Cysteine-specific ADP-ribosylation of actin.
Incubation of lysate from human polymorphonucleated neutrophils and human platelets with [32P]NAD resulted in the labeling of a 42-kDa protein. Phosphodiesterase (Crotalus durissus) released 5'-AMP from the radiolabeled protein. The 42-kDa protein was identified as actin by binding to DNAse-I, two-dimensional gel electrophoresis and partial proteolysis. The rate of ADP-ribosylation was greater with [32P]ADP-ribose than with [32P]NAD, indicating a non-enzymic modification. ADP-ribose also modified actin in the actin-DNAase-I complex, but denatured actin was not modified by ADP-ribose. Only cytoplasmic beta/gamma-actin isoforms were non-enzymically ADP-ribosylated but not muscle alpha-actin. The acceptor amino acid was identified as a cysteine residue whereas the bacterial ADP-ribosyltransferase C. perfringens iota toxin catalyzes incorporation of ADP-ribose to Arg177 of actin. Alkylation of cysteine residues of actin with N-ethylmaleimide prevented subsequent non-enzymic ADP-ribosylation but not the toxin catalyzed modification. Non-enzymically ADP-ribosylated actin was further modified by C. perfringens iota toxin. The F-actin stabilizing mycotoxin phalloidin blocked the non-enzymatic ADP-ribosylation and, conversely, ADP-ribosylation inhibited the phalloidin-induced polymerization of ADP-ribosylated actin. The data indicate that cytoplasmic actin is non-enzymically ADP-ribosylated by ADP-ribose at a cysteine residue to inhibit actin polymerization.
Just
I
I
Institut für Pharmakologie und Toxikologie, Universität des Saarlandes, Homburg-Saar, Germany.
Wollenberg
P
P
Moss
J
J
Aktories
K
K
D000199:Actins / Q000737:chemistry / Q000378:metabolism*; D000246:Adenosine Diphosphate Ribose / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D001792:Blood Platelets / Q000378:metabolism*; D003545:Cysteine / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D009243:NAD / Q000378:metabolism; D009504:Neutrophils / Q000378:metabolism*; D010449:Peptide Mapping; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism
D000199:Actins; D009243:NAD; D000246:Adenosine Diphosphate Ribose; D010727:Phosphoric Diester Hydrolases; D003545:Cysteine