* Division of Cellular Immunology, National Institute for Medical Research, London NW7 1AA, United Kingdom; Mitsubishi
Chemical Corporation, Mitsubishi Kasei Institute of Life Sciences, Machida, Tokyo 194, Japan; and § Howard Hughes Medical
Institute, Department of Medicine, and Department of Microbiology and Immunology, University of California, San Francisco,
California 94143
ZAP-70 is a nonreceptor protein tyrosine kinase that is essential for signaling via the T cell antigen receptor (TCR). ZAP-70 becomes phosphorylated and activated by LCK protein tyrosine kinase after interaction of its two NH2-terminal SH2 domains with tyrosine-phosphorylated subunits of the activated TCR. In this study, the localization of ZAP-70 was investigated by immunofluorescence and confocal microscopy. ZAP-70 was found to be localized to the cell cortex in a diffuse band under the plasma membrane in unstimulated T cells, and this localization was not detectably altered by TCR stimulation. Analysis of mutants indicated that ZAP-70 targeting was independent of its SH2 domains but required its active kinase domain. The specific compartmentalization of ZAP-70 suggests that it may interact with an anchoring protein in the cell cortex via its hinge or kinase domains. It is likely that the maintenance of high concentrations of ZAP-70 at the cell cortex, that only has to move a short distance to interact with phophorylated TCR subunits, facilitates rapid initiation of signaling by the TCR. In addition, as the major increase in tyrosine phosphorylation induced by the TCR also occurs at the cell cortex (Ley, S.C., M. Marsh, C.R. Bebbington, K. Proudfoot, and P. Jordan. 1994. J. Cell. Biol. 125:639-649), ZAP-70 may be localized close to its downstream targets.
The T cell antigen receptor (TCR)1 is a complex
oligomeric protein that may be considered as two
functionally linked modules. The polymorphic disulfide-linked TCR signaling is initiated by receptor clustering, which
results in the tyrosine phosphorylation of the cytoplasmic
domains of the CD3 complex subunits (Qian et al., 1993 Tyrosine phosphorylation of ITAMs of the CD3 complex and the Once activated, ZAP-70 becomes autophosphorylated
on multiple tyrosines (Watts et al., 1994 Earlier experiments from one of our laboratories demonstrated that LCK is predominantly targeted to the
plasma membrane in mature human T cells (Ley et al.,
1994a Cell Culture and Transfections
The E6.1 Jurkat T cell line was cultured in RPMI medium supplemented
with 5% FCS, 2 mM l-glutamine, 100 U/ml penicillin, and 100 µg/ml
streptomycin. Cells were maintained in a rapid growth phase before staining or transfection. Transient transfection was carried out by electroporation as described previously (Salmeron et al., 1996 3T3 fibroblasts were maintained in Dulbecco's minimal essential medium, supplemented with 10% newborn calf serum, and the above concentrations of l-glutamine and antibiotics. Cells were split 1/4-1/5 every
3 d and were not allowed to reach confluency. Before a transfection, cells
were plated at 1.25 × 105 cells/well of a 6-well plate (Costar Corp., Cambridge, MA) and cultured overnight. Cells were transfected using Lipofectamine (GIBCO BRL, Gaithersburg, MD) following the manufacturer's
instructions. 0.5 µg of DNA was mixed with 0.5 µl of Lipofectamine per
transfection, and cells were incubated for 6 h with this complex before
washing and transferring into complete medium. Cells were then cultured for an additional 18 h before harvesting and staining.
DNA Constructs
The generation of the NH2-terminal hemagglutinin (HA) epitope-tagged
ZAP-70, the COOH-terminal HA epitope-tagged ZAP-70 construct, the
kinase-inactive K369A mutant, and the phosphorylation site mutants
(Y292F, Y492F, Y493F, and YY492,493FF) has been described previously
(Chan et al., 1992 Antibodies
The ZAP-4 antiserum, which was raised against a synthetic peptide corresponding to residues 271-290 of human ZAP-70 (Huby et al., 1995 The fluorochrome-labeled antibodies used for immunofluorescent staining of cells were all obtained from Jackson Immunoresearch Labs (West
Grove, PA). Goat anti-rabbit Ig coupled to FITC was used for labeling
with ZAP-4 antibody. For labeling with 12CA5 mAb, goat anti-mouse Ig
coupled to either fluorescein or Texas red was used as a second-stage antibody. Goat anti-rat Ig coupled to Texas red was used for Immunofluorescence and Confocal Microscopy
To stain for endogenous ZAP-70, cells were first settled onto coverslips,
which had been pretreated with 3-aminopropyltriethoxy silane (TESPA;
Sigma Chemical Co.), for 30 min at RT and fixed in 2% paraformaldehyde
in PBS for 6 min. In experiments in which cells were double labeled for
PTyr, 1 mM Na3VO4 was added to the fixing buffer. Cells were then incubated with 0.5% BSA-PBS for 1 h and permeabilized with 0.2% saponin
in PBS for 1 h. Permeabilized cells were incubated with ZAP-4 antiserum
(1:1000 dilution) overnight and then washed three times in PBS. The cells
were then incubated with anti-rabbit Ig-FITC antibody for 1 h, washed
extensively in PBS, and mounted onto glass slides using a glycerol/PBS solution (Citifluor; Agar Scientific, UK).
For immunofluorescent staining of HA-ZAP-70, transiently transfected Jurkat T cells were washed with PBS and then fixed for 1 h in 3.7%
paraformaldehyde in PBS at RT. Fixed cells were then settled onto
TESPA-treated coverslips. Transfected 3T3 cells were directly cultured on
coverslips placed in the culture wells. Cells were fixed in 3.7% paraformaldehyde in PBS for 1 h at RT. Coverslips were then incubated with 0.1%
Triton X-100 in PBS for 5 min to permeabilize the attached cells and
blocked for 15 min with 0.5% fish skin gelatin. After blocking, the coverslips were washed in PBS and then incubated for 1-2 h with primary antibody at the appropriate concentration, followed by FITC or Texas red-conjugated second-stage antibody for 1 h. Nonspecifically bound antibody was removed by extensive washing in PBS, and the coverslips were then
mounted onto glass slides as above.
Confocal imaging was performed using a laser scanning head (MRC-600; BioRad Laboratories) fitted to an Olympus BH-2 microscope. Samples were excited at 488 and 568 nm and fluorescein and Texas red signals
detected through the K2 and K1 filter blocks, respectively. Images were
collected using the photon counting mode of the COMOS program (BioRad Laboratories). In double labeling experiments, bleedthrough of the
Texas red signal into the fluorescein channel was negligible. To visualize
cell nuclei, permeabilized cells were stained with 7-amino-actinomycin D
(Molecular Probes, Eugene, OR) at 50 µg/ml together with the primary
antibody. The stained cells were then excited at 568 nm, and images were
collected through the K1 filter. Separate images of fluorescein-stained antigens were then collected by exciting at 488 nm and a collection of images
through the K2 filter.
Cell Fractionation
To separate soluble and particulate cell fractions, 1 × 107 Jurkat T cells
were resuspended in 1 ml of hypotonic buffer (10 mM Tris, 2 mM EDTA,
and 1 mg/ml each of chymostatin, leupeptin, and pepstatin, pH 7.4) and
then subjected to two successive freeze/thaw cycles. Cells were then disrupted by homogenization on ice using a tight fitting Dounce homogenizer (40 strokes), and the salt concentration was then adjusted to 150 mM
NaCl. Intact cells, nuclei, and cytoskeleton were pelleted by two successive centrifugations at 480 g for 5 min. This pellet, termed P1, was washed
in hypotonic buffer and then resuspended in Laemmli sample buffer. The
supernatant from the low speed centrifugation was recentrifuged at
100,000 g for 30 min to generate a second pellet fraction, termed P2, which
was enriched for membranes. The supernatant from this high speed centrifugation, termed S2, contained soluble proteins. Equivalent portions of
the fractionated protein were resolved by SDS-PAGE and Western blotted with anti-ZAP-70 antiserum.
To determine the fraction of ZAP-70 that remained associated with
cells after paraformaldehyde fixation and permeabilization, 1 × 107 Jurkat
T cells were incubated for 1 h in 3.7% paraformaldehyde in PBS. Control
cells were incubated in PBS alone. The cells were then pelleted, permeabilized by addition of 1 ml of 0.1% Triton X-100 in PBS, and incubated for 5 min at RT. Insoluble material was pelleted by centrifugation and the supernatant aspirated (S fraction) and mixed 1:1 with reducing sample
buffer. The pellet was washed once in the lysis buffer and then solubilized
in 200 µl of reducing sample buffer (P fraction). Equivalent amounts of
the S and P fractions were then resolved by SDS-PAGE and Western blotted with anti-ZAP-70 antibody.
Immunoprecipitation and Western Blotting Analysis
Transfected Jurkat T cells were washed in serum-free RPMI and then
stimulated with OKT3 CD3 mAb for 5 min at 37°C. After pelleting by
centrifugation, cells were lysed with 1 ml of ice-cold immunoprecipitation buffer (IPB; 150 mM NaCl, 20 mM Tris-HCl, 1% NP-40, 100 mM Na3VO4, 5 mM NaF, 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 (Pharmacia Fine Chemicals, Piscataway, NJ) for 15 min at 4°C. For immunoprecipitation of HA-ZAP-70,
10 µg of 12CA5 mAb were coupled to 10 ml of protein-A Sepharose with
dimethylpimelimidate (Schneider et al., 1982 ZAP-70 Is Constitutively Targeted to the T Cell Cortex
in Jurkat T Cells
In initial experiments, the localization of endogenous
ZAP-70 was investigated using a polyclonal anti-ZAP-70
peptide antiserum, ZAP-4, to stain the endogenous protein
in Jurkat T cells. In Fig. 1 A, it can be seen that staining
was detected at the cell cortex and also in the cytoplasm.
Several other anti-ZAP-70 antisera and two anti-ZAP-70
mAb were also tested for immunofluorescence. However,
none of these produced any specific staining. The addition of specific immunizing ZAP-4 peptide during incubation
with the primary antibody reduced staining at the cell cortex to background levels, and staining was also markedly
reduced in the cytoplasm (Fig. 1 B). The cytoplasmic staining was also detected in a B cell line, which does not contain ZAP-70, suggesting that this resulted from cross-reaction with an irrelevant antigen (data not shown). Taken
together, these data suggested ZAP-70 was localized to
the cell cortex in unstimulated Jurkat T cells.
Since endogenous ZAP-70 was difficult to detect by immunofluorescence, it was important to confirm its cortical
localization. To do this, Jurkat T cells were transiently
transfected with a ZAP-70 cDNA in which an HA epitope
tag had been spliced onto its COOH terminus and then
stained using an anti-HA mAb. In Fig. 2 A, it can be seen
that transiently transfected ZAP-70-HA was concentrated at the cell cortex in a diffuse band. Counterstaining with
7-amino-actinomycin D, to reveal the position of the nucleus, clearly indicated that between the cell cortex and
the cell nucleus there was cytoplasm that contained only
low levels of ZAP-70-HA (Fig. 2 A). Similar results were
obtained using methanol/acetone to fix the Jurkat cells
(data not shown), indicating that the cortical localization of ZAP-70-HA was not an artifact of the paraformaldehyde-fixation procedure. Staining for transfected LCK
produced very sharp staining at the cell cortex in contrast
to transfected ZAP-70-HA (Fig. 2 C). This difference in
staining was also evident by measuring the pixel intensity
along a transect through a cell stained for ZAP-70-HA or
LCK (Fig. 2, B and D, respectively). The LCK staining profile produced a narrow peak that was symmetrical. In
contrast, the peak for ZAP-70-HA staining was broader
than for LCK and asymmetrically distributed, with highest
intensity towards the plasma membrane and reduced
staining moving into the cell. The asymmetric distribution of ZAP-70-HA suggested that the breadth of the ZAP-70-HA staining was not simply an artifact of its brightness.
By setting the background fluorescence levels against an
untransfected cell, it was also evident that ZAP-70-HA
staining was detected at low levels throughout the cytoplasm. However, the brightest staining was all concentrated at the cell cortex.
Endogenous ZAP-70 localized in a tight band at the cortex, in contrast to the broad staining pattern detected with
transfected ZAP-70-HA. However, the staining achieved
with the transfected ZAP-70-HA (Fig. 2 A) was considerably brighter than that with the endogenous protein (Fig. 1
A). By artificially decreasing the intensity of staining for
the transfected protein using the COMOS software, so
that only the highest levels of ZAP-70-HA were detected,
the staining pattern for transfected ZAP-70-HA became very similar to the endogenous protein (data not shown),
suggesting that the difference was quantitative rather than
qualitative. In addition, untagged ZAP-70 transfected into
Jurkat T cells had the same distribution as ZAP-70-HA,
revealed using the ZAP-4 antiserum (data not shown), indicating that the addition of an HA tag did not affect
ZAP-70 localization. Both endogenous ZAP-70 and transfected HA-ZAP-70 associated with phospho- The specific localization of ZAP-70 to the cell cortex suggested that it might be associated either with the plasma
membrane or cortical cytoskeleton. To determine the solubility of ZAP-70, Jurkat T cells were lysed in hypotonic
buffer by Dounce homogenization and then separated into
P1 (nuclei and cytoskeleton), P2 (membrane), and S2 (soluble) fractions (as described in Materials and Methods). Western blotting of these fractions demonstrated that virtually all ZAP-70 was soluble, and only a small fraction
was detected in the two particulate fractions (Fig. 3 A). No
change in the fractionation of ZAP-70 was detected after
TCR stimulation for 5 or 20 min. These data raised the
possibility that paraformaldehyde fixation might not have
rendered the majority of ZAP-70 insoluble, such that it
was lost from the cell after Triton X-100 extraction. In this
case, immunofluorescent staining would only have detected the small percentage that was already associated
with the particulate fraction. Alternatively, it was possible
that the putative interaction of ZAP-70 with the particulate fraction was not stable after cell lysis. To investigate
whether immunofluorescent staining detected all ZAP-70
after fixation, Jurkat T cells were fixed with 3% paraformaldehyde or left unfixed and were then extracted with
0.1% Triton X-100. Western blotting of soluble and insoluble protein confirmed that paraformaldehyde treatment
rendered essentially all ZAP-70 insoluble, confirming that
the cortical ZAP-70 staining corresponded to essentially all of ZAP-70 expressed in Jurkat T cells (Fig. 3 B). These
data were, therefore, consistent with the hypothesis that
interaction of ZAP-70 with the cell cortex was not stable
after cell lysis.
In summary, the data in this section indicated that ZAP-70
was not uniformly distributed throughout the cytoplasm
but rather was specifically targeted to the cell cortex in a
diffuse band under the plasma membrane in unstimulated
T cells.
Stimulation of the TCR Does Not Alter the Gross
Localization of ZAP-70
As outlined in the introduction, TCR stimulation induces
phosphorylation of
Localization of ZAP-70 Is Distinct from Cortical Actin
and Microtubule Cytoskeletons
The targeting of ZAP-70 to the cell cortex raised the possibility that it might be associated with the cortical actin cytoskeleton. However, double labeling for HA-ZAP-70
and F-actin, using rhodamine-coupled phalloidin, demonstrated that the patterns of staining were distinct (Fig. 5 A).
Thus, similar to the results with LCK, phalloidin staining
produced relatively sharp staining, in contrast to HA- ZAP-70 staining which was more diffuse. This difference
was also evident when pixel intensity was plotted along a
transect through a cell double labeled for ZAP-70-HA
and actin (Fig. 5 B). These data suggested that the cortical
localization of ZAP-70 was not mediated via its direct interaction with the cortical actin cytoskeleton.
Recent data from one of our laboratories has indicated
that a fraction of ZAP-70 is associated with Cortical Localization of ZAP-70 in Transfected
3T3 Fibroblasts
To investigate whether the cortical targeting of transfected
ZAP-70-HA was specific to T cells, 3T3 fibroblasts were
transiently transfected with a ZAP-70-HA plasmid vector.
ZAP-70-HA was detected exclusively in the cell cortex in
these cells (Fig. 6 A). Double labeling with phalloidin revealed that ZAP-70-HA had a similar distribution to the
cortical actin, albeit more diffuse, as in Jurkat T cells (Fig.
6 B). No ZAP-70-HA staining was detected on actin stress
fibers or on microtubules (data not shown). Disruption of
actin filaments with cytochalasin D did not delocalize
ZAP-70-HA from the cell cortex, although cell morphology was dramatically altered (Fig. 6, C and D). Cytochalasin D also had no effect on ZAP-70 localization in Jurkat
T cells (data not shown). Taken together, these data indicated that targeting of ZAP-70 to the cell cortex did not
require any other lymphoid-specific proteins and was not
determined via a direct interaction with cortical actin.
ZAP-70 Is Targeted to the Cell Cortex Independently of
its SH2 Domains
Since ZAP-70 interacts with ITAMs of the TCR via its two
SH2 domains after CD3 mAb stimulation (Weiss and Littman, 1994
To investigate whether the kinase domain of ZAP-70 was
involved in intracellular targeting, the localization of the
Mutation of the Major Inducible Sites of
Phosphorylation of ZAP-70 Does Not Affect its
Cortical Targeting
The requirement for an active kinase domain for ZAP-70
to localize to the cell cortex raised the possibility that autophosphorylation by ZAP-70 might be important for its
targeting. This could potentially create binding sites for
putative SH2-containing proteins in the cell cortex with
which it could interact and be targeted. After TCR stimulation, ZAP-70 is known to be inducibly phosphorylated on three tyrosine residues: Y292, Y492, and Y493 (Watts
et al., 1994 As an initial step to investigate the possible role of autophosphorylation of ZAP-70 in its localization, the distribution of several point mutants of HA-ZAP-70 was determined, in which the known phosphorylated tyrosines
described above were individually substituted for phenylalanine. In Fig. 8, it can be seen that all four of the point
mutants localized to the cell cortex both in transiently
transfected Jurkat T cells and 3T3 fibroblasts, similar to
the wild-type HA-ZAP-70. These data, therefore, indicated that phosphorylation of tyrosines 292, 492, and 493 on ZAP-70 was not required for its cortical targeting.
SYK Is Constitutively Targeted to the Cell Cortex in
Jurkat T Cells and 3T3 Fibroblasts
The protein tyrosine kinase SYK is 60% identical to ZAP-70 in humans (Law et al., 1994
This study demonstrates that ZAP-70 is localized at the
cell cortex in a diffuse band under the plasma membrane
in unstimulated Jurkat T cells. This specific targeting of
ZAP-70 suggests that an anchoring site exists in the cell
cortex, which concentrates it in this location. As ZAP-70
was also localized at the cell cortex when expressed in 3T3
fibroblasts, this putative anchoring site cannot be lymphoid cell specific. Comparison with phalloidin staining indicated that ZAP-70 distribution was distinct from that of
cortical actin, suggesting that the actin cytoskeleton is unlikely to be its anchoring site (Figs. 5 A and 6, A and B). This conclusion was substantiated by the observation that
the localization of ZAP-70 was unaffected by disruption of
the actin cytoskeleton with cytochalasin D (Fig. 6 C).
However, it is possible that ZAP-70 may interact with
other components of the cortical cytoskeleton that are
linked to the plasma membrane and, therefore, may be insensitive to the effects of cytochalasin D. Several examples exist of cortical cytoskeletal proteins which act as cross-linkers between the plasma membrane and the actin-based
cytoskeleton. These include ankyrin, dystrophin, One noticeable feature of HA-ZAP-70 localization was
its diffuse nature, which contrasted with the "tight" localization of LCK, which is directly bound to the plasma membrane via dual acylation of its NH2 terminus (Koegl et al.,
1994 No gross changes in the localization of ZAP-70 were detected after stimulation of the cells with CD3 mAb. However, biochemical experiments have indicated that only
~1% of ZAP-70 becomes associated with the TCR after
CD3 stimulation, which may be below the sensitivity of the
immunofluorescence technique against the background of 99% which does not move (Weill et al., 1995 Analysis of transfected mutant cDNAs indicated that
ZAP-70 cortical targeting was not mediated by its tandem
SH2 domains. Rather, distribution of the K369A mutant
suggested that an active kinase domain was required for
ZAP-70 localization. One model to account for this requirement is that autophosphorylation of ZAP-70, within
either the kinase domain or hinge, may create binding sites
for SH2-containing proteins which tether it in the cell cortex. It is unlikely that ZAP-70 is transphosphorylated by
ZAP-70 to target it to the cell cortex, as kinase-inactive
ZAP-70 was delocalized when expressed in Jurkat T cells
which express endogenous ZAP-70 that is cortically localized and enzymatically active. Analysis of point mutants
formally demonstrated that none of the known inducible
sites of phosphorylation (Y292, Y492, Y493) were required for cortical targeting (Fig. 8) which was perhaps not surprising since ZAP-70 is cortically targeted in unstimulated Jurkat T cells. Thus, this model requires that ZAP-70
is autophosphorylated on other sites. Both wild-type ZAP-70 and the As discussed in the introduction, biochemical and genetic experiments have indicated that ZAP-70 is recruited
to tyrosine-phosphorylated TCR subunits via its two SH2
domains. ZAP-70 is then itself tyrosine phosphorylated by
LCK and activated. This has suggested a model in which
ZAP-70 translocates from the cytoplasm to the plasma membrane to become activated (Weiss and Littman,
1994 Kong et al. (1996) Recent studies have indicated that phosphorylation
events in a cell are not only controlled by the balance of
kinase and phosphatase activity but also the subcellular location of these enzymes (Mochly-Rosen, 1995 heterodimer recognizes major histocompatibility complex molecules on the surface of antigen-presenting cells or target cells (Clevers et al., 1988
). Noncovalently associated with the
heterodimer is the CD3 complex of
polypeptides (
) and a disulfide-linked
homodimer.
These associated polypeptides are responsible for the signal transduction function of the TCR (Weiss, 1993
) and
are also required for efficient surface expression of the intact receptor (Weiss, 1991
). Stimulation of the TCR activates a cascade of biochemical events that triggers proliferation and differentiation of T cells into effector cells. The
induced tyrosine phosphorylation of multiple intracellular
proteins is one of the earliest detectable biochemical
events after TCR stimulation (Klausner and Samelson,
1991
). Pharmacologic and genetic experiments have established that the induction of protein tyrosine kinase (PTK) activity is essential for signaling via the TCR. However,
the primary sequences of the identified components of the
TCR contain no recognizable kinase domains. Rather, the
TCR stimulates protein tyrosine phosphorylation by interacting sequentially with two different families of cytoplasmic nonreceptor PTKs (Weiss and Littman, 1994
).
;
Sancho et al., 1993
; Straus and Weiss, 1993
) and
homodimers in specific motifs (Baniyash et al., 1988
), termed
immunoreceptor tyrosine-based activation motifs (ITAMs).
The ITAM sequence is defined as two tyrosine residues
spaced 9 to 11 residues apart with isoleucine or leucine residues positioned three residues COOH terminal to each tyrosine (YXXL X6-8 YXXL; Weiss, 1993
). Each of the CD3
subunits contains one ITAM, whereas the
chain contains
three tandemly arranged ITAMs. Studies in transfected
COS cells (Iwashima et al., 1994
) and in mutant T cell lines
have suggested that ITAM phosphorylation is mediated
by LCK, a member of the SRC family of cytoplasmic
PTKs (Karnitz et al., 1992
; Straus and Weiss, 1992
). Consistent with this hypothesis, both constitutive and inducible tyrosine phosphorylation of
is abolished in LCK null
thymocytes (van Oers et al., 1996a). Targeted disruption
of the lck gene also results in a substantial reduction in the
development of CD4+CD8+ thymocytes, demonstrating
its importance in T cell development (Molina et al., 1992
).
Gene knockout experiments in mice have also indicated
that FYN is functionally important in TCR signaling in
mature thymocytes, although it is not absolutely required
for
T cell development (Appleby et al., 1992
; Stein et
al., 1992
). However, combined disruption of lck and fyn
genes completely arrests
T cell development at the
CD4
CD8
stage, suggesting that the function of LCK
and FYN in T cell development is only partially redundant
(Groves et al., 1996
; van Oers et al., 1996b). Thus FYN
may also contribute to TCR ITAM phosphorylation in
certain circumstances.
homodimers recruits a second family of cytoplasmic PTKs to the TCR, comprising ZAP-70 and SYK
(Taniguchi et al., 1991
; Chan et al., 1994b
). The association
of these PTKs with the TCR is mediated by a high affinity
interaction between their two NH2-terminal SH2 domains
with the two phosphorylated tyrosines in an ITAM (Chan et al., 1992
, 1994a; Straus and Weiss, 1993
; Wange et al.,
1993
). This interaction requires both of the SH2 domains
(Bu et al., 1995
; Isakov et al., 1995
). After binding to the
TCR, ZAP-70 and SYK themselves become tyrosine phosphorylated and are then activated. For ZAP-70, this phosphorylation is thought to be primarily mediated by LCK
(Chan et al., 1995
; Wange et al., 1995
), whereas the binding of SYK to ITAMs itself has been shown to stimulate
autophosphorylation and subsequent activation (Rowley
et al., 1995a
; Shiue et al., 1995
). In thymocytes, ZAP-70 is
constitutively associated with the
chain but is not tyrosine phosphorylated and activated (van Oers et al.,
1994). In these cells, the triggering event in TCR signaling
does not appear to be the binding of ZAP-70 to the
chain. Rather, it is the colocalization of CD4-LCK complexes with the TCR after stimulation which then facilitates the phosphorylation of
-associated ZAP-70 by LCK
(Wiest et al., 1993
, 1996).
; Chan et al., 1995
).
This generates binding sites that recruit SH2-containing
signaling proteins to the plasma membrane, which include
LCK (Duplay et al., 1994
), Vav (Katzav et al., 1994
; Huby
et al., 1995
), Ras-GAP, abl (Neumeister et al., 1995
), and a
FAK-related PTK (Kanner et al., 1994
), where they can
become tyrosine phosphorylated or brought into proximity with their substrates. Subsequently, downstream effector functions are triggered, including the activation of Ras
and the mobilization of intracellular Ca2+ (Weiss and Littman, 1994
). Genetic studies in both murine and human
systems have confirmed the importance of ZAP-70 in TCR
signaling and T cell development (Hivroz and Fischer,
1994
; Negishi et al., 1995
). In contrast, SYK does not appear to be essential for
T cell development (Turner et al.,
1995
), although the development of epithelial
T cells is
disrupted in SYK-negative mice (Mallick-Wood et al., 1996
).
Recent experiments have also indicated that SYK does
not require either LCK or CD45 expression to function
in TCR signaling, unlike ZAP-70 (Chu et al., 1996
). Thus
the activation requirements for SYK are different from
ZAP-70, suggesting a distinct role in TCR signaling.
). In contrast, FYN was found to be localized adjacent to the microtubule cytoskeleton and was not detected
at the plasma membrane unless overexpressed by transfection, raising the possibility that it might carry out functions
that are distinct from LCK. The major increase in tyrosine phosphorylation that occurs after TCR stimulation was
found to take place in the T cell cortex coincident with
LCK localization. This suggested that LCK might contribute to the increase in cortical tyrosine phosphorylation,
consistent with its proposed role in phosphorylating TCR
ITAMs after stimulation. In this study, the intracellular
localization of ZAP-70 was investigated by immunofluorescence and confocal microscopy. These experiments indicated that ZAP-70 was also targeted to the T cell cortex,
in a constitutive fashion, independently of its SH2 domains. The implication of these results for the function of
ZAP-70 is discussed.
Materials and Methods
). Briefly, cells were
washed three times in serum-free RPMI medium and were then resuspended at 1 × 108 cells/ml. 250 µl was transferred into a gene pulser cuvette (BioRad Laboratories, Hertfordshire, UK) and 10 µg of the appropriate plasmid was added. Cells were pulsed at 960 µF and 250 V and left for 10 min at room temperature (RT). Cells were then transferred into 10 ml of RPMI medium supplemented with 10% FCS and cultured for 20 h
before harvesting.
, 1995; Iwashima et al., 1994
; Wange et al., 1995
). In the
SH2-ZAP-70 mutant both of the SH2 domains were deleted. To do
this the SacII-StuI DNA fragment encoding the SH2 domains (residues 220-964 of the original cDNA) was replaced with the following
oligonucleotides: (+)5
-GGCGCACCTGCCCTTCTACCCATACGATGTTCCAGATTACGCTG-3
and (-)5
-CGCCGCGTGGACGGGAAGATGGGTATGCTACAAGGTCTAATGCGAC-3
. The resulting construct encoded the first five amino acids of ZAP-70 followed by nine amino acids
from the HA epitope and then the remaining COOH-terminal part of
ZAP-70. The
Sph-ZAP-70 construct, in which the majority of the kinase domain was deleted (residues 1288-1988 of the cDNA), was generated by excising the Sph1 fragment from the NH2-terminally HA-tagged
ZAP-70 construct and religating. All of the ZAP-70 constructs were subcloned into the pcDNA3neo vector for transient transfection experiments.
HA epitope-tagged SYK was also subcloned into the pcDNA3neo vector for
transient transfection experiments (Rowley et al., 1995b
).
), was
used for staining and immunoprecipitation of endogenous ZAP-70. The
12CA5 mAb (Hsi et al., 1989
) was used for immunoprecipitation, immunoblotting (1 µg/ml), and immunohistological staining (0.3 µg/ml) of HA
epitope-tagged ZAP-70. The LCK-1 anti-peptide antibody was used for
immunofluorescent staining of LCK at a dilution of 1:1000 in PBS (Koegl
et al., 1994
). Tyrosine phosphorylated proteins were detected using the
4G10 anti-PTyr mAb at 1 µg/ml for both immunofluorescence and Western blotting (from B. Druker, Oregon Health Sciences University, Portland, OR). The rat anti-
-tubulin mAb, YOL34, was used at 1:20 dilution
and was obtained from Serotec (Oxford, UK). Polymerized actin was detected using rhodamine-coupled phalloidin at 1 µg/ml (Sigma Chemical
Co., Dorset, UK).
-tubulin staining with the YOL34 mAb.
) and incubated with precleared cell lysate for 4 h or overnight. Endogenous ZAP-70 was immunoprecipitated using ZAP-4 antiserum as described previously (Huby et al.,
1995
). After six washes with ice-cold IPB, immunoprecipitated protein was resolved by SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes. Western blotting was carried out as described previously (Ley et al., 1994b
). PVDF membranes were stripped of bound
antibody using the ECL protocol (Amersham, Buckinghamshire, UK) in
experiments in which blots were probed for multiple antigens.
Results
Fig. 1.
Localization of endogenous ZAP-70 in Jurkat T cells.
Staining with ZAP 4 anti-ZAP-70 antiserum in Jurkat T cells reveals specific cortical staining (A) which was abrogated by the addition of blocking peptide, to which the antiserum was raised during the primary staining step (B). Low levels of nonspecific
staining were also detected in the cytoplasm. Bar, 10 µm.
[View Larger Versions of these Images (104 + 103K GIF file)]
Fig. 2.
Localization of HA-tagged ZAP-70 after transient transfection into Jurkat T cells. Transfected cells were double stained with anti-HA tag mAb 12CA5 (green) and the nuclear stain 7-amino-actinomycin D (red), revealing that ZAP-70-HA expression is limited
primarily to the cell cortex, with maximal staining proximal to the plasma membrane (A). A gradient of ZAP-70-HA staining intensity
measured according to pixel brightness was quantified along a cell transect as shown in (B). In comparison, very bright LCK staining is
seen after transient transfection with an LCK expression plasmid, which nevertheless remains tightly and symmetrically associated with the plasma membrane (C), illustrated quantitatively by the corresponding cell transect (D). Transfected ZAP-70-HA is shown to behave similarly to endogenous ZAP-70 after TCR activation, becoming tyrosine phosphorylated and associated with phosphorylated
TCR (E). Bar, 10 µm.
[View Larger Versions of these Images (17 + 53 + 20K GIF file)]
and were
tyrosine phosphorylated after stimulation of the cells with
CD3 antibody (Fig. 2 E). Taken together, these data indicate that transfected ZAP-70-HA behaved in a similar
fashion to the endogenous protein in terms of its localization and also its inducible interaction with the TCR.
Fig. 3.
ZAP-70 is largely
soluble before fixation. Jurkat
T cells were disrupted by
Dounce homogenization (A)
and separated into three fractions; (P1) pellet recovered by 280 g centrifugation,
comprised primarily of nuclear debris and the actin
cytoskeleton; (P2) pellet recovered at 100,000 g comprised membranous fragments, protein aggregates, and the remaining soluble fraction S1. ZAP-70 compartmentalization was unaffected by stimulation of cells for 5 or
20 min with OKT3 F(ab)2 before homogenization. After fixation
for 5 min in 2% paraformaldehyde, essentially all ZAP-70 became insoluble (B) and was recovered in the pellet P rather than
the soluble fraction S after centrifugation for 2 min at 8,000 g.
[View Larger Version of this Image (20K GIF file)]
and CD3 ITAMs which creates docking sites for ZAP-70. To investigate whether the cortical
localization of ZAP-70 was altered after TCR stimulation,
Jurkat T cells were stimulated with CD3 mAb or left unstimulated and then fixed with paraformaldehyde. The
cells were then double labeled for endogenous ZAP-70
and PTyr. Stimulation of the TCR with CD3 antibody did
not alter the localization of ZAP-70 over a period of 5 min,
although there was a clear transient induction of PTyr proteins at the cell cortex (Fig. 4, A and B), and as reported
previously (Ley et al., 1994a
). The localization of transfected HA-ZAP-70 after CD3 mAb stimulation was also
unchanged after stimulation with CD3 antibody (data not
shown).
Fig. 4.
Localization of ZAP-70 is unaffected by TCR stimulation. Jurkat T cells were either unstimulated (A), or stimulated
for 5 min with F(ab)2 fragments of the CD3 mAb, OKT3 (B),
fixed, and double stained for endogenous ZAP-70 (left) and total
phosphotyrosine (right). Staining for endogenous ZAP-70 with
ZAP-4 antiserum also produced cytoplasmic fluorescence which
was nonspecific (Fig. 1). Bar, 10 µm.
[View Larger Version of this Image (75K GIF file)]
Fig. 5.
ZAP-70 does not
colocalize with actin or -tubulin. ZAP-70-HA transfected
Jurkat T cells were double
stained to reveal ZAP-70 and actin microfilaments in
the same cell (A). ZAP-70-
HA gave a broader staining
pattern, quantified by the
pixel density transect shown
in B. Double staining was
similarly used to reveal ZAP-70-HA and
-tubulin distribution within the same cells
(C). Bar, 10 µm.
[View Larger Versions of these Images (86 + 22K GIF file)]
tubulin in
cell lysates (Huby et al., 1995
). However, double labeling
for ZAP-70-HA and
-tubulin demonstrated that ZAP-70
localization was clearly distinct from that of microtubules,
which were detected throughout the cytoplasm and concentrated adjacent to the nucleus at the centrosome (Fig.
5 C). These data suggested that ZAP-70 was not targeted to the cell cortex via an association with the microtubule
cytoskeleton.
Fig. 6.
ZAP-70 localization is unaffected by disruption of the actin cytoskeleton. NIH 3T3 fibroblasts
were transfected with a plasmid encoding ZAP-70-HA,
fixed, and double stained to
reveal the distribution of
ZAP-70-HA (A) and actin
microfilaments (B) in the
same cells. Treatment of cells
with cytochalasin D before
fixation did not delocalize
ZAP-70-HA from the cell
cortex (C), although the actin cytoskeleton was disrupted
(D). Bar, 10 µm.
[View Larger Version of this Image (64K GIF file)]
), the localization of ZAP-70 to the cell cortex in
unstimulated Jurkat T cells suggested that this targeting
was not due to SH2 binding to tyrosine-phosphorylated
TCR. The cortical localization of ZAP-70 in 3T3 fibroblasts,
which do not express TCRs, supported this hypothesis
(Fig. 6). However, it was still possible that ZAP-70 was anchored to the cortical cytoskeleton via the binding of its
SH2 domains to other tyrosine-phosphorylated proteins. To investigate this possibility, the localization was tested of a COOH terminally HA-tagged mutant of ZAP-70,
SH2-ZAP-70-HA, in which both SH2 domains had been deleted. This mutant localized in a diffuse band at the cell cortex in both Jurkat T and 3T3 cells (Fig. 7, B and F, respectively) similarly to wild type ZAP-70-HA (Fig. 7, A and E).
Fig. 7.
ZAP-70 requires an active kinase domain for its localization. Jurkat T cells (A-D) or NIH 3T3 fibroblasts (E-H) were transfected with plasmids encoding either: wild-type ZAP-70-HA (A and E), SH2-ZAP-70-HA (B and F),
Sph-ZAP-70-HA (C and G),
or kinase-dead ZAP-70.K369A-HA. Transfected cells were stained with 12CA5 to reveal the HA-tagged ZAP-70 protein. Bars, 10 µm.
[View Larger Version of this Image (37K GIF file)]
Sph-ZAP-70-HA mutant was determined, in which the
majority of the kinase domain (residues 358 to 593) was
deleted.
Sph-ZAP-70-HA was detected throughout the
cytoplasm in both Jurkat T cells and 3T3 cells, suggesting
that the kinase domain played a role in ZAP-70 localization (Fig. 7, C and G). To investigate whether the kinase
activity of ZAP-70 was important in its intracellular targeting, the localization of the kinase-inactive K369A-ZAP-70-
HA mutant was determined. K369A-ZAP-70-HA had a
similar localization to the
Sph-ZAP-70-HA mutant (Fig.
7, D and H). Taken together, the data in this section suggested that cortical localization of ZAP-70 required its active kinase domain but was independent of its SH2 domains.
; Chan et al., 1995
; Wange et al., 1995
). Analysis
of point mutants has indicated that phosphorylation of
Y493 is required for the induction of ZAP-70 tyrosine kinase activity by the TCR (Chan et al., 1995
; Wange et al.,
1995
; Kong et al., 1996
). In contrast, tyrosine phosphorylation
of Y292 and Y492 appears to negatively regulate the activity of ZAP-70 (Wange et al., 1995
; Kong et al., 1996
; Zhao
and Weiss, 1996
). Current data suggest that phosphorylation of Y493 is mediated by LCK, whereas phosphorylation of Y292 and Y492 is the consequence of autophosphorylation (Wange et al., 1995
; Kong et al., 1996
).
Fig. 8.
Inducibly phosphorylated tyrosine residues are not involved in ZAP-70 localization to the cortex. Jurkat T cells (A-E) or NIH
3T3 fibroblasts (F-J) were transfected with plasmids encoding either wild-type ZAP-70 (A and F), Y292F (B and G), Y492F (C and H),
Y493F (D and I), or Y492F/Y493F (E and H). Unlike previous experiments, all constructs were HA-tagged at the NH2 terminus. Transfected cells were stained with 12CA5 mAb to reveal HA-tagged ZAP-70 protein (green) and in the case of Jurkat were counterstained with 7-amino-actinomycin D to reveal the position of the nucleus (red). Bars, 10 µm.
[View Larger Version of this Image (16K GIF file)]
). Similar to ZAP-70, SYK
can associate with phospho-
in murine thymocytes and in
Jurkat T cells (Chan et al., 1994a
; Thome et al., 1995
; van
Oers et al., 1995). Furthermore, ZAP-70 can complement
SYK function in a SYK-negative B cell line (Kong et al.,
1995
). The similarities of SYK to ZAP-70 raised the possibility that it might also be localized constitutively to the
cell cortex. E6.1 Jurkat T cells do not express SYK
(Fargnoli et al., 1995
). Therefore, HA epitope-tagged SYK
was transiently expressed by transfection in this cell line.
HA-SYK was detected in a band at the cell cortex in unstimulated cells (Fig. 9 A). A low level of staining was also
detected in the cytoplasm. HA-SYK also localized to the
cell cortex when transiently expressed in 3T3 fibroblasts
(Fig. 9 B). These data, therefore, indicated that the localization of SYK was similar to ZAP-70.
Fig. 9.
SYK localizes to the cell cortex. Jurkat T cells (A) or
3T3 fibroblasts (B) were transfected with a plasmid encoding NH2
terminally HA-tagged SYK, fixed, and the localization of the expressed protein determined, as described in Fig. 7. Bars, 10 µm.
[View Larger Version of this Image (31K GIF file)]
Discussion
-actinin, talin, and ERM family proteins such as ezrin (Hitt and
Luna, 1994
; Tsukita et al., 1997
).
; Zlatkine et al., 1997
). ZAP-70 was concentrated at
the cell cortex in a gradient that reached a maximum coincident with the cortical cytoskeleton and plasma membrane,
which could not be distinguished at the resolution of the
confocal microscope. It is possible that the anchoring site
may have a similar distribution to ZAP-70. However, a
more attractive model is that the putative anchoring molecule is located in the region where ZAP-70 staining was
highest and the interaction with ZAP-70 is very weak and
dynamic such that ZAP-70 is continually dissociating from
the anchor site and diffusing back into the cytoplasm. Consistent with this hypothesis, cell fractionation experiments
suggested that the interaction between ZAP-70 and its putative anchoring molecule, which would be predicted to be insoluble, was of low affinity such that this association was not stable to cell lysis. Cortactin, which is localized to the cortical actin cytoskeleton by binding directly to F-actin
via an internal 37-amino acid repeat (Wu and Parsons,
1993
), is also completely soluble after cell lysis (Wu et al.,
1991
). If Jurkat T cells were cultured for 48 h after transfection, rather than 24 h, HA-ZAP-70 was expressed at
much higher levels and was then found throughout the cytoplasm (data not shown). This suggested that the putative
anchor site in the cell cortex was saturable.
). The data in
this paper contrast with those in a recent study by Thome
et al. (1995)
in which an activation-inducible movement of
ZAP-70 from the cytoplasm to CD3 caps was reported.
However, those data imply that ZAP-70 quantitatively associates with the TCR after CD3 stimulation, which is unlikely based on the stoichiometry of association estimated from biochemical analyses (Weill et al., 1995
). The reason
for the discrepancies with this study is not clear.
SH2-ZAP-70 mutant, which were both targeted to the cell cortex, were tyrosine phosphorylated at
only very low levels in unstimulated Jurkat T cells (Fig. 2 E;
data not shown). The putative sites of basal autophosphorylation, therefore, would also have to turn over very rapidly. A more attractive model proposes that the K369A
mutation, which renders ZAP-70 kinase inactive by preventing ATP binding (Johnson et al., 1996
), is also unable
to bind to one of its substrates due to the alteration of the
catalytic binding site. In this model, therefore, ZAP-70 is
localized by interaction with one of its substrates which is present in the cell cortex. Interestingly, PKC
is localized to focal contacts by interaction with proteins that it phosphorylates, which include vinculin, talin, and the MARCKS
protein (Chapline et al., 1993
; Hyatt et al., 1994
; Liao et al.,
1994
). In this case, phosphorylation of these substrates
abolishes interaction with PKC
, thereby allowing the enzyme to phosphorylate other proteins. The identification
of the anchoring molecule or molecules for ZAP-70 will be
important to understand why the K369A mutant is delocalized.
). The data in this paper indicate that ZAP-70 is already localized very close to the plasma membrane in unstimulated Jurkat T cells. However, ZAP-70 only becomes
tyrosine phosphorylated and activated after CD3 mAb
stimulation. Thus it is likely that, although ZAP-70 is close
to or at the plasma membrane in unstimulated cells, it requires CD3 mAb stimulation to relocalize it to phospho-ITAMs, where it can then be phosphorylated by LCK.
Consistent with this hypothesis, a transmembrane chimera
of ZAP-70, which is anchored at the plasma membrane, is
constitutively active when expressed in Jurkat T cells but
requires functional LCK coexpression (Yamasaki et al., 1996
). This raises the question of why ZAP-70 is localized
to the cell cortex. One possibility is that this may increase
the rapidity with which signaling can be initiated via the
TCR by maintaining a high concentration of ZAP-70 that
has to move only a short distance to interact with phospho-ITAMs and become activated. The major increase in
tyrosine phosphorylation after TCR stimulation occurs at
the cell cortex (Ley et al., 1994a
). The targeting of ZAP-70, therefore, may also localize it close to its downstream targets. The similar localization of SYK to the cell cortex
(Fig. 9) suggests that compartmentalization may play an
analogous role in the function of this related protein tyrosine kinase.
have recently proposed a model for
ZAP-70 activation in which it is first phosphorylated by
LCK on Y493 after binding to phospho-ITAMs and then
autophosphorylates on Y292 and Y492. This model is
based on two observations. First, a kinase-inactive GST
fusion protein of ZAP-70 is phosphorylated only on Y493
when mixed with the isolated kinase domain of LCK, and
second, kinase-inactive ZAP-70 is phosphorylated at much
lower levels compared with the wild-type protein after BCR
stimulation, when expressed in SYK-negative DT40 B cells.
However, the results in this paper suggest an alternative
explanation for the latter result. Kinase-inactive ZAP-70 is
delocalized from the cell cortex and presumably, therefore, can less efficiently interact with the BCR ITAMs and
with PTKs at the plasma membrane. Thus the reduced phosphorylation of the kinase-inactive ZAP-70 in DT40 B
cells, after BCR stimulation, may result from its inaccessibility to PTKs in the cell cortex which can transphosphorylate it. Mege et al. (1996)
have recently reported that kinase-dead ZAP-70 is constitutively associated with the
phospho-
if sufficiently overexpressed in Jurkat T cells.
These authors have suggested a model in which, under
basal conditions, TCR ITAMs may be continually phosphorylated. However, the accumulation of phospho-ITAMs
is prevented by the action of a phosphatase that rapidly
dephosphorylates them. Overexpression of kinase-inactive
ZAP-70 has been postulated to prevent phospho-ITAM dephosphorylation by its binding to them, resulting in
their constitutive phosphorylation in unstimulated cells
(Mege et al., 1996
). Since kinase-inactive ZAP-70 is delocalized from the cell cortex, these results suggest that
ZAP-70 localization may not be important for interaction
with ITAMs if it is expressed at supraphysiological levels.
However, it is likely that, at physiological levels, the targeting of ZAP-70 to the cell cortex will be necessary for its efficient association with the TCR and subsequent interaction with LCK and downstream targets.
; Faux and
Scott, 1996b
). One emerging mechanism for such compartmentalization is the interaction of kinases and phosphatases with targeting proteins that localize enzymes close to
their site of action (Faux and Scott, 1996a
). This study
demonstrates that both ZAP-70 and SYK are constitutively localized to the T cell cortex where they are in close
proximity to the TCR with which they interact to become
activated (Weiss and Littman, 1994
; Chan and Shaw, 1996
).
These experiments raise the possibility that ZAP-70 and
SYK may also be compartmentalized via interaction with a targeting protein or proteins. Recent studies from one of
our laboratories have demonstrated that binding of LCK
to the plasma membrane via dual acylation of its NH2 terminus is essential for its function in TCR signaling (Kabouridis, P., A.I. Magee, and S.C. Ley, unpublished observations). It is anticipated that the constitutive cortical
localization of ZAP-70 and SYK will likewise be important for both their activation and access to downstream targets, which previous studies have indicated are also concentrated in the cell cortex (Takagi et al., 1991
; Ley et al.,
1994a
).
Received for publication 27 February 1997.
1. Abbreviations used in this paper: ITAM, immunoreceptor tyrosine-based activation motifs; PTK, protein tyrosine kinase; TCR, T cell antigen receptor.The authors would like to thank Larry Samelson, Jo Bolen, and Andy Chan for reagents used in this study. The authors are also grateful to Panos Kabouridis and Tony Magee for their critical review of the manuscript and to the Photo-Graphics department and Liz Hirst for their help with the figures.
This work was supported by the Medical Research Council.
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