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• W2020396988 abstract "Stathmin is an intrinsically disordered protein implicated in the regulation of microtubule dynamics and in the development of cancer. The microtubule destabilizing activity of stathmin is down-regulated by phosphorylation of four serine residues, Ser16, Ser25, Ser38, and Ser63. Here we have used calorimetric and spectroscopic methods, including nuclear magnetic resonance to analyze the properties of seven stathmin phosphoisoforms to bind tubulin and inhibit microtubule formation. We found that stathmin phosphorylation results in a substantial loss in hydration entropy upon tubulin-stathmin complex formation. Remarkably, a linear correlation between the free energy change of complex formation and the microtubule inhibition activities of stathmin phosphoisoforms was observed. This finding provides a biophysical basis for understanding the mechanism by which local stathmin activity gradients important for promoting localized microtubule growth are established. We further found that phosphorylation of Ser16 and Ser63 disrupts the formation of a tubulin-interacting β-hairpin and a helical segment, respectively, explaining the dominant role of these residues in regulating cell cycle progression. The insight into the tubulin-stathmin interaction offers a molecular basis for understanding the nature and the factors that control intrinsically disordered protein systems in general. Stathmin is an intrinsically disordered protein implicated in the regulation of microtubule dynamics and in the development of cancer. The microtubule destabilizing activity of stathmin is down-regulated by phosphorylation of four serine residues, Ser16, Ser25, Ser38, and Ser63. Here we have used calorimetric and spectroscopic methods, including nuclear magnetic resonance to analyze the properties of seven stathmin phosphoisoforms to bind tubulin and inhibit microtubule formation. We found that stathmin phosphorylation results in a substantial loss in hydration entropy upon tubulin-stathmin complex formation. Remarkably, a linear correlation between the free energy change of complex formation and the microtubule inhibition activities of stathmin phosphoisoforms was observed. This finding provides a biophysical basis for understanding the mechanism by which local stathmin activity gradients important for promoting localized microtubule growth are established. We further found that phosphorylation of Ser16 and Ser63 disrupts the formation of a tubulin-interacting β-hairpin and a helical segment, respectively, explaining the dominant role of these residues in regulating cell cycle progression. The insight into the tubulin-stathmin interaction offers a molecular basis for understanding the nature and the factors that control intrinsically disordered protein systems in general. Intrinsically disordered proteins have gained enormous interest not only because they are recognized to play key roles in many central cellular processes including cell cycle control, signal transduction, and transcriptional regulation but also because of their particular importance for cancer development and protein deposition diseases (1Dyson H.J. Wright P.E. Nat. Rev. Mol. Cell. Biol. 2005; 6: 197-208Crossref PubMed Scopus (2984) Google Scholar, 2Uversky V.N. Oldfield C.J. Dunker A.K. J. Mol. Recognit. 2005; 18: 343-384Crossref PubMed Scopus (679) Google Scholar, 3Tompa P. Szasz C. Buday L. Trends Biochem. Sci. 2005; 30: 484-489Abstract Full Text Full Text PDF PubMed Scopus (396) Google Scholar, 4Fink A.L. Curr. Opin. Struct. Biol. 2005; 15: 35-41Crossref PubMed Scopus (601) Google Scholar). Recent genome data base searches indicated that >30% of all eukaryotic proteins may be completely or partially disordered (5Ward J.J. Sodhi J.S. McGuffin L.J. Buxton B.F. Jones D.T. J. Mol. Biol. 2004; 337: 635-645Crossref PubMed Scopus (1591) Google Scholar). This high frequency of occurrence has provoked a change of the paradigm that stable tertiary structure is necessary for protein function.Intrinsically disordered polypeptide chain segments are primarily involved in molecular recognition and posttranslational modification including phosphorylation (6Iakoucheva L.M. Radivojac P. Brown C.J. O'Connor T.R. Sikes J.G. Obradovic Z. Dunker A.K. Nucleic Acids Res. 2004; 32: 1037-1049Crossref PubMed Scopus (1112) Google Scholar). Little is known, however, regarding the nature and mechanism of control of protein-protein interactions involving intrinsically disordered proteins. Stathmin is a key regulator of microtubule dynamics that lacks a stable three-dimensional structure (7Steinmetz M.O. Kammerer R.A. Jahnke W. Goldie K.N. Lustig A. van Oostrum J. EMBO J. 2000; 19: 572-580Crossref PubMed Scopus (86) Google Scholar, 8Honnappa S. Cutting B. Jahnke W. Seelig J. Steinmetz M.O. J. Biol. Chem. 2003; 278: 38926-38934Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). The soluble cytoplasmic protein destabilizes microtubules by binding tubulin dimers (7Steinmetz M.O. Kammerer R.A. Jahnke W. Goldie K.N. Lustig A. van Oostrum J. EMBO J. 2000; 19: 572-580Crossref PubMed Scopus (86) Google Scholar, 9Curmi P.A. Andersen S.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 (192) Google Scholar, 10Jourdain L. Curmi P. Sobel A. Pantaloni D. Carlier M.F. Biochemistry. 1997; 36: 10817-10821Crossref PubMed Scopus (213) Google Scholar, 11Ravelli R.B. Gigant B. Curmi P.A. Jourdain I. Lachkar S. Sobel A. Knossow M. Nature. 2004; 428: 198-202Crossref PubMed Scopus (1271) Google Scholar, 12Gigant B. Wang C. Ravelli R.B. Roussi F. Steinmetz M.O. Curmi P.A. Sobel A. Knossow M. Nature. 2005; 435: 519-522Crossref PubMed Scopus (544) Google Scholar) and stimulating catastrophes (referred to as the transition of microtubule growth to shortening) (13Howell B. Larsson N. Gullberg M. Cassimeris L. Mol. Biol. Cell. 1999; 10: 105-118Crossref PubMed Scopus (157) Google Scholar, 14Belmont L.D. Mitchison T.J. Cell. 1996; 84: 623-631Abstract Full Text Full Text PDF PubMed Scopus (582) Google Scholar, 15Manna T. Thrower D. Miller H.P. Curmi P. Wilson L. J. Biol. Chem. 2006; 281: 2071-2078Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar), playing a central role for cell proliferation, cell migration, and mitotic spindle formation (reviewed by Refs. 16Curmi P.A. Gavet O. Charbaut E. Ozon S. Lachkar-Colmerauer S. Manceau V. Siavoshian S. Maucuer A. Sobel A. Cell Struct. Funct. 1999; 24: 345-357Crossref PubMed Scopus (159) Google Scholar, 17Andersen S.S. Trends Cell Biol. 2000; 10: 261-267Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar, 18Walczak C.E. Curr. Opin. Cell Biol. 2000; 12: 52-56Crossref PubMed Scopus (149) Google Scholar, 19Cassimeris L. Curr. Opin. Cell Biol. 2002; 14: 18-24Crossref PubMed Scopus (363) Google Scholar, 20Rubin C.I. Atweh G.F. J. Cell. Biochem. 2004; 93: 242-250Crossref PubMed Scopus (309) Google Scholar). Interestingly, stathmin is expressed in high amounts in a wide variety of human malignancies, and its overexpression correlates with increased cell motility and invasion of human sarcomas in vivo (21Baldassarre G. Belletti B. Nicoloso M.S. Schiappacassi M. Vecchione A. Spessotto P. Morrione A. Canzonieri V. Colombatti A. Cancer Cell. 2005; 7: 51-63Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar). Moreover, stathmin is important in regulating innate and learned fear and is thus implicated in anxiety states of mental disorders (22Shumyatsky G.P. Malleret G. Shin R.M. Takizawa S. Tully K. Tsvetkov E. Zakharenko S.S. Joseph J. Vronskaya S. Yin D. Schubart U.K. Kandel E.R. Bolshakov V.Y. Cell. 2005; 123: 697-709Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). These findings underscore the crucial role of stathmin in a wide variety of central microtubule-dependent cellular processes.Characteristic of intrinsically disordered proteins, stathmin is devoid of stable tertiary structure in isolation (7Steinmetz M.O. Kammerer R.A. Jahnke W. Goldie K.N. Lustig A. van Oostrum J. EMBO J. 2000; 19: 572-580Crossref PubMed Scopus (86) Google Scholar, 8Honnappa S. Cutting B. Jahnke W. Seelig J. Steinmetz M.O. J. Biol. Chem. 2003; 278: 38926-38934Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 23Steinmetz M.O. Jahnke W. Towbin H. Garcia-Echeverria C. Voshol H. Muller D. van Oostrum J. EMBO Rep. 2001; 2: 505-510Crossref PubMed Scopus (51) Google Scholar, 24Wallon G. Rappsilber J. Mann M. Serrano L. EMBO J. 2000; 19: 213-222Crossref PubMed Scopus (60) Google Scholar), whereas its N-terminal moiety adopts little regular secondary structure the C-terminal domain populates an ensemble of transient helical conformations (see Fig. 1A). Upon binding of stathmin to two head-to-tail aligned α/β-tubulin heterodimers, the N terminus folds into a β-hairpin, and the C-terminal helical domain becomes stabilized (11Ravelli R.B. Gigant B. Curmi P.A. Jourdain I. Lachkar S. Sobel A. Knossow M. Nature. 2004; 428: 198-202Crossref PubMed Scopus (1271) Google Scholar, 12Gigant B. Wang C. Ravelli R.B. Roussi F. Steinmetz M.O. Curmi P.A. Sobel A. Knossow M. Nature. 2005; 435: 519-522Crossref PubMed Scopus (544) Google Scholar, 25Clement M.J. Jourdain I. Lachkar S. Savarin P. Gigant B. Knossow M. Toma F. Sobel A. Curmi P.A. Biochemistry. 2005; 44: 14616-14625Crossref PubMed Scopus (32) Google Scholar). The curved and capped structure of the ternary tubulin-stathmin complex (see Fig. 1B) provides a structural basis for understanding how stathmin family proteins destabilize microtubules.In vivo, the activity of stathmin is down-regulated by posttranslational phosphorylation in response to a number of signals on four serine residues, Ser16, Ser25, Ser38, and Ser63 (26Larsson N. Marklund U. Gradin H.M. Brattsand G. Gullberg M. Mol. Cell. Biol. 1997; 17: 5530-5539Crossref PubMed Scopus (168) Google Scholar, 27Horwitz S.B. Shen H.J. He L. Dittmar P. Neef R. Chen J. Schubart U.K. J. Biol. Chem. 1997; 272: 8129-8132Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 28Kuntziger T. Gavet O. Sobel A. Bornens M. J. Biol. Chem. 2001; 276: 22979-22984Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar, 29Wittmann T. Bokoch G.M. Waterman-Storer C.M. J. Biol. Chem. 2004; 279: 6196-6203Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar). In mitotic cells, for example, phosphorylation by an unknown kinase-phosphatase system allows creating local stathmin activity gradients, a process essential for regulating microtubule dynamics and spindle formation (30Andersen S.S. Ashford A.J. Tournebize R. Gavet O. Sobel A. Hyman A.A. Karsenti E. Nature. 1997; 389: 640-643Crossref PubMed Scopus (113) Google Scholar, 31Niethammer P. Bastiaens P. Karsenti E. Science. 2004; 303: 1862-1866Crossref PubMed Scopus (170) Google Scholar, 32Budde P.P. Kumagai A. Dunphy W.G. Heald R. J. Cell Biol. 2001; 153: 149-158Crossref PubMed Scopus (78) Google Scholar, 33Iancu C. Mistry S.J. Arkin S. Wallenstein S. Atweh G.F. J. Cell Sci. 2001; 114: 909-916Crossref PubMed Google Scholar). Phosphorylation of all four serine residues at the G2/M transition occurs sequentially; Ser25 and Ser38 are first phosphorylated by Cdk1, with subsequent phosphorylation of Ser16 and Ser63 by unknown kinase systems (26Larsson N. Marklund U. Gradin H.M. Brattsand G. Gullberg M. Mol. Cell. Biol. 1997; 17: 5530-5539Crossref PubMed Scopus (168) Google Scholar). Phosphorylation of Ser16 and Ser63 strongly down-regulates the microtubule destabilizing activity of stathmin (26Larsson N. Marklund U. Gradin H.M. Brattsand G. Gullberg M. Mol. Cell. Biol. 1997; 17: 5530-5539Crossref PubMed Scopus (168) Google Scholar, 27Horwitz S.B. Shen H.J. He L. Dittmar P. Neef R. Chen J. Schubart U.K. J. Biol. Chem. 1997; 272: 8129-8132Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 28Kuntziger T. Gavet O. Sobel A. Bornens M. J. Biol. Chem. 2001; 276: 22979-22984Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar, 29Wittmann T. Bokoch G.M. Waterman-Storer C.M. J. Biol. Chem. 2004; 279: 6196-6203Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar;34;35Amayed P. Pantaloni D. Carlier M.F. J. Biol. Chem. 2002; 277: 22718-22724Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). In contrast, phosphorylation of Ser25 and Ser38 has only a moderate effect on down-regulation but is a prerequisite for allowing phosphorylation of Ser16 and Ser63 in vivo (26Larsson N. Marklund U. Gradin H.M. Brattsand G. Gullberg M. Mol. Cell. Biol. 1997; 17: 5530-5539Crossref PubMed Scopus (168) Google Scholar).The current knowledge of the tubulin-stathmin interaction provides a unique basis to gain detailed insight into factors regulating intrinsically disordered protein systems. To define how multiple phosphorylation sites control stathmin function, we here have explored seven stathmin phosphoisoforms by using biochemical and biophysical methods.EXPERIMENTAL PROCEDURESProtein Preparation—For the production of high amounts of pure and specific human stathmin phosphoisoforms, seven serine-to-alanine mutants were constructed: S25A,S38A,S63A (for p16), S16A,S25A,S38A (for p63), S25A,S38A (for p16,63), S16A,S63A (for p25,38), S63A (for p16,25,38), S16A (for p25,38,63), and S16A,S25A,S38A,S63A (4A). The microtubule-polymerization inhibition and tubulin binding activities, the secondary structure content, and the thermal stability of the quadruple mutant 4A were indistinguishable from the wild-type, justifying the validity of the approach (Table 1). To produce the mutant molecules, two silent nucleotide mutations, G66C and G141C, were introduced by PCR into the human pET-16b (Novagen) stathmin clone (7Steinmetz M.O. Kammerer R.A. Jahnke W. Goldie K.N. Lustig A. van Oostrum J. EMBO J. 2000; 19: 572-580Crossref PubMed Scopus (86) Google Scholar) generating SacI and XhoI recognition sites between the codons for Ser16 and Ser25 and Ser38 and Ser63, respectively. This new plasmid provided the basis for subsequent cloning of all mutants by using synthetic oligonucleotide adapter primers at either NdeI/SacI, SacI/XhoI, or XhoI/BbvCI recognition sites. The production of the ΔN mutant (stathmin residues 41–149) is described in Ref. 23Steinmetz M.O. Jahnke W. Towbin H. Garcia-Echeverria C. Voshol H. Muller D. van Oostrum J. EMBO Rep. 2001; 2: 505-510Crossref PubMed Scopus (51) Google Scholar.TABLE 1Thermodynamic binding parameters derived from the titration of tubulin with stathmin variants and phosphoisoformsKD,T2SΔHTΔSΔGM2kcal/molkcal/molkcal/molWild type5.3E-1331.246.8-15.64A4.6E-1329.845.5-15.7p169.0E-1213.427.5-14.1ΔN1.0E-1113.627.5-14.0S16E3.2E-1222.236.8-14.6p636.0E-1116.029.0-13.0S63E2.0E-1233.448.3-14.9p16,631.2E-1012.625.2-12.6p25,382.0E-1216.431.3-14.9p16,25,381.4E-1115.028.8-13.8p25,38,637.2E-1115.528.4-12.9p16,25,38,63NDNDNDND Open table in a new tab Recombinant unlabeled and 15N uniformly labeled stathmin proteins were bacterially expressed, purified, and processed as described (7Steinmetz M.O. Kammerer R.A. Jahnke W. Goldie K.N. Lustig A. van Oostrum J. EMBO J. 2000; 19: 572-580Crossref PubMed Scopus (86) Google Scholar, 8Honnappa S. Cutting B. Jahnke W. Seelig J. Steinmetz M.O. J. Biol. Chem. 2003; 278: 38926-38934Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). Specific phosphorylation by protein kinase A (for phosphorylation of Ser16 and Ser63) and a mixture of MAPK 2The abbreviations used are: MAPK, mitogen-activated protein kinase; PIPES, 1,4-piperazinediethanesulfonic acid; HSQC, heteronuclear single quantum correlation; SLD, stathmin-like domain. and CdC2 (for phosphorylation of Ser25 and Ser38) was achieved by incubating the proteins with the respective kinases (2.8, 2.0, and 0.5 units of protein kinase A, MAPK, and/or CdC2, respectively, per μg of stathmin) in 50 mm Tris-HCl, pH 7.5, 10 mm MgCl2, 5 mm EGTA, 2 mm dithiothreitol, 500 μm ATP for 6–8 h at 30 °C. Product formation was assessed by native-PAGE. Kinases were heat-inactivated at 75 °C for 10 min. Phosphoisoforms were purified to high homogeneity (>93%) by anion exchange chromatography. Highly pure bovine brain GTP-tubulin was obtained from Cytoskeleton Inc.The identities of stathmin proteins were assessed by mass spectral analyses. Concentrations of protein samples were determined by the Advanced Protein Assay (Cytoskeleton Inc.).Tubulin-polymerization Assay—In vitro polymerization of tubulin was performed according to Ref. 26Larsson N. Marklund U. Gradin H.M. Brattsand G. Gullberg M. Mol. Cell. Biol. 1997; 17: 5530-5539Crossref PubMed Scopus (168) Google Scholar. Briefly, 4 μm tubulin in G buffer (80 mm PIPES-KOH, pH 6.8, 1 mm MgCl2, 1 mm EGTA, 1 mm GTP) supplemented with 4 mm MgCl2 was preincubated with 4 μm of stathmin (in the same buffer) for 30 min at room temperature in a total reaction volume of 100 μl. Polymerization was initiated by adding 1 μlofa400 μm taxol stock solution and incubating at 37 °C for 1.5 h. Microtubules were separated from tubulin-stathmin oligomers by sedimentation at 300,000 × g for 15 min at 37 °C in an Optima TLX ultracentrifuge (Beckman Instruments). The protein contents of supernatants and pellets were analyzed with the bicinchoninic acid protein assay reagent (Pierce).Biophysical Analysis—High sensitivity isothermal titration calorimetry experiments were carried out on a VP-ITC calorimeter (Microcal Inc., Northampton, MA). For each experiment, the temperature-controlled sample cell (volume 1.4 ml) was filled with either ∼10 μm (for WT, 4A, p25,38, S16E, and S63E) or ∼20 μm (for p16, p63, p16,63, p16,25,38, p25,38,63, and p16,25,38,63) GTP-tubulin in G buffer (80 mm PIPES-KOH, pH 6.8, 1 mm MgCl2, 1 mm EGTA, 1 mm GTP). Either 5 μl (for WT, 4A, p25,38, S16E, and S63E) or 10 μl (for p16, p63, p16,63, p16,25,38, p25,38,63, and p16,25,38,63) of ∼100 μm stathmin aliquots (present in the same buffer as tubulin) were injected into the sample cell. Binding isotherms were fitted using a nonlinear least squares minimization method assuming two independent and equal binding sites on stathmin for tubulin (8Honnappa S. Cutting B. Jahnke W. Seelig J. Steinmetz M.O. J. Biol. Chem. 2003; 278: 38926-38934Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). The apparent molar heat capacity change of the binding reaction, ΔCp, corresponds to the slope of the linear regression obtained form the fitting of the apparent ΔH values at different temperatures.Protein samples (0.35 mg/ml) for CD were in phosphate-buffered saline (10 mm sodium phosphate, pH 7.4, 150 mm NaCl). Far-ultraviolet CD spectra and thermal unfolding profiles were recorded on a Jasco J-810 spectropolarimeter (Jasco Inc.) equipped with a temperature-controlled quartz cell of 0.1-cm path length. A ramping rate of 1 °C·min–1 was used to record the thermal unfolding profiles.15N,1H-HSQC NMR experiments of 19 mg/ml protein samples in G buffer were carried out at 25 °C on a Varian UnityPlus 600 spectrometer operating at 600 MHz proton frequency. For resonance assignment, three-dimensional 15N-edited TOCSY-HSQC using a clean DIPSI-2 mixing sequence and three-dimensional 15N-HSQC-TOCSY-NOESY-HSQC were recorded.Modeling—The 3.5 Ä resolution x-ray crystal structure of the tubulin-RB3 stathmin-like domain (SLD) complex (PDB entry 1SA0), and the PyMol (De-Lano Scientific LLC, San Carlos, CA) and Moloc (40Gerber P.R. Muller K. J. Comput. Aided Mol. Des. 1995; 9: 251-268Crossref PubMed Scopus (497) Google Scholar) software packages were used for modeling studies. The accuracy of the conformations of key residue side chains was verified by inspecting the electron density map for PDB entry 1SA0. Solvent-accessible area calculations were carried out with the program NACCESS V2.1.1 with the default set of atomic radii and parameters. The stathmin residues 29–45 were taken from PDB entry 1SA1. Tyr63 of the RB3-SLD was replaced by a serine to reflect the human stathmin sequence. Stathmin and RB3-SLD share 72% sequence identity, and 82 and 92% of all tubulin-contacting RB3-SLD residues are invariant or similar, respectively, in stathmin.RESULTS AND DISCUSSIONFor the following studies, milligram amounts of pure and specific single (denoted p16 and p63), double (denoted p25,38 and p16,63), triple (denoted p16,25,38 and p25,38,63), and quadruple (denoted p16,25,38,63) stathmin phosphoisoforms were produced (Fig. 2A). The activities of the proteins were assessed in vitro by a microtubule polymerization assay. Under the experimental conditions applied the efficiency to inhibit microtubule formation decreased from 90 to 0% with a differential combination of stathmin phosphorylation (Fig. 2B). These findings are consistent with stathmin sequestering tubulin dimers into assembly-incompetent complexes (9Curmi P.A. Andersen S.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 (192) Google Scholar, 10Jourdain L. Curmi P. Sobel A. Pantaloni D. Carlier M.F. Biochemistry. 1997; 36: 10817-10821Crossref PubMed Scopus (213) Google Scholar), a process controlled by phosphorylation. In agreement with the microtubule polymerization data obtained in vivo (26Larsson N. Marklund U. Gradin H.M. Brattsand G. Gullberg M. Mol. Cell. Biol. 1997; 17: 5530-5539Crossref PubMed Scopus (168) Google Scholar, 27Horwitz S.B. Shen H.J. He L. Dittmar P. Neef R. Chen J. Schubart U.K. J. Biol. Chem. 1997; 272: 8129-8132Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 28Kuntziger T. Gavet O. Sobel A. Bornens M. J. Biol. Chem. 2001; 276: 22979-22984Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar, 29Wittmann T. Bokoch G.M. Waterman-Storer C.M. J. Biol. Chem. 2004; 279: 6196-6203Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar), phosphorylation of Ser16 and Ser63 contributes most to stathmin inactivation.FIGURE 2Tubulin-binding properties of stathmin and phosphoisoforms. A, Coomassie-stained native-PAGE of purified stathmin phosphoisoforms. The number of phosphoryl groups incorporated in each stathmin phosphoisoform is given on the left. B, taxol-driven in vitro tubulin polymerization in the presence of stathmin variants and phosphoisoforms. C, thermodynamics of T2S complex formation for unmodified stathmin as a function of temperature. The apparent reaction enthalpies, ΔH(open circles), and TΔS(closed circles), were fitted with a linear regression. The solid line describing the reaction free energy, ΔG(closed squares), is the theoretical prediction taking into account ΔCp,T2S,obs. D, reaction enthalpies (open symbols) and TΔS(closed symbols) of phosphoisoforms relative to unmodified stathmin upon T2S complex formation. WT, wild type.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The thermodynamics of the tubulin-stathmin interaction was assessed by isothermal titration calorimetry (supplemental Fig. 1). Between 6 and 25 °C, stathmin binds two tubulin subunits and all thermodynamic parameters are thus referred to the ternary tubulin-stathmin T2S complex (supplemental Table 1). As shown in Fig. 2C, the binding reaction is predicted to be driven by both enthalpy and entropy at physiological temperatures. The large apparent negative heat capacity change of ΔCp,T2S,obs =–1504 ± 206 cal mol–1 K–1 suggests that the hydrophobic effect (removal of non-polar surface from water) promotes T2S complex formation (36Spolar R.S. Record Jr., M.T. Science. 1994; 263: 777-784Crossref PubMed Scopus (1367) Google Scholar). As a consequence, the apparent entropic and enthalpic contributions to the free energy change of complex formation vary with temperature in a linear and nearly parallel manner, changing sign at ∼28 and ∼44 °C, respectively.Empirical studies on proteins showed that the removal of hydrophobic and polar surface from water contributes –45 and 26 cal mol–1/100 Ä2, respectively, to ΔCp (37Luque I. Freire E. Methods Enzymol. 1998; 295: 100-127Crossref PubMed Scopus (114) Google Scholar). We have calculated the total buried hydrophobic and polar surface areas from the 3.5 Ä resolution x-ray crystal structure of the ternary complex formed between the stathmin homologue RB3 and tubulin (denoted T2R, see Ref. 11Ravelli R.B. Gigant B. Curmi P.A. Jourdain I. Lachkar S. Sobel A. Knossow M. Nature. 2004; 428: 198-202Crossref PubMed Scopus (1271) Google Scholar) as 5316 and 3074 Ä2, respectively.Accordingly, the estimated heat capacity change ΔCp,T2S,cal amounts –1593 cal mol–1 K–1, in good agreement with the experimentally obtained value for the tubulin-stathmin complex (see above). These findings are consistent with a mechanism in which dehydration of the protein-protein interface is the major driving force of T2S complex formation.Isothermal titration calorimetry showed that each stathmin phosphoisoform bound two tubulin dimers as observed for unmodified stathmin (Table 1 and supplemental Fig. 1). The equilibrium dissociation constant, KD,T2S, of T2S for unmodified stathmin is 5.3 × 10–13 m2 under the conditions applied. This relatively high value underscores the dynamic nature of the tubulin-stathmin equilibrium observed in cellular systems. Single phosphorylation of Ser16 or Ser63 increases KD,T2S 17- and 113-fold, respectively. A strong effect was found with p16,63, which displays a 233-fold reduced binding affinity. Dual phosphorylation of Ser25 and Ser38 reduced KD,T2S only 4-fold, and the down-regulating effect of the single phosphoisoforms, p16 and p63, was only marginally enhanced (on average 1.4-fold) by additional phosphorylation of Ser25 and Ser38. The isothermal titration calorimetry data of p16,25,38,63 could not be evaluated because binding was too weak. For all measured stathmin phosphoisoforms, a reduced binding entropy that is partially offset by an increased binding enthalpy is observed (Fig. 2D).The finding that phosphorylation of Ser16 and Ser63 contributes most to the reduced binding of stathmin explains the dominant role of these residues for in vivo inactivation (26Larsson N. Marklund U. Gradin H.M. Brattsand G. Gullberg M. Mol. Cell. Biol. 1997; 17: 5530-5539Crossref PubMed Scopus (168) Google Scholar, 27Horwitz S.B. Shen H.J. He L. Dittmar P. Neef R. Chen J. Schubart U.K. J. Biol. Chem. 1997; 272: 8129-8132Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 28Kuntziger T. Gavet O. Sobel A. Bornens M. J. Biol. Chem. 2001; 276: 22979-22984Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar, 29Wittmann T. Bokoch G.M. Waterman-Storer C.M. J. Biol. Chem. 2004; 279: 6196-6203Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar). The moderate effect obtained with phosphorylation of Ser25 and Ser38 correlates with their location in the proline/serine-rich loop segment of stathmin (Fig. 1), which is poorly ordered in the T2R complex (11Ravelli R.B. Gigant B. Curmi P.A. Jourdain I. Lachkar S. Sobel A. Knossow M. Nature. 2004; 428: 198-202Crossref PubMed Scopus (1271) Google Scholar, 12Gigant B. Wang C. Ravelli R.B. Roussi F. Steinmetz M.O. Curmi P.A. Sobel A. Knossow M. Nature. 2005; 435: 519-522Crossref PubMed Scopus (544) Google Scholar). The local perturbation caused by phosphorylated Ser25 and Ser38, however, is expected to facilitate phosphorylation of the adjacent Ser16 and Ser63 residues as suggested from in vivo data (26Larsson N. Marklund U. Gradin H.M. Brattsand G. Gullberg M. Mol. Cell. Biol. 1997; 17: 5530-5539Crossref PubMed Scopus (168) Google Scholar).Remarkably, a linear correlation between the free energy of T2S complex formation and the tubulin polymerization inhibition activities of stathmin phosphoisoforms is observed (Fig. 3). In agreement with cell biological data (26Larsson N. Marklund U. Gradin H.M. Brattsand G. Gullberg M. Mol. Cell. Biol. 1997; 17: 5530-5539Crossref PubMed Scopus (168) Google Scholar, 27Horwitz S.B. Shen H.J. He L. Dittmar P. Neef R. Chen J. Schubart U.K. J. Biol. Chem. 1997; 272: 8129-8132Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 28Kuntziger T. Gavet O. Sobel A. Bornens M. J. Biol. 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