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.
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D000818:Animals; D001923:Brain Chemistry; D002417:Cattle; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002772:Cholera Toxin / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D005910:Glioma; D006160:Guanosine Triphosphate / Q000494:pharmacology; D009243:NAD / Q000378:metabolism; D009702:Nucleoside Diphosphate Sugars / Q000378:metabolism*; D051381:Rats; D014404:Tubulin / Q000378:metabolism*
D009702:Nucleoside Diphosphate Sugars; D014404:Tubulin; D009243:NAD; D000246:Adenosine Diphosphate Ribose; D006160:Guanosine Triphosphate; D002772:Cholera Toxin