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Biochemistry 1983, 22, 4814-4822
Taxol Effect on Tubulin Polymerization and Associated Guanosine 5’-Triphosphate Hydrolysis+ Marie-France Carlier* and Dominique Pantaloni
ABSTRACT:
Taxol has been used as a tool to investigate the relationship between microtubule assembly and guanosine 5’-triphosphate (GTP) hydrolysis. The data support the model previously proposed [Carlier, M.-F., & Pantaloni, D. (198 1) Biochemistry 20, 19181 that GTP hydrolysis is not tightly coupled to the polymerization process but takes place as a monomolecular process following polymerization. The results further indicate that the energy liberated by GTP hydrolysis is not responsible for the subsequent blockage of GDP on polymerized tubulin. When tubulin is polymerized in the presence of 10-100 pM taxol, the rapid formation of a large number of very short microtubules (I < 1 pm) is accompanied by the development of turbidity to a lesser extent than what is observed when the same weight amount of longer micro-
tubules (1 = 5 pm) is formed. A slower subsequent turbidity increase corresponds to the length redistribution of these short microtubules into 3-5-fold longer ones without any change in the weight amount of polymer. The evolution of the rate of length redistribution with the concentration of taxol suggests a model within which taxol would bind to dimeric tubulin and to tubulin present at the ends of microtubules with a somewhat 10-fold lower affinity than to polymerized tubulin embedded in the bulk of microtubules. In agreement with this model, binding of taxol to the tubulin-colchicine complex in the dimeric form could be measured from the increase in the GTPase activity of the tubulin-colchicine complex accompanying taxol binding.
A m o n g the antitumor agents that interact with tubulin, taxol (Wani et al., 1971) exhibits a unique behavior since, in contrast to other antimitotic alkaloids that inhibit microtubule formation, it acts as a promotor of tubulin polymerization and stabilizes microtubules to depolymerization by various agents, both in vitro (Schiff et al., 1979) and in vivo (Schiff & Horwitz, 1980; Masurovsky et al., 1980). It has been shown that taxol bound to polymerized tubulin with a 111 stoichiometry and a high affinity (Parness & Horwitz, 1981) and that taxol promoted microtubule formation in conditions under which no polymerization could otherwise be observed, Le., in the absence of microtubule-associated proteins (MAPS) or of exogenous GTP (Schiff & Horwitz, 1981) or at low temperature (Thompson et al., 1981). Taxol binds to a site different from the colchicine and vinblastine sites (Kumar, 1981; Schiff & Horwitz, 1981); the opposite effects of these drugs are not caused by a direct competition for the same site but probably result from the displacement of the association-dissociation equilibrium of tubulin in opposite directions, colchicine and vinblastine preventing the tubulin-tubulin interactions that take place in the microtubule while taxol would stabilize such interactions. Such a view would account for the observation of enhanced nucleation events and correlated decreased microtubule length in the presence of taxol (Schiff & Horwitz, 1981). Although the binding parameters of taxol to dimer tubulin have never been approached, this model would suggest that the affinity of taxol for the dimer is lower than for the polymer. The fact that taxol-bound microtubules cannot be significantly depolymerized upon dilution or upon cooling suggests that taxol slows down the dissociation rate constant of tubulin from microtubules; this is corroborated by studies of the dynamics of microtubules at steady state in the presence of taxol (Kumar, 1981; Caplow & Zeeberg, 1982).
The GDP-tubulin complex was found able to nucleate and elongate microtubules in the presence of taxol; however, when the polymerization was performed in the presence of both GTP and taxol, GDP was found bound to microtubules at steady state (Schiff & Horwitz, 1981). These two observations raised the problem of the interaction between GTP and taxol on tubulin and, more generally, of the role of GTP hydrolysis in tubulin polymerization. We had previously shown that GTP hydrolysis was a kinetic process subsequent to the polymerization of tubulin but kinetically uncoupled from the polymerization process (Carlier & Pantaloni, 1981). The data mentioned above are consistent with this analysis since they suggest that although either GDP- or GTP-tubulin can be assembled into microtubules in the presence of taxol, GTP is nevertheless hydrolyzed following polymerization. This peculiar property of taxol should also enable one to answer the question of the conformation change following polymerization and leading to the blockage of GDP in the E site: is the reaction of GTP hydrolysis energetically necessary to obtain blockage of GDP, or is the conformation change merely triggered by the presence of GDP in the E site? The other interesting property of taxol is stabilization of microtubules at steady state by decreasing the dissociation rate constant and maintaining a very low, if measurable, critical concentration. It had been shown previously (David-Pfeuty et al., 1977, 1978; Carlier & Pantaloni, 1981) that a steadystate hydrolysis of GTP was catalyzed by the ends of microtubules at steady state. This GTP hydrolysis is likely to be the sum of two components: (i) GTP hydrolysis by a “GTP cap” formed at the end of the microtubule as a result of the steady-state diffusional incorporation of GTP-tubulin molecules followed by GTP hydrolysis; (ii) GTP hydrolysis catalyzed by the last molecules of tubulin at the very end of the microtubules that have been shown to be able to bind and exchange GTP or GDP (Carlier & Pantaloni, 1982); Engelborghs & Van Houtte, 1981). The exact contributions of both reactions are unknown, but the inhibition of the diffusional process by taxol should eliminate the first reaction and permit the analysis of the overall GTP hydrolysis at steady state. Taxol is used, in the experiments reported here, as a tool to
‘From the Laboratoire d’Enzymologie, C.N.R.S., 91 190 Gif-surYvette, France. Received January 13, 1983. This work was supported in part by the Centre National de la Recherche Scientifique, by the Institut National de la Sante et de la Recherche MEdicale, by the Mission des Biotechnologies, and by the Ligue Nationale Franpaise contre le Cancer.
0006-2960/83/0422-48 14$01.50/0
0 1983 American Chemical Society
TUBULIN POLYMERIZATION IN THE PRESENCE OF TAXOL
V O L . 22, N O . 20, 1983
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answer these questions and get a better understanding of the essential reactions that govern microtubule assembly. Materials and Methods Chemicals. Guanosine di- and triphosphate nucleotides and guanylate kinase from hog brain were from Boehringer. Alkaline phosphatase was from Sigma and colchicine from Prolabo. All salts used in buffers were analytical grade. 3Hor 32P-labelednucleotides were from Amersham. Taxol was purified from Taxus baccata in Dr. Potier’s laboratory at the Institut de Chimie des Substances Naturelles and kindly given to us by Dr. Daniel Guinard. Tubulin Purification. Unless otherwise specified, tubulin used in this study was purified by three cycles of polymerization according to Shelanski et al. (1973), followed by chromatography on phosphocellulose according to Weingarten et al. (1975). The protein was stored at 5-8 mg/mL in 50 mM 4-morpholineethanesulfonic acid (Mes) buffer, pH 6.6, containing 4 M glycerol, 0.5 mM ethylene glycol bis(@aminoethyl ether)-N,N,N’,N’-tetraaceticacid (EGTA), and 0.25 mM Mg2+. Polymerization was usually carried out at 37 OC in the same buffer supplemented with 6 mM Mg2+and guanine nucleotides or taxol as specified in the text. Polymerization Measurements. Tubuline polymerization was monitored turbidimetrically at 350 nm as previously described (Carlier & Pantaloni, 1978). When polymerization was carried out in the presence of taxol, the drug was added to the solution of tubulin at 0 OC 1-2 s before the temperature of the solution was raised to 37 OC in the thermostated cell. Microtubules could be rapidly pelleted by centrifugation for 8 min at 160000g in the Airfuge. GTP-Hydrolysis Measurements. GTP hydrolysis accompanying microtubule formation and steady state was measured according to Avron (1960) and as previously described (Carlier & Pantaloni, 1981). Incorporation of labeled nucleotides in microtubules was performed as previously described (Carlier & Pantaloni, 1981). Electron Microscopy Studies and Microtubule-Length Distribution. Histograms of microtubule-length distribution and microtubule average length were determined from electron micrographs at a 7500-fold magnification of negatively stained samples. A drop of microtubule solution was rapidly fixed, at a given time of the polymerization process, in 1% glutaraldehyde at 37 OC, then diluted, deposited on a carbon-coated grid, and stained with 2% uranyl acetate. Observation was carried out in a Hitachi HUIIB electron microscope. Acquisition of the data was performed by tracing over the picture of a few hundred microtubules with a stylus digitizer connected to a Kontron Q80 computer. Memorized data were processed by the computer to generate the histograms of length distribution. Results Tubulin Polymerization and Associated GTP Hydrolysis in the Presence of Substoichiometric Concentrationsof Taxol. Tubulin was polymerized at a concentration high enough (30 pM) to ensure a large disconnection between GTP hydrolysis and microtubule formation (Carlier & Pantaloni, 198 1). While about 80-90% polymerization was completed in 3 min, at the same time only 50% bound GTP had been hydrolyzed on polymerized tubulin. When the assembly was performed under the same conditions and in the presence of substoichiometric concentrations of taxol (
