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 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.
Chaudhuri
A R
AR
Department of Biochemistry, University of Texas Health Science Center, San Antonio 78284-7760, USA. Asish@bioC09.uthscsa.edu
Tomita
I
I
Mizuhashi
F
F
Murata
K
K
Ludueña
R F
RF
D000818:Animals; D000970:Antineoplastic Agents / Q000378:metabolism*; D001665:Binding Sites; D001921:Brain / Q000378:metabolism; D002417:Cattle; D011485:Protein Binding; D011728:Pyridones / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D014404:Tubulin / Q000737:chemistry / Q000378:metabolism*
D000970:Antineoplastic Agents; D011728:Pyridones; D014404:Tubulin; C067096:IKP 104