From the Division of Cellular Immunology, National
Institute for Medical Research, Mill Hill, London NW7 1AA, United
Kingdom and the § Howard Hughes Medical Institute,
Departments of Medicine and of Microbiology and Immunology, University
of California-San Francisco, San Francisco, California 94143
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ABSTRACT |
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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 by in 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.
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INTRODUCTION |
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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).
The earliest detectable biochemical event following stimulation of the
TCR is the induction of protein tyrosine kinase (PTK) activity, which
is essential to couple it to downstream signaling pathways (5).
However, none of the component subunits of the TCR contain any
intrinsic tyrosine kinase domains. Rather, the TCR activates
intracellular protein tyrosine phosphorylation by interacting
sequentially with two different types of cytoplasmic PTKs (6).
Following TCR oligomerization, the cytoplasmic domains of the CD3
complex subunits (7-9) and homodimers (10) become rapidly
phosphorylated within 16 amino acid motifs
(YXXLX6-8YXXL), termed
immunoreceptor tyrosine-based activation motifs (ITAMs) (3, 11). ITAM
phosphorylation is mediated by members of the SRC-family PTKs,
primarily LCK (12-14) and, under some circumstances, FYN (15, 16).
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 Ca2+ (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).
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MATERIALS AND METHODS |
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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 µg/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 × 108 cells/ml. 250 µl were transferred into a Bio-Rad gene pulser cuvette, and 5 µg 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 Me2SO. Control cells were treated with equivalent volumes of Me2SO 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 SRC-family 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) epitope-tagged 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, Fab2 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
epitope-tagged ZAP-70 constructs in the pcDNA3neo
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 µg/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 × 107-cell aliquots, with Fab2 fragments of the OKT3 CD3 mAb (final concentration, 0.9 µg/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 Na3VO4, 10 mM Na4P2O7, 5 mM NaF, 5 mM EDTA, 5 mM EGTA, and 5 µg/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 × g for 15 min at 4 °C and then precleared once by incubation with 10 µl of protein A-Sepharose (Amersham Pharmacia Biotech) for 15 min at 4 °C. ZAP-70, LCK, and FYN were immunoprecipitated using 5 µl of ZAP-4 or LCK-1 antiserum and 1 µg of FYN-15 mAb, respectively, coupled covalently to 10 µl 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 MnCl2, 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 µl of kinase buffer containing 10 µM ATP, 1 µCi of [32P]ATP and 20 µM synthetic peptide substrate (AEEEIYGVLFAKKK for LCK and FYN (43); EELQQDDYEMMEENLKKK 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 µl of kinase buffer containing 20 mM EDTA. 47.5 µl of the kinase reaction were bound to p85 ion-exchange paper (Whatman), and free [32P]ATP was washed off in four changes of 60 mM phosphoric acid. Bound 32P-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 × 107 aliquots of Jurkat T cells were incubated with the indicated drugs for 30 min, and then washed in warm Ca2+, Mg2+-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 × 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 forCalcium Flux Analysis-- RPMI-washed cells were resuspended to 5 × 106/ml in complete medium supplemented with 3 mM of the Ca2+ 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 CaCl2, 1 mM MgSO4, 5 mM KCl, 10 mM glycine, 15 mM HEPES, pH 7.4) resuspended in the same buffer to a concentration of 1 × 106 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. Ca2+ fluctuations, before and after the addition of 0KT3 mAb at 10 µg/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 Ca2+-associated Indo-1, respectively, to give an absorbance ratio.
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RESULTS |
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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 Me2SO 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 FAb2 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.
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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 (Me2SO-treated) 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 phosphorylation 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 Ca2+ 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 Ca2+ 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 Ca2+ fluxing and ERK MAP kinase phosphorylation (data not shown), consistent with their effects on TCR stimulation of tyrosine phosphorylation (Fig. 1).
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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.
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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 phospho-
became 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.
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DISCUSSION |
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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 Ca2+ 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
Ca2+, 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 Ca2+ 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 dependent 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
baculovirus2 confirmed that
the inhibitory effect of the nocodazole on LCK activity is mediated
directly. The potency of nocodazole in vivo (measuring
ZAP-70 phosphorylation; IC50 = 12.5-25 µM)
was reduced relative to kinase inhibition of immunoprecipitated LCK
in vitro (IC50 = 2.5-5 µM). 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.
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ACKNOWLEDGEMENTS |
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We thank Panos Kabouridis and Tony Magee for their critical review of the manuscript and the Photo-Graphics Department for help with the figures. We are also grateful to Sheldon Ratnovsky (BASF Bioresearch Corp.) for some helpful suggestions for experiments.
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FOOTNOTES |
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* This work was supported by the United Kingdom Medical Research Council.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed. Fax: 44-181-906-4477; E-mail: s-ley@uk.ac.nimr.mrc.
1 The abbreviations used are: TCR, T cell antigen receptor; HA, hemagglutinin; ITAM, immunoreceptor tyrosine-based activation motif; MAP, mitogen-activated protein; PTyr, phosphotyrosine; PTK, protein tyrosine kinase; mAb, monoclonal antibody; RT, room temperature; PIPES, 1,4-piperazinediethanesulfonic acid; PM2G, 0.1% Nonidet P-40, 0.1 M PIPES, pH 6.9, 2 M glycerol, 5 mM MgCl2, 2 mM EGTA, 0.5% Nonidet P-40, 100 µM vanadate, 1 µg/ml each of chymotrypsin, leupeptin, and pepstatin; LOCKS, 150 mM NaCl, 1 mM CaCl2, 1 mM MgSO4, 5 mM KCl, 10 mM glycine, 15 mM HEPES, pH 7.4.
2 S. Ratnovsky, J. Kamens, H. Allen, and R. Munschauer, personal communication.
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REFERENCES |
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