosylated by the botulinum exoenzyme C3 [4,[9][10][11][12][13].…”

Section: Introductionmentioning

confidence: 99%

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Interaction of recombinant rho A GTP‐binding proteins with photoexcited rhodopsin

et al. 1990

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The small molecular mass GTP-binding proteins rho A, B and C are targets for ADP-ribosyltransferase activity of the botulinum exoenzyme C3. The possible interaction of recombinant rho A proteins expressed in E. coli with photoexcited rhodopsin was studied by reconstitution with bovine rod outer segment (ROS) membranes depleted of endogenous GTP-binding proteins by treatment with urea. As reported for C3 substrates present in untreated ROS membranes, ADP-ribosylation of recombinant rho A proteins, both normal and Val-14 mutant, by C3 was inhibited when reconstituted with illuminated compared to dark-adapted ROS membranes pretreated with urea. GDP reduced the light-induced inhibition, while GTP [S] and light inhibited ADP-ribosylation of rho A proteins in a synergistic manner.Rho protein; GTP-binding protein; Rhodopsin; Botulinum C3 ADP-ribosyltransferase; Bovine rod outer segment

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The rho gene product expressed in E. Coli is a substrate of botulinum ADP-ribosyltransferase C3

Cited by 248 publications

References 13 publications

The role of RhoA in the regulation of cell morphology and motility

The role of RhoA in the regulation of cell morphology and motility

Bombesin-induced gastrin release from canine G cells is stimulated by Ca2+ but not by protein kinase C, and is enhanced by disruption of rho/cytoskeletal pathways.

Interaction of recombinant rho A GTP‐binding proteins with photoexcited rhodopsin

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