Isolation of the native assembled and nonassembled tubulin fractions from cells treated and not treated with okadaic acid, and subsequent in vitro assay of the carboxypeptidase activity,
Trang 1Inhibitors of protein phosphatase 1 and 2A decrease the level of tubulin carboxypeptidase activity associated with microtubules
Marı´a A Contı´n, Silvia A Purro, C Gasto´n Bisig, He´ctor S Barra and Carlos A Arce
Centro de Investigaciones en Quı´mica Biolo´gica de Co´rdoba, CIQUIBIC (UNC-CONICET), Departamento de Quı´mica Biolo´gica, Facultad de Ciencias Quı´micas, Universidad Nacional de Co´rdoba, Argentina
The association of tubulin carboxypeptidase with
micro-tubules may be involved in the determination of the
tyrosi-nation state of the microtubules, i.e their proportion of
tyrosinated vs nontyrosinated tubulin We investigated the
role of protein phosphatases in the association of
carb-oxypeptidase with microtubules in COS cells Okadaic acid
and other PP1/PP2A inhibitors, when added to culture
medium before isolation of the cytoskeletal fraction,
pro-duced near depletion of the carboxypeptidase activity
asso-ciated with microtubules Isolation of the native assembled
and nonassembled tubulin fractions from cells treated and
not treated with okadaic acid, and subsequent in vitro assay
of the carboxypeptidase activity, revealed that the enzyme
was dissociated from microtubules by okadaic acid
treat-ment and recovered in the soluble fraction There was no
effect by nor-okadaone (an inactive okadaic acid analogue)
or inhibitors of PP2B and of tyrosine phosphatases which do
not affect PP1/PP2A activity When tested in an in vitro system, okadaic acid neither dissociated the enzyme from microtubules nor inactivated it In living cells, prior stabili-zation of microtubules with taxol prevented the dissociation
of carboxypeptidase by okadaic acid indicating that dynamic microtubules are needed for okadaic acid to exert its effect On the other hand, stabilization of microtubules subsequent to okadaic acid treatment did not reverse the dissociating effect of okadaic acid These results suggest that dephosphorylation (and presumably also phosphorylation)
of the carboxypeptidase or an intermediate compound occurs while it is not associated with microtubules, and that the phosphate content determines whether or not the carboxypeptidase is able to associate with microtubules Keywords: microtubules; PP1; PP2A; tubulin carboxypepti-dase; tyrosination state
Microtubules are dynamic structures formed by tubulin
and associated proteins, and are involved in chromosome
segregation, morphogenesis, intracellular transport and
other cell functions [1] We showed previously [2–4] that
the alpha chain of tubulin can be modified by enzymatic
removal of the C-terminal tyrosine residue by tubulin
carboxypeptidase, and by re-addition of this tyrosine by a
distinct enzyme, tubulin tyrosine ligase The physiological
role of this cyclic detyrosination/tyrosination reaction has
not been clarified, but is believed to be crucial for normal
microtubule functioning We are studying the mechanisms
that determine the tyrosination state of microtubules, i.e
the proportions of tyrosinated vs nontyrosinated tubulin
(Tyr- and Glu-tubulin, respectively) that constitute a
particular microtubule Our biochemical studies have
shown that the tyrosination reaction occurs rapidly and exclusively on nonassembled tubulin, whereas detyrosina-tion occurs more slowly, and mainly in microtubules [4,5] These findings were confirmed by studies in living cells [6,7] A striking correlation was observed between tyros-ination state and dynamics of microtubules: Glu- and Tyr-microtubules are stable and dynamic structures, respectively [8,9] On the basis of this concept, supported
by a variety of experiments in different laboratories [10,11], identification of Tyr- and Glu-microtubules is used at present as a marker of, respectively, dynamic and stable microtubules
We showed in vitro that tubulin carboxypeptidase is associated with microtubules, and that the association is modulated by phosphorylation/dephosphorylation reac-tions [12,13] Microtubules were reconstituted from soluble rat brain extracts, and carboxypeptidase activity present in sedimentable (microtubules) and nonsedimentable fractions was measured Preincubation of extracts under conditions favouring either phosphorylation or dephosphorylation led
to, respectively, lower and higher proportions of carboxy-peptidase activity associated with microtubules Total carboxypeptidase activity was not significantly modified
by conditions favouring phosphorylation Microtubules were not the target of the kinase(s) and phosphatase(s) presumably involved in this phenomenon We demonstra-ted recently that the association of carboxypeptidase with microtubules also occurs in living cells [14,15] In this paper,
we present evidence that the serine/threonine phosphatases
Correspondence to C A Arce, Departamento de Quı´mica Biolo´gica,
Facultad de Ciencias Quı´micas, Ciudad Universitaria, 5000-Co´rdoba,
Argentina Fax: +54 351433 4074, Tel.: +54 351433 4168,
E-mail: caecra@dqb.fcq.unc.edu.ar
Abbreviations: CPA, pancreatic carboxypeptidase A;
Glu-micro-tubules, microtubules composed mainly of Glu-tubulin; Glu-tubulin,
detyrosinated tubulin or tubulin whose a-subunit lacks a C-terminal
tyrosine residue; MAP, microtubule-associated protein; OA, okadaic
acid; Tyr-microtubules, microtubules composed mainly of
Tyr-tubu-lin; Tyr-tubulin, tubulin with C-terminal tyrosine residue a-subunit.
(Received 21 August 2003, revised 15 October 2003,
accepted 23 October 2003)
Trang 2PP1 and/or PP2A are involved in regulation of the degree of
tubulin carboxypeptidase activity associated with
micro-tubules, and that microtubule dynamics is necessary to this
regulatory mechanism
Materials and methods
Chemicals
Nitrocellulose membrane, pancreatic carboxypeptidase A
(CPA), phenylmethanesulfonyl fluoride, EGTA, aprotinin,
benzamidine, nocodazole, Paclitaxel (taxol),
4-chloro-naph-th-1-ol, Triton X-100, and Mes were from Sigma-Aldrich
Co Okadaic acid (OA), calyculin A, 1-nor-okadaone,
cantharidin, deltamethrin, and phenylarsine oxide were
from Alomone Laboratories (Israel)
Antibodies
Antibody against Glu-tubulin (anti-Glu) was prepared in
our laboratory as described by Gundersen [16], with
specificity and titre similar to those of samples provided
by the original author Rat monoclonal YL 1/2 antibody
specific to Tyr-tubulin (anti-Tyr) was from Sera-Lab
Rhodamine-conjugated goat rabbit secondary
anti-body, fluorescein-conjugated goat anti-mouse secondary
antibody, and peroxidase-conjugated Protein A were from
Sigma-Aldrich Co
Cell culture
COS-7 cells were grown in Dulbecco’s modified Eagle’s
medium (Sigma) supplemented with 10% (v/v) foetal bovine
serum (Serono) at 37°C in an air/CO2(19 : 1) incubator
Cells were plated on plastic Petri dishes (60 mm diameter)
and grown for 2 days until reaching the desired final
density Culture medium was renewed at 24 h Cells were
suspended in culture medium by careful scraping and then
transferred to conical plastic tubes When used, effectors
were maintained in cell suspension by gentle agitation
Unless stated otherwise, all cell procedures were performed
at 37°C
Isolation of cytoskeletal fraction
Cell suspensions (obtained from 60 mm-dishes) were
centrifuged at 600 g for 2 min to remove culture medium
Sedimented cells were resuspended in 0.5 mL
micro-tubule-stabilizing buffer [90 mM Mes pH 6.7, 1 mM
EGTA, 1 mMMgCI2, 10% (v/v) glycerol] and centrifuged
again Pelleted cells were resuspended in 0.5 mL
micro-tubule-stabilizing buffer containing 10 lM taxol, 0.5%
(v/v) Triton X-100, and protease inhibitors (10 lgÆmL)1
aprotinin, 0.5 mM benzamidine, 5 lgÆmL)1
o-phenanthro-line, 0.2 mMphenylmethanesulfonyl fluoride) at 37°C for
2 min with frequent agitation The tubes were centrifuged
at 8000 g for 2 min and the soluble fraction discarded
To eliminate residual Triton X-100 and cytosolic fraction,
the pelleted cytoskeletons were rapidly washed twice (by
resuspension and centrifugation) with
microtubule-stabil-izing buffer containing 10 lMtaxol Finally, the
cytoskele-tons were suspended in microtubule-stabilizing buffer
containing 10 lM taxol and the protease inhibitor mixture
Isolation of microtubular and soluble tubulin fractions from living cells under microtubule-stabilizing conditions The isolation of native microtubules and nonassembled tubulin was performed by the method of Pipeleers et al [17] Sedimented cells (0.5 mL) were suspended in 5 mL warm (37°C) microtubule-stabilizing buffer [20 mM
sodium phosphate pH 7, containing 40% (v/v) glycerol, 5% (v/v) dimethylsulfoxide, 0.1 mM GTP] and disrupted with a glass-Teflon homogenizer (20 strokes) and centri-fuged at 100 000 g for 1 h at 27°C The supernatant fraction was collected and kept at 0°C The pellet was resuspended in 2.5 mL cold disassembling buffer (20 mM
sodium phosphate buffer pH 7, containing 0.4M NaCl and 0.1 mM GTP) and kept at 0°C for 30 min after which it was centrifuged at 100 000 g for 30 min at 2–4°C The soluble fraction (disassembled microtubules) was collected, diluted with 1 vol 20 mM sodium phos-phate buffer pH 7 to decrease saline concentration and kept on ice The first supernatant (soluble tubulin pool) and the second supernatant (microtubular pool) fractions were loaded onto small (0.1 mL-bed volume) columns of cellulose phosphate P11 (Whatman) activated according
to the manufacturer’s instructions and equilibrated with
20 mM phosphate buffer pH 7 Tubulin carboxypeptidase
is retained by the resin [13] Elution is performed with 0.4 mL equilibration buffer containing 0.8MNaCl After dilution with 4 vols 20 mM phosphate buffer in order to decrease saline concentration, proteins were concentrated
by centrifuging the samples in Centricon-3 devices (Amicon) After reducing volumes to 0.1 mL, carboxy-peptidase activity was assayed immediately
Measurement of tubulin carboxypeptidase activity
We used two different methods The carboxypeptidase activity associated with the isolated cytoskeletal fraction was quantified as the increase in Glu-tubulin amount as
a function of incubation time Immediately after isolation, cytoskeletons (contained in conical plastic tubes) were incubated at 37°C in 0.25 mL microtubule-stabilizing buffer containing taxol and protease inhibitors as above After various incubation times, the Glu-tubulin content was determined by immunoblotting
When the activity of tubulin carboxypeptidase was determined in the microtubule and soluble tubulin fractions isolated under microtubule-stabilizing conditions, a method based on the release of [14C]tyrosine from [14C]tyrosinated tubulin was used [18] In brief, varying aliquots of the enzyme preparations were loaded onto nitrocellulose circles containing adsorbed [14C]tyrosinated tubulin ( 4000 c.p.m.) and after addition of 100 lL albumin solution (10 mgÆmL)1), the systems were incubated at
37°C for 1 h Then, soluble fractions which contain released [14C]tyrosine, were transferred to vials and radio-activity determined in a liquid scintillation counter In several independent experiments, time curves performed using 20 lL of each enzyme preparation showed linearity
up to 1 h
Trang 3Following incubation as above, tubes were centrifuged at
1600 g for 2 min and the supernatant discarded Pellets
were dissolved in 60 lL sample buffer [19] by heating at
90°C for 2 min Samples were subjected to SDS/PAGE
(10% gel) by the method of Laemmli [19] and transferred
to nitrocellulose sheets [20] Two identical gels were run in
parallel One sheet was treated with 10 lgÆmL)1 CPA
(30 min, 37°C) and extensively washed Both sheets were
then blocked for 1 h with 5% (w/v) fat-free dried milk
dissolved in NaCl/Tris containing 0.1% (v/v) Triton
X-100, and blots were treated for 3 h at room temperature
with anti-Glu antibody diluted 1 : 200, and washed
Sheets were incubated for 1 h at room temperature in
the presence of horseradish peroxidase conjugated to
Protein A (dilution 1 : 1000), and washed Colour was
developed using 4-chloro-naphth-1-ol as chromogen
Because CPA converts all Tyr-tubulin to the Glu form
[14], total tubulin amount was determined from the
CPA-treated sheet
Quantification of Glu-tubulin
Immunoblots were scanned and amount of Glu-tubulin in
cytoskeletal preparation was determined as in Contin et al
[14] The amount of Glu-tubulin in a particular sample is
expressed as a percentage of total detyrosinable tubulin,
calculated as 100 (Ano CPA/ACPA), where Ano CPAand ACPA
are the absorbances of the control and CPA-treated
samples, respectively Provided that the numerator and
the denominator correspond to identical samples, this
expression is independent of the amount of protein loaded
The method is described more fully in Contin et al [14]
Within a particular independent experiment, each value is
the average of two samples run in parallel In some cases,
values are expressed as the mean ± SD of three to five
independent experiments
Immunofluorescence
After defined durations of incubation of cytoskeletons,
samples on coverslips were fixed with methanol at)20 °C
for 5 min, and stored at 2–4°C in NaCl/Pi containing
0.2% sodium azide until use Fixed cytoskeletons were
incubated with 2% (w/v) BSA in NaCl/Pifor 60 min and
stained by double indirect immunofluorescence using
anti-Glu and anti-Tyr (dilution 1 : 200 and 1 : 500,
respect-ively) Secondary antibodies were used simultaneously at
1 : 200 dilution in NaCl/Pi/BSA Coverslips were
moun-ted in FluorSave and epifluorescence was observed on an
Axioplan microscope (Zeiss) Images were captured with
a sensitive, digital camera (Princeton Instrument) and
stored on a CD for subsequent analysis Estimations of
Glu- and Tyr-microtubules present in fields selected at
random were obtained by measuring the integrated
intensity of the corresponding immunostaining with the
aid of the METAMORPH IMAGING SYSTEM (Version 4.6r5)
For a determined field, the value of integrated intensity of
Glu-microtubules was divided by that of
Tyr-micro-tubules to estimate the relative proportion of Glu- with
respect to Tyr-microtubules
Treatment of cells with effectors Cells were treated at 37°C with various effector drugs and maintained in an incubator until the time of cytoskeletal fraction isolation Stock solutions of effectors were prepared in dimethylsulfoxide such that final solvent concentration in the growth medium did not exceed 0.5% (v/v) Controls were performed by adding 0.25% (v/v) dimethylsulfoxide to the medium This concentration
of dimethylsulfoxide had no effect on distribution or level
of Glu-tubulin in cells
Results Exposure of cells to okadaic acid induces decrease
in the activity of tubulin carboxypeptidase associated with microtubules in living cells
The level of association of tubulin carboxypeptidase activity with microtubules was determined by measuring enzyme activity present in cytoskeletons freed of soluble components Isolated cytoskeletons were incubated in vitro, and carboxypeptidase activity was inferred from the increase of the reaction product, detyrosinated tubulin (Glu-tubulin), as a function of incubation time The slope
of the time curves provides an estimate of the amount of the associated carboxypeptidase This method showed such
an association in several cell lines [14] Now, we investi-gated the effect of protein phosphatase inhibitors on the association of carboxypeptidase with microtubules in COS cells We first tested the effect of OA [21,22], which produces a marked increase in phosphorylation of many proteins in living cells When OA was added (1 lM final concentration) to culture medium 1 h before isolation of cytoskeletons, production of Glu-tubulin during in vitro incubation was significantly reduced (Fig 1) This indicates that OA treatment of the cells induced a decrease in carboxypeptidase activity associated with microtubules Replacement of OA by its inactive analogue, 1-nor-okadaone, resulted in activity associated with microtubules similar to that of control
The effect of OA on the carboxypeptidase/microtubule association was analysed by double immunofluorescence using cells cultured on glass coverslips Fig 2 shows images representative of many fields observed in each case Freshly isolated cytoskeletons from untreated cells con-tained minor amounts of Glu-microtubules (Fig 2A), whereas Tyr-microtubules were observed as brightly stained structures (Fig 2B) Similar results were obtained with 1-norokadaone-treated cells (data not shown) When isolated cytoskeletons were incubated at 37°C for 2 h, Glu-microtubules were clearly stained (Fig 2C), whereas
in cytoskeletons from OA-treated cells the staining revealed
no Glu-microtubules (Fig 2E), indicating lack of carb-oxypeptidase activity in these microtubules In 1-nor-okadaone treated cells, microtubules were brightly stained after 2 h in vitro incubation (Fig 2G) These results, again, indicate that the effect of OA on the carboxypeptidase activity associated with microtubules is based on its inhibitory effect on protein phosphatase activities Fluor-escence intensity measurements of Glu-microtubules relat-ive to Tyr-microtubules (see statistical values in the legend
Trang 4of Fig 2) confirmed conclusions drawn from direct
visualization
Exposure of cells to OA induces redistribution of tubulin
carboxypeptidase activity between the
microtubule-associated and nonmicrotubule-associated states
The scarce (or null) tubulin carboxypeptidase activity
associated with the cytoskeletons of OA-treated cells could
be attributed to: (a) inhibition of the enzyme while
remaining associated; or (b) dissociation of the enzyme
from microtubules To clarify this point, we investigated the
enzyme activity associated and nonassociated with
micro-tubules in cells treated and nontreated with OA Since the
detergent-extracting method used in this work to isolate
the cytoskeleton fraction produces a great dilution of the
soluble fraction, determination of carboxypeptidase activity
in this fraction was not possible Therefore, to perform this
study we disrupted cells under microtubule-stabilizing
conditions and separated the microtubular and soluble
fractions by centrifugation as described by Pipeleers et al
[17] Carboxypeptidase present in the assembled and nonassembled tubulin fractions from cells treated and nontreated with 1 lMOA was concentrated on phospho-cellulose columns (for details see Materials and methods) and enzyme activity determined As shown in Fig 3A, in control cells (nontreated with OA), higher carboxypeptidase activity was found in the microtubule fraction as compared with the soluble fraction In contrast, when cells were treated with OA, the major proportion of activity was present in the soluble fraction (Fig 3B) Another observa-tion from Fig 3 is that the sum of the activities recovered in both fractions is approximately the same when compared control and OA-treated cells These results clearly indicate
Fig 1 Effect of OA treatment on level of tubulin carboxypeptidase
activity associated withmicrotubules in living cells COS cells were
grown in Petri dishes to 60–70% confluence OA (1 l M final
concen-tration), 1-nor-okadaone (1 l M ), or no compound (control) was added
to the culture medium and incubation continued for 1 h Cytoskeletal
fractions were then isolated, incubated for the stated times, and
sub-jected to Western blotting with anti-Glu to determine the tubulin
carboxypeptidase activity associated with microtubules as described in
Materials and methods Upper panel: before immunostaining, the
nitrocellulose membranes were treated (+CPA) or not (–CPA) with
pancreatic carboxypeptidase A which produces full detyrosination of
tubulin [14] Lower panel: blots shown in the upper panel were used
to quantify Glu-tubulin Results are expressed as percentage of
total detyrosinable tubulin s, control; ,, + okadaic acid; h,
+1-nor-okadaone Results are mean ± SD of four independent
experiments.
Fig 2 Visualization of Glu- and Tyr-microtubules by double immuno-fluorescence, showing the effect of OA on the activity of tubulin carb-oxypeptidase associated withmicrotubules COS cells were grown on glass coverslips and treated with effectors as in Fig 1 After isolation, cytoskeletons were incubated for 2 h at 37 °C and processed for double immunofluorescence using anti-Glu (A,C,E,G) and anti-Tyr (B,D,F,H) Igs (A,B) Freshly isolated cytoskeletons (t ¼ 0 incub-ation) At this time, pictures similar to (A) and (B) were obtained for OA- and nor-okadaone-treated cells (not shown) (C–H) Cytoskele-tons incubated in vitro for 2 h (C,D) Control cells (E,F) OA-treated cells (G,H) Nor-okadaone-treated cells Scale bar, 10 lm For each panel, fluorescence intensity was measured by using the METAMORPH IMAGING SYSTEM and, for each condition, the ratio Glu/Tyr was cal-culated A/B ¼ 0.17 ± 0.03; C/D ¼ 1.21 ± 0.15; E/F ¼ 0.28 ± 0.04; G/H ¼ 1.12 ± 0.17 Each value represents the mean ± SE of four independent experiments.
Trang 5that the effect of the PP1/PP2A inhibitor was to induce
redistribution of the enzyme between the
microtubule-associated and nonmicrotubule-associated states rather than to inhibit it
Type of phosphatase(s) involved in regulation
of the carboxypeptidase activity associated with
microtubules
To determine the type of phosphatase(s) involved, we tested
effects of various compounds that specifically inhibit
different phosphatases Among these compounds, only
calyculin A and cantharidin, two well-known inhibitors of
PP1 and PP2A [23,24], showed effects similar to that of OA
(Fig 4) Deltamethrin, a specific inhibitor of PP2B [25], and
phenylarsine oxide, a putative inhibitor of tyrosine
phos-phatases [26], had no effect, even though the concentrations
used in our experiments (10 lM) were higher than those
reported to inhibit the corresponding phosphatases (100 p
and 5 lM, respectively) [25,26] These results suggest that the effects of OA, calyculin A, and cantharidin on activity of carboxypeptidase associated with microtubules are due to their inhibition of phosphatase activity, rather than to a side effect The phosphatases involved seem to be PP1 and/or PP2A although it is difficult at this time to distinguish between them
In vitro effect of OA on tubulin carboxypeptidase activity associated with microtubules
The possibility that OA causes dissociation of the enzyme from microtubules through direct interaction was ruled out
by the following experiment Cytoskeletal fraction of nontreated cells was incubated in the presence or absence (control) of OA to determine its effect on associated carboxypeptidase activity OA had no effect on the enzyme activity, and renewal of incubation medium 30 min after addition of OA did not alter subsequent detyrosination, indicating that OA does not cause direct dissociation of carboxypeptidase from microtubules (Fig 5) If such dissociation had occurred, the enzyme would have been eliminated during removal of medium and detyrosination would have stopped The incubated cytoskeletons represent only a part of the cell components, and they contain microtubules that were stabilized with taxol during the isolation procedure; therefore, these results suggest that intact cells and/or dynamic microtubules are required for phosphatase inhibitor to exert its inhibitory effect on the carboxypeptidase activity associated with microtubules The experiments shown below address this point
Fig 4 Effect of protein phosphatase inhibitors on the carboxypeptidase activity associated withmicrotubules in living cells COS cells were grown, treated with the effectors indicated below and processed as in Fig 1 The following effectors were tested separately by addition into culture medium: OA (1 l M final concentration); calyculin A (5 l M ); cantharidin (40 l M ); calyculin A plus cantharidin (5 and 40 l M , respectively); deltamethrin (10 l M ); phenylarsine oxide (10 l M ) Glu-tubulin was determined in cytoskeletons at t ¼ 0 and after 2 h of incubation Results are mean ± SD of three independent experiments Fig 3 Effect of OA on the distribution of tubulin carboxypeptidase
activity between the microtubule-associated and nonassociated states.
Confluent COS cells from twenty 100 mm Petri dishes were collected,
suspended in incubation medium and separated into two fractions
(5 mL each) The fractions were incubated at 37 °C in the presence or
absence of 1 l M OA for 1 h with gentle agitation Cells were
sedi-mented, washed once with microtubule-stabilizing buffer, and
homo-genized to isolate the microtubular and soluble tubulin fractions Both
fractions were concentrated and assayed as described in Materials and
methods Carboxypeptidase activities corresponding to the
micro-tubule-associated (s) and nonassociated (d) states are shown for
control (upper panel) and OA-treated (lower panel) cells Results are
mean ± SD of four independent experiments.
Trang 6Effect of stabilization of microtubules with taxol on the
dissociation of carboxypeptidase from microtubules
induced by OA
We investigated the possible involvement of microtubule
dynamics in the mechanism by which the OA treatment of
the cells results in a low activity of carboxypeptidase
associated with microtubules Microtubules in living cells
were stabilized by addition of 10 lM taxol 10 min before
addition of OA One hour later, cytoskeletons were isolated
and incubated to determine associated carboxypeptidase
activity Treatment with taxol prior to OA addition
preven-ted the dissociating effect of the phosphatase inhibitor
(Fig 6A, j and ; and d) This result supports the idea
that dynamic microtubules are necessary for OA to decrease
the activity of carboxypeptidase associated with
micro-tubules The possibility that this result is due to a neutralizing
effect of taxol on phosphatase inhibitory activity was ruled
out by the following experiment Using an in vitro assay in
which phosphatase activity present in soluble rat brain
extract is partially inhibited by OA, we found that taxol had
no effect on such inhibition (data not shown)
On the other hand, when taxol was added following OA
treatment, the decrease in the activity of tubulin
carboxy-peptidase associated with microtubules was not reverted
(Fig 6B) This reveals that, once the enzyme has been
dissociated from microtubules by the phosphatase inhibitor,
it cannot be re-associated even when microtubules are
stabilized
Detyrosination of tubulin in living cells can proceed even
when tubulin carboxypeptidase is not associated with
microtubules
There is increasing evidence of an association of tubulin
carboxypeptidase with microtubules and energy
consump-tion, which regulates its distribution between the micro-tubule-associated and nonassociated states ([13–15] and this study) We therefore investigated whether this association is
a necessary event for detyrosination of microtubules, taking advantage of the fact that once the phosphatases have been inhibited by OA, subsequent addition of taxol does not reverse the dissociation of carboxypeptidase from micro-tubules (Fig 6) We treated living cells with OA to induce dissociation of carboxypeptidase from microtubules, and then added taxol to stabilize microtubules, and continued the culture of intact cells The amount of Glu-tubulin in cells was measured as a function of time in culture following addition of taxol The amount of Glu-tubulin was directly correlated with incubation time (Fig 7), indicating that
Fig 5 In vitro effect of OA on tubulin carboxypeptidase activity
asso-ciated withmicrotubules COS cells were grown to 60–70% confluence,
and cytoskeletons were isolated and incubated in vitro for the stated
durations At the end of the incubation period, Glu-tubulin was
determined and expressed as in Fig 1 s, control (incubation without
added compound); ,, at t ¼ 0, OA (1 l M final concentration) was
added to incubation medium; h, at t ¼ 0, OA was added, and at
t ¼ 30 min (arrow) incubation medium was removed and replaced by
fresh medium lacking OA.
Fig 6 Effect of stabilization of microtubules previous or subsequent to
OA treatment on the inhibition of tubulin carboxypeptidase activity associated withmicrotubules (A) COS cells were grown to 60–70% confluence Taxol (10 l M final concentration) was added to culture medium After 10 min, OA (1 l M ) was added and culturing continued for a further 1 h Cytoskeletal fractions were isolated and incubated for the stated times At the end of the incubation period Glu-tubulin was determined and expressed as in Fig 1 ., Cells treated with OA alone for 1 h prior to isolation of cytoskeletons; j, cells treated with taxol for 10 min and subsequently with OA for 1 h longer; d, control (nontreated) cells (B) Cells were treated with 1 l M OA for 1 h, and then with 10 l M taxol for 10 min Cytoskeletons were isolated and incubated for the stated times to determine the amount of tubulin carboxypeptidase activity associated with microtubules ., cells trea-ted with OA alone for 70 min; j, cells treatrea-ted with OA for 1 h and then with taxol for 10 min; d, control (nontreated) cells.
Trang 7detyrosination of tubulin within intact cells proceeded even
when carboxypeptidase was not associated with
micro-tubules In comparative experiments, cells treated with OA
alone (+OA) or no treatment (control) showed no increase
of Glu-tubulin, and cells treated with taxol alone (+taxol)
were detyrosinated faster than cells treated with OA and
then with taxol Evaluation of the slope of the curves
indicated that detyrosination can occur when
carboxypepti-dase is not associated with microtubules within the cell,
although at a rate 2.5 times lower than when it is
associated with microtubules
Discussion
The amount of tubulin carboxypeptidase activity
associated with microtubules is regulated by
phosphorylation/dephosphorylation events
in living cells
The results described above show that tubulin
carboxy-peptidase activity associated with microtubules was very
low when cells were treated with 1 lMOA (Figs 1 and 2)
The possibility that the decrease was due to a lower amount
of the enzyme associated with microtubules) with a
corresponding increase in the cytosolic fraction) was
tested and confirmed by biochemical assay of enzyme
activity in the microtubule-associated and soluble fractions,
as isolated by a properly established method that preserves
native microtubules [17] In effect, carboxypeptidase in
control cells was associated mainly with microtubules,
whereas in OA-treated cells, the higher proportion of
enzyme activity was in the soluble fraction (Fig 3) As the
sum of the activities of both fractions is practically the same
for OA-treated and untreated cells (Fig 3), it appears that
the effect of OA is not to inhibit the carboxypeptidase but to
redistribute it Complementary observations also support this conclusion: (a) enzyme activity was unchanged when cytoskeletons were incubated in vitro with 1 lMOA (Fig 5); (b) enzyme activity was not modified by OA in living cells previously treated with taxol (Fig 6A); (c) no alteration of tubulin carboxypeptidase activity was reported previously
in fibroblasts and epithelial cells treated with OA [27]; (d) a previous in vitro study [13] showed that tubulin carboxy-peptidase activity of a rat brain soluble fraction was unchanged regardless of incubation conditions favouring
vs not favouring high phosphorylation
OA by itself did not disrupt the association of tubulin carboxypeptidase with microtubules (Fig 5), and the inac-tive OA analogue 1-nor-okadaone also had no effect on this association (Figs 1 and 2) These observations suggest that the effect of OA on the carboxypeptidase activity associated with microtubules is mediated by its capacity to inhibit protein phosphatases Other phosphatase inhibitors (caly-culin A and cantharidin) showed a similar effect on the association (Fig 4) These drugs are structurally unrelated, and it is unlikely that all of them would produce the same side effect OA, calyculin A, and cantharidin are all serine/ threonine-specific protein phosphatase inhibitors specific for PP1 and PP2A but, at the concentrations tested they are not inhibitors of PP2B, PP2C, or tyrosine phosphatases Other compounds such as deltamethrin and phenylarsine oxide which inhibit, respectively, PP2B and tyrosine phospha-tases, did not affect the carboxypeptidase activity associated with microtubules (Fig 4) These results confirm that the
OA effect on the carboxypeptidase activity associated with microtubules is mediated by its capacity to inhibit protein phosphatases, and indicate that PP1 and/or PP2A are probably the phosphatases involved in regulation of this phenomenon in living cells
It remains unclear whether the target of phosphoryla-tion/dephosphorylation is tubulin carboxypeptidase itself
or an intermediary compound [for example, microtubule-associated protein (MAP) or microtubule-based motor protein] which, according to its phosphate content, could interact with the enzyme and allow it (or not) to become associated with microtubules The presence of most MAPs on the microtubule surface is known to be modulated by phosphate group content of their serine and threonine residues [28–30]; a high phosphate MAP content precludes association, and vice versa Alternat-ively, one can imagine a cascade of biochemical events (at least one of them controlled by phosphorylation/dephos-phorylation) which eventually allows (or not) the enzyme
to associate with microtubules In any case, phosphory-lation/dephosphorylation events are clearly involved in association of carboxypeptidase with microtubules in living cells
Phosphorylation/dephosphorylation of tubulin carboxypeptidase (or an intermediary compound)
is dependent on disassembly of microtubules The fact that nondynamic microtubules (stabilized with taxol) retain associated tubulin carboxypeptidase activity when cells are subsequently treated with OA (Fig 6A) indicates that: (a) OA does not dissociate the enzyme from microtubules by direct interaction or through its
Fig 7 Detyrosination of microtubules by nonassociated
carboxypepti-dase in intact cells COS cells were treated with or without 1 l M OA for
1 h and subsequently with or without 10 l M taxol Time of taxol
addition was defined as zero Cells were incubated for the indicated
times, and cytoskeletons were isolated and immediately processed to
measure amount of Glu-tubulin as described in Materials and
meth-ods s, Control (nontreated) cells; d, cells treated with OA alone; ,,
cells treated with taxol alone; , cells treated first with OA and then
with taxol Data shown are mean ± SD of five experiments.
Trang 8phosphatase inhibitory activity; and (b) the dynamics of
microtubules is required for OA to reduce association of
carboxypeptidase with microtubules The requirement for
dynamic microtubules while OA is exerting its effect agrees
with the idea that the disassembly phase is an obligatory
step for carboxypeptidase to become a soluble entity
Disassembly of microtubules, during normal equilibrium, is
presumably the means by which carboxypeptidase becomes
a soluble entity After disassembly, the target molecule
could be subjected to phosphorylation/dephosphorylation
by the respective kinases and phosphatases Then, according
to the resulting phosphate content, the carboxypeptidase
could coassemble with tubulin in the assembly phase of
equilibrium, or associate directly on the surface of
micro-tubules This view is supported by the finding that OA
treatment prior to stabilization led to formation of
micro-tubules without carboxypeptidase activity (Fig 6B)
Is association of tubulin carboxypeptidase with
microtubules necessary to catalyse detyrosination?
One might initially hypothesize that this association results
in rapid production of detyrosinated microtubules
How-ever, in confluent cells, where carboxypeptidase is
maxi-mally associated with microtubules, they remain mostly
tyrosinated [14] The mere association of the enzyme with
microtubules therefore does not seem to guarantee rapid
detyrosination A plausible hypothesis is that the association
is a necessary but not sufficient condition Stabilization of
microtubules could be the complementary factor required
for effective detyrosination This is the basis for the
generally accepted definition of stable and dynamic
micro-tubules as Glu- and Tyr-micromicro-tubules, respectively There
seems to be no doubt that Glu-microtubules are always
stable On the other hand, Tyr-microtubules are not
necessarily always dynamic structures—they may be stable
when lacking associated carboxypeptidase, e.g cultured
nerve cells contain a nocodazole- and cold-resistant subset
of microtubules having a higher content of Tyr-tubulin than
the mean population [15] Although these prior studies
suggest that association of carboxypeptidase with
micro-tubules is necessary for their detyrosination, the alternative
possibility that nonassociated carboxypeptidase also
cata-lyses detyrosination is supported by findings in the present
study
Our findings suggest that even though association of
tubulin carboxypeptidase with microtubules results in faster
detyrosination (Fig 7), this association is not a requirement
for detyrosination, i.e any microtubule may undergo
detyrosination regardless of presence vs absence of
associ-ated carboxypeptidase If true, this concept would imply
that the association/dissociation phenomenon is not a
regulatory factor determining the tyrosination state of
microtubules However, because of the variety and
com-plexity of cellular physiological processes, we hesitate to
state this conclusion definitively without further
experimen-tal confirmation Even though nonassociated
carboxypepti-dase can catalyse detyrosination, one can speculate that,
within the cell, all (or most) carboxypeptidase, in response
to certain signals (perhaps enzyme phosphate content),
could be associated with microtubules, i.e no enzyme is in
the nonassociated state In this case, the only microtubules
capable of undergoing detyrosination would be those having associated enzyme Studies to resolve this point are underway
Acknowledgements
We thank C.A Argaran˜a and C.R Ma´s for critical reading of the manuscript; S.N Deza and M.G Schachner for technical assistance, and S Anderson for English editing of the manuscript This work was supported partly by grants from Agencia Nacional de Promocio´n Cientı´fica y Tecnolo´gica de la Secretarı´a de Ciencia y Tecnologı´a del Ministerio de Cultura y Educacio´n en el marco del Programa
de Modernizacio´n Tecnolo´gica (BID 802/0C-AR), Consejo Nacional
de Investigaciones Cientı´ficas y Te´cnicas (CONICET), Secretarı´a de Ciencia y Te´cnica de la Universidad Nacional de Co´rdoba y Agencia Co´rdoba Ciencia del Gobierno de la Provincia de Co´rdoba, Argentina.
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