Cysteine‐specific ADP‐ribosylation of actin

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Abstract
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–DNAse‐I complex, but denatured actin was not modified by ADP‐ribose. Only cytoplasmic β/γ‐actin isoforms were non‐enzymically ADP‐ribosylated but not muscle α‐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‐enzymic 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.