Microtubule protein ADP-ribosylation in vitro leads to assembly inhibition and rapid depolymerization.
Bovine brain microtubule protein, containing both tubulin and microtubule-associated proteins, undergoes ADP-ribosylation in the presence of [14C]NAD+ and a turkey erythrocyte mono-ADP-ribosyltransferase in vitro. The modification reaction could be demonstrated in crude brain tissue extracts where selective ADP-ribosylation of both the alpha and beta chains of tubulin and of the high molecular weight microtubule-associated protein MAP-2 occurred. In experiments with purified microtubule protein, tubulin dimer, the high molecular weight microtubule-associated protein MAP-2, and another high molecular weight mirotubule-associated protein which may be a MAP-1 species were heavily labeled. Tubulin and MAP-2 incorporated [14C]ADP-ribose to an average extent of approximately 2.4 and 30 mol of ADP-ribose/mol of protein, respectively. Assembly of microtubule protein into microtubules in vitro was inhibited by ADP-ribosylation, and incubation of assembled steady-state microtubules with ADP-ribosyltransferase and NAD+ resulted in rapid depolymerization of the microtubules. Thus, the eukaryotic enzyme can ADP-ribosylate tubulin and microtubule-associated proteins to much greater extents than previously observed with cholera and pertussis toxins, and the modification can significantly modulate microtubule assembly and disassembly.
Scaife
R M
RM
Fred Hutchinson Cancer Research Center, Seattle, Washington 98104.
Wilson
L
L
Purich
D L
DL
D036002:ADP Ribose Transferases; D000818:Animals; D001923:Brain Chemistry; D002417:Cattle; D004912:Erythrocytes / Q000201:enzymology; D005260:Female; D066298:In Vitro Techniques; D008868:Microtubule Proteins / Q000378:metabolism*; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism*; D011247:Pregnancy; D014404:Tubulin / Q000378:metabolism; D014422:Turkeys
D008868:Microtubule Proteins; D008869:Microtubule-Associated Proteins; D014404:Tubulin; D036002:ADP Ribose Transferases; D011065:Poly(ADP-ribose) Polymerases