title: Inhibition of PMA-induced, LFA-1-dependent lymphocyte aggregation by ADP ribosylation of the small molecular weight GTP binding protein, rho.
abstract: Botulinum C3 exoenzyme specifically ADP-ribosylates a group of ras-related small molecular weight GTP-binding proteins, rho, and inhibits their biological activity. Using this enzyme, we examined the function of rho in PMA-induced activation of lymphocyte function-associated antigen-1 (LFA-1) in a B lymphoblastoid cell line, JY. Northern blot analysis revealed that among the three rho genes, rhoA mRNA was predominantly expressed in JY cells. Consistently, only one [32P]ADP-ribosylated band was found when the lysate of the cells was subjected to ADP ribosylation by C3 exoenzyme. When the cells were cultured with C3 exoenzyme, this substrate was ADP-ribosylated in situ in a time- and concentration-dependent manner. Concomitant with this ADP ribosylation, PMA-induced LFA-1/intercellular adhesion molecule (ICAM)-1-dependent aggregation of JY cells was inhibited. This inhibition was blocked by prior treatment of the enzyme with an anti-C3 monoclonal antibody, and overcome by stimulation with higher concentrations of PMA. The C3 exoenzyme-induced inhibition was not affected by shaking of the cell suspension, while inhibition of aggregation by cytochalasin B was abolished by this procedure, suggesting that the inhibitory effect of the C3 exoenzyme treatment was not due to decrease in cell motility. The C3 exoenzyme treatment affected neither protein phosphorylation in JY cells before and after PMA stimulation, nor affected surface expression of LFA-1 and ICAM-1. These results suggest that rhoA protein works downstream of protein kinase C activation linking PMA stimulation to LFA-1 activation and aggregation in JY cells.
authors.affiliation: Department of Pharmacology, Kyoto University Faculty of Medicine, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Tominaga
authors.forename: K
authors.initials: K
authors.lastname: Sugie
authors.forename: M
authors.initials: M
authors.lastname: Hirata
authors.forename: N
authors.initials: N
authors.lastname: Morii
authors.forename: J
authors.initials: J
authors.lastname: Fukata
authors.forename: A
authors.initials: A
authors.lastname: Uchida
authors.forename: H
authors.initials: H
authors.lastname: Imura
authors.forename: S
authors.initials: S
authors.lastname: Narumiya
mesh_terms: D036002:ADP Ribose Transferases / Q000037:antagonists & inhibitors / Q000276:immunology / Q000378:metabolism; D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D000911:Antibodies, Monoclonal; D001402:B-Lymphocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D015152:Blotting, Northern; D001905:Botulinum Toxins; D015815:Cell Adhesion Molecules / Q000032:analysis; D002449:Cell Aggregation / Q000187:drug effects; D002461:Cell Line, Transformed; D015180:Electrophoresis, Gel, Two-Dimensional; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004854:Herpesvirus 4, Human / Q000235:genetics; D006801:Humans; D018799:Intercellular Adhesion Molecule-1; D016169:Lymphocyte Function-Associated Antigen-1 / Q000032:analysis / Q000378:metabolism*; D010750:Phosphoproteins / Q000302:isolation & purification / Q000378:metabolism; D011061:Poly A / Q000235:genetics / Q000302:isolation & purification; D011493:Protein Kinase C / Q000378:metabolism; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000276:immunology / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D020742:rhoA GTP-Binding Protein
chemical_list: D000911:Antibodies, Monoclonal; D015815:Cell Adhesion Molecules; D016169:Lymphocyte Function-Associated Antigen-1; D010750:Phosphoproteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; D018799:Intercellular Adhesion Molecule-1; D000246:Adenosine Diphosphate Ribose; D011061:Poly A; D012313:RNA; D036002:ADP Ribose Transferases; C063007:exoenzyme C3, Clostridium botulinum; D011493:Protein Kinase C; D001905:Botulinum Toxins; D019204:GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein; D013755:Tetradecanoylphorbol Acetate