Nocodazole Inhibits Signal Transduction by the T Cell Antigen Receptor*

The potential role of the cytoskeleton in signaling via the T cell antigen receptor (TCR) was investigated using pharmacological agents. In Jurkat T cells, disruption of the actin-based cytoskeleton with cytochalasin D or disruption of the microtubules with colchicine did not affect TCR induction of proximal signaling events triggered by CD3 mAb. Polymerized actin and tubulin, therefore, were not required for TCR-mediated signal transduction. Nocodazole, however, was found to inhibit dramatically TCR signaling, independently of its ability to depolymerize microtubules. This effect was TCR-specific, because signaling via the human muscarinic acetylcholine receptor 1 in the same cells was unaffected. A mechanism for the inhibition of TCR signaling by nocodazole was suggested byin vitro assays, which revealed that the drug inhibited the kinase activity of LCK and, to a lesser extent, FYN. The kinase activity of ZAP-70 in vitro, however, was unaffected. These results, therefore, suggested that nocodazole prevented initial phosphorylation of the TCR by LCK after stimulation, and as a result, it blocked activation of downstream signaling pathways. Immunofluorescence analyses also revealed that nocodazole and the specific SRC-family kinase inhibitor PP1 delocalized ZAP-70 from its constitutive site at the cell cortex. These effects did not require the SH2 domains of ZAP-70. The localization of ZAP-70 to the cell cortex is, therefore, regulated by the activity of SRC-family kinases, independently of their ability to phosphorylate immunoreceptor tyrosine-based activation motifs of the TCR.

The ␣␤ disulfide-linked heterodimer of the T cell antigen receptor (TCR) 1 interacts with an antigenic peptide bound to a major histocompatibility complex molecule on the surface of an antigen-presenting cell (1). Together with signals from accessory surface receptors, such as CD28 (2), this interaction promotes the proliferation and differentiation of resting T cells into effector T cells. The signaling function of the TCR is mediated by the CD3 complex of polypeptides (␥␦⑀) and disulfide-linked homodimers, which are both noncovalently associated with ␣␤ heterodimers (3) and required for efficient surface expression of the intact TCR (4).
Tyrosine phosphorylation of ITAMs by LCK (or FYN) recruits a second family of cytoplasmic PTKs to the TCR, which comprises ZAP-70 and SYK (17,18). Both ZAP-70 and SYK are present in T cells, although only the former is critical for T cell development and function, and also is more abundant (19 -21). These PTKs bind to the TCR via binding of their two N-terminal SH2 domains with the doubly phosphorylated ITAMs (9,17,22,23). The association of ZAP-70 and SYK with the TCR facilitates their phosphorylation and subsequent activation. For ZAP-70, phosphorylation is thought to be mediated, in part, by LCK (24,25), with which it associates following engagement of the TCR (26). In contrast, binding to the doubly phosphorylated ITAM appears to activate SYK directly (27,28). Once activated, ZAP-70 and SYK autophosphorylate multiple tyrosines, generating SH2 binding sites for other signaling proteins, including Vav (29,30), Cbl (31), Ras-GAP, abl (32), and FakB (33). In this way, ZAP-70 and SYK may act as scaffolding proteins, recruiting other signaling molecules to the activated TCR and into close proximity with their upstream regulators and downstream targets (11). Subsequent to ZAP-70 and SYK activation, downstream effector functions are triggered, which include the activation of Ras and consequent activation of ERK1/2 MAP kinases (34,35) and the mobilization of intracellular Ca 2ϩ (6).
Two recent reports have indicated that a small percentage of the subunit of the TCR is associated with a Triton X-100 detergent-insoluble fraction in T cells, and this increases after TCR stimulation with CD3 mAbs (36,37). Association between and the insoluble fraction is broken by treatment of T cells with the actin-disrupting drugs, cytochalasins D and B, suggesting that the subunit might interact directly with the actin cytoskeleton. Based on these data, it has been suggested that the actin cytoskeleton might play a role in signal transduction via the TCR (36,37). Earlier experiments from one of our laboratories demonstrated that ZAP-70 is associated with ␣␤tubulin heterodimers in cell lysates (29). Furthermore, ␣-tubulin, which is tyrosine-phosphorylated in vivo (38), is efficiently phosphorylated by ZAP-70 in vitro (39). These data have raised the possibility that the microtubule cytoskeleton might also be involved in TCR signal transduction (29,39). In this study, the possible role of the actin-based cytoskeleton and microtubules in TCR signaling was investigated using pharmacological agents that disrupt these two cytoskeletons. These experiments indicated that polymerized actin and tubulin were not essential for TCR activation of proximal signaling events following CD3 mAb stimulation. However, the microtubule-disrupting drug nocodazole did specifically inhibit TCR signaling, which could be attributed to its ability to act as a SRC-family kinase inhibitor. Inhibition of SRC-family kinases was found to have a secondary effect, causing delocalization of ZAP-70 from its normal site in the cell cortex, independently of its SH2 domains (40).

MATERIALS AND METHODS
Cell Culture and Transfections-The E6.1 Jurkat T cell line and a subline stably transfected to express the HM-1 receptor (HM-1 Jurkat) (41) were cultured in RPMI medium supplemented with 5% fetal calf serum, 2 mM L-glutamine, 100 units/ml penicillin, and 100 g/ml streptomycin. Cells were maintained in a rapid growth phase prior to use in experiments. Transient transfection was carried out by electroporation, as described previously (42). Briefly, cells were washed three times in serum-free RPMI medium and resuspended at 1 ϫ 10 8 cells/ml. 250 l were transferred into a Bio-Rad gene pulser cuvette, and 5 g of the appropriate plasmid were added. Cells were pulsed at 960 microfarads and 250 V (Gene pulser, Bio-Rad laboratories), left for 10 min at RT, and then transferred to 25 ml of RPMI medium supplemented with 5% fetal calf serum and cultured for 14 h before harvesting.
All drugs were made up as stocks in Me 2 SO. Control cells were treated with equivalent volumes of Me 2 SO alone. Nocodazole was obtained from Sigma-Aldrich and was determined to be over 99% pure by the manufacturers (data not shown). Nocodazole repurified by reverse phase high performance liquid chromatography was found to have inhibitory effects on TCR signaling identical to those of the starting material (data not shown). Thus, the inhibitory effects of nocodazole were not due to a low level of contaminating material in preparations of the drug. Nocodazole stocks were prepared at 20 mM. Colchicine (Fluka Biochemicals; 20 mM stock solution), cytochalasin D (Sigma-Aldrich; 5 mM stock solution), and Taxol (Calbiochem; 20 mM stock solution) were also over 99% pure, as determined by the manufacturers. The SRCfamily kinase inhibitor, PP1, was a gift of Yajun Xu and Rainer Munshauer (BASF Bioresearch Corp., Worcester, MA). The concentrations of drugs and times of pretreatment used are indicated in the figures and their legends. Cells were pretreated with nocodazole for either 5 or 30 min before stimulation in biochemical experiments. No difference was detected between these two different times of incubation with the drug. The maximal inhibitory effect on TCR signaling was detected within 1 min of pretreatment with nocodazole (data not shown).
Antibodies and DNA Constructs-The generation of ZAP-4 and LCK-1 antisera has been described previously (29,43). Anti-FYN mAb (FYN-15) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Immunofluorescent staining of hemagglutinin (HA) epitopetagged ZAP-70 was carried out using 12CA5 mAb (44) and fluorescein isothiocyanate-conjugated goat anti-mouse Ig (Jackson Laboratories, Avondale, PA). Tyrosine-phosphorylated proteins were detected using 4G10 (from Brian Druker, Oregon Health Sciences University, Portland, OR). The rat anti-␣-tubulin mAb, YOL34, was obtained from Serotec. The TCR chain was recognized in Western blots using N39 antiserum (8), kindly provided by Jaime Sancho (Granada, Spain). To stimulate T cells via their TCR, Fab 2 fragments of the OKT3 mAb (American Type Culture collection, Rockville, MD) were used to avoid binding of the stimulating antibody to the protein A-Sepharose beads used for immunoprecipitation. This antibody preparation was kindly provided by M. Glennie and A. Tutt (Tenovus, Southampton, United Kingdom). For Western blotting, horseradish peroxidase-coupled antibodies, and protein A were obtained from Amersham Pharmacia Biotech. The wild-type and ⌬SH2 (amino acids 6 -251 deleted) HA epitopetagged ZAP-70 constructs in the pcDNA3 neo vector have been described previously (17,40).
Immunofluorescence and Confocal Microscopy-Transfected Jurkat T cells were washed three times with RPMI medium, pretreated with drugs as appropriate for 1 h, and then fixed for 1 h in 3.7% paraformaldehyde in PBS at RT. Fixed cells were settled onto coverslips and coated with 3-aminopropyltriethoxy silane (Sigma) for 30 -60 min at RT. After rinsing, cells were permeabilized with 0.1% Triton X-100 for 5 min, rinsed, and blocked for 15 min with 0.5% fish skin gelatin (Sigma). Cells were then incubated for 1 h with 12CA5 anti-HA mAb at 0.25 g/ml, rinsed, and incubated with goat anti-mouse fluorescein isothiocyanate for 1 h. After washing, coverslips were mounted onto glass slides using a glycerol/PBS solution (Citifluor). A Leica TCS NT confocal microscope was used to visualize single optical sections through the center of stained transfected cells.
Immunoprecipitation, Western Blotting Analysis, and in Vitro Kinase Assays-Jurkat T cells were washed in serum-free RPMI and then stimulated, in 1-2 ϫ 10 7 -cell aliquots, with Fab 2 fragments of the OKT3 CD3 mAb (final concentration, 0.9 g/ml) at 37°C for the times indicated in figure legends. After pelleting by centrifugation, cells were lysed with 1 ml of ice-cold immunoprecipitation buffer (150 mM NaCl, 25 mM Tris-HCl, 1% Nonidet P-40, 100 mM Na 3 VO 4 , 10 mM Na 4 P 2 O 7 , 5 mM NaF, 5 mM EDTA, 5 mM EGTA, and 5 g/ml each of chymostatin, leupeptin, and pepstatin; pH 7.4) for 15 min at 4°C. Cell lysates were cleared of insoluble debris by centrifugation at 13,000 ϫ g for 15 min at 4°C and then precleared once by incubation with 10 l of protein A-Sepharose (Amersham Pharmacia Biotech) for 15 min at 4°C. ZAP-70, LCK, and FYN were immunoprecipitated using 5 l of ZAP-4 or LCK-1 antiserum and 1 g of FYN-15 mAb, respectively, coupled covalently to 10 l of protein A-Sepharose (Amersham Pharmacia Biotech) with dimethylpimelimidate (45). Tyrosine-phosphorylated (PTyr) proteins were immunoprecipitated using 4G10 mAb coupled covalently to protein A-Sepharose at 1 mg/ml. Immunoprecipitation was carried out by incubating cell lysates with coupled antibody for 4 h or overnight at 4°C. Following six washes with ice-cold immunoprecipitation buffer, precipitated protein was resolved by SDS-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes (Millipore). Western blotting was carried out as described previously (38). Polyvinylidene difluoride membranes were stripped of bound antibody using the Amersham Pharmacia Biotech ECL protocol, enabling blots to be probed for multiple antigens. Western blots were quantified by laser densitometry using a Molecular Probes Personal Densitometer.
For in vitro kinase assays, immunoprecipitates prepared in duplicate as above were washed four times in lysis buffer and twice in kinase buffer (50 mM PIPES, pH 6.5, 5 mM MnCl 2 , 1 mM DTT, 0.1 mg/ml BSA for LCK/FYN; the same buffer with the addition of 150 mM NaCl for ZAP-70). Immunoprecipitates were resuspended in 25 l of kinase buffer containing 10 M ATP, 1 Ci of [ 32 P]ATP and 20 M synthetic peptide substrate (AEEEIYGVLFAKKK for LCK and FYN (43); EE-LQQDDYEMMEENLKKK for ZAP-70) (gifts from Yajun Xu and Rainer Munschauer, BASF Bioresearch Corp.), and incubated at RT for 10 min with agitation. Reactions were stopped by adding 25 l of kinase buffer containing 20 mM EDTA. 47.5 l of the kinase reaction were bound to p85 ion-exchange paper (Whatman), and free [ 32 P]ATP was washed off in four changes of 60 mM phosphoric acid. Bound 32 P-labeled peptide was quantified using a Wallac 1410 scintillation counter to measure Cherenkov radiation. Protein recovered from the protein A-Sepharose beads in reducing sample buffer was resolved by SDS-polyacrylamide gel electrophoresis and Western blotted to confirm that equal amounts of protein had been immunoprecipitated in each assay.
To quantify in vivo tubulin polymerization, 1 ϫ 10 7 aliquots of Jurkat T cells were incubated with the indicated drugs for 30 min, and then washed in warm Ca 2ϩ , Mg 2ϩ -free PBS. Cells were then extracted at RT for 3 min with the PM2G buffer described by Solomon (46). Cell extracts were then centrifuged at 14,000 ϫ g for 3 min, and the pellets were washed twice in 1 ml of PM2G buffer. Pellet protein was then solubilized in reducing sample buffer, resolved by SDS-polyacrylamide gel electrophoresis, and Western blotted for ␣-tubulin.
Calcium Flux Analysis-RPMI-washed cells were resuspended to 5 ϫ 10 6 /ml in complete medium supplemented with 3 mM of the Ca 2ϩ indicator Indo-1 and incubated at 37°C for 30 min. Cells were then washed in ice-cold LOCKS buffer (150 mM NaCl, 1 mM CaCl 2 , 1 mM MgSO 4 , 5 mM KCl, 10 mM glycine, 15 mM HEPES, pH 7.4) resuspended in the same buffer to a concentration of 1 ϫ 10 6 cells/ml, and then kept on ice and protected from light until use. For analysis, cells were warmed to 37°C for 5 min in the presence of the indicated drugs. Ca 2ϩ fluctuations, before and after the addition of 0KT3 mAb at 10 g/ml, were monitored using an LS50 Perkin-Elmer luminescence spectrometer. Cells were excited at 355 nm, and emission was measured at 480 and 405 nm, representing free versus Ca 2ϩ -associated Indo-1, respectively, to give an absorbance ratio.

Nocodazole Inhibits TCR-induced Tyrosine
Phosphorylation of ZAP-70 -As discussed under "Introduction," the initial event that occurs after TCR cross-linking is phosphorylation of CD3 and ITAMs and their association with ZAP-70, which then itself becomes tyrosine-phosphorylated and activated (6). To investigate whether intact microtubules were important in this proximal signaling pathway, Jurkat T cells were preincubated with Me 2 SO control (which was used as the vehicle to solubilize all of the drugs tested), nocodazole, or colchicine for 30 min. The cells were then stimulated for 5 min with FAb 2 fragments of the OKT3 CD3 mAb, lysed, and ZAP-70 immunoprecipitated. Western blotting with an anti-PTyr mAb demonstrated that nocodazole pretreatment dramatically reduced both basal and OKT3-induced ZAP-70 phosphorylation (Fig.  1A). The amount of ZAP-70-associated phospho-in stimulated cells was also reduced by nocodazole. In contrast, colchicine pretreatment had no effect on basal and induced ZAP-70 or associated phosphorylation. Nocodazole and colchicine treatment both completely depolymerized microtubules, as determined by Western blotting for insoluble ␣-tubulin after extraction of cells with the microtubule stabilizing buffer PM2G (Fig.  1A, bottom panel) or by immunofluorescence (data not shown).
Pretreatment of the cells with cytochalasin D, which disrupted the actin cytoskeleton as determined by phalloidin staining and fluorescence microscopy (data not shown) or Taxol, which promoted microtubule polymerization (see Fig. 1B), also had no effect on TCR-stimulated ZAP-70 and associated -chain tyrosine phosphorylation.
Because tyrosine phosphorylation of ZAP-70 is involved in its activation (24), the inhibition by nocodazole of TCR-induced phosphorylation of ZAP-70 suggested that the drug might also inhibit the tyrosine phosphorylation of downstream intracellular proteins. To investigate this possibility, PTyr proteins were immunoprecipitated with an anti-PTyr mAb from lysates of cells pretreated with the panel of drugs and Western blotted with the same antibody. In Fig. 1B, it can be seen that nocodazole, but not colchicine, cytochalasin D, or Taxol, dramatically inhibited both basal and OKT3-induced tyrosine phosphorylation of multiple intracellular proteins. Taken together, the data in Fig. 1 suggested that polymerization of the microtubules (result of colchicine) or actin cytoskeleton (result of cytochalasin D) was not essential for TCR induction of ZAP-70 phosphorylation and subsequent tyrosine phosphorylation of intracellular proteins. However, TCR stimulation of these events was sensitive to nocodazole pretreatment.
In Fig. 1C, Jurkat T cells were pretreated with a range of nocodazole concentrations, and ZAP-70 tyrosine phosphoryla- tion was determined before and after CD3 mAb stimulation by Western blotting. It can be seen that the effect of nocodazole on ZAP-70 phosphorylation was dose-dependent, with an IC 50 of between 12.5 and 25 M (a similar IC 50 was obtained in 5 separate experiments). At saturating concentrations of nocodazole (100 M), CD3 mAb-induced ZAP-70 phosphorylation was inhibited by 90% (S.E., 1.3%; n ϭ 12). Basal levels of ZAP-70 phosphorylation were reduced to undetectable levels. Similarly, 100 M nocodazole reduced the amount of ZAP-70-associated phosphoby 92% (S.E., 0.7%; n ϭ 7) in stimulated cells and by 98% (S.E., 1.6%; n ϭ 7) in unstimulated cells ( Fig. 2A). Direct immunoprecipitation of the TCR also demonstrated that there was a dose-dependent inhibition of CD3 mAb-induced "total" tyrosine phosphorylation by nocodazole (data not shown).
The inhibitory effect of nocodazole was not the result of a change in the kinetics of ZAP-70 phosphorylation, and the drug inhibited CD3 mAb-induced ZAP-70 phosphorylation over a time course from 0.5 min to 1 h post-stimulation (data not shown). Kinetic experiments also demonstrated that the maximum inhibitory effect of nocodazole on ZAP-70 phosphorylation was attained by preincubation of cells with the drug for only 1 min (data not shown).
Inhibitory Effect of Nocodazole on ZAP-70 Phosphorylation Is Not Reversed by Taxol-The observation that ZAP-70 phosphorylation was inhibited by nocodazole but not by colchicine suggested that the former drug might have inhibited TCR signaling independently of its effects on microtubule cytoskeleton polymerization. To address this question directly, Jurkat T cells were pretreated with nocodazole, Taxol, or a combination of these drugs for 30 min. The cells were then stimulated for 5 min with CD3 mAb and lysed, and ZAP-70 immunoprecipitates Western blotted for PTyr. In a parallel experiment, the effect of these drug treatments was tested on the polymerization of tubulin using PM2G buffer extraction. In Fig. 2B, it can be seen that Taxol completely blocked the depolymerization of microtubules by nocodazole and that the amount of polymerized ␣-tubulin was actually increased relative to control (Me 2 SOtreated) cells. However, the inhibition of ZAP-70 phosphorylation by nocodazole was unaffected by the simultaneous addition of Taxol (Fig. 2A). These data, therefore, demonstrated that nocodazole mediated its effects on TCR-induced ZAP-70 phos-phorylation independently of its ability to depolymerize microtubules.
Signaling via the HM1 Receptor Expressed on Jurkat T Cells Is Unaffected by Nocodazole-It was important to determine whether the inhibitory effect of nocodazole on TCR signaling was due to some pleiotropic effect on cell metabolism or was affecting a specific component of signal transduction machinery of the TCR. Trypan blue dye exclusion ruled out the trivial possibility that nocodazole was simply killing the Jurkat T cells (data not shown). Furthermore, the inhibitory effect of nocodazole was reversed by extensive washing of pretreated cells with control medium (data not shown). To investigate this question further, the effect of nocodazole was determined on signaling via a G protein-coupled receptor, the human muscarinic acetylcholine receptor 1 (HM1), expressed on stably transfected Jurkat T cells (41). The HM1 receptor activates both increases in intracellular free Ca 2ϩ and ERK MAP kinase but does not require LCK or ZAP-70 to activate these signaling pathways, unlike the TCR (13,47).
As in the E6.1 parental Jurkat cell line, nocodazole inhibited TCR-stimulated tyrosine phosphorylation in HM1-Jurkat T cells (data not shown). The induction of intracellular signaling events downstream from activation of the TCR were also inhibited. Thus, nocodazole effectively inhibited induction of intracellular Ca 2ϩ fluxing (Fig. 3A) and phosphorylation of ERK MAP kinase (Fig. 3B), which normally occur following TCR stimulation. Nocodazole had no effect on the fluxing of calcium following treatment with the calcium ionophore ionomycin (data not shown), confirming that the cells were correctly loaded with Indo-1. Pretreatment of cells with colchicine or cytochalasin D had no effect on TCR stimulation of intracellular Ca 2ϩ fluxing and ERK MAP kinase phosphorylation (data not shown), consistent with their effects on TCR stimulation of tyrosine phosphorylation (Fig. 1).
Stimulation of the HM-1 receptor with carbachol induced increases in intracellular free Ca 2ϩ (Fig. 3A) and ERK phosphorylation (Fig. 3B), as expected. In contrast to stimulation via the TCR, however, nocodazole had no effect on the stimulation of either of these signaling pathways. These data supported the hypothesis that nocodazole was able to inhibit proximal signaling events specifically associated with the TCR but not the G protein-coupled HM-1 receptor.
Nocodazole Inhibits LCK and FYN Kinase Activity-The HM-1 Jurkat experiments (Fig. 3) suggested that nocodazole specifically inhibited a proximal component of the TCR signaling machinery, involved in the induction of cytoplasmic PTyr proteins. The level of tyrosine-phosphorylated -chain is governed by its phosphorylation by LCK and its dephosphorylation by undefined protein tyrosine phosphatases. Although ZAP-70 is unable to phosphorylate the chain directly (39) it may nevertheless promote the accumulation of phospho-by binding to phospho-ITAMs and preventing their dephosphorylation by constitutively active protein tyrosine phosphatases (12,48). Inhibition of phosphorylation by nocodazole after TCR stimulation, therefore, might have resulted either directly, from preventing the action of LCK, or indirectly, from preventing ZAP-70 binding to phospho-ITAMs.
Initial experiments investigated whether nocodazole affected the activity of LCK and FYN in vitro. These kinases were individually immunoprecipitated from Jurkat T cell lysates and then tested for their ability to phosphorylate a synthetic peptide substrate, which is the optimal substrate for SRC kinases (43,49), in the presence of a range of concentrations of nocodazole. The kinase activity of LCK was found to be profoundly inhibited by nocodazole (Fig. 4). FYN kinase activity was also inhibited, although to a lesser extent. The IC 50 for The inhibitory effect of nocodazole on SRC kinase activity was apparently specific, because the in vitro kinase activity of ZAP-70, tested by its ability to phosphorylate a synthetic peptide substrate (Fig. 4) or to autophosphorylate (data not shown), was completely unaffected by nocodazole. These data suggested that the inhibitory effect of nocodazole on TCR signaling primarily resulted from its ability to prevent LCK phosphorylation of ITAMs, thereby blocking the activation of all signaling pathways downstream of the TCR.
Nocodazole Delocalizes ZAP-70 from the T Cell Cortex-As discussed above, it was possible that decreased TCR-induced and ZAP-70 phosphorylation, after nocodazole pretreatment of Jurkat T cells, was due to inhibition of the interaction of ZAP-70 with the TCR after TCR stimulation, such that phosphobecame dephosphorylated. Our laboratories have recently shown that ZAP-70 is constitutively targeted to the T cell cortex in a diffuse band beneath the plasma membrane (40). Thus, ZAP-70 is held close to the TCR, with which it must interact to become activated. During the course of these studies, therefore, the effect of nocodazole on ZAP-70 localization was also assessed to investigate whether alteration of ZAP-70 localization might also contribute to the inhibitory effects of the drug.
To investigate this possibility, Jurkat T cells were transiently transfected with a C-terminally HA epitope-tagged ZAP-70 cDNA, cultured for 14 h, pretreated with control Me 2 SO or 100 M nocodazole for 1 h, fixed, and then immunofluorescently stained with an anti-HA mAb. ZAP-70 was found to be delocalized in the majority of cells and redistributed throughout the cytoplasm in cells pretreated with nocodazole (Fig. 5A). Treatment with colchicine, however, had no effect on ZAP-70 localization (data not shown), suggesting that delocalization was not mediated by the ability of nocodazole to disrupt microtubules. Consistent with this hypothesis, simultaneous addition of Taxol did not prevent delocalization of ZAP-70 by nocodazole (data not shown). In order to quantify the effect of nocodazole on ZAP-70 localization, cells were treated with a range of nocodazole concentrations, and ZAP-70-HA in transfected cells was scored as either localized (discrete cortical rim) or delocalized (cortical structure lost). The resulting titration curve for the effect of nocodazole on ZAP-70 localization indicated an IC 50 of approximately 10 M (Fig. 5C). Kinetic experiments indicated that a detectable effect on ZAP-70 localization was evident after pretreatment of cells with nocodazole for 15-30 min (data not shown).
To determine whether ZAP-70 delocalization was mediated by inhibition of SRC-family kinase activity, the effect of nocoda- In vitro kinase assays were performed on the relevant peptide substrates in the presence of nocodazole at the concentrations indicated, and 32 P radioactivity was incorporated into peptides measured. Data represent the mean of independent duplicate samples S.E. Western blotting confirmed that equal amounts of the relevant proteins had been precipitated in each assay (data not shown). Dose-response assays were carried out in three or four separate experiments for each immunoprecipitated kinase, with essentially identical results.
zole was compared with that of PP1, a specific SRC-family kinase inhibitor (50). PP1, like nocodazole, was able to delocalize ZAP-70 from the cell cortex (Fig. 5A), with an IC 50 of approximately 0.3 M (Fig. 5B), comparable to that for its inhibition of TCR-induced tyrosine phosphorylation in vivo (data not shown). Unlike nocodazole, PP1 had no effect on microtubule polymerization (data not shown). Taken together, these data suggested that SRC-family kinase activity is required for the efficient localization of ZAP-70 in the cell cortex. However, even high concentrations of PP1 or nocodazole failed to completely delocalize ZAP-70 in all cells, suggesting that SRC-family kinase activity is not an absolute requirement for ZAP-70 cortical localization.
ZAP-70 is localized to the cell cortex independently of its SH2 domains (40). However, it remained possible that the SH2 domains could be involved in the active delocalization of ZAP-70 following inhibition of SRC-family kinase activity, because this would inhibit any low levels of constitutive phosphorylation of ITAM, to which the tandem SH2 domains could bind. To test this possibility, Jurkat T cells were transiently transfected with the ⌬SH2-HA-ZAP-70 mutant, in which both SH2 domains had been removed, and the effects of nocodazole and PP1 on its localization were determined. Both drugs were able to induce significant delocalization of the ⌬SH2-HA-ZAP-70 mutant (Fig. 5). These data indicated that ZAP-70 was delocalized independently of its SH2 domain function. DISCUSSION It has recently been reported that the and CD3 ⑀ subunits of the TCR are physically associated with the actin cytoskeleton (36,37). Analysis of chimera mutants has suggested that this association might play a role in signal transduction (36). However, the experiments in this study, in which Jurkat T cells were pretreated with cytochalasin D, failed to demonstrate a requirement for actin polymerization for TCR induction of tyrosine phosphorylation following ligation with CD3 mAb (Fig.  1). TCR-induced increases in intracellular free Ca 2ϩ and ERK MAP kinase activation were also insensitive to cytochalasin D pretreatment (data not shown). These data, therefore, indicated that association of the TCR with the actin cytoskeleton is not essential for its signal transduction. It is likely, however, that the actin cytoskeleton does play an important role in mediating the changes in cell polarity and shape that occur when a T cell is activated by an antigen-presenting cell (51,52). The association of the TCR with the actin cytoskeleton may also be involved in its endocytosis (53,54).
One of our laboratories and that Geahlen have recently demonstrated that ZAP-70 and SYK PTKs are associated with tubulin heterodimers in cell lysates (29,55). Furthermore, ␣-tubulin is efficiently phosphorylated by ZAP-70 and SYK in vitro (39,55) and is tyrosine-phosphorylated in activated T cells (38). Taken together, these data raised the possibility that microtubules might be important for signaling via the TCR, which requires ZAP-70 function (56 -58). However, experiments in this study using colchicine to disrupt the microtubules failed to reveal an effect on TCR stimulation of tyrosine phosphorylation (Fig. 1), increases in intracellular free Ca 2ϩ , or activation of ERK MAP kinases (data not shown). Similarly, another microtubule disrupting drug, vincristine, had no detectable effect on TCR signaling (data not shown). Thus, intact microtubules did not appear to be important for TCR activation of these proximal signaling events. The association of ZAP-70 and SYK with tubulin may nevertheless be important for other signaling events triggered by the TCR, perhaps directly influencing microtubule polymerization (59).
Although the experiments in this study failed to reveal a role for the microtubules in TCR signaling, a novel biological effect of nocodazole was demonstrated: it was found to inhibit significantly TCR-induced ZAP-70 phosphorylation and activation of downstream signaling pathways (Fig. 1). This effect persisted when microtubule integrity was maintained by the simultaneous addition of Taxol (Fig. 2), suggesting that an unrelated biological activity had been revealed, probably mediated by binding to a target molecule distinct from ␤-tubulin (60). Nocodazole inhibited Ca 2ϩ mobilization and ERK phosphorylation stimulated by the TCR but not by the G protein-coupled HM-1 receptor in the same Jurkat T cell subline (Fig. 3). Thus, nocodazole appeared to be affecting a component of the TCR signaling machinery that was not involved in HM-1 receptor activation of these signaling pathways.
The ability of nocodazole to inhibit -chain phosphorylation after TCR ligation (Fig. 1) raised the possibility that it mediated its inhibitory effects on TCR signaling by preventing LCK from phosphorylating the TCR. Consistent with this hypothesis, nocodazole was found to profoundly inhibit the in vitro kinase activity of LCK, assayed using an exogenous peptide substrate (Fig. 4). LCK autophosphorylation in vitro was also suppressed by nocodazole, although this was found to be a less robust assay for LCK specific activity (data not shown). The kinase activity of FYN was inhibited to a lesser extent, whereas ZAP-70 kinase activity was completely insensitive to the drug (both assayed using exogenous peptide substrates (Fig. 4)). Because TCR signaling in Jurkat T cells is completely depend- Staining was scored as localized or fully delocalized. Graphs represent data from a typical experiment, which was repeated on two other occasions with similar results. ent on functional LCK (13), these data provided a plausible mechanism to explain how nocodazole blocked TCR signaling and also identified a second target molecule for the drug. Experiments with purified recombinant LCK protein produced in baculovirus 2 confirmed that the inhibitory effect of the nocodazole on LCK activity is mediated directly. The potency of nocodazole in vivo (measuring ZAP-70 phosphorylation; IC 50 ϭ 12.5-25 M) was reduced relative to kinase inhibition of immunoprecipitated LCK in vitro (IC 50 ϭ 2.5-5 M). This difference may be attributed to permeability of the compound and its distribution within cells relative to LCK or to metabolism of the drug in vivo.
Previously, our laboratories have shown that ZAP-70 is constitutively targeted to the cell cortex and that this localization requires its active kinase domain (40). Although nocodazole did not affect the kinase activity of ZAP-70 (Fig. 4), it was found to delocalize ZAP-70 from the cell cortex in the majority of the transfected cells (Fig. 5). This effect could be attributed to inhibition of SRC-kinase activity, because PP1, which is a specific inhibitor of this class of PTKs (50), also delocalized ZAP-70. These data indicate that the efficient localization of ZAP-70 to the cell cortex requires SRC kinase activity. However, because the ⌬SH2-ZAP-70 mutant was also delocalized by both nocodazole and PP1 (Fig. 5), this effect did not result from inhibition of the low level of constitutive phosphorylation of ITAMs by LCK in Jurkat T cells. Delocalization of ZAP-70 from the cell cortex would be expected to significantly reduce the efficiency with which it could bind to phospho-ITAMs to become phosphorylated and activated by LCK. However, given the slow kinetics of delocalization after nocodazole or PP1 treatment (data not shown), these effects may not contribute significantly to ability of these drugs to inhibit TCR signaling, which are mediated very rapidly (data not shown).
LCK phosphorylates ZAP-70 after TCR stimulation, thereby activating its kinase activity (11). It was possible, therefore, that nocodazole inhibition of LCK kinase activity might indirectly affect the kinase activity of ZAP-70 and thereby its localization. However, this possibility is unlikely, because it has been previously been shown, by analysis of point mutants, that the major sites of phosphorylation on ZAP-70 are not required for cortical localization (40). Furthermore, the ⌬SH2-ZAP-70 mutant, which is cortically localized, is not detectably tyrosine-phosphorylated (data not shown). Localization of ZAP-70 through a direct interaction with LCK is also unlikely, because expression of a delocalized C3A/C5A-LCK mutant (43) does not affect the cortical targeting of co-expressed ZAP-70 (data not shown). Furthermore, when ZAP-70 is expressed in the Jurkat subline JCam 1.6 (data not shown) or in 3T3 cells (40), neither of which express LCK, ZAP-70 is localized to the cell cortex in this majority of cells. Thus, LCK is not absolutely required for ZAP-70 targeting. Presumably, in these cells, other SRC-family members can substitute for LCK to regulate ZAP-70 localization. Consistent with this hypothesis, ZAP-70 is delocalized by both nocodazole and PP1 in transfected 3T3 fibroblasts (data not shown).
Our laboratories have speculated that the specific cortical targeting of ZAP-70 implies the existence of an anchoring molecule, which tethers its in this location (40). Thus, an attractive hypothesis for the effects of nocodazole and PP1 on ZAP-70 localization is that the anchor protein requires phosphorylation by a SRC kinase for efficient ZAP-70 binding. Clearly, the identification of the anchor protein for ZAP-70 will be important to determine whether this model is correct.
The benzimidazole drug nocodazole was originally generated synthetically and was identified by virtue of its ability to inhibit the growth of a panel of model tumor cell lines (61,62). Subsequently, it was shown that the drug's antitumor activity resulted from its ability to bind to tubulin and inhibit its polymerization (63,64). In this study, a novel biological activity of nocodazole has been revealed that is unrelated to its effects on the microtubule cytoskeleton. Thus, nocodazole was found to inhibit the activity of the SRC-family kinases LCK and FYN, thereby inhibiting signal transduction via the TCR, which depends on LCK activity (13). Nocodazole is structurally distinct from PP1, a specific SRC-family kinase inhibitor (59). Unlike, PP1, nocodazole shows specificity within the SRC-family. Thus, LCK is approximately 7-fold more sensitive to nocodazole than FYN (Fig. 4). LYN, a SRC-family kinase that is essential for signaling via the B cell antigen receptor (60), is also significantly less sensitive to nocodazole inhibition than LCK in vitro (data not shown). In the future, it may be possible to generate nocodazole analogs that inhibit SRC-family kinases without disrupting the microtubules. If such analogs can be generated that are completely selective for LCK, they might be useful clinically to suppress T cell function in disease.