IKP104-induced decay of tubulin: Role of the A-ring binding site of colchicine

Tubulin, the major subunit protein of microtubules, has a tendency to lose its ability to assemble or to interact with ligands in a time-dependent pro cess known as decay. Decay involves the increase in exposure of sulfhydryl groups and hydrophobic areas. The antimitotic drug IKP104 [2-(4-fluoropheny l)-1-(2-chloro-3,5-dimethoxyphenyl)-3-methyl-6-phenyl-4(1H)-pyridinone] acc elerates the decay of tubulin [Luduena et al. (1995) Biochemistry 34, 15751 -15759]. In the presence of colchicine, however, IKP104 stabilizes tubulin against decay. We have shown that the stability and the acceleration of the decay of tubulin are mediated respectively by the high- and low-affinity b inding site(s) of IKP104 [Chaudhuri et al. (1998) J, Protein Chem, 17, 303- 309]. To better understand the mechanism by which colchicine protects tubul in from IKP104-induced decay, we examined the effect of colchicine and its analogues on this process. We found that IKP104 unfolds tubulin in a proces s involving a specific domain where colchicine interacts, although the bind ing sites of these two drugs are distinctly different. 2-Methoxy-5-(2',3',4 '-trimethoxyphenyl) tropolone (MTPT), the bicyclic analogue of colchicine t hat lacks the B-ring, can also protect tubulin from IKP104-induced decay. A n A-ring analogue of colchicine, 3,4,5-trimethoxybenzaldehyde (TMB), can al so stop IKP104-induced unfolding of tubulin significantly. Interestingly, t he C-ring analogue of colchicine, tropolone methyl ether (TME), does not pr event this process. Our results thus suggest that neither the B-ring nor th e C-ring binding regions of colchicine are involved in the IKP104-induced d ecay and that the A-ring binding site of colchicine on tubulin plays a cruc ial role in IKP104-induced decay.