Tubulin adenosine diphosphate ribosylation is catalyzed by cholera toxin

Cholera toxin catalyzed the transfer of radioactive label from [adenine-2,8-3H2]NAD+ or ((32P]NAD+ to rat C6 glioma cell membrane and cytosolic proteins. Labeled proteins were resolved by polyacrylamide-NaDodSO4 gel or two-dimensional gel electrophoresis and stained with Coomassie blue, and the gels were subjected to fluorography or autoradiography. Autoradiograms of gels revealed labeled Mr 42000 and 46000-48000 membrane proteins that are putative subunits of the regulatory component (G/F) of the C6 cell hormone-sensitive adenylate cyclase. Cholera toxin also catalyzed the labeling of several cytosolic proteins including a Mr 54000 protein that was observed in autoradiograms of two-dimensional gels to migrate as an acidic satellite relative to Coomassie-stained C6 cell tubulin. Tubulin modified by ADP-ribosylation would undergo an acid shift relative to the stained unmodified tubulin in two-dimensional gels. The data led us to postulate that tubulin undergoes cholera toxin catalyzed ADP-ribosylation. Bovine brain tubulin prepared by three cycles of warm/cold polymerization/depolymerization was incubated with [32P]NAD+, GTP, and cholera toxin and then subjected to two-dimensional gel electrophoresis. Autoradiograms of the gels revealed the presence of [32P]ADP-ribosylated proteins that migrated as acidic satellites relative to the Coomassie-stained brain alpha and beta tubulin. Peptide maps of bovine brain tubulin and the associated [32P]ADP-ribosylated proteins showed a correspondence between the autoradiographic images and the stained peptide fragments. The data demonstrate that cholera toxin catalyzes the ADP-ribosylation of tubulin.