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

Abstract

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...

Sphingosylphosphorylcholine Activation of Mitogen-activated Protein Kinase in Swiss 3T3 Cells Requires Protein Kinase C and a Pertussis Toxin-sensitive G Protein

“…The concentration of pertussis toxin required to block PKC activation has previously been demonstrated to ADPribosylate a 40-kDa G i protein in intact Swiss 3T3 cells (47 raf-1 activation in MEF. Confluent secondary cultures of MEF were treated with 10 M SPC or 20 ng/ml PDGF for 3 min and lysed; p74 raf-1 activity was then assayed as described under "Experimental Procedures."…”

Endogenous ADP-ribosylation of the G Protein β Subunit Prevents the Inhibition of Type 1 Adenylyl Cyclase

“…Since NAD ϩ is a membrane-impermeant compound, it must either be injected into the cell or metabolically radiolabeled in order to study endogenous ADP-ribosylation. It has been previously shown that the labeling with [ 3 H]adenine permits the study of toxin-catalyzed G proteins ADP-ribosylation in intact cells (30) and the in vivo ADPribosylation of GRP78/BiP protein (49). Thus the mono-ADPribosylation of the ␤ subunit was analyzed by in vivo labeling of intact CHO cells employing [ 3 H]adenine to label metabolically the cellular NAD ϩ pool, followed by separation of the labeled ␤ subunit with an affinity column (Fig.…”

The potent mitogen Pasteurella multocida toxin is highly resistant to proteolysis but becomes susceptible at lysosomal pH

“…PMT is encoded as a 146 kDa singlechain bacterial protein, which shares partial homology with the multinucleating toxins, cytotoxic necrotising factors types 1 and 2 (CNF-1, CNF-2), produced by some strains of E. coli [10,11]. Although PMT contains a putative ADP-ribosylation motif [6], recent data have shown that PMT is unlikely to ADP ribosylate its substrate [12,13]. There is evidence that PMT facilitates the coupling of a G protein to PI-PLC [14].…”