Báo cáo khoa học: Inhibitors of protein phosphatase 1 and 2A decrease the level of tubulin carboxypeptidase activity associated with microtubules pptx

Inhibitors 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) Eur. J. Biochem. 270, 4921–4929 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03893.x PP1 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 anti-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 °Cinanair/CO 2 (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 m M Mes pH 6.7, 1 m M EGTA, 1 m M MgCI 2 , 10% (v/v) glycerol] and centrifuged again. Pelleted cells were resuspended in 0.5 mL micro- tubule-stabilizing buffer containing 10 l M taxol, 0.5% (v/v) Triton X-100, and protease inhibitors (10 lgÆmL )1 aprotinin, 0.5 m M benzamidine, 5 lgÆmL )1 o-phenanthro- line, 0.2 m M phenylmethanesulfonyl fluoride) at 37 °Cfor 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 l M taxol. Finally, the cytoskele- tons were suspended in microtubule-stabilizing buffer containing 10 l M 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 m M sodium phosphate pH 7, containing 40% (v/v) glycerol, 5% (v/v) dimethylsulfoxide, 0.1 m M 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 m M sodium phosphate buffer pH 7, containing 0.4 M NaCl and 0.1 m M GTP) and kept at 0 °Cfor30minafter 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 m M 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 m M phosphate buffer pH 7. Tubulin carboxypeptidase is retained by the resin [13]. Elution is performed with 0.4 mL equilibration buffer containing 0.8 M NaCl. After dilution with 4 vols 20 m M 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 [ 14 C]tyrosine from [ 14 C]tyrosinated tubulin was used [18]. In brief, varying aliquots of the enzyme preparations were loaded onto nitrocellulose circles containing adsorbed [ 14 C]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 [ 14 C]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. 4922 M. A. Contı ´ n et al. (Eur. J. Biochem. 270) Ó FEBS 2003 Immunoblotting Following 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 (A no CPA /A CPA ), where A no CPA and A CPA 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 for5min,andstoredat2–4°CinNaCl/P i containing 0.2% sodium azide until use. Fixed cytoskeletons were incubatedwith2%(w/v)BSAinNaCl/P i for 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/P i /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 l M 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 °Cfor2h, 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 Ó FEBS 2003 Tubulin carboxypeptidase/microtubule association 1 (Eur. J. Biochem. 270) 4923 of Fig. 2) confirmed conclusions drawn from direct visualization. Exposure of cells to OA induces redistribution of tubulin carboxypeptidase activity between the microtubule- associated and nonassociated 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 l M OA 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 with microtubules 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; ,,+okadaicacid;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 with microtubules. 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. 4924 M. A. Contı ´ n et al. (Eur. J. Biochem. 270) Ó FEBS 2003 that the effect of the PP1/PP2A inhibitor was to induce redistribution of the enzyme between the microtubule- associated and nonassociated 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 l M ) were higher than those reported to inhibit the corresponding phosphatases (100 p M and 5 l M , 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 with microtubules 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 0andafter2hof 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. Ó FEBS 2003 Tubulin carboxypeptidase/microtubule association 1 (Eur. J. Biochem. 270) 4925 Effect 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 l M 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 with microtubules. 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); ,,att¼ 0, OA (1 l M final concentration) was added to incubation medium; h,att¼ 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 with microtubules. (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 treated with OA for 1 h and then with taxol for 10 min; d, control (nontreated) cells. 4926 M. A. Contı ´ n et al. (Eur. J. Biochem. 270) Ó FEBS 2003 detyrosination 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 l M OA (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 l M OA (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. Ó FEBS 2003 Tubulin carboxypeptidase/microtubule association 1 (Eur. J. Biochem. 270) 4927 phosphatase 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-microtubules, 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 ˜ aandC.R.Ma ´ s for critical reading of the manuscript; S.N. Deza and M.G. Schachner for technical assistance, and S. 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