THE JOURNAL OF BIOLOGICAL CHEMISTRY
© 1997 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 272, No. 40, Issue of October 3, pp. 25029 –25036, 1997
Printed in U.S.A.
The Stathmin/Tubulin Interaction in Vitro*
(Received for publication, June 26, 1997, and in revised form, July 29, 1997)
Patrick A. Curmi‡§, Søren S. L. Andersen¶, Sylvie Lachkar‡, Olivier Gavet‡, Eric Karsenti¶,
Marcel Knossowi, and André Sobel‡
From ‡INSERM U440, 17 rue du Fer à Moulin, 75005 Paris, France, ¶EMBL, Cell Biology Program, D-69012 Heidelberg,
Germany, and iLaboratoire d’Enzymologie et Biologie Structurale UPR 9063 CNRS, 91198 Gif sur Yvette, France
Stathmin (1, 2), also designated Op18, p18, p19, prosolin,
and metablastin (3– 6), is a ubiquitous cytosolic phosphoprotein
highly conserved in vertebrates (7, 8) and specifically abundant
in neurons (9 –11). Expression and phosphorylation of stathmin
are modulated in various situations related to the control of
cellular activities, and it has been proposed that it may act as
a relay integrating various intracellular signaling pathways
(1). Expression of stathmin was shown to be regulated in vivo
during development (7, 12–14), during tissue regeneration (15,
16), and in cell culture by cell/cell interactions (17). Stathmin is
also up-regulated in many malignant cell types and tumors (5,
18, 19). Phosphorylation of stathmin is observed in response to
hormones (20), cytokines (21), neurotransmitters (22), and
growth and differentiation factors (23). Moreover, progression
through the cell cycle appears to require multisite phosphorylation of stathmin (24). Actually, overexpression of a nonphos* This work was supported by funds from the Institut National de la
Santé et de la Recherche Médicale, l’Association Française contre les
Myopathies, l’Association pour la Recherche contre le Cancer, and the
Ligue Nationale Française Contre le Cancer. The costs of publication of
this article were defrayed in part by the payment of page charges. This
article must therefore be hereby marked “advertisement” in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: INSERM U440, 17
rue du Fer à Moulin, 75005 Paris, France. Tel.: 33 1 45 87 61 30; Fax:
33 1 45 87 61 32; E-mail: curmi@infobiogen.fr.
This paper is available on line at http://www.jbc.org
phorylatable mutant of stathmin resulted in a large population
of cells blocked in G2/M with a high DNA content (24, 25).
Finally, stathmin is the generic element of a protein family
whose other members most probably play distinct roles related
to the control of neuronal differentiation or to the expression of
neuron-specific traits (8, 26).
The molecular mechanism(s) by which stathmin acts in these
processes remain largely unknown. Two domains can be distinguished in the primary structure of stathmin, an N-terminal
“regulatory” domain that contains the four phosphorylation
sites that account for all of the electrophoretic forms of stathmin observed in vivo (27) and a C-terminal “interaction” domain that includes a predicted coiled-coil forming a-helical
structure thought to interact with other proteins (28). Various
stathmin protein partners have been identified in our laboratory (28) including a novel type of protein kinase, KIS (29).
Recently, stathmin has been identified as a major microtubule
(MT)1-destabilizing factor in Xenopus egg extracts (30). It was
proposed that stathmin binds to unpolymerized tubulin subunits and regulates the dynamic instability of MTs by increasing the frequency of catastrophes. Stathmin also inhibited
spontaneous polymerization of tubulin in a substoichiometric
fashion. Stathmin is thus the second factor identified that acts
as a negative regulator of tubulin polymerization (31). Overexpression of wild type stathmin or of a CDK target site mutant
both elicited rapid depolymerization of MTs in vivo (32).
Horwitz et al. (33) showed that, in vivo, microinjection of recombinant stathmin induced a loss of MTs in COS-7 cells and,
in vitro, that stoichiometric amounts of stathmin were able to
either prevent assembly or promote disassembly of MTs. Interestingly, this latter result was also noted with the stathmin
domain of SCG10, a neuron-specific member of the stathmin
family (34). However, the molecular mechanisms by which
stathmin destabilizes MTs still remain unclear.
In this study, we assessed quantitatively the conditions of
the stathmin/tubulin interaction and determined some of the
binding parameters of the interaction, using gel filtration chromatography, plasmon resonance, and analytical ultracentrifugation. Furthermore, we examined the influence of stathmin on
the polymerization kinetics of tubulin using video-enhanced
contrast, differential interference contrast microscopy. We
demonstrate that stathmin interacts directly with tubulin,
with a maximum affinity of 0.5 mM around pH 6.5. This interaction leads to formation of a 217-kDa complex consisting of the
association of one stathmin with two tubulin heterodimer molecules. Furthermore, we observed that the sole effect of stathmin on MT dynamics in our conditions, as analyzed by video
1
The abbreviations used are: MT, microtubule; BSA, bovine serum
albumin; Fc, flow cell; fcat, catastrophe frequency; RS, Stokes radius;
RU, resonance unit; Vg, growth rate; Pipes, 1,4-piperazinediethanesulfonic acid; WT, recombinant wild type; bWT, boiled recombinant wild
type; FPLC, fast protein liquid chromatography.
25029
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Stathmin is a highly conserved ubiquitous cytoplasmic protein, phosphorylated in response to extracellular signals and during the cell cycle. Stathmin has recently been shown to destabilize microtubules, but the
molecular mechanisms of this function remained unclear. We show here that stathmin directly interacts
with tubulin. We assessed the conditions of this interaction and determined some its quantitative parameters
using plasmon resonance, gel filtration chromatography, and analytical ultracentrifugation. The stathmin/
tubulin interaction leads to the formation of a 7.7 S
complex with a 60-Å Stokes radius, associating one
stathmin with two tubulin heterodimer molecules as
determined by direct quantification by Western blotting. This interaction is sensitive to pH and ionic environment. Its equilibrium dissociation constant, determined by plasmon resonance measurement of kinetic
constants, has an optimum value of 0.5 mM at pH 6.5. The
affinity was lowered with a fully “pseudophosphorylated” 4-Glu mutant form of stathmin, suggesting that it
is modulated in vivo by stathmin phosphorylation. Finally, analysis of microtubule dynamics by video microscopy shows that, in our conditions, stathmin reduces the growth rate of microtubules with no effect on
the catastrophe frequency. Overall, our results suggest
that the stathmin destabilizing activity on microtubules
is related to tubulin sequestration by stathmin.
25030
Stathmin/Tubulin Interaction
microscopy, was a reduction of the MT growth rate, independently of any effect on the catastrophe frequency.
EXPERIMENTAL PROCEDURES
dR/dt 5 k9aC ~Rmax 2 Rt! 2 k9dRt
(Eq. 1)
assuming a single site interaction between stathmin and tubulin, where
dR/dt is the rate of formation of surface complexes (i.e. the derivative of
R 5 R0 z e2k9d~t2t0!
(Eq. 2)
where R is the observed response, R0 is the response at the start of the
dissociation, t is time in seconds, and t0 is the start time for the
dissociation. The association rate constant, k9a, was then calculated by
fitting data to the equation,
R 5 Req~1 2 e2~k9aCn1k9d!~t2t0!!
(Eq. 3)
where Req is the steady state response level, C is the molar concentration of tubulin, n is the steric interference factor (default 1, assuming a
homogeneous single-site interaction between stathmin and tubulin), t0
is the start time of association, and k9d is the dissociation rate constant.
The fit was assessed by comparing constants obtained at different
tubulin concentrations and by examining the x2 and the residual plots
for the fitting. A good fit was considered when x2 was ,1.
Video Microscopy—We used video-enhanced contrast, differential interference contrast microscopy (38) to follow MT dynamics as described
below. Chambers with a volume of 3 ml were used. Two strips of
double-sided tape were fixed on a microscope slide (Select Micro Slide,
76 3 22 mm, 0.8 mm thick, Chance Propper Ltd, United Kingdom;
catalog number BS3836 –1975) approximately 3 mm apart and perpendicular to the long axis of the slide. A 22 3 22-mm coverslip (Gold Seal
cover glass, Clay Adams, United Kingdom; catalog number 3306) was
then placed on top of the two tape strips and firmly pressed to attach it
well. Slide and coverslip were ethanol- and water-washed prior to use.
The channel formed by the tape strips, the slide, and the coverslip made
up the chamber. Prior to use, centrosomes (3 3 103/ml) with a working
concentration of tubulin in BRB80 (80 mM K-Pipes, 1 mM EGTA, 1 mM
MgCl2) were injected into the chamber and left for 5 min on ice. The
sample (always 30-ml total volume) was mixed on ice and consisted of
BRB80, 1 mM GTP, tubulin, and stathmin or control buffer (the sample
contained 1 ml of control buffer or 1 ml of concentrated stathmin (30-fold
dilution)). Using a paper towel and capillary forces, the 30-ml sample
was flowed through the chamber, which was then sealed at both ends
with grease, and placed on the stage of a Zeiss Axioskop (Zeiss GmbH,
Oberkochen, Germany), equipped with a Zeiss 3 100 Achrostigmat 3
100/1.25 oil objective and a Hamamatsu Phototonics (Japan) C3077
CCD camera. Objective and condenser were heated to 37 °C, and the
temperature inside the chamber was 37 °C (measured with a thermocouple). Measurements of MT dynamics were performed as described
previously (39) using NIH 1.617b and Microsoft Excel 5 programs. No
sample was observed for more than 40 min.
Sedimentation Velocity—Sedimentation velocity experiments were
performed at 20 °C in a Beckman Optima XL-A ultracentrifuge
equipped with a Ti60 titanium four-hole rotor with two-channel 12-mm
path length centerpieces. Sample volumes of 400 ml were centrifuged at
45,000 rpm, and radial scans were taken at 280 nm at 5-min intervals.
At this wavelength, tubulin is the only species detected, since the
extinction coefficient of stathmin is very low due to its lack of aromatic
residues. Data analysis was performed using the program dc/dt (40).
The partial specific volume of the complex was estimated to be 0.73
ml/g, the solvent density taken as 1.005 g/cm3, and its viscosity as 1.002
centipoise.
Gel Electrophoresis and Western Blots—One-dimensional electrophoresis was performed on 13% SDS-polyacrylamide gels (41). Proteins
were either silver-stained on fixed gels as described previously (2) or
immunoblotted as follows. Proteins were transferred onto 0.2-mm nitrocellulose filters (Schleicher & Schuell, Germany) in a semidry electroblotting apparatus (transfer buffer: 48 mM Tris, 39 mM glycine, 20%
isopropyl alcohol). The membrane was saturated with 5% nonfat dry
milk in immunoblot solution (12 mM Tris-HCl, pH 7.4, 160 mM NaCl,
0.1% Triton X-100). Membranes were probed with the stathmin antiserum directed against its C-terminal peptide (7) at 1:5000 dilution and
a mouse monoclonal antibody against a-tubulin (N356, Amersham, UK)
at 1:3000 dilution. Bound antibodies were detected by chemiluminescence (ECL, Amersham, UK) with the appropriate secondary antibodies
and the membranes were exposed to XAR5 film (Eastman Kodak Co.).
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Purification of Tubulin and Stathmin—Tubulin was prepared from
calf brain by two cycles of polymerization and depolymerization followed by chromatography on phosphocellulose and an additional cycle
of polymerization and depolymerization (35). The product was stored at
280 °C at a concentration of 8.2 mg/ml in 20 mM K-Pipes, pH 6.8, 0.25
mM EGTA, 0.25 mM MgCl2, until use.
Recombinant wild type (WT) stathmin, E mutants (16E, 25E, 38E,
25/38E, or 4E in which Ser-16, Ser-25, Ser-38, Ser-25/38, or Ser-16/25/
38/63 were, respectively, substituted for Glu) and 4A mutant (Ser-16/
25/38/63 substituted for Ala) were prepared as described previously
(36). Boiled recombinant wild type (bWT) stathmin was prepared as
follows. After sonication of WT stathmin expressing bacteria in extraction buffer, a low speed supernatant was complemented with 100 mM
NaCl and heated at 100 °C for 3 min and then ultracentrifuged, and the
supernatant was recovered. It was used to purify stathmin to homogeneity in a two-step procedure: anion exchange chromatography and
FPLC gel filtration on a Superose 12 HR 10/30 column (Pharmacia,
Uppsala, Sweden). Pure stathmin was concentrated to about 20 mg/ml
in water on Centriprep-10 (Amicon) and stored at 280 °C. Protein
concentrations were achieved by amino acid determination.
Gel Filtration Chromatography—To estimate the Stokes radius (RS)
of tubulin, stathmin, and the stathmin-tubulin complex, proteins were
subjected to gel filtration on an FPLC Superose 12 HR 10/30 column.
Samples (100 ml) were run on the column after equilibration in AB
buffer (80 mM K-Pipes, pH 6.5, 1 mM EGTA, 5 mM MgCl2) at 0.5 ml/min.
Standard proteins used to calibrate the column were ribonuclease A (RS
5 16.4 Å), ovalbumin (RS 5 30.5 Å), bovine serum albumin (RS 5 35.5
Å), aldolase (RS 5 48.1 Å), and ferritin (RS 5 61 Å) (gel filtration
calibration kit (Pharmacia)). Gel filtration data are presented either as
elution volumes or as the molecular sieve coefficient, Kav, a parameter
calculated as (Ve 2 V0)/(Vt 2 V0), where Ve represents the elution
volume corresponding to the peak concentration of a protein, V0 is void
volume of the column, and Vt is total volume of the gel bed. The void
volume was determined by measuring the elution volume with blue
dextran. The Stokes radius of the stathmin-tubulin complex was determined graphically on a (2log Kav)1⁄2 versus Stokes radius plot, according
to Siegel and Monty (37), constructed with the above mentioned standard proteins. Spectrophotometer recordings were performed at 280 nm
to detect tubulin and the stathmin-tubulin complex, because at this
wavelength stathmin is not absorbing (36).
Plasmon Resonance Experiments—BIAcorey 2000 system, sensor
chip CM5, HBS buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 3.4 mM
EDTA, 0.005% (v/v) surfactant p20), and an amine-coupling kit containing N-hydroxysuccinimide, N-ethyl-N9-(3-diethyl-aminopropyl)-carbodiimide, and 1 M ethanolamine-hydrochloride, pH 8.5, were obtained
from Pharmacia Biosensor AB (Uppsala, Sweden).
Flow cells (Fc) were prepared with the various stathmin forms: WT,
bWT, 16E, 25E, 38E, 25/38E, 4E, and 4A. Immobilization of the different forms of stathmin to the CM5 sensor chip was performed in the
BIAcore system as follows. A continuous flow of HBS buffer over the
sensor surface at 10 ml/min was maintained. The carboxylated dextran
matrix was activated by the injection of 70 ml of a solution containing
0.2 M N-ethyl-N9-(3-diethyl-aminopropyl)-carbodiimide and 0.05 M Nhydroxysuccinimide. Next, 40 – 80 ml of stathmin (30 mg/ml in 10 mM
sodium acetate, pH 5) were injected, followed by 70 ml of ethanolamine
to block remaining N-hydroxysuccinimide ester groups. The immobilization levels were 1000 resonance units (RU) and 2000 RU for WT, 1000
RU for bWT, 570 RU for 4E, and 1000 RU for 4A. Control flow cells
consisted either of an activated-deactivated flow cell or of a flow cell
coupled with bovine serum albumin (1000 RU).
To follow the interaction of tubulin with sensor chip-coupled stathmin, an analytical cycle consisted in the injection of 14 ml of tubulin at
increasing concentrations (1, 2, 3, 6, 8, 10, and 20 mM) in AB buffer at
the indicated pH (pH 6 –7.5, with 0.25 pH unit steps) and at a flow rate
of 7 ml/min at 25 °C. Analytical cycles were preprogrammed, and the
entire analysis was completely automated.
Kinetic Analysis of Plasmon Resonance Results—Interaction curves
were obtained, and data were evaluated as shown in Fig. 3. We used the
rate equation,
the observed response curve), C is the concentration of tubulin (constant), Rmax is the total amount of immobilized ligand expressed as
surface plasmon resonance response, Rt is the response observed at
time t, and Rmax 2 Rt is the amount of remaining free binding sites at
time t. (Please note that all the rate and derived equilibrium constants
calculated here are reported with a prime to indicate that they are not
solution constants). To calculate the dissociation rate constant, k9d, data
obtained with a high flow of buffer passing over the flow cell (assuming
no rebinding) were fitted to the equation,
Stathmin/Tubulin Interaction
For the determination of the stoichiometry of the stathmin-tubulin
complex, samples of unknown concentration were compared with stathmin and tubulin standards whose concentrations were determined by
amino acid analysis. The blots were probed with rabbit stathmin antiserum at 1:5000 dilution mixed, respectively, with either mouse monoclonal antibodies against a-tubulin or b-tubulin at 1:3000 dilution
(N356 and N357, respectively, Amersham, UK). Appropriate secondary
antibodies used were 35S-labeled whole antibody to rabbit Ig (SJ434,
Amersham, UK) and 35S-labeled whole antibody to mouse Ig (SJ431,
Amersham, UK). Quantification of stathmin, a-tubulin, or b-tubulin
was achieved by direct counting of the radioactivity in each relevant
35
S-labeled band with an Instant Imager apparatus (Packard Instruments, Meriden, CT).
RESULTS AND DISCUSSION
Stathmin Interacts Directly with Tubulin—The stathmin/
tubulin interaction was revealed by using the plasmon resonance technology. This method allows a real time observation
of binding of molecules to surface immobilized ligands with no
need to label either of the two (42). Mass variations in the
vicinity of the surface are detected. Upon the addition of a
molecule in solution to a surface bound ligand, the signal measured, which is proportional to the mass detected, provides a
sensorgram, which allows to monitor the interaction.
Fig. 1A shows the net binding of pure tubulin to immobilized
WT stathmin. 3 mM tubulin in AB buffer, pH 7.5, was injected
at point A, and the injection was terminated at point B when
the buffer was allowed to flow over the sensor surface. We then
varied the pH of the buffer to screen for the best conditions of
this interaction. Fig. 1B shows an overlay plot of net interaction curves obtained with 3 mM tubulin injected in AB buffer at
pH varying between 7.5 and 6.5, with 0.25 pH unit steps.
Comparison of the binding curves allowed us to evaluate the
effect of pH on the affinity of the stathmin/tubulin interaction;
the higher the steady state level, the higher the affinity (42). It
was of the same order at pH 7.5 or 7.25 and rose sharply at pH
7 or below, being maximal at pH 6.5. The affinity dramatically
fell at lower pH (not shown). All the following experiments
were therefore performed in buffer AB at pH 6.5.
The specificity of the interaction was ascertained by the
absence of tubulin binding to flow cells coupled with BSA (Fig.
2A) or simply activated-deactivated (not shown), the reduction
of binding in buffer without Mg21 (Fig. 2B), and the possibility
of competing for tubulin binding to the surface immobilized
stathmin by adding soluble stathmin to the tubulin solution
prior to injection (Fig. 2C). Furthermore, this competition assay showed that tubulin binding to immobilized stathmin was
abolished when 2 mM stathmin was added to a 4 mM tubulin
flowing solution. This latter result suggested that at these
stathmin and tubulin concentrations, more than 90% of the
tubulin was complexed with stathmin in the solution, indicating that the solution KD is at least 1 order of magnitude below
this concentration range.
Affinity Determination from Kinetic Measurements—Buffer
AB, pH 6.5, was used throughout this study. Interaction of
tubulin with immobilized stathmin was studied over a tubulin
concentration range of 1–20 mM. Fig. 3 shows representative
net sensorgrams of association, steady state binding, and dissociation using various tubulin concentrations. For the determination of the dissociation rate constant, k9d, rebinding of any
released tubulin to the immobilized stathmin was prevented by
raising the flow rate to 100 ml/min at the end of injection (point
B). The dissociation phase was fitted according to the model
AB 5 A 1 B, assuming a single site interaction between stathmin and tubulin. k9d was equal to 5 6 0.15 3 1023 s21 as
calculated by fitting the data to Equation 2 (see “Experimental
Procedures”). An association constant k9a was then obtained by
fitting the association phase with Equation 3 and was equal to
8.9 6 1 3 103 M21 s21. The equilibrium dissociation constant k9D
was then calculated as k9d/k9a and was found to be 0.56 6 0.05 3
1026 M. A k9D in the micromolar range reflects a relatively low
affinity for the stathmin/tubulin interaction. This low affinity
is in agreement with our earlier attempts in solution where we
failed to retain tubulin by affinity onto a stathmin-Sepharose
column or to co-immunoprecipitate tubulin with stathmin-antistathmin-protein A-Sepharose beads.2 Furthermore, this relatively low affinity is in agreement with the intracellular concentration of tubulin in the cell also found in the micromolar
range and gives weight to the idea that this stathmin/tubulin
interaction is of physiological relevance (43).
Rate constants were found similar to those measured with
WT stathmin when the sensor chip was coupled with bWT,
with an unphosphorylatable form of stathmin (4A), or with a
series of “pseudophosphorylated” stathmin mutants aimed to
mimic stathmin phosphorylated by Ca21/calmodulin-depend-
2
P. Curmi and V. Manceau, unpublished data.
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FIG. 1. Plasmon resonance monitoring of tubulin interaction
with stathmin surface. A, net sensorgram (relative response in RU
after background subtraction versus time in seconds) of tubulin injected
over WT stathmin surface at a flow rate of 7 ml/min in buffer AB, pH 7.5.
Injection of 3 mM tubulin started at point A and ended at point B. The
interaction between tubulin and stathmin is indicated by the increase of
the bound response units during the injection of protein. For background correction, we subtracted the signal obtained with a control flow
cell coupled with BSA and injected with the same sample. B, net
sensorgrams showing injection of 3 mM tubulin diluted in AB buffer at
varying pH over the same WT stathmin surface as in panel A. Affinities,
reflected by maximal binding levels, increased with the lower pH
values.
25031
25032
Stathmin/Tubulin Interaction
FIG. 2. Specificity of the stathmin tubulin interaction. A, sensorgrams showing injection of 3 mM tubulin in AB buffer, pH 6.5, over
the WT stathmin and BSA control Fc. Sensorgrams demonstrate the
specificity of tubulin binding to the stathmin Fc; changes in the signal
observed with the BSA Fc were due to protein affecting the bulk refractive index of the running buffer. These large changes are also observed
at the start and end of injection over the stathmin Fc and are subtracted
as background in the net sensorgrams. B, net sensorgrams showing
injection of 3 mM tubulin over the WT stathmin Fc in AB buffer, pH 6.5,
with or without 5 mM MgCl2. Specificity of the interaction is evidenced
by the reduction of binding observed in the absence of MgCl2. C, net
sensorgrams showing injection of 4 mM tubulin over the WT stathmin Fc
in AB buffer, pH 6.5, preincubated (5 min, room temperature) or not
preincubated with competing concentrations of stathmin in solution. A
dose-dependent reduction of surface binding was observed with increasing concentrations of stathmin. Surface binding was totally abolished
when tubulin was preincubated with 2 mM stathmin, suggesting a two
stathmin-one tubulin heterodimer complex in the flowing solution. On
each curve, the start and end of injections of proteins are indicated with
arrows A and B, respectively.
ent kinase II or IV on serine 16 (16E), by mitogen-activated
protein kinase on serine 25 (25E), by Cdc2 kinase on serine 38
(38E), or by mitogen-activated protein kinase and Cdc2 on
serines 25 and 38 (25/38E).
On the other hand, when the sensor chip was coupled with
the fully pseudophosphorylated 4E mutated stathmin (where
all four phosphorylation sites of stathmin were replaced by
Glu), k9d and k9a were 8 6 0.1 3 1023 s21 and 5 6 0.1 3 103 M21
s21, respectively, leading to a calculated k9D of 1.6 6 0.2 3 1026
M. This result corresponds to about a 3-fold lowered affinity of
4E stathmin as compared with WT stathmin. Horwitz et al. (33)
observed no difference between WT and a 4D mutant of stathmin (where the four serines were replaced by Asp) in their
ability to inhibit polymerization or elicit depolymerization of
MTs in vitro. This may be due to the relatively modest difference in k9D between WT and 4E (and probably 4D mutants).
However, it is likely that differences in k9D may be more important at other working pH levels or different ionic conditions
from those existing in the cell, since we observed in our preliminary attempts that at pH 7.5 no binding of tubulin was
observed on a 4E-coupled sensor chip surface. It thus appears
that a direct modulation of the stathmin/tubulin interaction
may be the consequence of stathmin phosphorylation. Our result is indeed in agreement with that of Melander Gradin et al.
(44), who recently showed with a cross-linking procedure that
stathmin, immunopurified from K562 cells, interacts with tubulin, while this interaction was dramatically reduced when a
mixture of stathmin phosphoforms was immunopurified from
cells transfected with a constitutively active mutant of Ca21/
calmodulin-dependent kinase IV/Gr. This modulation may explain the absence of MT destabilization observed with pseudophosphorylated stathmin mutants in vivo and is possibly due
to a conformational change of stathmin upon phosphorylation
modifying the accessibility of its C-terminal interaction domain
without the need of a third intervening protein partner as
suggested by Horwitz et al. (33).
Stathmin and Tubulin form a 60-Å Stokes Radius Complex—
The Stokes radius of the stathmin-tubulin complex was determined by gel filtration chromatography. Pure stathmin and
pure tubulin were applied separately or immediately after mixing to a column equilibrated with AB buffer, pH 6.5, at room
temperature. Tubulin and stathmin-tubulin complex elutions
were monitored at 280 nm with no interference of free stathmin, since it absorbs very poorly at 280 nm due to the lack of
aromatic residues. Fig. 4 shows representative elution profiles
for pure tubulin or tubulin mixed with WT stathmin. Pure
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FIG. 3. Plasmon resonance kinetic analysis of tubulin binding
to WT stathmin surface. Representative net sensorgrams illustrating
the real time binding of tubulin to WT stathmin immobilized on the
sensor chip at various tubulin concentrations. A indicates the start of
association by injecting tubulin in AB buffer, pH 6.5. B indicates the
start of dissociation by changing to buffer AB, pH 6.5, containing no
tubulin. Analyses were performed as described under “Experimental
Procedures.” k9d was 5 6 0.15 3 1023 s21, k9a was 8.9 6 1 3 103 M21 s21,
and k9D calculated as k9d/k9a was 0.56 6 0.05 3 1026 M.
Stathmin/Tubulin Interaction
25033
FIG. 4. Gel filtration analysis of tubulin and of the stathmintubulin complex. Representative FPLC elution profiles are shown of
tubulin and of the stathmin-tubulin complex applied at room temperature to a Superose-12 HR 10/30 gel filtration column equilibrated with
AB buffer, pH 6.5. Records were achieved at 280 nm, since at this
wavelength only tubulin causes absorbance. 100-ml samples containing
10 mM tubulin alone or 10 mM tubulin premixed with 2, 10, or 20 mM
stathmin for 5 min at room temperature (A) or 2 mM stathmin premixed
for 5 min at room temperature, with 6, 10, or 15 mM tubulin (B) were
applied to the column. Elution was performed with the same buffer.
Arrow S indicates the elution volume of the stathmin peak as determined by Western blotting (Fig. 5), and arrow T indicates the elution
volume of the tubulin peak observed in the absence of stathmin. The
mixture of stathmin with tubulin provoked a variable shift of the
tubulin peak toward a mean minimum elution volume value of 11.1 6
0.1 ml (arrow S/T). The shifted peaks correspond to the stathmintubulin complex as evidenced by Western blotting (Fig. 5). Intermediate
peaks reflected dissociation of stathmin-tubulin complexes.
tubulin eluted at about 12.7 6 0.1 ml. The addition of stathmin
provoked a variable shift of the tubulin peak toward a minimum value of the elution volume of about 11.1 6 0.1 ml. This
shifted peak, as analyzed by one-dimensional Western blotting
was always composed of stathmin and tubulin and thus represented the stathmin-tubulin complex (Fig. 5). The relative importance of the shift and intensity of the shifted peak were
dependent both on the absolute concentrations of tubulin and
stathmin and on the molar ratio of the two proteins in the
loaded sample, as demonstrated by the elution profiles obtained with 10 mM tubulin and variable amounts of stathmin
(Fig. 4A) and with 2 mM stathmin and variable amounts of
tubulin (Fig. 4B). To better analyze the conditions of the stathmin/tubulin interaction, we plotted (Fig. 6) the molecular sieve
coefficient, Kav ((Ve 2 V0)/(Vt 2 V0); see “Experimental Procedures”) of the shifted peak versus protein concentration. Shifts
reached a Kav plateau of 0.26 – 0.27 above a concentration of 2
mM stathmin for 10 mM tubulin (Fig. 6A). Values observed with
stathmin concentrations under the threshold of 2 mM stathmin
for 10 mM tubulin were most probably due to dissociation of the
complex during the run, since the morphology of the peak was
clearly asymmetrical in these conditions (see Fig. 4). Similarly,
we found that Kav reached a plateau of comparable value over
a 5 mM tubulin to 2 mM stathmin ratio (Fig. 6B). The stoichiometry was not directly accessible from the analysis of these
curves, since a variable amount of tubulin aggregated and
migrated in the void volume, apparently without associated
stathmin as assessed by one-dimensional Western blotting. We
thus determined directly by Western blotting the stathmin
tubulin stoichiometry of the complex in peak fractions collected
at the Kav plateau (see “Experimental Procedures”). A stoichiometry of one stathmin for two tubulin ab-heterodimers was
steadily measured through the Kav plateau (Table I), indicating
the formation of a 217-kDa (calculated value) molecular
complex.
The Stokes radius of the complex was determined by reporting minimal Kav values of the stathmin-tubulin complex peak
on a (2log Kav)1⁄2 versus Stokes radius graph constructed with a
series of reference proteins. The Stokes radius of stathmin was
37 Å, slightly different from the value of 33 Å found with the
native bovine brain protein (45) and that of tubulin (41.5 Å),
and the Stokes radius of the stathmin-tubulin complex was
60 6 1 Å (Fig. 7). The comparison of the calculated molecular
weight of the one stathmin-two tubulin complex to that of a
globular 60-Å protein (about 430 kDa), indicates that the stathmin-tubulin complex presents an asymmetrical conformation.
Sedimentation Analysis—Sedimentation velocity experiments were performed to assess the sample heterogeneity as
well as to measure directly the molecular weights of stathmin,
tubulin, and the complex in the native state. All experiments
were performed in AB buffer, pH 6.5. The sedimentation of
tubulin was found to be 5.66 S in these conditions, as reported
(46). When tubulin and stathmin were mixed at respective
concentrations of 10 and 20 mM, so that all tubulin would be
complexed to stathmin, a single symmetrical boundary of tu-
Downloaded from http://www.jbc.org/ by guest on May 21, 2020
FIG. 5. Interaction between stathmin and tubulin evidenced
by gel filtration chromatography. Western blot analyses of stathmin, tubulin, and stathmin-tubulin were applied to a FPLC Superose
12 HR 10/30 gel filtration column equilibrated with AB buffer, pH 6.5 as
in Fig. 4. Elution was performed with the same buffer, and fractions of
80 ml each were collected. A, a 20 mM stathmin sample was applied to
the column. Elution volume of the stathmin peak was evaluated at
12.8 –12.9 ml. B, a 10 mM tubulin sample was applied to the column.
Elution volume of the tubulin peak was found at 12.7 6 0.1 ml. C, a
sample containing 20 mM stathmin premixed with 10 mM tubulin was
applied to the column. Two peaks were observed, a peak eluting at ;11
ml, representing the stathmin-tubulin complex, and a residual stathmin peak at ;12.8 ml. Note the small intermediate stathmin-tubulin
peak at ; 11.6 ml due to a partial dissociation of the stathmin-tubulin
complex.
25034
Stathmin/Tubulin Interaction
FIG. 7. The stathmin-tubulin complex has a 60-Å Stokes radius.
A Superose 12 HR 10/30 FPLC gel filtration column was calibrated by
using a series of standard proteins: ribonuclease A (R), ovalbumin (O),
bovine serum albumin (B), aldolase (A), and ferritin (F). Upon gel
filtration, a linear relationship (y 5 0.35 1 6.8 3 1023x, r 5 0.99) was
noted between the Stokes radii of standard proteins and (2log Kav)1⁄2
(E). Stokes radii of stathmin (S), tubulin (T), and the stathmin-tubulin
complex (S/T) were then determined graphically and found to be 37,
41.5, and 60 Å, respectively (1).
TABLE I
Direct determination of the stoichiometry of the stathmin-tubulin
complex
Molar concentrations of stathmin (S), a-tubulin (a-T) and b-tubulin
(b-T) were determined by quantitative Western blotting (as described
under “Experimental Procedures”) in stathmin-tubulin complexes isolated by gel filtration chromatography. Gel filtration runs were performed with 10 mM tubulin mixed to 2 (experiment I), 10 (experiment II)
and 20 (experiment III) mM stathmin, respectively, and the fractions
analyzed were collected at the beginning (a), middle (b), and at the top
(c) of the ascending slope of the stathmin-tubulin complex peak. A mean
tubulin:stathmin molar ratio of about 2 was found with both a- and
b-tubulin throughout the Kav plateau (see Fig. 6).
Tubulin:stathmin molar ratio
Exp I
Exp. II
Exp. III
[a-T]/S
[b-T]/S
a
b
c
a
b
c
a
b
c
2.13
2.30
1.87
1.90
2.23
1.75
1.80
2.27
1.97
1.78
1.85
2.10
2.06
1.80
2.27
2.17
1.87
2.08
Mean
S.D.
2.02
0.20
1.99
0.16
bulin-containing species was observed; the data could be fitted
with a species sedimenting at 7.7 S and accounting for more
than 98% of the total tubulin. Taking into account our evaluation of the Stokes radius, the molecular mass of the corresponding complex can be calculated from the Svedberg equation (47) to be 197 kDa, which, given the uncertainties of the
Stokes radius and of other physical constants, is consistent
FIG. 8. Stathmin reduces the growth rate of microtubules. We
used video-enhanced contrast, differential interference contrast microscopy to follow MT dynamics. The experiments were performed in a
chamber at 37 °C with BRB80 plus 1 mM GTP (see “Experimental
Procedures”). MTs were analyzed during growth off centrosomes in the
absence or presence of stathmin. The addition of stathmin resulted in a
dose-dependent reduction of Vg. Moreover, curves relating Vg to tubulin
concentration were found parallel in the presence or absence of stathmin, suggesting that stathmin sequesters free tubulin heterodimers.
with the calculated mass (217 kDa) for a one stathmin-two
tubulin ab-heterodimer complex.
Stathmin Impedance of Microtubule Growth May Be Partly
Due to Tubulin Sequestration in the Stathmin-Tubulin Complex—The effect of stathmin on MT dynamics was investigated
on centrosome-nucleated MTs by video microscopy (39, 48).
Tubulin concentrations in this study varied between 11 and 17
mM where MTs grew at a rate (Vg) of 1.48 –2.64 mm/min, respectively (Fig. 8 and Table II). The number of catastrophes
was low and varied between 0 and 4 in these conditions, which
led to a catastrophe frequency (fcat) of 0 – 0.039 min21. As
evidenced on Fig. 8, the addition of stathmin resulted in a
reduction of Vg. Moreover, the Vg dependence on tubulin concentration observed in the presence of two different stathmin
concentrations was parallel to that observed in the absence of
stathmin.
At Vg 5 1.5 mm/min, the displacement of the curves by 1.7
and 2.7 mM stathmin corresponds to 2 and 3.8 mM tubulin,
respectively. If the curve displacement reflects a sequestration
of tubulin by its interaction with stathmin, these results would
indicate a 1:1.2 and 1:1.4 stathmin:tubulin heterodimer molar
ratio. This result is not identical with but fits the stoichiometry
determined by immunodetection after chromatography. It is
therefore most likely that stathmin indeed influences MT
Downloaded from http://www.jbc.org/ by guest on May 21, 2020
FIG. 6. Kav of tubulin and of the stathmin-tubulin complex. Kav
values were determined from elution positions of the shifted peak
observed with 10 mM tubulin or 10 mM tubulin premixed with various
concentrations of stathmin upon FPLC chromatography on a Superose
12 HR 10/30 gel filtration column (A). Kav reached a plateau over a
stathmin:tubulin molar ratio of about 2/10. B, 2 mM stathmin was mixed
with varying concentrations of tubulin. A Kav plateau was observed for
a stathmin:tubulin molar ratio of about 2:5.
25035
Stathmin/Tubulin Interaction
TABLE II
Stathmin reduces Vg and does not alter the catastrophe frequency
MT dynamics was followed with video-enhanced contrast, differential interference contrast microscopy (see “Experimental Procedures”). The
tubulin and stathmin concentrations inside the video chamber are indicated. In the experiments concerning fcat, MTs grown at the same growth
rate in the presence or absence of stathmin were compared, and the fcat values were obtained for the indicated total time.
Tubulin
13 mM
14 mM
15 mM
17 mM
1.48 6 0.59
0.039
25
84
102
4
NDb
ND
ND
ND
ND
ND
2.01 6 0.58
0.039
14
47
50
2
ND
ND
ND
ND
ND
ND
2.64 6 0.49
0
13
55
45
0
1.03 6 0.85
0.18
6
22
17
3
1.48 6 0.40
0.02
12
30
47
1
ND
ND
ND
ND
ND
ND
1.8 6 0.90
0.028
14
32
36
1
2.22 6 0.52
0
15
47
44
0
NPh
NP
NP
NP
NP
NP
ND
ND
ND
ND
ND
ND
1.37 6 0.76
0.077
12
48
52
4
ND
ND
ND
ND
ND
ND
1.79 6 0.60
0.029
16
69
69
2
a
Vg, growth rate 6 S.D. (mm/min).
ND,not determined.
c
fcat, catastrophes/min.
d
Number of MT measured.
e
Number of individual measurements.
f
Total time of observation (min).
g
Number of catastrophes.
h
NP, no polymerization.
b
growth by sequestering tubulin heterodimers. However, the
effect of stathmin on MT polymerization may be not simply due
to tubulin sequestration, since at 11 mM tubulin and 2.7 mM
stathmin, no polymerization occurred, whereas simple curve
extrapolation should have led to a significant residual polymerization level. This may reflect an additional nonlinear component in the effect of stathmin on MT polymerization.
Importantly, we found that at similar Vg, fcat was unchanged
in presence of stathmin (Table II). Our result is clearly different from that reported by Belmont and Mitchison (30), who
found in a similar assay that the catastrophe rate is 5–10-fold
higher in the presence of stathmin. This might be an important
point still to be investigated, since in our study the sequestration of tubulin by stathmin in a stathmin-tubulin complex, by
reducing the available tubulin concentration, seems to account
for a large part to the reduction of net MT polymerization.
Reasons for this divergence are not clear. Part of it may be
explained by differences in the source of the protein or in the
buffers used in the two studies, possibly conditioning the dynamic instability of MTs. Belmont and Mitchison (30) used
purified stathmin from calf in a pH 7.5 buffer containing 5 mM
MgCl2, whereas our study was performed with recombinant
human stathmin in BRB80 buffer, pH 6.8, the reference buffer
for studies on MT dynamics in vitro.
Conclusion—We have demonstrated in the present work that
a direct interaction between stathmin and tubulin occurs in
vitro and gives rise to a complex formed by one stathmin
molecule and two tubulin ab-heterodimers. The stathmin/tubulin interaction has a k9D of about 0.5 mM and is sensitive to
ionic variations; it occurs preferentially at an optimum pH of
about 6.5. We found that the 4E pseudophosphorylated mutant
of stathmin has an altered affinity for tubulin in vitro, which
may indicate that, in vivo, stathmin phosphorylation directly
regulates the stathmin/tubulin interaction without intervening
protein partners. Our results further indicate that one of the
mechanisms of the stathmin destabilization of MTs might be
the sequestration of free tubulin heterodimers in a stathmintubulin complex.
Acknowledgments—We thank G. Batelier from Laboratoire
d’Enzymologie et Biologie Structurale UPR9063 CNRS, Gif sur Yvette,
France, for help in performing the ultracentrifugation experiments, M.
Dutreix from the Institut Curie, Paris, France, for skillful help in
performing the BIAcore experiments, and M. F. Carlier for critical
reading of this manuscript.
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The Stathmin/Tubulin Interaction in Vitro
Patrick A. Curmi, Søren S. L. Andersen, Sylvie Lachkar, Olivier Gavet, Eric Karsenti,
Marcel Knossow and André Sobel
J. Biol. Chem. 1997, 272:25029-25036.
doi: 10.1074/jbc.272.40.25029
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