Effects of Clostridium difficile toxin A and toxin B on phospholipase D activation in human promyelocytic leukemic HL60 cells.
The possible involvement of Rho family GTP-binding proteins in the regulation of phospholipase D (PLD) activity has recently been demonstrated. In the present study, to further examine the role of Rho family proteins in PLD activation of human promyelocytic leukemic HL60 cells, we used toxin A and toxin B from the anaerobic bacterium Clostridium difficile, which was shown to glucosylate Rho family proteins and inhibit their interaction with effectors. Pretreatment of [3H]oleic acid-labeled HL60 cell lysates with either one of the toxins resulted in a remarkable inhibition of membrane PLD activity stimulated by guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS). The magnitude of inhibition of PLD activity was correlated well with the extent of toxin A- or B-induced glucosylation of 22-kDa RhoA in HL60 cells, toxin B being more effective than toxin A. GTPgammaS-stimulated PLD activation measured with the exogenous substrate containing phosphatidylinositol 4,5-bisphosphate was also inhibited by toxin B. Toxin B had no effect on GTP-gammaS-induced translocation of RhoA from cytosol to membranes. Furthermore, the toxin B pretreatment also suppressed PLD activation induced by 4beta-phorbol 12-myristate 13-acetate in HL60 cell lysates. Thus, it was indicated that Rho family proteins play a key role in GTPgammaS- and 40-phorbol 12-myristate 13-acetate-induced PLD activity in HL60 cells. In addition, the results obtained here indicate that C. difficile toxins are a useful tool for researching the regulation of the Rho family protein-mediated PLD activation and also provide a clue toward understanding the pathogenic background of pseudomembranous colitis from the viewpoint of signal transduction.
Ohguchi
K
K
Department of Biochemistry, Gifu University School of Medicine, Japan.
Banno
Y
Y
Nakashima
S
S
Kato
N
N
Watanabe
K
K
Lyerly
D M
DM
Nozawa
Y
Y
D020727:ADP-Ribosylation Factors; D001426:Bacterial Proteins; D001427:Bacterial Toxins / Q000494:pharmacology*; D016360:Clostridioides difficile; D004768:Enterotoxins / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D019204:GTP-Binding Proteins / Q000378:metabolism; D006031:Glycosylation; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D018922:HL-60 Cells; D006801:Humans; D010739:Phospholipase D / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D020742:rhoA GTP-Binding Protein
D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004768:Enterotoxins; D011994:Recombinant Proteins; C041348:tcdA protein, Clostridium difficile; C031815:toxB protein, Clostridium difficile; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D010739:Phospholipase D; D019204:GTP-Binding Proteins; D020727:ADP-Ribosylation Factors; D020742:rhoA GTP-Binding Protein; D013755:Tetradecanoylphorbol Acetate