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INTRODUCTION |
Microtubules (MT)1
constitute a crucial part of the cytoskeleton and are involved in a
variety of cell functions such as maintenance of shape, organization of
intracellular transport, motility, and cell division. The
polymerization dynamics of MT is under strict control (1). In addition
to small molecules (inorganic ions, guanine nucleotides, and drugs),
numerous proteins are known to interact with MT as positive regulators
of MT assembly (MAPs) either by promoting the polymerization of tubulin
or by stabilizing MT (1, 2). Several other proteins including
Op18/stathmin, katanin, and some kinesin-like proteins act as
destabilizers (1, 2).
As shown by various methods including co-sedimentation assays and
immunostaining, many glycolytic enzymes can interact transiently with
the actin and/or microtubular network either in vitro or in vivo; the properties of associated partners
(e.g. the stability of cytoskeleton and the activity of the
enzyme) are deeply influenced by the mutual interactions (Refs. 3-5;
for review see Ref. 6). Recently, we found that two of the glycolytic
enzymes, the M1 isoform of pyruvate kinase (PK) and Dictyostelium
discoideum phosphofructokinase, can act as
microtubule-destabilizing factors by inhibiting paclitaxel-induced polymerization of tubulin and by promoting disassembly of microtubules into thread-like oligomers (7, 8).
In the present study we have further characterized the PK-tubulin/MT
interaction with purified proteins using surface plasmon resonance
technology, and we present data on the heterologous association of PK
with MTs in brain extract, the protein composition of which
approximates that of the living cells. We present evidence on the
crucial role of acidic C-terminal fragments of MTs in the PK binding.
We show the modulating role of phosphoenolpyruvate (PEP) on tubulin-PK
as well as on MT-PK interaction at different organization levels and
demonstrate that the distribution of PK in mouse fibroblasts is highly
dependent on the integrity of microtubular network.
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EXPERIMENTAL PROCEDURES |
Chemicals--
Rabbit muscle PK, ATP, EGTA, GDP, GTP, lactate
dehydrogenase, MES, NADH, PEP, PK, phenylmethylsulfonyl fluoride,
subtilisin, paclitaxel, and Tris were purchased from Sigma. Biotin-XX
SSE (catalog no. B6352) was from Molecular Probes. All other chemicals were reagent grade commercial preparations. Solutions were prepared using Millipore's Milli-Q ultra-pure water.
Protein Determination--
Protein concentration was measured by
the Bradford method (9) using the Bio-Rad Protein Assay Kit. To
determine the composition of pellets and supernatants in sedimentation
experiments, proteins were separated on 9% SDS-PAGE. Stained gels as
well as immunoblots were scanned using the Bio-Rad GelDoc 1000 densitometer system and quantitated using NIH Image software run on a
Macintosh desktop computer.
Tubulin and MT Preparation--
MAP-depleted tubulin was
purified from bovine brain by the method of Na and Timasheff (10) and
stored in 10 mM phosphate buffer, pH 7.0, containing 1 M sucrose, 0.5 mM MgCl2, and 0.1 mM GTP at
80 °C. Purified tubulin showed no
contamination with MAPs on overloaded SDS-PAGE. Before use, stored
tubulin was dialyzed against 50 mM MES buffer, pH 6.8, at
4 °C for 3 h and then centrifuged at 100,000 × g for 20 min at 4 °C. MT was assembled by adding 20 µM paclitaxel to 10 mg/ml tubulin, followed by incubation
for 30 min at 37 °C.
Enzyme Activity Assay--
The activity of PK was followed by
monitoring the NADH consumption at 340 nm with a Cary 50 or a Jasco
V-550 spectrophotometer utilizing a coupled enzymatic reaction with
lactate dehydrogenase at 25 °C. The conditions for PK activity
measurement corresponds to that described previously (7).
Polymerization Assays--
10 µM tubulin was
assembled to MT at 37 °C in 50 mM MES buffer, pH 6.6, containing 2 mM dithioerythritol, 5 mM
MgCl2, 1 mM EGTA (polymerization buffer), and
KCl in concentrations as indicated. The polymerization was initiated
with 20 µM paclitaxel at 37 °C. PK was either
incubated with tubulin for 20 min at 4 °C before starting the
polymerization or added to the paclitaxel-stabilized MTs. For turbidity
measurements, absorbance was monitored at 350 nm with a Cary 50 spectrophotometer. The error rate of determinations of turbidity
measurements was ±5% within a series of experiments, i.e.
using the same tubulin stock solution and polymerizing buffer or the
same freshly prepared cell-free extract. In another set of experiments
the absolute value of the rate of turbidity increase could be
different; however, the relative inhibitory effect caused by PK was
within ±5% error. The standard error of fitting of the polymerization
curves for determination of the rates was ±2%.
Limited Proteolysis of MT--
The acidic C-terminal
fragments of paclitaxel-stabilized MTs were selectively removed
by limited subtilisin digestion as described by Wang and Sheetz (11),
and the product was analyzed by SDS-PAGE, which provided evidence for
the cleavage of the fragments from both tubulin subunits.
Pelleting Experiments--
MAP-free tubulin or
subtilisin-treated MTs at final concentrations of 10 µM
were incubated with or without 2 µM PK in polymerizing buffer for 30 min at 37 °C. The samples were centrifuged at
100,000 × g for 20 min at 30 °C. Under these
conditions MTs and MT-bound PK are completely pelleted, whereas
uncomplexed PK remains in the supernatant (7). The partition of the
proteins into pellet and supernatant fractions was analyzed by SDS-PAGE
separation in 9% polyacrylamide gels. To study the influence of PEP on
the partition of PK, a set of pelleting experiments was performed in
the presence of 100 µM PEP.
Preparation of Cell-free Cytosolic Fraction--
Cell-free
cytosolic fraction containing the soluble proteins of cells including
tubulin and MAPs (30 mg/ml total protein concentration) was prepared
from bovine brain as described previously (12). To initiate MT
formation, the fraction was warmed to 37 °C in the presence of 1 mM GTP and 20 µM paclitaxel.
Immunoblot of PK--
Serum against rabbit muscle PK was raised
by immunizing rats as described previously (7). The IgG fraction was
purified according to Tracey et al. (13). Proteins from the
brain cell-free extract and from the pellet and supernatant fractions
of the co-sedimentation experiments were separated on SDS-PAGE and
transferred to nitrocellulose membranes. The anti-PK IgG was used at a
500-fold dilution from a 20 mg/ml stock solution. The secondary
antibody-alkaline phosphatase conjugate (Sigma) was used at a dilution
of 1:5000. The immunocomplex was visualized by the
5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (BCIP/NBT)
chromogenic detection system.
Cell Culture--
L929 cells were pleated on glass
coverslips and maintained in Dulbecco's modified Eagle's medium
containing 10% fetal calf serum. For testing the effect of
anti-microtubular drugs, the cells were incubated for 2 h in the
presence of 10 µg/ml paclitaxel or 1 µg/ml vinblastine. For
immunostaining, the cells were fixed in cold methanol. PK-antiserum,
prepared as described above, was used to label PK at 1:50 dilution
followed by fluorescein isothiocyanate-conjugated anti-rat IgG
(Sigma) to visualize the immunocomplex under a fluorescent microscope.
Transmission Electron Microscopy--
MT-containing samples were
pelleted by centrifugation, and the pellets were pre-fixed with 2%
glutaraldehyde, 0.2% tannic acid in 0.1 M sodium
cacodylate, pH 7.4, for 60 min and then washed with 0.1 M
sodium cacodylate, post-fixed with 0.5% OsO4 in 0.1 M sodium cacodylate, stained en bloc with 1%
uranyl acetate, dehydrated in graded ethanol, and embedded in Durcupan
(Fluka). Thin sections, contrasted with uranyl acetate and lead
citrate, were examined and photographed in a JEOL CX 100 electron microscope.
Surface Plasmon Resonance (SPR)--
The binding kinetics of PK
to tubulin was monitored in real-time with a BIAcore X instrument
(BIAcore AB, Uppsala, Sweden). Tubulin at a concentration of 10 mg/ml
was biotinylated with a sulfosuccinimidyl ester derivative of biotin,
and the biotinylated tubulin at a concentration of 0.5 µM
was immobilized onto a streptavidin-coated sensor chip (Sensor Chip SA,
product code BR-1000-32, BIAcore) as described previously (12). All
experiments were performed at 25 °C. Sensorgrams were recorded at a
flow rate of 5 µl/min for 3 min at different concentrations of PK in
the absence and presence of PEP. In some cases the injected sample
contained 100 mM KCl in addition to PK. Dissociation
and association rate constants were calculated using the Langmuir (1:1)
binding model by the BIAevaluation 3.0 software supplied by the
manufacturer or by the linearization methods based upon Equation 1
(14),
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(Eq. 1)
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where R is the resonance signal in RU at time
t, dR/dt is the rate of change of the
SPR signal, C is the concentration of the analyte (PK), and
Rmax is the maximum analyte binding capacity in
RU. The slope of the plot of ln(dR/dt)
versus t is
(konC + koff), which we designated
kobs, and the tangent of
kobs versus C gives
kon (the knowledge of
Rmax is not necessary). For linear fitting we
used Microcal Origin version 5.0 software (Microcal Software Inc.). The
error of determination was ±10%.
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RESULTS |
Quantitative Characterization of PK-Tubulin Interaction and the
Effect of PEP on the Interaction--
To obtain direct quantitative
data on the binding of PK to tubulin, the association and dissociation
kinetics of the interaction were followed by surface plasmon resonance
measurements. Fig. 1A shows
typical sensorgrams obtained at 65, 86, 108, and 129 nM PK
concentrations. The kinetic constants obtained by nonlinear fitting
are: kon = 2.44 × 105
M
1s
1;
koff = 1.02 × 10
2
s
1. The dissociation constant, Kd = koff/kon, is 4.18 × 10
8 M. The kon
value was also calculated from the slope of kobs
versus C (Fig. 1C) as described under
"Experimental Procedures." The value of kon
is (1.94 × 105 ± 2.9 × 103) M
1s
1,
which is in good agreement with the value obtained from nonlinear direct fitting of SPR sensorgrams. In the presence of 100 mM KCl, virtually no PK binding could be detected (data not
shown).

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Fig. 1.
PK-tubulin interaction monitored by surface
plasmon resonance and the effect of PEP. Sensorgrams of injections
of PK alone (A: 65 nM (a), 86 nM (b), 108 nM (c), and
129 nM (d)) and 65 nM PK
in the presence of various PEP concentrations (C: 0 mM (a), 0.1 mM (b), 0.2 mM (c), 0.4 mM (d), 0.6 mM (e), 0.8 mM (f), and 1 mM (g)). B, determination of
kon by straight line fitting of the
kobs versus C (PK
concentration) graph. D, dependence of RUeq
values on PEP concentrations.
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To study the effect of PEP on tubulin-PK interaction, a solution of 65 nM PK containing different concentrations of PEP was injected over the tubulin-coated surface. The rate constant of the dissociation process evaluated from the sensorgrams obtained at
various PEP concentrations is independent of the PEP concentration (data not shown), however, the RUequ values, which are
characteristic for the amount of PK bound to tubulin, decreased with
increasing PEP concentrations (see Fig. 1D). These
data suggest that PEP competes with tubulin for PK binding.
PK Targets Negatively Charged C-terminal "Tails" on
MTs--
Previously we demonstrated by differential centrifugation
that PK induces the formation of less sedimentable tubulin oligomers and binds preferentially and substoichiometrically to this tubulin oligomer fraction (7). The finding that the kinase is present in the
high speed pellet (100,000 × g) provided evidence for
its binding to the tubulin oligomer/MT. Here we have
investigated the effect of ionic strength on the binding of PK to MTs.
2 µM PK was added to paclitaxel-stabilized MTs in the
presence of either 50 or 150 mM KCl in the polymerizing
buffer, and then the samples were centrifuged at 100,000 × g, and the pellets were analyzed by transmission electron
microscopy. Long smooth-surfaced intact MTs are seen in the control
samples (Fig. 2A). When PK was
added to these MTs at low ionic strength, the samples became crowded with large thread-like aggregates. Among these aggregates,
bundled MTs arranged in parallel and connected by rows of small dense particles could be observed (Fig. 2B). Bundled MTs and the
aggregates were practically absent in samples prepared at high ionic
strength (Fig. 2D), which is in agreement with our SPR data
obtained with unpolymerized tubulin, showing that high salt
concentration inhibits the complex formation of tubulin with PK.

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Fig. 2.
Electron microscopy of PK-MT
interaction. Microtubules assembled as described under
"Experimental Procedures" were incubated without (A) or
with the addition of PK (B and D) or PK + PEP
(C) in the presence of 50 mM KCl (B
and C) or 150 mM KCl (D). Note the
thread-like aggregates and bundled MTs in B. Their presence
is less conspicuous in C, and they are absent in
A and D. Bar, 125 nm.
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The aggregates and bundled MTs were also conspicuous in samples
containing PK and 1 mM pyruvate at low salt concentration (data not shown). By contrast, if the sample contained 1 mM
PEP instead of pyruvate, the amount of both bundled MTs and thread-like aggregates was greatly reduced (Fig. 2C). The effect of PEP
on the association of PK to MTs was demonstrated by pelleting
experiments as well. As shown in Fig.
3A, after centrifugation of
samples containing MT + PK practically all MT appeared in the pellet
together with significant amounts of PK. The addition of 1 mM PEP reduced the amount of PK co-sedimented with MTs.
These observations indicate that PEP specifically inhibits the
association of PK to MTs.

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Fig. 3.
Pelleting experiments with
paclitaxel-stabilized intact (A) and tail-free
(B) MTs. Conditions for MT assembly induced by
paclitaxel and for the pelleting experiments are described under
"Experimental Procedures." A, SDS-PAGE images of pellets
(MT alone (lane 1), MT + PK (lane 3), MT + PK + PEP (lane 4)) and of supernatant (PK alone (lane
2)). B, time dependence of MT digestion by subtilisin
(cf. "Experimental Procedures") at: 0 min (lane
2), 2 min (lane 3), 10 min (line 5), 30 min (lane
6), and 60 min (lane 7). Lane 1, control (no
subtilisin added); lane 8, tail-free MT + PK.
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To study the role of the acidic C-terminal tail of tubulin
exposed on the surface of MTs in the salt-sensitive binding of PK, the
pelleting experiments were also performed with "tail-free" MT
prepared by limited subtilisin digestion as suggested by Wang and Sheetz (11). The release of the C-terminal tails of tubulin subunits was monitored by SDS-PAGE, and as shown in Fig 3B,
after a 60-min digestion the limited proteolysis was completed. When PK
was added to the tail-free MTs under the conditions used for intact
MTs, and the pellet fractions were analyzed, no PK was detected in the
pellet fraction. Examination of the subtilisin-digested MTs by
transmission electron microscopy revealed that the tail-free MTs are
significantly shorter than their intact counterparts in accordance with
the observation of Wang and Sheetz (11), and neither the
oligomerization nor the bundling of MTs could be observed in the
PK-containing samples prepared even at low salt concentration (data
not shown). These data are in contrast to those obtained with intact
MTs (cf. Fig. 3A) and reveal the crucial role of
the negatively charged C-terminal tails of MT in the PK-MT interaction.
Characterization of PK-MT Interaction in Brain Cell-free
Extract--
The experiments with purified proteins described above
clearly show that both tubulin and MT bind PK and the binding occurs at
relatively low protein concentrations. To obtain direct evidence for
the occurrence of this hetero-association in a complex system, the
interaction of PK with tubulin/MT was investigated in brain extract,
which mimics the cellular conditions concerning the composition of
soluble proteins. In one set of experiments the co-pelleting of
endogenous PK with MTs formed by paclitaxel-induced polymerization was
tested by analyzing the partition of PK in the supernatant and the
pellet fractions in the absence and presence of assembled MTs. The MT
assembly was induced by adding GTP plus paclitaxel to the concentrated
cell-free extract (30 mg/ml), and the pellet and supernatant fractions
were analyzed for the partition of PK by activity measurements as well
as by immunoblotting using anti-PK antibody. As shown in Table
I, both methods revealed that about 20%
of the endogenous PK co-sedimented with the MTs. It has to be noted
that less than 5% of the extract proteins, including endogenous PK,
appeared in the pellet prepared without the addition of GTP + paclitaxel.
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Table I
Effect of MT assembly on the distribution of PK in brain cell-free
extract
Brain cell-free extract (30 mg/ml total protein concentration) was
warmed to 37 °C, and MT formation was initiated by adding 1 mM GTP and 20 µM paclitaxel. After
centrifugation at 100,000 × g, the amount of PK in the
pellet and supernatant was analyzed by activity measurements and
densitometry of immunoblots as described under "Experimental
Procedures." The data are the average of at least three independent
measurements. Relative error of determinations is less than 10%.
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In another set of experiments, MT assembly in brain extract was also
initiated by adding paclitaxel and followed by turbidimetry in the
absence and presence of exogenous PK or PK plus PEP added to the
extract prior to starting the polymerization of endogenous tubulin. As
shown in Fig. 4A, PK decreases
the rate of polymerization. However, the simultaneous addition of PEP
with exogenous PK partially suspends this effect. Fig. 4B
shows the counteracting effect of PEP on PK-induced inhibition of
tubulin polymerization with purified proteins as well. These data
reveal that PK and MT can mutually recognize and interact with each
other not only in purified systems but in extract containing a number
of other microtubular (e.g. MAPs) and cytosolic proteins,
which suggests the binding of PK to microtubular apparatus in
complex biological systems.

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Fig. 4.
Polymerization of endogenous tubulin in brain
extract (A) and in a solution of purified tubulin
(B) in the absence (a) or
presence of PK (b) and PK + PEP (c).
The concentrations of PEP, PK, and tubulin in B are
100, 2, and 10 µM, respectively.
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Localization of PK in L929 Cells--
Previously we demonstrated
the localization of PK on tubulin polymers by immunoelectron microscopy
in an in vitro system using anti-PK immunogold
labeling (7). Here we aimed to visualize the distribution of PK, known
as cytosolic enzyme, in living cells. The distribution of the enzyme
was studied by immunohistochemical method in L929 cells. As shown in
Fig. 5A, although the intact L929 cells have an elongated shape, PK as shown by the bright fluorescence is concentrated in a juxtanuclear part of their cytoplasm. The nucleus and the extensions of the cells are not stained.

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Fig. 5.
Immunohistochemistry of PK in intact L929
cells (A) and following treatment with paclitaxel
(B) or vinblastine (C).
The PK is concentrated in a domain near the nucleus, as
indicated by arrowheads in the control cells. Lanes
1 and 2 are the low power and high power
magnifications, respectively. Bar, 100 µm
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To obtain information about the role of the microtubular network in the
localization of PK, cells treated with the anti-microtubular drugs
paclitaxel or vinblastine were also studied. The paclitaxel-treated cells maintained the elongated shape characteristic of intact L929
cells, showing, however, an altered staining pattern in most cells
(Fig. 5B). Although cells similar to the untreated cells are
found because of the inhomogeneity of the cells, however, the major
fraction of these cells behaved differently than the control cells; the
distinct juxtanuclear concentration of the stain was no longer
visible, and instead diffuse staining over extended areas of cytoplasm
could be observed (Fig. 5B). The difference in PK
localization is more evident in the colored images with high power magnification.
The shape of the vinblastine-treated cells was changed
dramatically because of the collapse of the microtubular network, and the round cells showed an unlocalized distribution of the fluorescently labeled PK (Fig. 5C). Although colocalization of PK and
individual MTs was not examined in these experiments, our observations
show that the intracellular localization of the kinase is strongly influenced by the organization of the MT network.
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DISCUSSION |
Our data demonstrate that both the polymerized and unpolymerized
tubulin interacts with PK. SPR technique was used here for the first
time to characterize the kinetic and affinity parameters of this
interaction. The high affinity of PK to tubulin, characterized by a low
dissociation constant, made it possible to use low protein concentrations in the experiments with purified proteins for
association studies and to detect this hetero-association in cell-free
extract. As shown by electron microscopy, the bundling of MTs and the
appearance of thread-like oligomeric aggregates are the morphologic
manifestations of this interaction. Both processes were suppressed
under high salt conditions and were absent in subtilisin-treated MT
samples. These data are in good agreement with earlier observations
demonstrating the sensitivity of PK-tubulin interaction to salt
concentration (15) and to proteolytic cleavage of the C-terminal
segment of tubulin (16). The mechanism of formation of oligomeric
aggregates needs further elucidation. These aggregates accumulated in
the presence of PK in samples during paclitaxel-induced tubulin
polymerization as well as in samples of preassembled
paclitaxel-stabilized MTs, which are practically free of nonsedimenting
tubulin dimers. This suggests that the enzyme induces partial
depolymerization of MTs. The presence of PK in the oligomeric
aggregates was demonstrated earlier by SDS-PAGE analysis as well as by
immunocytochemistry (7). It is tempting to speculate that occupation of
the C-terminal tails of tubulins by PK may weaken the contacts
necessary for the ordered arrangement of tubulin dimers in the
cylindrical wall of MTs but do not suppress their propensity to bind
each other and form atypical tubulin assemblies as observed in our experiments.
An important finding of our study is that PEP can suppress the binding
of PK to tubulin/MTs. This effect was demonstrated by four independent
methods, SPR, pelleting, electron microscopy, and turbidimetry, in a
system containing pure proteins only and in brain extracts as well. PK
is a key glycolytic enzyme, present in the brain in micromolar
concentrations (17), which is within the range used in our experiments.
Our data suggest that in living cells a significant fraction of the PK
could be in an associated form around the nucleus. The
localization of PK could be the result of the hetero-association of the
kinase in which the microtubular network might be involved. The
organization of the microtubular network is an important issue in the
formation of macromolecular superstructure, as indicated by the results
obtained using various drug treatments. Under physiological
conditions, however, the PEP could modulate the distribution of PK
within the cytoplasm. The binding is counteracted by PEP, a crucial
intermediary in glycolysis, the concentration of which is controlled by
the metabolic state of the cells.