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Tubulin stability and decay: mediation by two distinct classes of IKP104-binding sites
A R Chaudhuri1, I Tomita, F Mizuhashi
1Department of Biochemistry, University of Texas Health Science Center, San Antonio 78284-7760, USA. Asish@bioC09.uthscsa.edu
Abstract:
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 alpha and beta subunits of tubulin with subtilisin. We have found that the selective removal of the C-terminal ends from both the alpha and beta subunits of alphabeta 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 (alpha(s)beta(s)). Moreover, IKP104 lowers the sulfhydryl titer of alpha(s)beta(s) tubulin approximately by 1 mol/mol and appears to inhibit completely the time-dependent decay of alpha(s)beta(s) tubulin. The cleavage at the C-terminal ends of both alpha and beta modulates the effect of IKP104 on the beta subunit, but not on the alpha subunit. Fluorometric binding data analysis suggests that IKP104 binds to the alpha(s)beta(s) tubulin only at the high-affinity site; the low-affinity site(s) disappear almost completely. The sulfhydryl titer data for alpha and beta and 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 alpha and beta and the effect of the high-affinity site is restricted largely to the alpha subunit, while the low-affinity-site binding is modulated by the C-terminal domain of beta. 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.
Insights
IKP104, an antimitotic drug, interacts with tubulin differently based on binding sites. Its effects on tubulin decay and stabilization depend on interactions with specific tubulin subunits and their C-terminal domains.
Area of Science:
- Biochemistry
- Molecular Biology
- Pharmacology
Background:
- IKP104 is a novel antimitotic drug targeting tubulin.
- Tubulin has two classes of binding sites for IKP104 with varying affinities.
- IKP104 exhibits contradictory effects: enhancing tubulin decay alone but stabilizing it with colchicine or podophyllotoxin.
Purpose of the Study:
- To dissect the dual effects of IKP104 on tubulin.
- To investigate the role of C-terminal domains of alpha and beta tubulin subunits in IKP104 binding and activity.
- To elucidate the distinct mechanisms underlying tubulin stabilization and decay mediated by IKP104.
Main Methods:
- Selective cleavage of C-terminal ends of alpha and beta tubulin subunits using subtilisin.
- Sulfhydryl titer analysis to assess changes in tubulin structure.
- Fluorometric binding assays to determine IKP104 affinity for modified tubulin.
Main Results:
- Cleavage of C-terminal ends reduced tubulin sulfhydryl titer.
- IKP104 did not increase sulfhydryl titer or hydrophobic area exposure in subtilisin-treated tubulin (α(s)β(s)).
- IKP104 lowered sulfhydryl titer and inhibited decay of α(s)β(s) tubulin, binding only to the high-affinity site.
- C-terminal cleavage modulated IKP104's effect on the beta subunit, not the alpha subunit.
Conclusions:
- High-affinity binding of IKP104 is independent of C-terminal domains and primarily affects the alpha subunit.
- Low-affinity binding is modulated by the beta subunit's C-terminal domain.
- Tubulin stabilization and accelerated decay are mediated by distinct IKP104 interactions with high- and low-affinity sites, respectively.