Pasteurella multocida toxin, a potent mitogen, increases inositol 1,4,5-trisphosphate and mobilizes Ca2+ in Swiss 3T3 cells.

Staddon, James M.; Barker, Christopher J.; Murphy, Anne C.; Chanter, N.; Lax, Alistair J.; Michell, Robert H.; Rozengurt, Enrique

doi:10.1016/s0021-9258(19)67725-2

Cited by 73 publications

(8 citation statements)

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“…Phospholipids were labeled with [2-3H]inositol and after appropriate incubations the total cellular [3H]inositol phosphate content was determined by ion-exchange chromatography, as described previously (Staddon et al ., 1991). DNA synthesis was assayed by [3H]thymidine incorporation (Rozengurt et al ., 1990).…”

Section: Inositol Phosphate Release and Dna Synthesis Assaysmentioning

confidence: 99%

“…P. multocida toxin is an extremely potent mitogen for Swiss 3T3 and other cultured mesenchymal cells (Rozengurt et al ., 1990) . This toxin activates phospholipase C as indicated by the stimulation of inositol phosphate release (Staddon et al ., 1991) and activation of protein kinase C (Staddon et al ., 1990) . Intriguingly, the toxin enters the cells to elicit mitogenesis (Rozengurt et al, 1990) and activate phospholipase C (Staddon et al ., 1990(Staddon et al ., , 1991 .…”

Section: Irreversibility Of Toxin Actionmentioning

confidence: 99%

“…This toxin activates phospholipase C as indicated by the stimulation of inositol phosphate release (Staddon et al ., 1991) and activation of protein kinase C (Staddon et al ., 1990) . Intriguingly, the toxin enters the cells to elicit mitogenesis (Rozengurt et al, 1990) and activate phospholipase C (Staddon et al ., 1990(Staddon et al ., , 1991 . The mechanism whereby P. multocida toxin activates phospholipase C is not known .…”

Section: Irreversibility Of Toxin Actionmentioning

confidence: 99%

“…The mechanism whereby P. multocida toxin activates phospholipase C is not known . However, the inositol phosphate species formed in response to P. multocida toxin and bombesin are remarkably similar (Staddon et al ., 1991) . The bombesin receptor is believed to couple to phospholipase C via a pertussis toxin-insensitive and yet to be identified G protein (Zachary et al ., 1987;Erusalimsky et al ., 1988;Fisher and Schonbrunn, 1988 ;Hasegawa-Sasaki et al ., 1988 ;Cattaneo and Vicentini, 1989;Coffer et al ., 1990 ;Sinnett-Smith et al, 1990;Plevin et al ., 1990;Battey et al ., 1991) .…”

Section: Irreversibility Of Toxin Actionmentioning

confidence: 99%

“…The approach that we describe should be applicable to the study of the cellular action of other ADP-ribosylatiog toxins and also to explore the role of ADP-ribosylation in the action of recently identified bacterial toxins. We describe our findings with Pasteurella multocida toxin, a potent mitogen (Rozengurt et al ., 1990) that activates phospholipase C to stimulate the hydrolysis of polyphosphoinositides (Staddon et al ., 1990(Staddon et al ., , 1991 .…”

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A novel approach to detect toxin-catalyzed ADP-ribosylation in intact cells: its use to study the action of Pasteurella multocida toxin.

Staddon

Bouzyk

1991

The Journal of Cell Biology

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Abstract. Certain microbial toxins are ADP-ribosyltransferases, acting on specific substrate proteins. Although these toxins have been of great utility in studies of cellular regulatory processes, a simple procedure to directly study toxin-catalyzed ADPribosylation in intact cells has not been described . Our approach was to use [2?H]adenine to metabolically label the cellular NAD+ pool. Labeled proteins were then denatured with SDS, resolved by PAGE, and detected by fluorography. In this manner, we show that pertussis toxin, after a dose-dependent lag period, [3H]-labeled a 40-kD protein in intact cells. Furthermore, incubation of the gel with trichloroacetic acid at 95°C before fluorography caused the release of label from bands other than the pertussis toxin substrate, thus, allowing its selective visualization . The modification of the 40-kD protein was ascribed to ADP-ribosylation of a T HE pathological basis of the action of certain bacteria is accounted for by their ability to produce toxins that enter eukaryotic cells and subvert cellular regulatory processes. Some ofthese toxins catalyze the transfer of ADP-ribose from NAD+ to specific target proteins, resulting in a protein that may be either inactive or altered in properties. Diphtheria toxin and Pseudomonas aeruginosa exotoxin A block protein synthesis by ADP-ribosylatiog elongation factor-2 (Collier, 1990;Wick and Iglewski, 1990). Clostridium botulinum C2 toxin ADP-ribosylates nonmuscle actin ) and the C3 toxin ADP-ribosylates rho, a ras-related GTPase (Aktories and Just, 1990) . P. aeruginosa exoenzyme S preferentially ADP-ribosylates p21a-"-s in vitro (Coburn et al., 1989b) and has also been described to ADP-ribosylate vimentin (Coburn et al ., 1989a). Pertussis toxin, cholera toxin, and Escherichia coli heat-labile enterotoxin ADP-ribosylate the a subunits of heterotrimeric signal transducing G proteins (Moss and Vaughan, 1988 ;Pfeuffer and Helmreich, 1988;Ui, 1990). The specificity of the action of these toxins and the modification of target protein function can be exploited to make them extremely useful as probes of cellular regulatory processes .The classic approach to investigate the role of ADPribosylation in toxin action has been to identify ADPribosylated toxin substrates in cell extracts using radiola- Pasteurella multocida toxin has recently been described as a novel and potent mitogen for Swiss 3T3 cell and acts to stimulate the phospholipase C-mediated hydrolysis of polyphosphoinositides . The basis of the action of the toxin is not known. Using the methodology described here, P. multocida toxin was not found to act by ADP-ribosylation . beled NAD+ . This requires activation of the holotoxin in vitro and its use at concentration orders ofmagnitude greater than those used on intact cells . Furthermore, this approach can be thwarted by the absence of ADP-ribosylation factors and excessive NADase activity in the cell extracts. It can also be sensitive to the composition of the reaction mixture (see, e.g., Ribeiro-Neto et al., 1985, andWoo...

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Section: Inositol Phosphate Release and Dna Synthesis Assaysmentioning

confidence: 99%

Section: Irreversibility Of Toxin Actionmentioning

confidence: 99%

Section: Irreversibility Of Toxin Actionmentioning

confidence: 99%

Section: Irreversibility Of Toxin Actionmentioning

confidence: 99%

mentioning

confidence: 99%

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A novel approach to detect toxin-catalyzed ADP-ribosylation in intact cells: its use to study the action of Pasteurella multocida toxin.

Staddon

Bouzyk

1991

The Journal of Cell Biology

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Pasteurella multocida toxin stimulates mitogenesis and cytoskeleton reorganization in Swiss 3T3 fibroblasts

et al. 1996

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Pasteurella multocida toxin (PMT) causes cytoplasmic retraction in epithelial cells, activates osteoclast neoformation, and is a potent mitogen for Swiss 3T3 fibroblasts. In the present study designed to further investigate the effects of PMT on cell shape and proliferation, we report that the mitogenic effect of affinitypurified PMT on quiescent 3T3 cells was even superior at 5 ng/ml to that of fetal bovine serum or bombesin. This positive effect was inhibited by heat denaturation and methylamine treatment (this agent blocks internalization). Preincubation of PMT with gangliosides GM1, GM2, or GM3 counteracted its effect on DNA synthesis, suggesting that the toxin binds to GM-type ceramides on target cells. The distribution of F-actin was analyzed in control/treated cells using FITC-conjugated phalloidin. In comparison with FBS and bombesin, PMT triggered a more rapid and profound reorganization of cortical actin into prominent stress fibers after only 5-10 min. This event lead to the retraction of cells after only 30 min and ultimately to the induction of mitotic figures. Interestingly, methylamine blocked the effects of PMT on stress fiber formation and cell retraction but not the ruffling response, suggesting that some early events may not require toxin internalization. In summary, these findings indicate that PMT concomitantly exerts a strong mitogenic activity and a rapid stimulation of cytoskeletal rearrangements, possibly after binding to membrane gangliosides and subsequent internalization. We propose that this toxin could be used in the future as a defined inducer of transduction signals involved in cellular proliferation and control of cell shape.

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Molecular Biology of Pasteurella multocida Toxin

Orth

Aktories

2012

Current Topics in Microbiology and Immunology

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Pasteurella multocida toxin, a potent mitogen, increases inositol 1,4,5-trisphosphate and mobilizes Ca2+ in Swiss 3T3 cells.

Cited by 73 publications

References 50 publications

A novel approach to detect toxin-catalyzed ADP-ribosylation in intact cells: its use to study the action of Pasteurella multocida toxin.

A novel approach to detect toxin-catalyzed ADP-ribosylation in intact cells: its use to study the action of Pasteurella multocida toxin.

Pasteurella multocida toxin stimulates mitogenesis and cytoskeleton reorganization in Swiss 3T3 fibroblasts

Molecular Biology of Pasteurella multocida Toxin

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