Colchicine photosensitizes covalent tubulin dimerization

Wolff, J.; Hwang, Jean; Sackett, Dan L.; Knipling, Leslie

doi:10.1021/bi00131a007

Cited by 9 publications

(9 citation statements)

References 26 publications

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“…Whether these bands are precursor peptides or provide other binding surfaces remains to be determined. Moreover, the region of a-tubulin that is near enough to the colchicine-binding site on ,B-tubulin to become labeled under mild denaturing conditions (6,9,10) remains to be identified. Nonetheless, our findings identify two regions of 3-tubulin that are important structural elements of the colchicine-binding site.…”

Section: Resultsmentioning

confidence: 99%

“…These results suggested the possibility that the colchicine-binding domain on 3-tubulin might be near a-tubulin. That proposal was strengthened by the finding that colchicine photosensitized the formation of covalent tubulin dimers, trimers, and tetramers when the colchicine-tubulin complex was irradiated (10). Predominant labeling of (3-tubulin has also been demonstrated with chloroacetates of 2-or 3-demethylthiocolchicines (11).…”

Section: Example Low-mentioning

confidence: 99%

See 1 more Smart Citation

Localization of the colchicine-binding site of tubulin.

Uppuluri

Knipling

Sackett

et al. 1993

Proc. Natl. Acad. Sci. U.S.A.

142

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We have previously shown that rat brain tubulin, a heterodimer consisting of an a and fi monomer, can be covalently labeled with [3H]colchicine by near UV irradiation. Most of the label appears in (&tubulin. We show here that (3-tubulin can be separated and purified from SDS preparative gels and analyzed by proteolysis. Chymotrypsin yielded a labeled -4-kDa band that contained two peptides. Tryptic digestion also yielded an =4-kDa band containing two peptides. Sequence analysis revealed a peptide ofresidues 1-36 and 213-242 for chymotrypsin and a peptide of residues 1-46 and 214-241 for trypsin. To identify which peptide carried the label, limited hydrolysis of -tubulin was done with trypsin; this procedure yielded a labeled 16-kDa N-terminal peptide and a 35-kDa C-terminal peptide, as identified by antibodies. Isolation of these peptides and extensive digestion with trypsin yielded two labeled peptides corresponding to residues 1-46 from the 16-kDa N-terminal fragment and residues 214-241 from the 35-kDa C-terminal fragment. These results show that at least two regions in P-tubulin are specifically involved in colchicine binding and that the span of the colchicine molecule, s11 A, bridges these two regions in the native /3 monomer.Colchicine is the most studied of all antimitotic agents. It binds to tubulin with a stoichiometry of one and inhibits microtubule assembly substoichiometrically. Colchicine binding to tubulin exhibits pseudoirreversible kinetics; it displays a fast step followed by a slow step involving conformational changes of both ligand and tubulin. The latter, in turn, promote fluorescence characteristic of the tropolone moiety of colchicine. Many analogs have been studied, resulting in the proposal that the A and C rings of colchicine both bind to specific loci, whereas the B ring is primarily a regulator of binding kinetics (see refs. 1 and 2 and the references therein).Attempts to localize the binding site(s) in the tubulin dimer have led to conflicting results. Studies with bromacetylcolchicine indicated binding to a-tubulin (3), but these results have been criticized on the basis of the nonspecificity of the alkylation reaction. Proteolytic studies of the colchicinetubulin complex yielded a-tubulin-derived, colchicinebearing peptides of 16-18 kDa; because z90% of the label bound to the protein was lost during processing, conclusions are uncertain (4). Others (5) have also shown colchicine localization to a-tubulin by using a photoaffinity label with a long spacer arm. A shorter spacer arm led to colchicine binding on both a-and ,B-tubulins (6), suggesting that localization was a function ofthe spacer length. On the other hand, Luduena and Roach (7) [3H]colchicine that the label is localized almost exclusively on 3-tubulin. In late stages of irradiation, or with "damaged" tubulin, label also appeared in a-tubulin (9). These results suggested the possibility that the colchicine-binding domain on 3-tubulin might be near a-tubulin. That proposal was strengthened by the finding that co...

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Section: Resultsmentioning

confidence: 99%

Section: Example Low-mentioning

confidence: 99%

Localization of the colchicine-binding site of tubulin.

Uppuluri

Knipling

Sackett

et al. 1993

Proc. Natl. Acad. Sci. U.S.A.

142

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“…solution with an ultraviolet source at 89 W lamp power (Wolff et al, 1992). Irradiated samples were separated by SDS gel electrophoresis.…”

Section: Methodsmentioning

confidence: 99%

“…Ice-water cooled samples were irradiated under 2.5 cm of 20% CuS04-5H20 (as far ultraviolet and infrared filter) for 5 min at 95-W lamp power. Stained 8% gels were quantified on a Beckman DU-8 gel scanner as described (Wolff et al" 1992).…”

Section: Urea Mmentioning

confidence: 99%

Local Unfolding and the Stepwise Loss of the Functional Properties of Tubulin

Sackett¹,

Bhattacharyya²,

Wolff³

1994

Biochemistry

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Tubulin exhibits a number of characteristic functions that can be used to identify it. They include the ability to polymerize to microtubules, GTPase activity, and the binding of numerous antimitotic drugs and fluorophores. These functions can be differentially modified by low (0.1-1.0M) urea concentrations, and such urea-induced modifications are stable over time periods of minutes to hours. These intermediate states suggest the existence of restricted regions in the protein each of which is associated with a function and its own urea sensitivity. In order of decreasing sensitivity to urea these effects are decreased rate of polymerization of tubulin to microtubules > decreased extent of polymerization approximately decreased GTPase activity > enhanced fluorescence of a rapidly binding analogue of colchicine-MTPT [2-methoxy-5-(2',3',4'-trimethoxyphenyl)tropone] approximately decreased proteolysis by trypsin (after alpha Arg339) and by chymotrypsin (after beta Tyr281) > enhanced fluorescence of 1-anilino-8-naphthalenesulfonic acid (ANS). Additional evidence for the independent behavior of the restricted regions stems from the markedly different time dependence of the response to urea. These low urea concentrations do not induce significant changes in tryptophan fluorescence, suggesting that the observed effects are due to local unfolding. At higher urea concentrations (2-4 M), the enhanced fluorescence of the ligands is abolished; MTPT fluorescence decreases at lower urea concentrations than ANS fluorescence. Moreover, tubulin becomes highly susceptible to proteolysis at multiple sites, and tryptophan emission shows a red-shift, as expected. Multistep unfolding in response to denaturants has been reported for some other proteins. Tubulin appears to be an extreme example of such local responses that proceed under milder conditions than the global transition to the unfolded state.

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“…Such ligands include, for instance, the peptidoleukotrienes (3740). methyltrienolone (41,42), colchicine and related compounds (43,44), the vitamin D3 series ( 4 9 , cytochalasin (46) and steroid hormones (47-49). Finally, the retinoic acid-binding proteins were photolabeled by unmodified all-trans-retinoic and related derivatives (50).…”

Section: Of Photoaffinity Labeling and Site-mentioning

confidence: 99%

Photochemical Labeling: Can Photoaffinity Labeling be Differentiated from Site‐Directed Photochemical Coupling?

Bouchet

Goeldner

1997

Photochem & Photobiology

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Site-directed photochemical labeling is a methodology designed to irreversibly and specifically label, through the action of light, a ligand binding site of a biological macromolecule. Photoaffinity labeling, a widely used site-directed labeling methodology, uses photosensitive ligand analogs generally obtained after chemical modification of the ligand by introducing an appropriate photoactivatable moiety. This methodology can be applied to natural ligands showing inherent photosensitivity, without any additional modification, and which can be linked efficiently to their receptor binding site by direct photoactivation. The emergence of an alternative methodology that links nonphotosensitive ligands to their receptors has raised the question of their potential use and their mechanisms of photocoupling. This article presents a series of examples that are meant to compare the general characteristics of the different site-directed labeling reactions and proposes distinct photochemical activation processes between photoaffinity labeling and site-directed photochemical coupling reactions. We suggest in particular that the former is necessarily a ligand-mediated activation process while the latter might involve a receptormediated mechanism.

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Colchicine photosensitizes covalent tubulin dimerization

Cited by 9 publications

References 26 publications

Localization of the colchicine-binding site of tubulin.

Localization of the colchicine-binding site of tubulin.

Local Unfolding and the Stepwise Loss of the Functional Properties of Tubulin

Photochemical Labeling: Can Photoaffinity Labeling be Differentiated from Site‐Directed Photochemical Coupling?

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