Tubulin stability and decay: Mediation by two distinct classes of IKP104-binding sites

IKP104, a novel antimitotic drug, has two classes of binding sites on bovine brain tubulin with different affinities. IKP104, by itself, enhances the decay of tubulin, but in the presence of colchicine or podophyllotoxin, it stabilizes tubulin instead of opening up the hydrophobic areas [Luduena et al. (1995), Biochemistry 34, 15751-15759). Here, we have dissected these two apparently contradictory effects of IKP104 by cleaving the C-terminal ends of both α and β subunits of tubulin with subtilisin. We have found that the selective removal of the C-terminal ends from both the α and β subunits of αβ tubulin lowers the sulfhydryl titer by approximately 1.5 mol/mol of dimer. Interestingly, IKP104 does not increase either the sulfhydryl titer or the exposure of hydrophobic areas of this subtilisin-treated tubulin (α(s)β(s)). Moreover, IKP104 lowers the sulfhydryl titer of α(s)β(s) tubulin approximately by 1 mol/mol and appears to inhibit completely the time-dependent decay of α(s)β(s) tubulin. The cleavage at the C-terminal ends of both α and β modulates the effects of IKP104 on the β subunit, but not on the α subunit. Fluorometric binding data analysis suggests that IKP104 binds to the α(s)β(s) tubulin only at the high-affinity site; the low-affinity site(s) disappear almost completely. The sulfhydryl titer data for α and β the fluorometric data therefore suggest that the interaction of IKP104 at the high-affinity site on tubulin is not regulated by the C- terminal domains of α and β and the effect of the high-affinity site is restricted largely to the α subunit, while the low-affinity-site binding is modulated by the C-terminal domain of β. It also appears that the stabilization and the acceleration of the decay of tubulin are mediated by distinct interactions of IKP104 with its high- and low-affinity sites on tubulin, respectively.