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• W2006895265 abstract "Stathmin is a phosphorylation-regulated tubulin-binding protein. In vitro and in vivostudies using nonphosphorylatable and pseudophosphorylated mutants of stathmin have questioned the view that stathmin might act only as a tubulin-sequestering factor. Stathmin was proposed to effectively regulate microtubule dynamic instability by increasing the frequency of catastrophe (the transition from steady growth to rapid depolymerization), without interacting with tubulin. We have used a noninvasive method to measure the equilibrium dissociation constants of the T2S complexes of tubulin with stathmin, pseudophosphorylated (4E)-stathmin, and diphosphostathmin. At both pH 6.8 and pH 7.4, the relative sequestering efficiency of the different stathmin variants depends on the concentration of free tubulin,i.e. on the dynamic state of microtubules. This control is exerted in a narrow range of tubulin concentration due to the highly cooperative binding of tubulin to stathmin. Changes in pH affect the stability of tubulin-stathmin complexes but do not change stathmin function. The 4E-stathmin mutant mimics inactive phosphorylated stathmin at low tubulin concentration and sequesters tubulin almost as efficiently as stathmin at higher tubulin concentration. We propose that stathmin acts solely by sequestering tubulin, without affecting microtubule dynamics, and that the effect of stathmin phosphorylation on microtubule assembly depends on tubulin critical concentration. Stathmin is a phosphorylation-regulated tubulin-binding protein. In vitro and in vivostudies using nonphosphorylatable and pseudophosphorylated mutants of stathmin have questioned the view that stathmin might act only as a tubulin-sequestering factor. Stathmin was proposed to effectively regulate microtubule dynamic instability by increasing the frequency of catastrophe (the transition from steady growth to rapid depolymerization), without interacting with tubulin. We have used a noninvasive method to measure the equilibrium dissociation constants of the T2S complexes of tubulin with stathmin, pseudophosphorylated (4E)-stathmin, and diphosphostathmin. At both pH 6.8 and pH 7.4, the relative sequestering efficiency of the different stathmin variants depends on the concentration of free tubulin,i.e. on the dynamic state of microtubules. This control is exerted in a narrow range of tubulin concentration due to the highly cooperative binding of tubulin to stathmin. Changes in pH affect the stability of tubulin-stathmin complexes but do not change stathmin function. The 4E-stathmin mutant mimics inactive phosphorylated stathmin at low tubulin concentration and sequesters tubulin almost as efficiently as stathmin at higher tubulin concentration. We propose that stathmin acts solely by sequestering tubulin, without affecting microtubule dynamics, and that the effect of stathmin phosphorylation on microtubule assembly depends on tubulin critical concentration. 4-morpholineethanesulfonic acid 1,4-piperazinediethanesulfonic acid Microtubules are dynamic polymers that play a role in cell morphology and cell division. The dynamics of microtubule assembly is finely regulated during the cell cycle (1Inoue S. Salmon E.D. Mol. Biol. Cell. 1995; 6: 1619-1640Crossref PubMed Scopus (442) Google Scholar, 2Desai A. Mitchison T.J. Annu. Rev. Cell Dev. Biol. 1997; 13: 83-117Crossref PubMed Scopus (1978) Google Scholar, 3Joshi H.C. Curr. Opin. Cell Biol. 1998; 10: 35-44Crossref PubMed Scopus (118) Google Scholar). In interphase, microtubules are mainly organized in a radial array, with minus ends anchored at the centrosome; however, a fraction of the population has two free ends (4Meads T. Schroer T.A. Cell Motil. Cytoskeleton. 1995; 32: 273-288Crossref PubMed Scopus (157) Google Scholar, 5Keating T.J. Peloquin J.G. Rodionov V.I. Momcilovic D. Borisy G.G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5078-5083Crossref PubMed Scopus (223) Google Scholar, 6Rodionov V.I. Borisy G.G. Science. 1997; 275: 215-218Crossref PubMed Scopus (140) Google Scholar, 7Saoudi Y. Fotedar R. Abrieu A. Dorée M. Wehland J. Margolis R.L. Job D. J. Cell Biol. 1998; 142: 1519-1532Crossref PubMed Scopus (16) Google Scholar). Upon entry into mitosis, microtubules disassemble and then reassemble into a highly dynamic mitotic spindle, with minus ends at the poles. Finally, at the end of mitosis, disassembly of the mitotic microtubules is balanced by the formation of the interphase array in the daughter cells (8Zhai Y. Kronebusch P.J. Simon P.M. Borisy G.G. J. Cell Biol. 1996; 135: 201-214Crossref PubMed Scopus (161) Google Scholar). In living cells, two main processes, treadmilling (6Rodionov V.I. Borisy G.G. Science. 1997; 275: 215-218Crossref PubMed Scopus (140) Google Scholar, 7Saoudi Y. Fotedar R. Abrieu A. Dorée M. Wehland J. Margolis R.L. Job D. J. Cell Biol. 1998; 142: 1519-1532Crossref PubMed Scopus (16) Google Scholar, 9Margolis R.L. Wilson L. Cell. 1978; 13: 1-8Abstract Full Text PDF PubMed Scopus (356) Google Scholar) and dynamic instability (10Mitchison T.J. Kirschner M.W. Nature. 1984; 312: 237-242Crossref PubMed Scopus (2369) Google Scholar), are responsible for monomer-polymer exchange reactions leading to microtubule turnover. Both are driven by the hydrolysis of GTP linked to tubulin assembly. Treadmilling derives from the energetic imbalance between the plus and the minus ends of microtubules and operates when the two ends are free. The steady-state concentration of dimeric GTP-tubulin allows equal net rates of assembly at the plus end and disassembly from the minus end. Dynamic instability concerns microtubules that have either two or only one free end (the plus end, generally), which switches infrequently between a rapidly depolymerizing state and a growing state. The transitions between the two states, called “catastrophe” and “rescue,” describe the stochastic loss and gain of a GTP cap at the end of the microtubule. The steady-state concentration of dimeric GTP-tubulin in this case is determined in part by the frequencies of catastrophe and rescue. Many cellular factors affect the dynamics of monomer-polymer exchange (see Ref. 11Cassimeris L. Curr. Opin. Cell Biol. 2002; 14: 18-24Crossref PubMed Scopus (366) Google Scholar for a review). Microtubule-associated proteins slow down microtubule depolymerization in a phosphorylation-controlled fashion (12Kowalski R.J. Williams R.C., Jr. J. Biol. Chem. 1993; 268: 9847-9855Abstract Full Text PDF PubMed Google Scholar). Kinesins of the KIF family bind to microtubule ends and catalyze depolymerization (13Desai A. Verna S. Mitchison T.J. Walczak C.E. Cell. 1999; 96: 69-78Abstract Full Text Full Text PDF PubMed Scopus (587) Google Scholar), whereas end stabilizers like XMAP215 prevent depolymerization (14Vasquez R.J. Gard D.L. Cassimeris L. J. Cell Biol. 1994; 127: 985-993Crossref PubMed Scopus (187) Google Scholar). As a result of these activities, the steady-state concentration of dimeric tubulin coexisting with microtubules is regulated. The microtubule-severing factor katanin modulates the fraction of microtubules with one or two free ends (15Mc Nally F.J. Thomas S. Mol. Biol. Cell. 1998; 9: 1847-1861Crossref PubMed Scopus (92) Google Scholar,16Quarmby L.M. Lohret T.A. Cell Motil. Cytoskeleton. 1999; 43: 1-9Crossref PubMed Scopus (0) Google Scholar). Microtubule dynamics in a living cell depends on the proportion of microtubules with one or two free ends (17Vorobjev I.A. Rodionov V.I. Maly I.V. Borisy G.G. J. Cell Sci. 1999; 112: 2277-2289PubMed Google Scholar, 18Rodionov V. Nadezhdina E. Borisy G.G. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 115-120Crossref PubMed Scopus (131) Google Scholar), hence katanin also affects the steady-state concentration of free GTP-tubulin by changing the relative contributions of treadmilling and dynamic instability. Thus far, it has not been technically possible to evaluate the steady-state concentration of GTP-tubulin in cells with great accuracy. Nonetheless, when the fraction of free minus ends increases,e.g. by detachment of microtubules from centrosomes, the concentration of free GTP-tubulin is expected to increase from a value close to the critical concentration of the plus end to a value closer to the critical concentration of the minus end. This shift has actually been observed (18Rodionov V. Nadezhdina E. Borisy G.G. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 115-120Crossref PubMed Scopus (131) Google Scholar), supporting the view that the concentration of GTP-tubulin may vary in vivo. In contrast with regulatory factors that control microtubule assembly dynamics, tubulin-sequestering factors bind tubulin in a nonpolymerizable complex. These proteins establish a pool of unassembled tubulin, built at the expense of the microtubule pool and in equilibrium with free tubulin at its steady-state concentration. The concentration of sequestered tubulin is therefore governed by the dynamic state of microtubules. Op18/stathmin is a 17-kDa protein that has been recently revealed to bind tubulin and destabilize microtubules (19Belmont L.D. Mitchison T.J. Cell. 1996; 84: 623-631Abstract Full Text Full Text PDF PubMed Scopus (591) Google Scholar) and is negatively regulated by phosphorylation by a variety of kinases (20Marklund U.N. Larsson H.M. Gradin G. Brattsand G. Gullberg M. EMBO J. 1996; 15: 5290-5298Crossref PubMed Scopus (249) Google Scholar, 21Di Paolo G. Antonsson B. Kassel D. Riederer B.M. Grenningloh G. FEBS Lett. 1997; 416: 149-152Crossref PubMed Scopus (78) Google Scholar, 22Anderssen S.S. Ashford A.J. Tournebize R. Gavet O. Sobel A. Hyman A.A. Karsenti E. Nature. 1997; 389: 640-643Crossref PubMed Scopus (114) Google Scholar, 23Gradin H.M. Larsson N. Marklund U. Gullberg M. J. Cell Biol. 1998; 140: 131-141Crossref PubMed Scopus (125) Google Scholar, 24Gradin H.M. Marklund U. Larsson N. Chatila T.A. Gullberg M. Mol. Biol. Cell. 1997; 6: 3459-3467Crossref Scopus (126) Google Scholar, 25Daub H. Gevaert K. Vandekerckhove J. Sobel A. Hall A. J. Biol. Chem. 2001; 276: 1677-1680Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar). Stathmin plays a crucial role in cell division (see Ref. 26Andersen S. Trends Cell Biol. 2000; 10: 261-267Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar for review). It is phosphorylated to a low basal level in interphase and becomes hyperphosphorylated by cyclin-dependent kinases (27Larsson N. Marklund U. Gradin H.M. Brattsand G. Gullberg M. Mol. Cell. Biol. 1997; 17: 5530-5539Crossref PubMed Scopus (169) Google Scholar) and a polo-like kinase (28Budde P.P. Kumagai A. Dunphy W.G. Heald R. J. Cell Biol. 2001; 153: 149-157Crossref PubMed Scopus (78) Google Scholar) upon entry into mitosis. Progression through the cell cycle requires phosphorylation of all four serines (Ser-16, -25, -38, and -63). The phosphorylation level is regulated by protein phosphatase 2A phosphatase during mitosis (29Tournebize R. Andersen S.S. Verde F. Dorée M. Karsenti E. Hyman A.A. EMBO J. 1997; 16: 5537-5549Crossref PubMed Scopus (163) Google Scholar), and dephosphorylation occurs at the end of mitosis (30Mistry S.J. Atweh G.F. J. Biol. Chem. 2001; 276: 31209-31215Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Stathmin sequesters tubulin in a T2S complex in which it interacts with two αβ-tubulin heterodimers (31Jourdain L. Curmi P. Sobel A. Pantaloni D. Carlier M.-F. Biochemistry. 1997; 36: 10817-10821Crossref PubMed Scopus (213) Google Scholar) in a polar αβ-αβ arrangement (see Ref. 11Cassimeris L. Curr. Opin. Cell Biol. 2002; 14: 18-24Crossref PubMed Scopus (366) Google Scholar for review; Refs. 32Steinmetz M.O. Kammerer R.A. Jahnke W. Goldie K.N. Lustig A. van Oostrum J. EMBO J. 2000; 19: 572-580Crossref PubMed Scopus (88) Google Scholar and 33Gigant B. Curmi P.A. Martin-Barbey C. Charbaut E. Lachkar S. Lebeau L. Siavoshian S. Sobel A. Knossow M. Cell. 2000; 102: 809-816Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar). Recent chemical cross-linking data indicate that the NH2-terminal region of stathmin is at the α end of the αβ-αβ dimer (34Muller D.R. Schindler P. Towbin H. Wirth U. Voshol H. Hoving S. Steinmetz M. Anal. Biochem. 2001; 73: 1927-1934Google Scholar). Whether the biological function of stathmin is supported by its tubulin-sequestering activity only or by some additional catastrophe-promoting activity, independent from tubulin binding, is not understood yet (see Ref. 35Walczak C.E. Curr. Opin. Cell Biol. 2000; 12: 52-56Crossref PubMed Scopus (151) Google Scholar for review). Conflicting results have been obtained using pseudophosphorylated mutated stathmin (4E-stathmin) in which all four phosphorylatable serines were replaced by glutamate. The 4E-stathmin showed unaltered tubulin sequestering activity, yet it failed to destabilize microtubules in vivo (36Larsson N. Segerman B. Gradin H.M. Wandzioch E. Cassimeris L. Gullberg M. Mol. Cell. Biol. 1999; 19: 2242-2250Crossref PubMed Scopus (35) Google Scholar, 37Horwitz S.B. Shen H.J., He, L.F. Dittmar P. Neef R. Chen J.H. Schubart U.K. J. Biol. Chem. 1997; 272: 8129-8132Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 38Gavet O. Ozon S. Manceau V. Lawler S. Curmi P. Sobel A. J. Cell Sci. 1998; 111: 3333-3346Crossref PubMed Google Scholar). Mutants affected in the coiled-coil domain of stathmin interacted with tubulin like wild-type stathmin but failed to destabilize microtubules in leukemia cells (36Larsson N. Segerman B. Gradin H.M. Wandzioch E. Cassimeris L. Gullberg M. Mol. Cell. Biol. 1999; 19: 2242-2250Crossref PubMed Scopus (35) Google Scholar). When injected in living cells, NH2- and COOH-terminal-truncated fragments of stathmin also had different effects on the microtubule lattice, suggesting that different activities of stathmin were carried by different regions of the protein (39Howell B. Larsson N. Gullberg M. Cassimeris L. Mol. Biol. Cell. 1999; 10: 105-118Crossref PubMed Scopus (159) Google Scholar, 40Larsson N. Segerman B. Howell B. Fridell K. Cassimeris L. Gullberg M. J. Cell Biol. 1999; 146: 1289-1302Crossref PubMed Scopus (60) Google Scholar). Finally, 4E-stathmin, which seems to lack catastrophe-promoting activity and retain unaltered tubulin-sequestering activity, is able to disrupt interphase microtubules but not mitotic microtubules (41Holmfeldt P. Larsson N. Segerman B. Howell B. Morabito J. Cassimeris L. Gullberg M. Mol. Biol. Cell. 2001; 12: 73-83Crossref PubMed Scopus (56) Google Scholar). Consistently, 4E-stathmin does not prevent normal development of Xenopusembryo (42Küntziger T. Gavet O. Sobel A. Bornens M. J. Biol. Chem. 2001; 276: 22979-22984Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). Quantitative measurements of tubulin binding to wild-type stathmin and mutated stathmin (4E-stathmin) or phosphorylated stathmin, using plasmon resonance (43Curmi P. Andersen S. Lachkar S. Gavet O. Karsenti E. Knossow M. Sobel A. J. Biol. Chem. 1997; 272: 25029-25036Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar) or a pull-down assay (40Larsson N. Segerman B. Howell B. Fridell K. Cassimeris L. Gullberg M. J. Cell Biol. 1999; 146: 1289-1302Crossref PubMed Scopus (60) Google Scholar, 41Holmfeldt P. Larsson N. Segerman B. Howell B. Morabito J. Cassimeris L. Gullberg M. Mol. Biol. Cell. 2001; 12: 73-83Crossref PubMed Scopus (56) Google Scholar), showed that the affinity of stathmin for tubulin was decreased only 3–4-fold by the serine to glutamate mutation and that phosphorylated stathmin had <2-fold lower affinity for tubulin than 4E-stathmin. It was thought (40Larsson N. Segerman B. Howell B. Fridell K. Cassimeris L. Gullberg M. J. Cell Biol. 1999; 146: 1289-1302Crossref PubMed Scopus (60) Google Scholar, 41Holmfeldt P. Larsson N. Segerman B. Howell B. Morabito J. Cassimeris L. Gullberg M. Mol. Biol. Cell. 2001; 12: 73-83Crossref PubMed Scopus (56) Google Scholar) that these modest differences in affinity could not account for the large differences in the effects of the various stathmin variants on microtubules in cells. In contrast with the above-mentioned studies, much larger differences in affinity for tubulin were observed between wild-type, 4E-mutated, and phosphorylated stathmins when their effects on nucleotide exchange on tubulin were measured (44Amayed P. Carlier M.F. Pantaloni D. Biochemistry. 2000; 39: 12295-12302Crossref PubMed Scopus (22) Google Scholar). We thought that the discrepancies regarding stathmin function might originate from the lack of a quantitative evaluation of the tubulin-sequestering activity of the different stathmin derivatives. Here we set up a sequestration assay in which the concentration of free GTP-tubulin coexisting with microtubules at steady state is buffered to any desired value using Taxotere. Using this assay, the affinity of tubulin for the different stathmins differs to a greater extent than in previous measurements. Phosphorylation exerts a regulatory effect on stathmin depending on the concentration of free tubulin. We conclude that the simple sequestering activity of stathmin can support its effects on microtubules in vivo. We tentatively propose that the changes in microtubule dynamics during the cell cycle are associated with variations in the concentration of free tubulin that coexists with microtubules. Changes in the concentration of free tubulin in turn modulate the effect of phosphorylation of stathmin on its sequestering activity. Phosphocellulose-purified bovine brain tubulin (44Amayed P. Carlier M.F. Pantaloni D. Biochemistry. 2000; 39: 12295-12302Crossref PubMed Scopus (22) Google Scholar) was used. The tubulin used in this work had been kept at −80 °C for at most 3 weeks. Older preparations showed a measurable amount of nonpolymerizable material, which increased with aging. Before each experiment, tubulin was recycled by polymerization at 37 °C in M buffer (50 mmMES1-KOH, pH 6.8, 4m glycerol, 0.5 mm EGTA, 0.5 mmGTP, and 6 mm MgCl2). Microtubule pellets were resuspended in M buffer containing 0.5 mm MgCl2on ice and centrifuged at 200,000 × g at 4 °C for 10 min to remove aggregates. Tubulin was equilibrated in the desired buffer by Sephadex G-25 gel filtration (PD-10; Amersham Biosciences). Experiments were performed immediately after the above-mentioned recycling procedure to avoid denaturation of tubulin. Before each experiment, it was verified that tubulin polymerized in microtubules that depolymerized at least 95% at 4 °C. Recombinant wild-type stathmin and 4E-stathmin were expressed inEscherichia coli and purified as described previously (43Curmi P. Andersen S. Lachkar S. Gavet O. Karsenti E. Knossow M. Sobel A. J. Biol. Chem. 1997; 272: 25029-25036Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar). Stathmin was phosphorylated on serines 16 and 63 by protein kinase A (Sigma) as described previously (44Amayed P. Carlier M.F. Pantaloni D. Biochemistry. 2000; 39: 12295-12302Crossref PubMed Scopus (22) Google Scholar). Spontaneous polymerization of tubulin in microtubules was monitored turbidimetrically at 350 nm in a Cary 1 spectrophotometer using a 1-cm path, 120-μl cuvette thermostated at 37 °C. Experiments were carried out in either glycerol-containing M buffer or glycerol-free P buffer (0.1 m PIPES-KOH, pH 6.8, 0.5 mm EGTA, 0.5 mm GTP, and 6 mmMgCl2) or glycerol-free H buffer (100 mmHEPES-KOH, pH 7.4, 0.5 mm EGTA, 0.5 mm GTP, and 6 mm MgCl2) in the absence or presence of Taxotere or stathmin as indicated. In preliminary assays, the range of stathmin concentrations was selected to cause at most a 10% decrease in the mass of microtubules (see “Results”). Polymerization was started by the addition of MgCl2 and Taxotere to the tubulin + stathmin solution that was immediately brought into the prewarmed cuvette. The temperature reached 37 °C in less than 15 s. Critical concentration plots were derived from turbidity measurements as described previously (31Jourdain L. Curmi P. Sobel A. Pantaloni D. Carlier M.-F. Biochemistry. 1997; 36: 10817-10821Crossref PubMed Scopus (213) Google Scholar). Parallel samples were polymerized identically in centrifuge tubes placed in a water bath at 37 °C and then centrifuged at 300,000 × g for 15 min at 37 °C in a TL 100 ultracentrifuge (Beckman). The supernatants were denatured and subjected to SDS-PAGE electrophoresis for evaluation of the amount of unassembled tubulin. In the absence of stathmin, the concentration of unassembled tubulin equaled the critical concentration [T]SS. In the presence of stathmin, the concentration of unassembled tubulin was [T]U = [T]SS + 2 [T2S]. A series of tubulin standards in the appropriate range were electrophoresed on the same gel. Gels were stained with either Coomassie Blue or silver (45Morrissey J.H. Anal. Biochem. 1981; 117: 307-310Crossref PubMed Scopus (2942) Google Scholar), depending on the amounts of tubulin present in the supernatants. Gels were scanned and analyzed using NIH Image software. The amounts of nonassembled tubulin in the samples were determined by interpolation using the calibration curve obtained with standards spanning the range of tubulin concentrations found in the samples. The value of the equilibrium dissociation constant for the T2S complex was determined as follows at different total concentrations of stathmin [S]0. KD=[T]SS2×[S]/[T2S]Equation 1 [T2S]=([T]U−[T]SS)/2Equation 2 [S]=[S]0−([T]U−[T]SS)/2Equation 3 Stathmin interacts with two αβ-tubulin heterodimers in a T2S complex (31Jourdain L. Curmi P. Sobel A. Pantaloni D. Carlier M.-F. Biochemistry. 1997; 36: 10817-10821Crossref PubMed Scopus (213) Google Scholar, 32Steinmetz M.O. Kammerer R.A. Jahnke W. Goldie K.N. Lustig A. van Oostrum J. EMBO J. 2000; 19: 572-580Crossref PubMed Scopus (88) Google Scholar, 33Gigant B. Curmi P.A. Martin-Barbey C. Charbaut E. Lachkar S. Lebeau L. Siavoshian S. Sobel A. Knossow M. Cell. 2000; 102: 809-816Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar). At 5 μm tubulin and at concentrations of stathmin as high as 50 μm, the T2S complex was the only complex observed in the analytical ultracentrifuge; no evidence was obtained for an intermediate 1:1 TS complex (31Jourdain L. Curmi P. Sobel A. Pantaloni D. Carlier M.-F. Biochemistry. 1997; 36: 10817-10821Crossref PubMed Scopus (213) Google Scholar, 44Amayed P. Carlier M.F. Pantaloni D. Biochemistry. 2000; 39: 12295-12302Crossref PubMed Scopus (22) Google Scholar). This result, corroborated by structural (33Gigant B. Curmi P.A. Martin-Barbey C. Charbaut E. Lachkar S. Lebeau L. Siavoshian S. Sobel A. Knossow M. Cell. 2000; 102: 809-816Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar) and biochemical studies (40Larsson N. Segerman B. Howell B. Fridell K. Cassimeris L. Gullberg M. J. Cell Biol. 1999; 146: 1289-1302Crossref PubMed Scopus (60) Google Scholar, 41Holmfeldt P. Larsson N. Segerman B. Howell B. Morabito J. Cassimeris L. Gullberg M. Mol. Biol. Cell. 2001; 12: 73-83Crossref PubMed Scopus (56) Google Scholar), suggested that tubulin bound cooperatively to stathmin, i.e. tubulin-tubulin interactions as well as stathmin-tubulin interactions were responsible for the high stability of the T2S complex. The lateral interaction of the two αβ-tubulin molecules with a tandem of two related consecutive α-helices in stathmin (33Gigant B. Curmi P.A. Martin-Barbey C. Charbaut E. Lachkar S. Lebeau L. Siavoshian S. Sobel A. Knossow M. Cell. 2000; 102: 809-816Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar) stabilizes the longitudinal interactions in the αβ-αβ tubulin dimer, under ionic conditions where tubulin exists only in the αβ-tubulin form. The general binding scheme of tubulin to stathmin can therefore be described by an isoenergetic square model (Fig.1) in which the intermediate 1:1 complexes TS and ST interact strongly with a second tubulin molecule, leading to T2S. The TS and ST complexes account for the binding of tubulin to the NH2-terminal or the COOH-terminal α-helix of stathmin, hence they have different structures (Fig.1 a). Whether only one of the two α-helices is sufficient to induce dimerization of tubulin in a T2S complex is not known. Alternatively (Fig. 1 b), stathmin can formerly bind strongly to a poorly represented αβ-αβ tubulin dimer (TT), leading to T2S. The kinetic mechanism of binding of tubulin to stathmin is not known. The TS, ST, and TT species are putative kinetic intermediates that play an important role in the pathway leading to T2S but are not detected at equilibrium because of their low stability (44Amayed P. Carlier M.F. Pantaloni D. Biochemistry. 2000; 39: 12295-12302Crossref PubMed Scopus (22) Google Scholar). Equilibrium binding of tubulin to stathmin is described by an extremely cooperative scheme.2T+S⇔T2SREACTION1The overall equilibrium dissociation constant of the T2S complex is as follows:KD=[T]2×[S]/[T2S]Equation 4 where [T] and [S] are the concentrations of free tubulin and stathmin, and [T2S] is the concentration of the tubulin-stathmin complex. Irrespective of the pathway leading to T2S, K D has the dimension of a square concentration, expressed in m2. A physically significant parameter is the concentration of free tubulin at which half of total stathmin is in complex with tubulin at equilibrium ([S] = [T2S]), called [T]12. Eq. 4 shows that [T]12 = KD 12. Because of the strong cooperativity in stathmin-tubulin interactions, the ratio of the free tubulin concentrations at which 90% and 10% of stathmin is in complex with tubulin ([T]90%/[T]10%) equals 9, whereas it would have a value of 81 if binding was noncooperative. In a solution of microtubules at steady state, the concentration of free tubulin is maintained at a steady-state value [T]SS (often called critical concentration) that depends on the dynamics of microtubules under these solution conditions. When stathmin is added to microtubules at steady state, it binds to tubulin, and because the resulting T2S complex is nonpolymerizable, microtubules depolymerize to maintain the concentration of free tubulin equal to [T]SS. The T2S complex is formed at the expense of the microtubule pool, without affecting the value of [T]SS. Depolymerization stops, and steady state is again established when both the remaining microtubules and T2S complex coexist with free tubulin at the unchanged concentration [T]SS. The amount of T2S complex at steady state is determined by the values of K D and [T]SS as follows. [T2S]=[S]0×[T]SS2/(KD+[T]SS2)Equation 5 The total concentration of stathmin is [S]0 = [S] + [T2S]. Note that if stathmin is added at a concentration high enough to cause complete depolymerization of microtubules, the concentration of free tubulin is no longer buffered by microtubules, hence Eq. 5 is no longer valid. The relevant description then is the binding and saturation of tubulin by stathmin, and a cubic equation describes the dependence of [T2S] on the concentrations of total tubulin and total stathmin. According to Eq. 5, the value of K D can easily be derived from measurements of the amount of sequestered tubulin upon addition of stathmin to a solution of microtubules at steady state (see “Materials and Methods”). In fact, in the conventional polymerization buffers used for microtrubule assembly in vitro, all the added stathmin or 4E-stathmin or diphosphostathmin was found in complex with tubulin (data not shown), in agreement with previous reports (31Jourdain L. Curmi P. Sobel A. Pantaloni D. Carlier M.-F. Biochemistry. 1997; 36: 10817-10821Crossref PubMed Scopus (213) Google Scholar). This result indicates that the value of [T]SS2 is much higher than the value of K D for all stathmin derivatives, in either glycerol-containing M buffer ([T]SS = 2.5 μm) or glycerol-free P buffer ([T]SS = 15 μm), therefore [T2S] = [S]0in Eq. 5. Differences in the value ofK D for the different stathmin variants would be revealed if the value of [T]SS could be lowered to a value closer to KD12. Taxotere can fulfill this function because this microtubule-stabilizing drug binds specifically to microtubules with an affinity of 2 × 107m−1 at 37 °C (46Diaz J.F. Strobe R. Engelborghs Y. Souto A.A. Andreu J.M. J. Biol. Chem. 2000; 275: 26265-26276Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar) and lowers the critical concentration. To obtain the value of K D for the different stathmin variants, the dependence of [T]SS on Taxotere concentration was established. Experiments were done in glycerol-free P buffer, in which the critical concentration can be varied from 15 μm in the absence of Taxotere to <0.25 μmat high Taxotere concentrations. Tubulin was polymerized at 40 and 15 μm in P buffer in the presence of different concentrations of Taxotere, [X]0. The relationship between the concentration of tubulin in the supernatants of sedimented microtubules, [T]SS, and the concentration of free Taxotere, [X], was established as follows. The concentration of assembled tubulin ([MT]0) was derived from the difference between the total ([T]0) and free ([T]SS) tubulin concentrations. The concentrations of microtubule-bound Taxotere [MTX] and free Taxotere [X] were calculated as follows:[MT]0=[MT]+[MTX]Equation 6 KX=[MT]×[X]/[MTX]Equation 7 [MTX]=1/2{[X]0+[MT]0Equation 8 +KX±(([X]0+[MT]0+KX)2−4[MT]0×[X]0)½}[X]=[X]0−[MTX]Equation 9 where K X represents the equilibrium dissociation constant for Taxotere binding to tubulin in microtubules. The value of [T]SS decreased with free Taxotere (Fig.2). The curves representing [T]SS versus [X] obtained at the two concentrations of tubulin tested (○ and ●) were superimposable, as expected for the dependence of an equilibrium constant on free ligand concentration, which should be independent of the total concentration of tubulin. The fact that the curves obtained at 40 and 15 μm tubulin are superimposable, within an experimental error of" @default.
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• W2006895265 date "2002-06-01" @default.
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• W2006895265 title "The Effect of Stathmin Phosphorylation on Microtubule Assembly Depends on Tubulin Critical Concentration" @default.
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• W2006895265 doi "https://doi.org/10.1074/jbc.m111605200" @default.
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