ADP-ribosylation of the ras-related, GTP-binding protein RhoA inhibits lymphocyte-mediated cytotoxicity.
The Rho proteins are identified as a subgroup of the Ras superfamily of low molecular weight GTP-binding proteins. We have studied the expression of these proteins in human cytotoxic natural killer cells and found that RhoA is the most abundantly expressed member of the Rho family. The Rho proteins are specific substrates for ADP-ribosylation catalyzed by the C3 exoenzyme from Clostridium botulinum. We report here that introduction of recombinant C3 in electropermeabilized natural killer cells or in cytotoxic T lymphocytes resulted in a dose-dependent inhibition of their cytolytic function. Furthermore, a single substrate is efficiently ADP-ribosylated by C3 in extracts from cytotoxic cells. Biochemical analyses indicate that this substrate is RhoA, and subcellular fractionation experiments demonstrate that it is essentially present in the cytosol of the cells. Western blot analysis, however, revealed that a small proportion of the Rho protein can be found associated with the cell membrane as well as with the cytotoxic granules. These results indicate that the low molecular weight GTP-binding protein RhoA is present in cytotoxic lymphocytes and plays a critical role in cell-mediated cytotoxicity.
Lang
P
P
Institut National de la Santé et de la Recherche Médicale (INSERM), Unité 333, Institut Gustave Roussy, Villejuif, France.
Guizani
L
L
Vitté-Mony
I
I
Stancou
R
R
Dorseuil
O
O
Gacon
G
G
Bertoglio
J
J
D036002:ADP Ribose Transferases / Q000235:genetics / Q000378:metabolism; D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D015152:Blotting, Northern; D015153:Blotting, Western; D001905:Botulinum Toxins; D002478:Cells, Cultured; D003014:Clostridium botulinum / Q000201:enzymology; D003602:Cytotoxicity, Immunologic; D015180:Electrophoresis, Gel, Two-Dimensional; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D007694:Killer Cells, Natural / Q000276:immunology; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D013379:Substrate Specificity; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*; D020742:rhoA GTP-Binding Protein
D000246:Adenosine Diphosphate Ribose; D006160:Guanosine Triphosphate; D036002:ADP Ribose Transferases; C063007:exoenzyme C3, Clostridium botulinum; D011065:Poly(ADP-ribose) Polymerases; D001905:Botulinum Toxins; D019204:GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein