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Mol Cell Biol, May 1998, p. 2855-2866, Vol. 18, No. 5
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Genetic Evidence of a Role for Lck in T-Cell Receptor Function
Independent or Downstream of ZAP-70/Syk Protein Tyrosine
Kinases
Jane
Wong,1,2,3
David
Straus,4 and
Andrew C.
Chan1,3,5,6,7,*
Center for
Immunology,1
Divisions of
Nephrology2 and
Rheumatology,5
Department of
Medicine,3
Department of
Pathology,6 and
Howard Hughes Medical
Institute,7 Washington University School of
Medicine, St. Louis, Missouri 63110, and
Division of
Gastroenterology, Department of Internal Medicine, University of
Chicago, Chicago, Illinois 606374
Received 23 October 1997/Returned for modification 5 December
1997/Accepted 13 February 1998
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ABSTRACT |
T-cell antigen receptor (TCR) engagement results in sequential
activation of the Src protein tyrosine kinases (PTKs) Lck and Fyn and
the Syk PTKs, ZAP-70 and Syk. While the Src PTKs mediate the
phosphorylation of TCR-associated signaling subunits and the phosphorylation and activation of the Syk PTKs, the lack of a constitutively active Syk PTK has prohibited the analysis of Lck function downstream of these initiating signaling events. We describe here the generation of an activated Syk family PTK by substituting the
kinase domain of Syk for the homologous region in ZAP-70 (designated as
KS for kinase swap). Expression of the KS chimera resulted in its
autophosphorylation, the phosphorylation of cellular proteins, the
upregulation of T-cell activation markers, and the induction of
interleukin-2 gene synthesis in a TCR-independent fashion. The KS
chimera and downstream ZAP-70 or Syk substrates, such as SLP-76, were
still phosphorylated when expressed in Lck-deficient JCaM1.6 T cells.
However, expression of the KS chimera in JCaM1.6 cells failed to rescue
downstream signaling events, demonstrating a functional role for Lck
beyond the activation of the ZAP-70 and Syk PTKs. These results
indicate that downstream TCR signaling pathways may be differentially
regulated by ZAP-70 and Lck PTKs and provide a mechanism by which
effector functions may be selectively activated in response to TCR
stimulation.
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INTRODUCTION |
Signaling through the T-cell
antigen receptor (TCR) requires the sequential activation of the
Src and Syk families of protein tyrosine kinases (PTKs) (for reviews,
see references 8, 11, and 70).
The Src PTKs Lck and Fyn phosphorylate the two conserved tyrosine
residues in the immunoreceptor tyrosine-based activation motif
(designated as ITAM) (7), which is present in each
of the TCR signaling subunits. In turn, phosphorylation of the ITAMs mediates the interaction of the Syk family PTKs ZAP-70 and Syk with the
receptor. Both ZAP-70 and Syk have tandemly arranged amino (N)-terminal
Src homology 2 (SH2) domains that are separated from their carboxy
(C)-terminal catalytic domain by a hinge domain. These two PTKs have
~55% amino acid identity and share overlapping functions in a
variety of cell lines. Moreover, the coexpression of ZAP-70 and Syk in
the thymus enables these two PTKs to play overlapping roles in T-cell
development and TCR activation (2, 9, 12, 19, 24, 28).
Lck, a member of the Src family of PTKs, is also required for both
T-cell development and TCR function (34, 47, 61). The
catalytic activity of Lck is required for phosphorylation of the TCR
ITAM sequences and ZAP-70 (32). In addition, the SH2 domain
of Lck plays an important role in stabilization of the
CD4/CD8-TCR-major histocompatibility complex and is required for
efficient 
-chain phosphorylation, TCR-mediated intracellular Ca2+ concentration ([Ca2+]i)
mobilization, and interleukin-2 (IL-2) synthesis (18, 44, 60, 65, 76). The ability of isolated Lck SH2 and SH3 domains to
interact with tyrosine-phosphorylated or proline-rich effector molecules, respectively, suggests a potential role for Lck either downstream or independent of ZAP-70 (14, 22, 53, 63,
77). However, one of the effector molecules bound to the Lck SH2
domain is ZAP-70, and expression of an Lck molecule with a
nonfunctional SH2 domain fails to induce ZAP-70 phosphorylation
(44, 60). Hence, analysis of the functional roles of
Lck downstream of ZAP-70 activation has been hampered by the lack of a
constitutively activated form of a Syk family kinase.
We report here the generation of such a constitutively active form of a
Syk family kinase PTK, accomplished by substituting the kinase domain
of Syk for the kinase domain of ZAP-70 (designated as the KS [for
kinase swap] chimera). Expression of the KS chimera results in
constitutive T-cell activation through signaling pathways normally
mediated by activation of ZAP-70 and Syk following TCR cross-linking.
By utilizing this constitutively active chimera, we obtained genetic
evidence of a role for Lck, subsequent to or independent of the
activation of ZAP-70, in TCR function.
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MATERIALS AND METHODS |
Cells, antibodies, and FACS analysis.
HeLa cells, Jurkat
cells, and Jurkat cell derivatives JCaM1.6, J.RT3-T3.5, and J449.3 were
maintained as previously described (27, 38, 73). Antibodies
used included 12CA5, an antihemagglutinin (anti-HA) epitope monoclonal
antibody (MAb; Boehringer Mannheim); 2F3.2, an anti-ZAP-70 MAb (Upstate
Biotechnology, Inc. [UBI]); 4G10, an antiphosphotyrosine MAb
(pY; UBI); PY20, an anti-pY MAb (Santa Cruz Biotechnology), an
anti-PLC
1 antiserum (UBI); an anti-Vav antiserum (Santa Cruz
Biotechnology); anti-human CD69 MAb (Becton Dickinson); an anti-SLP-76
antiserum (6); an anti-ZAP-70 antiserum, raised against a
peptide encoding amino acids 327 to 343; and an anti-Syk antiserum,
raised against a peptide encoding amino acids 308 to 335. Analysis of
cell surface markers was performed by fluorescence-activated cell
sorter (FACS) analysis with a FACSCALIBUR (Becton Dickinson).
Construction of cDNAs.
Chimeric and truncated PTKs were
produced by PCR-directed mutagenesis and confirmed by dideoxynucleotide
sequencing. Epitope-tagged versions of the PTKs were produced by
appending the HA epitope (YPYDVPDYA) to their N termini
(21). IL-2- and nuclear factor of activated T cells
(NFAT)-luciferase constructs and cytomegalovirus-chloramphenicol acetyltransferase (CMV-CAT) cDNAs were gifts from K. Murphy and T. Chatila (Washington University, St. Louis, Mo.).
Expression and analysis of proteins.
Infection of HeLa cells
with recombinant vaccinia virus was performed as previously described
(49). Following an 8-h infection, cells (5 × 105) were lysed in 0.5 ml of 1% Nonidet P-40-150 mM
NaCl-10 mM Tris-HCl (pH 8.0) containing protease and phosphatase
inhibitors. Cellular debris was removed by centrifugation at
10,000 × g for 10 min at 4°C. Clarified supernatants
were then used for protein analysis.
Protocols for transfection of Jurkat cells and derivatives thereof have
been previously reported (6, 38). For stable transfections,
107 cells were electroporated with 25 µg of DNA at 270 V
and 1,060 µF at a resistance setting of 6 (R6) (BTX ElectroCell
Manipulator 600). Forty-eight hours after electroporation, J449.3 cells
were selected in medium containing 1 µg of hygromycin, 10 µg of
tetracycline, and 2 mg of neomycin per ml. Selection of Jurkat cells
with pApuro vectors was accomplished with medium containing 0.5 µg of
puromycin per ml. Transient transfections were performed under the
electroporation conditions of 250 V, 960 µF, and R6 (BTX). In brief,
2 × 107 cells were electroporated with 60 µg of
IL-2-luciferase or NF-AT-luciferase and 80 µg of empty vector or
the PTK cDNA. Cells were harvested at 48 h and replated at a
concentration of 106/ml in medium alone, in medium
containing phorbol myristate acetate (PMA) and either an anti-TCR MAb
(C305 or 235) or phytohemagglutinin (PHA), or in medium containing PMA
and ionomycin. Following 6 h of stimulation, luciferase assays
were performed as previously described (62). Transfection
efficiency was determined by cotransfection of 5 µg of a CMV-CAT
reporter as previously described (38).
T-cell activation, immunoprecipitation, kinase assays, and
Western blot analysis.
T-cell clones were analyzed under resting
and TCR-stimulated conditions as previously described (6).
Concentrations of anti-TCR MAbs used for stimulation were 1:250 for
both 235 (anti-CD3 MAb, courtesy of Shu Man Fu) and C305 (anti-Ti
MAb, courtesy of Arthur Weiss). For biochemical analysis, cells were
stimulated for 2 min at 37°C prior to lysis. Lysates were clarified
by centrifugation and subsequently incubated with the appropriate
antiserum for 90 min at 4°C and then with protein A-Sepharose
(Pharmacia) for 60 min at 4°C. Samples were washed three times with
lysis buffer and boiled in 3× Laemmli sample buffer.
For analysis of enzymatic activity, washed immunoprecipitates were
incubated with 10 µCi of [
-
32P]ATP and 1 µg of a
glutathione
S-transferase (GST)-band III exogenous
substrate in kinase buffer for 10 min at room temperature as previously
described (
10,
56). Proteins were analyzed by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis transferred to Immobilon
(Millipore, Bedford, Mass.) or nitrocellulose membranes, blotted
with
the appropriate antibodies, and developed by enhanced chemiluminescence
(Amersham) in accordance with the manufacturer's recommendations.
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RESULTS |
Generation and expression of chimeric PTKs.
To investigate the
functional and structural homologies between ZAP-70 and Syk, we
generated three chimeric PTKs in which various domains of human ZAP-70
were replaced with the homologous regions of human Syk (Fig.
1A). These included the following: (i) a
chimera in which the ZAP-70 kinase domain, amino acids (aa) 331 to 619, was replaced with the Syk kinase domain, aa 358 to 630 (designated as
the KS chimera); (ii) a chimera in which the hinge region located
between the C-terminal SH2 and catalytic domains of ZAP-70, aa 254 to
326, was replaced with the homologous region within Syk, aa 255 to 354 (designated as H); and (iii) a chimera in which a region encompassing
the ZAP-70 transactivation domain, aa 476 to 523, was replaced with a
homologous region encompassing the Syk transactivation domain, aa 505 to 552 (designated as TAD).
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FIG. 1.
Expression of PTKs in HeLa cells. (A) Schematic
representation of chimeric PTKs. Chimeras of ZAP-70 and Syk PTKs were
generated by PCR-directed mutagenesis. Three chimeric PTKs, KS, H, and
TAD, were generated by exchange of the kinase domains, the hinge
regions, and the transactivation domains, respectively. (B)
Autophosphorylation of the KS chimera. HeLa cells were infected with
recombinant vaccinia virus encoding the chimeric and wild-type PTKs
individually as described in Materials and Methods. Cells (2 × 105) were lysed, and lysates were used for
immunoprecipitation or immunoblotting studies with an anti-pY MAb (top
panel) and anti-ZAP-70 or anti-Syk antiserum (bottom panel). Lanes: 1, wild-type (WT) ZAP-70; 2, TAD chimera; 3, KS chimera; 4, wild-type Syk;
5, H chimera. To ensure that equimolar amounts of each of the PTKs were
analyzed, expression of each of the wild-type and chimeric PTKs was
determined by blotting with anti-ZAP-70 or anti-Syk antibodies and
normalized to standardized amounts of baculovirus-encoded GST-ZAP-70
or GST-Syk protein (data not shown) (6, 10). No differences
between tagged and untagged versions of Syk were observed (data not
shown). The experiment shown here is representative of five independent
experiments. Molecular weight standards (in thousands) are depicted at
the left margin. (C) Autoactivation and phosphorylation of HeLa cell
proteins by the KS chimera. HeLa cell lysates infected with chimeric or
wild-type PTK as described in for panel B were analyzed for tyrosine
phosphorylation of cellular proteins. MW, molecular weight standards.
(D) The tyrosine residues within the transactivation loop contribute
to, but are not absolutely required for, autoactivation and
autophosphorylation. HeLa cells (2 × 105) were
infected with wild-type Syk (lanes 1), the KS chimera (lanes 2), or the
KS chimera in which Tyr 518 and Tyr 519 in Syk were mutated to
phenylalanine [KS(YYFF)] (lanes 3). Anti-HA immunoprecipitates of
each PTK were analyzed in an in vitro kinase assay using an exogenous
substrate (top panel) and immunoblotting with an anti-pY MAb (third
panel from top). Coomassie blue staining demonstrates comparable levels
of the GST-band III exogenous substrate (second panel from top), and
immunoblotting analysis with an anti-HA MAb demonstrates comparable
levels of the expressed PTK (bottom panel).
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ZAP-70, Syk, and the three chimeric PTKs were expressed in HeLa cells.
Each PTK was immunoprecipitated with either anti-ZAP-70
or anti-Syk
antiserum and analyzed by immunoblotting with an anti-pY
MAb. Only the
KS chimera was phosphorylated on tyrosine residues
(Fig.
1B, top, lane
3). In contrast, no tyrosine phosphorylation
was observed with
wild-type ZAP-70, wild-type Syk, the TAD chimera,
or the H chimera
(Fig.
1B, top, lanes 1, 2, 4, and 5). All chimeric
PTKs retained
enzymatic activity, which indicated that the exchanging
of homologous
regions within ZAP-70 and Syk had not disrupted
their overall
structures (data not shown).
Since the KS chimera could undergo autophosphorylation, we also
analyzed the ability of the KS chimera to undergo autoactivation
to
phosphorylate cellular proteins. Again, only the expression
of the KS
chimera resulted in significant tyrosine phosphorylation
of HeLa
cellular proteins (Fig.
1C, lane 3); tyrosine phosphorylation
of
cellular proteins was substantially diminished (>50-fold difference)
in HeLa cells expressing wild-type ZAP-70, Syk, the TAD chimera,
or the
H chimera (Fig.
1C, lanes 1, 2, 4, and 5). In addition,
analysis of
HeLa cells expressing the KS chimera at 1/10 the level
of the wild-type
or other chimeric PTKs still demonstrated significantly
augmented
phosphorylation of cellular proteins (data not shown).
Together, these
data demonstrate that expression of the KS chimera
in HeLa cells
induces greater than a 10-fold increase in tyrosine
phosphorylation of
cellular proteins compared to wild-type ZAP-70
or Syk.
Since the tyrosine residues within the transactivation loop of Syk are
required for Syk activation (
40), we analyzed the
enzymatic
activity and phosphorylation status of wild-type Syk,
the KS chimera,
and the KS chimera in which both tyrosine residues
(i.e., Tyr 518 and
519) were mutated to phenylalanine [designated
as KS(YYFF) in Fig.
1D]. Both the KS and KS(YYFF) chimeras were
tyrosine
phosphorylated and demonstrated increased enzymatic activity
compared
to wild-type Syk (Fig.
1D). Enzymatic activation of the
KS(YYFF) mutant
was slightly decreased compared to that of the
KS chimera, consistent
with the ability of Syk to contribute to
autoactivation
(
20). However, the enzymatic activity of the
KS(YYFF)
chimera was still substantially greater than that of
the Syk
holoenzyme, suggesting that the increased phosphorylation
of cellular
proteins observed in HeLa cells expressing the KS
chimera (Fig.
1B to
D) is, in large part, independent of the tyrosine
residues within the
transactivation loop that are phosphorylated
by Src PTKs or by
autophosphorylation (
20,
40).
Characterization of the KS chimeric PTK in T cells.
To analyze
the functional effects of the KS chimera, stable clones were
established following transfection of the Jurkat leukemic T-cell line
(J6) with plasmids which express wild-type ZAP-70, wild-type Syk, or
the KS chimera. Each PTK was appended with a common HA epitope tag
to permit direct comparison of their levels of expression. Since
constitutive activation of T cells may result in activation-induced
cell death, the KS chimeric and Syk PTKs were expressed via a
tetracycline-regulated promoter. Multiple clones of the KS chimera,
wild-type ZAP-70, or wild-type Syk PTKs were isolated, and their
levels of expression were analyzed (Fig. 2A and data not shown). Data for
two representative clones (2E4 and 3G6) which differed in their basal
levels of expression of the KS chimera are shown in Fig. 2A (lanes 2 to
5). While the two clones demonstrated different levels of expression of
the KS chimera under nonpermissive conditions (i.e., in the presence of
tetracycline [lanes 2 and 4]), withdrawal of tetracycline resulted in
increased expression of the KS chimera (lanes 3 and 5). The KS chimera
was consistently expressed at less than one-third the level of
overexpressed wild-type ZAP-70 or Syk PTK (lanes 6 to 8).
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FIG. 2.
Biochemical analysis of cells expressing the KS chimera.
(A) Expression of KS, Syk, and ZAP-70. Stable transfectants of the KS
chimera (2E4 and 3G6) and Syk (5G4) were expressed under the control of
a tetracycline-regulated promoter in Jurkat cells (lanes 2 to 5, 7, and 8) (37). In addition, ZAP-70 was
overexpressed under the control of an actin promoter (lane 6)
(38). Parental Jurkat 449 cells are included as a control
(C) (lane 1). Expression of the HA-epitope-tagged PTKs was
analyzed by Western blotting. Since the KS chimera was expressed
at approximately one-third the level of wild-type ZAP-70
or Syk, immunoprecipitates from 4 × 107 cells of the
KS-expressing clones (lanes 2 to 5) were analyzed,
compared to 2 × 107 cells expressing HA-Syk
or HA-ZAP-70 (lanes 6 to 8). Expression of the KS chimera was maximal
at 24 h following tetracycline withdrawal and did not change for
the 72-h period of analysis (data not shown). Lanes: 1, parental cells
(449); 2; KS clone 2E4 with tetracycline (nonpermissive conditions); 3, KS clone 2E4 without tetracycline (permissive conditions); 4, KS clone
3G6, nonpermissive conditions; 5, KS clone 3G6, permissive conditions;
6, ZAP-70 (wt24); 7, Syk clone 5G4, nonpermissive conditions; 8, Syk clone 5G4, permissive conditions. (B) Phosphorylation of
KS in T cells. The epitope-tagged KS (clone 2E4; 4 × 107 cells), wild-type ZAP-70 (clone wt24; 107
cells), and wild-type Syk (clone 5G4; 107 cells) were
immunoprecipitated in resting or TCR-activated cells and
analyzed by Western blotting with an anti-pY MAb (top) or an anti-HA
MAb (bottom). Both KS- and Syk-expressing clones were analyzed
under permissive conditions. These data are representative of three
independent experiments. (C) Tyrosine phosphorylation of cellular
proteins by the KS chimera. Lysates (5 × 106
cells/lane) from stable transfectants were analyzed by immunoblotting
with an anti-pY MAb. Lanes: 1, control (C) Jurkat cells (449); 2, KS
cells (clone 2E4) under nonpermissive conditions; 3, KS cells (clone
2E4) under nonpermissive conditions; 4, control parental Jurkat cells
(449); 5, KS cells (clone 2E4) under permissive conditions; 6, KS
cells (clone 2E4) under permissive conditions. Lanes 1 to 3 represent resting cells, while lanes 4 to 6 represent cells stimulated
with an anti-CD3 MAb (235) for 2 min at 37°C. All lanes were derived
from the same gel and exposure, although the molecular weight markers
originally placed between lanes 3 and 4 were cropped from the final
photograph. These data are representative of three independent
experiments and of three independent clones expressing the KS
chimera. (D) Tyrosine phosphorylation of SLP-76, an in vivo
downstream substrate of ZAP-70. SLP-76 was immunoprecipitated
from control (C) parental cells (clone 449; 2 × 107
cells/lane) (lanes 1 and 2) or KS cells (clone 2E4; 2 × 107 cells/lane) (lanes 3 and 4) and analyzed by Western
blotting with an anti-pY MAb (top panel) or an anti-SLP-76 MAb (H3 MAb)
(bottom panel). Unstimulated cells are represented in lanes 1 and 3, while TCR-activated cells are represented in lanes 2 and 4. These data
are representative of a minimum of three independent experiments and of
two independent clones expressing the KS chimera. (E) Tyrosine
phosphorylation of Vav. Vav was immunoprecipitated from control
(C) parental 449 cells (2 × 107 cells/lane) (lanes 1 and 2) or KS cells (clone 2E4; 2 × 107 cells/lane)
(lanes 3 and 4) and analyzed by Western blotting with an anti-pY MAb
(top panel) or an anti-Vav MAb (bottom panel). Unstimulated cells are
represented in lanes 1 and 3, while TCR-activated cells are
represented in lanes 2 and 4. These data are representative of a
minimum of three independent experiments and of two
independent clones expressing the KS chimera.
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To analyze the biochemical characteristics of these transfected PTKs,
we immunoprecipitated the KS chimera, wild-type ZAP-70,
or wild-type
Syk PTK from resting or TCR-activated cells. Both
wild-type ZAP-70 and
wild-type Syk were phosphorylated on tyrosine
residues following TCR
cross-linking, although a very low level
of phosphorylation of ZAP-70
and Syk was observed in unstimulated
cells after longer exposures (Fig.
2B, lanes 5 to 8). In contrast,
the KS chimera immunoprecipitated from
cells (2E4) under permissive
conditions was phosphorylated on tyrosine
residues in unstimulated
cells, and its level of phosphorylation was
only slightly augmented
following TCR cross-linking (Fig.
2B, compare
lanes 3 and 4).
Analysis of the biochemical pathways activated by the KS
chimera.
To determine if the KS chimera would activate the
biochemical pathways mediated by wild-type ZAP-70 and Syk, we first
analyzed the pattern of tyrosine phosphorylation of cellular proteins
in cells expressing the KS chimera (Fig. 2C). Minimal phosphorylation of cellular proteins was observed in parental (449) cells or in cells
(2E4) under the nonpermissive conditions (Fig. 2C, lanes 1 and 2). Upon
tetracycline withdrawal, expression of the KS chimera resulted in the
induction of tyrosine phosphorylation of a number of cellular proteins
(Fig. 2C, lane 3). Moreover, the pattern of phosphorylation of these
proteins was qualitatively similar to, though quantitatively less than,
the pattern induced following TCR cross-linking of parental or
KS-expressing cells (Fig. 2C, lanes 4 to 6).
Since the pattern of phosphorylation of cellular proteins by the KS
chimera appeared similar to that of proteins phosphorylated
following
TCR cross-linking, we assessed the phosphorylation status
of specific
cellular proteins implicated as effector proteins
downstream of ZAP-70.
We and others have previously demonstrated
that SLP-76 represents a
downstream in vivo substrate of both
ZAP-70 and Syk (
6,
55).
Whereas SLP-76 was inducibly tyrosine
phosphorylated following TCR
cross-linking in parental cells (Fig.
2D, lanes 1 and 2), SLP-76 was
constitutively tyrosine phosphorylated
in cells expressing the KS
chimera (Fig.
2D, lane 3). A 72,000-molecular-weight
tyrosine
phosphoprotein representing the KS chimera coimmunoprecipitated
with
SLP-76 in the basal state, consistent with our previous demonstration
of the association of SLP-76 with activated ZAP-70 following TCR
cross-linking (
6). Consistent with the further increase in
tyrosine phosphorylation of the KS chimera following receptor
activation, SLP-76 also demonstrated a small increase in tyrosine
phosphorylation following TCR cross-linking (Fig.
2D, lane 4).
Interestingly, the association of the KS chimera with SLP-76 decreased
following TCR engagement, although the decrease was not a consistent
result; this may reflect activation of protein tyrosine phosphatases
by
TCR activation.
In addition to SLP-76, Vav was also phosphorylated on tyrosine
residues in unstimulated cells expressing the KS chimera (Fig.
2E, lane
3). Consistent with the ability of SLP-76 to interact
with Vav
following TCR stimulation of parental cells (
51,
66,
75)
(Fig.
2E, lanes 1 and 2), cells expressing the KS chimera
demonstrated constitutive association of tyrosine-phosphorylated
SLP-76
with Vav. Additional cellular proteins, including phospholipase
C
1
(PLC
1) and, to a lesser extent, cbl (Casitas B-lineage lymphoma),
were also found to be phosphorylated on tyrosine residues in
unstimulated
cells expressing the KS chimera and were all further
phosphorylated
following TCR cross-linking (data not shown).
Analysis of the functional parameters activated by the KS
chimera.
In addition to analyzing the phosphorylation status of
cellular proteins, we also assessed the ability of the KS chimera
to upregulate the CD69 T-cell activation marker. Resting parental T cells (clone 449) demonstrated minimal (3.6%) CD69 expression (Fig. 3A, top left panel). Unstimulated
cells examined under nonpermissive conditions also demonstrated a low
level of CD69 expression which was comparable to that of
resting parental 449 cells (data not shown). In contrast, when
examined under permissive conditions, two representative clones
which express the KS chimera demonstrated increased expression of CD69
(70.3% for clone 2E4.1 and 53.8% for clone 3G6) in the absence of TCR
stimulation (Fig. 3A, top middle and right panels). Activation of all
cells with PMA or following TCR cross-linking resulted in
further upregulation of CD69 expression (Fig. 3A, bottom panels,
and data not shown).
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FIG. 3.
Functional analysis of cells expressing the KS chimera.
(A) Upregulation of CD69, an early TCR activation marker, by the KS
chimera. Cells expressing the KS chimera (clones 2E4.1 and 3G6) were
analyzed for CD69 expression by FACS analysis. Parental 449 T cells are
also shown (left panels). A total of 106 cells were
analyzed under each set of experimental conditions. The top panels
represent resting cells examined under permissive conditions. The
bottom panels represent cells examined following stimulation with PMA.
These data are representative of five independent experiments. (B)
Receptor-independent IL-2 gene synthesis in cells expressing the KS
chimera. Jurkat T cells were transiently transfected with wild-type or
chimeric PTKs and a reporter plasmid encoding the IL-2 promoter. Cells
were harvested after 36 h and replated in medium alone, medium
containing PMA and an anti-TCR MAb (235), or medium containing PMA and
PHA. Luciferase activity was assessed following 6 h of stimulation
and normalized for CAT activity as previously described
(62). The experiment shown here is representative of at
least five independent experiments. Similar results were obtained for
stable clones.
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Finally, we analyzed the ability of cells overexpressing ZAP-70, Syk,
or the KS chimera to regulate IL-2 synthesis. A reporter
plasmid
consisting of the IL-2 promoter fused to luciferase was
transiently
transfected with each PTK into Jurkat cells. The activity
of the IL-2
promoter was analyzed in resting cells, in cells stimulated
with a
combination of PMA and an anti-CD3 MAb (235), or in cells
stimulated
with a combination of PMA and PHA. While expression
of wild-type ZAP-70
or wild-type Syk resulted in activation of
the IL-2 promoter only
following TCR stimulation, activation of
the IL-2 promoter in cells
expressing the KS chimera was observed
without receptor engagement
(Fig.
3B). In fact, the degree of
basal activity observed with
expression of the KS chimera was
comparable to the level of IL-2
promoter activity in receptor-activated
cells overexpressing wild-type
ZAP-70 or wild-type Syk. IL-2 gene
transcription was augmented
following TCR engagement in cells
expressing the KS chimera,
indicating that the TCR signaling pathway
remained intact in these
cells. Expression of the TAD or H chimeric
PTKs had no effect on basal
IL-2 promoter activity (data not shown).
Similar data were
observed when a reporter construct consisting
of the NF-AT promoter
element was used (data not shown). Together,
these data demonstrate
that expression of the KS chimera results
in activation of
biochemical pathways utilized by wild-type ZAP-70
and Syk in TCR
signaling that culminate in CD69 expression and
IL-2 gene
synthesis.
Signaling by the KS chimera is independent of the TCR.
Since
expression of other PTKs in T cells, such as the epidermal growth
factor receptor, can induce TCR signaling through cross-talk mechanisms
between the epidermal growth factor and T-cell receptors
(35), we undertook studies to determine whether the
transcriptional activation of the IL-2 promoter observed with the KS
chimera was mediated through a receptor or via a receptor-independent mechanism. Hence, we transiently cotransfected the KS chimera with an IL-2 reporter gene into a Jurkat cell derivative devoid of a
surface TCR (J.RT3-T3.5 [73]). Consistent with
the ability of the KS chimeric PTK to activate the IL-2 promoter in
Jurkat T cells without engagement of the TCR,
transcriptional activation of the IL-2 promoter was observed in the
TCR-negative Jurkat derivative at a level comparable to that in
TCR-positive Jurkat T cells (Fig. 3B and
4A).
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FIG. 4.
Constitutive activation of T cells by the KS chimera
does not require a surface TCR complex. (A) IL-2 gene synthesis induced
by the KS chimera does not require a surface TCR. The KS chimera was
transiently transfected into a TCR-negative variant of the Jurkat cell
line with an IL-2-luciferase construct. Cells were analyzed as
described in the legend to Fig. 3B. Luciferase activity induced by PMA
and ionomycin was comparable to the level induced in TCR-positive T
cells (Fig. 3B). These data are representative of two independent
experiments. (B) The TCR chain is not tyrosine phosphorylated by
the KS chimera. The TCR chain was immunoprecipitated from 4 × 107 parental Jurkat cells (C) (lanes 1 and 2), from
KS-expressing cells under nonpermissive conditions (clone 2E4) (lanes 3 and 4), and from KS-expressing cells under permissive conditions (clone
2E4) (lanes 5 and 6). Immunoprecipitates from 4 × 107
resting (lanes 1, 3, and 5) or TCR-stimulated (lanes 2, 4, and 6) cells
were analyzed by immunoblotting with an anti-pY MAb (top panel) or an
anti- antiserum (bottom panel). These data are representative of two
independent experiments.
|
|
To further confirm the ability of the KS chimera to activate T cells in
a receptor-independent fashion, we assessed the tyrosine
phosphorylation status of the TCR
chain in TCR-positive cells
(Fig.
4B). Similar to the TCR-induced phosphorylation of the TCR
chains
in parental cells or cells analyzed under nonpermissive
conditions
(Fig.
4B, lanes 1 to 4), cells expressing the KS chimera
also
demonstrated TCR-inducible
phosphorylation (Fig.
4B, upper
panel,
compare lanes 5 and 6). No phosphorylation of the
chain
was
observed in unstimulated cells under the permissive conditions
(Fig.
4B, upper panel, lane 5). Together, these data demonstrate
that the
ability of the KS chimera to activate signaling pathways
downstream of
ZAP-70 and Syk, culminating in IL-2 gene transcription,
is independent
of the signaling events proximal to ZAP-70, including
the requirement
of a functional TCR and ITAM phosphorylation.
Functional role for Lck downstream of ZAP-70 and Syk PTKs in TCR
activation.
Since our studies in HeLa cells indicated that the KS
chimera could function in an Lck-independent fashion, we addressed
whether the KS chimera could bypass the signaling defects observed in Lck-deficient T cells. Stable clones expressing wild-type Syk or the KS
chimera were established in the Lck-deficient JCaM1.6 leukemic cell
line (27, 61). To ensure that the KS chimera could undergo
autoactivation and autophosphorylation in an Lck-independent fashion,
we first analyzed the phosphorylation status of wild-type Syk or the KS
chimera expressed in JCaM1.6 cells. Consistent with our observations in
HeLa cells, the KS chimera, when expressed in JCaM1.6 cells, was
tyrosine phosphorylated and occurred independently of receptor
engagement (Fig.
5A,
lanes 3 to 6). In contrast, wild-type Syk was not tyrosine
phosphorylated in resting or TCR-activated JCaM1.6 cells (clone 3H3),
despite being expressed at higher levels than the KS chimera in clone
5F7 (Fig. 5A, lanes 5 to 8). Hence, consistent with our results in HeLa
cells, the KS chimera can undergo autoactivation independently of Lck.
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|
FIG. 5.
Functional evidence for Lck acting downstream or
independently of ZAP-70 activation. (A) Tyrosine phosphorylation of the
KS chimera is receptor and Lck independent. Immunoprecipitates of KS
(lanes 1 to 6) or wild-type Syk (lanes 7 and 8) from Jurkat cells
(clone 2E4) (lanes 1 and 2) or JCaM1.6 cells (clones 4D11, 5F7, and
3H3) (lanes 3 to 8) were analyzed by immunoblotting with an
antiphosphotyrosine MAb (top) or an anti-HA MAb (bottom). A total of
4 × 107 cells were analyzed per immunoprecipitate.
The JCaM1.6 clones were expressed under the control of the actin
promoter. The 2E4 clone was examined under permissive conditions. (B)
Tyrosine phosphorylation of SLP-76 by the KS chimera is receptor and
Lck independent. Immunoprecipitates of SLP-76 from Jurkat cells (lanes
1 to 3 and 6 to 8) or JCaM1.6 cells (lanes 4, 5, 9, and 10) expressing
the KS chimera (clone 2E4 [lanes 3 and 8] and clone 4D11 [lanes 4 and 9]), wild-type Syk (clone 5G4 [lanes 2 and 7]) and clone 3H3,
[lanes 5 and 10]), or wild-type ZAP-70 (clone wt24 [lanes 1 and 6])
were analyzed by immunoblotting with an anti-pY MAb (top panel) or an
anti-SLP-76 (H3) MAb (bottom panel). A total of 2 × 107 cells were analyzed per immunoprecipitate. Clones 5F4
and 2E4 were analyzed under permissive conditions. These data are
representative of four independent experiments. (C) Tyrosine
phosphorylation of Vav by the KS chimera. Immunoprecipitates of Vav
from JCaM1.6 cells (lanes 1 and 2), JCaM1.6 cells expressing Syk (clone
5G4) (lanes 3 and 4), or JCaM1.6 cells expressing the KS chimera (clone
4D11) (lanes 5 and 6) were analyzed by immunoblotting with an anti-pY
MAb (top panel) or an anti-Vav MAb (bottom panel). A total of 2 × 107 cells were analyzed per immunoprecipitate. These data
are representative of two independent experiments. On substantially
longer exposures, two minor nonspecific bands migrating above and below
Vav were observed in lanes 4 and 5. (D) Lck is required downstream of
ZAP-70 activation for CD69 expression. JCaM1.6 cells, Jurkat cells
expressing the KS chimera (clone 2E4), or JCaM1.6 cells expressing the
KS chimera (clones 4D11 and 7E7) were analyzed for CD69 expression as
described in the legend to Fig. 3A. A total of 106 cells
were analyzed under each set of experimental conditions. The top panels
represent resting cells, while the bottom panels represent cells
examined following treatment with PMA as described in Materials and
Methods. The percentage of CD69+ cells is quantitated above
each bracket. These data are representative of three independent
experiments. (E) Lck is required downstream of ZAP-70 activation for
IL-2 gene synthesis. Wild-type and chimeric PTKs were individually
transiently transfected with an IL-2 promoter into the Lck-deficient
variant Jurkat T-cell line JCaM1.6. Luciferase activity was detected in
unstimulated and TCR-activated cells as described in Materials and
Methods. A parallel experiment was performed in parental Jurkat
cells for comparison purposes. These data are representative of at
least three independent experiments.
|
|
Analysis of the phosphorylation status of SLP-76 demonstrated similar
results. While SLP-76 was phosphorylated in a receptor-dependent
fashion in Jurkat cells expressing wild-type ZAP-70 or wild-type
Syk
(Fig.
5B, lanes 1, 2, 6, and 7), it was constitutively phosphorylated
in Jurkat (clone 2E4 [lanes 3 and 8]) and JCaM1.6 (clone 4D11
[lanes
4 and 9]) cells expressing the KS chimera. Hence, phosphorylation
of
SLP-76 occurs independently of receptor stimulation and Lck.
In
contrast, SLP-76 is not phosphorylated in resting or TCR-activated
JCaM1.6 cells expressing Syk or ZAP-70 (Fig.
5B, lanes 5 and 10,
and
data not shown). Hence, the KS chimera still retains its ability
to
phosphorylate, in vivo, downstream substrates of ZAP-70 and
Syk in the absence of Lck.
Vav was also phosphorylated in resting JCaM1.6 cells expressing the KS
chimera (clone 4D11 [Fig.
5C, top panel, lanes 5 and
6]), but it was
not phosphorylated in resting JCaM1.6 parental
cells (lanes 1 and 2),
although its level of phosphorylation in
the 4D11 cells was lower
than that of Jurkat cells expressing
the KS chimera (Fig.
2E, lane
3, and data not shown). Intriguingly,
Vav phosphorylation was further
increased following TCR cross-linking
in JCaM1.6 cells expressing
the KS chimera (Fig.
5C, top panel,
compare lanes 5 and 6),
suggesting that both the Src and Syk families
of PTKs likely contribute
to Vav phosphorylation. In contrast,
phosphorylation of Vav was not
detected in JCaM1.6 parental cells
or in JCaM1.6 cells expressing
Syk, under resting conditions or
following TCR cross-linking
(Fig.
5C, top panel, lanes 1 to 4).
To determine if expression of the KS chimera could restore downstream
signaling defects in Lck-deficient cells, we analyzed
the ability of
the KS chimera to upregulate CD69 in JCaM1.6 cells.
While
Lck-sufficient cells expressing the KS chimera (2E4.1)
demonstrated
increased expression of CD69 in unstimulated cells (70%),
CD69
expression in two representative JCaM1.6 clones expressing the
KS
chimera was indistinguishable from that of parental JCaM1.6
cells (Fig.
5D, top panel). Hence, while expression of the KS
chimera in
Lck-deficient cells is sufficient for tyrosine phosphorylation
of KS,
SLP-76, and, to a lesser degree, Vav, expression is not
sufficient to
bypass the absence of Lck in upregulating CD69.
Finally, we analyzed the ability of the KS chimera to bypass the
absence of Lck to upregulate IL-2 synthesis. Consistent with
the
inability of the KS chimera to upregulate CD69, expression
of the KS
chimera in JCaM1.6 cells resulted in a sevenfold-lower
level of basal
IL-2 gene transcription than that in Jurkat cells
(Fig.
5E). The
residual basal function observed may be due to
the ability of Fyn to
partially compensate for the absence of
Lck (
30,
67). In
addition, overexpression of neither wild-type
ZAP-70 nor wild-type Syk
in JCaM1.6 cells resulted in basal or
receptor-dependent IL-2 gene
transcription. Together, these data
suggest that in addition to the
proximal signaling functions established
for Lck, which include
phosphorylation of the receptor signaling
subunit-encoded ITAMs and
ZAP-70, Lck plays roles in TCR function
downstream of the ZAP-70 and
Syk PTKs.
| |
DISCUSSION |
We have described the generation of a constitutively active form
of the Syk family of PTKs by exchange of the catalytic domains of
ZAP-70 and Syk. Expression of this chimeric PTK in HeLa and Jurkat T
cells resulted in tyrosine phosphorylation of the KS chimera that was
independent of the Src PTKs and TCR activation, respectively. In
addition, expression of the KS chimera in stably transfected Jurkat T-cell clones resulted in
constitutive tyrosine phosphorylation of a number of cellular
proteins normally phosphorylated following TCR cross-linking
that reside downstream (e.g., SLP-76, Vav, PLC1, and cbl),
but not upstream (e.g., the TCR -chains), of ZAP-70. Phosphorylation
of these substrates, in turn, resulted in
transcriptional activation of both the NF-AT and IL-2 promoter elements and expression of T-cell activation markers in the
absence of receptor engagement. Finally, the KS chimera
did not exert any aberrant effects on the endogenous TCR
signaling machinery, since TCR cross-linking still resulted in
tyrosine phosphorylation of and endogenous ZAP-70. While
we cannot exclude the possibility that the KS chimera may
activate biochemical pathways not normally activated by
wild-type ZAP-70 and Syk PTKs, these data together support the hypothesis that the KS chimera is mediating its effects through the normal TCR-mediated signaling pathways downstream of
ZAP-70.
The ability of the KS chimera to autoactivate implies that ZAP-70
and/or Syk PTKs may possess some basal intrinsic inhibitory conformation that is disrupted with the generation of the KS chimera. Multiple lines of evidence suggest that such inhibitory constraints may
exist. First, the p42 tryptic fragment of Syk that encodes solely its
catalytic domain has an approximately twofold-greater enzymatic
activity than the holoenzyme (79). Second, an antibody directed to the C terminus of Syk inhibits the ability of doubly phosphorylated ITAMs to activate Syk and recognizes only a
subpopulation of activated Syk (36, 56). Third, the
activated and nonactivated forms of Syk exhibit different trypsin
sensitivities (36). Conformational changes, as determined by
biophysical and biostructural methods, have also been described for
ZAP-70 (41). Alternatively, but not exclusively, the KS
chimera may have lost inhibitory elements that normally regulate ZAP-70
and Syk in their basal states. Consistent with this latter possibility
is the inability of the SHP-1 protein tyrosine phosphatase to
efficiently dephosphorylate and downregulate the catalytic activity of
the KS chimera in insect Sf9 cells (data not shown). In contrast,
coinfection of SHP-1 with activated ZAP-70 or Syk resulted in the
downregulation of PTK activity (52) (data not shown). Hence,
the resultant substitution of the Syk domain for the ZAP-70 kinase
domain may alleviate intrinsic inhibitory effects, resulting in its
dysregulation. Similar inhibitory mechanisms have been demonstrated for
the Src PTK Hck. The resolution of the Hck holoenzyme structure has
revealed inhibitory constraints, mediated by both its SH2 and SH3
domains, that also affect Hck enzymatic activity (46, 57).
Additional molecular dissections of potential inhibitory domains within
ZAP-70 and Syk are presently under way.
Functional role for Lck downstream of ZAP-70.
The generation
of constitutively active mutants permits one to complement biochemical
studies with genetic analyses. While expression of the KS chimera in
Jurkat T cells resulted in constitutive activation of a number of
signaling pathways, expression of the KS chimera in JCaM1.6 T cells
failed to bypass the deficiency in Lck in mediating IL-2 gene
expression or upregulation of activation markers. We and others have
demonstrated that membrane localization and transphosphorylation of
ZAP-70 at Tyr 493 by Lck are required for efficient T-cell function
(10, 29, 37, 38, 69, 71, 78). Studies in a variety of
cellular systems have demonstrated that ZAP-70 is required for both
receptor-mediated calcium mobilization and Ras activation (38, 39,
50, 54). The data presented in this paper provide genetic
evidence for a functional role of Lck downstream of ZAP-70 or
independent of the phosphorylation and activation of ZAP-70. The
constitutive phosphorylation of SLP-76 by the KS chimera in
Lck-deficient cells favors a model in which the TCR signaling pathway
diverges, with ZAP-70 and Lck mediating distinct subsets of signaling
pathways downstream of ZAP-70. However, the convergence of these
distinct signaling pathways is required for efficient T-cell function.
This additional level of regulation provides mechanisms by which the
interaction of Lck with other signaling molecules may affect Lck
activity through SH2 and/or SH3 interactions or recompartmentalizes Lck
into distinct subcellular localizations and modulates the biological
response of a given TCR-activating event. Studies by Mustelin and
colleagues have suggested that Syk may phosphorylate the SH2 domain of
Lck to mediate downstream signaling functions (16). Both the
SH2 and SH3 domains of Lck have been described to interact with a variety of signaling molecules, including the TCR chain, ZAP-70, phosphatidylinositol 3-kinase, the HS1 protein, the Lck-binding protein
1 (LckBP1), Nef, and GTPase-activating protein (5, 14, 18, 22, 53,
60, 63, 65, 77). Recent studies suggest that the SH3 domain of
Lck plays a role in T-cell function independent of ZAP-70 activation
(16a). Mutation of the SH3 domain of Lck inhibited
TCR-mediated Erk activation but did not affect ITAM or ZAP-70
phosphorylation. Studies have also indicated the involvement of the Lck
SH2 domain in stabilization of the interaction of CD4 with the class II
major histocompatibility complex (76), colocalization of CD4
with the activated TCR complex (18), and regulation of Lck
enzymatic activity through interactions mediated via its C-terminal
tyrosyl residue (1, 58, 68, 72). In addition, Lck may
mediate TCR-independent signaling pathways and molecules such as CD28
and Itk to effect full T-cell function (3, 4, 25, 26, 31).
The ability of the KS chimera to bypass the proximal signaling
functions of Lck (i.e., and ZAP-70 phosphorylation) will permit us
to determine if these additional functions attributable to the SH2
domain of Lck are upstream or downstream of ZAP-70 activation.
Src dependence of the Syk PTK.
Our inability to bypass the
deficiency in Lck by overexpression of Syk, as measured by SLP-76, Vav,
or Syk tyrosine phosphorylation, CD69 induction, or IL-2 gene synthesis
(Fig. 5 and data not shown), is in contrast with recent studies showing
that transient expression of Syk in JCaM1.6- or CD45-deficient J45.01 T
cells was sufficient to reconstitute TCR-mediated NF-AT transcriptional
activation (13, 74). In addition, expression of Syk in COS
cells under certain conditions has been demonstrated to result in ITAM
phosphorylation independently of Src PTKs (43, 80). Our
stable clones derived from JCaM1.6 T cells that express Syk or ZAP-70
demonstrate no basal or TCR-induced phosphorylation of these kinases.
However, higher levels of Syk expression in fibroblasts and insect Sf9 cells have been demonstrated to result in Syk autoactivation (6, 15, 42). A recent study of Syk activation demonstrated that while
Syk could be activated through autophosphorylation, Src-dependent transphosphorylation was still required for the
initiation of activation while autophosphorylation appeared to function
in signal amplification (20). Moreover, expression of a Syk
transgene which is sufficient to reconstitute thymocyte development in
zap-70
/ mice does not restore the
developmental defects observed in CD45/ mice
(28). Finally, while syk/ mice do
not exhibit any T-cell developmental abnormalities, recent studies
indicate that ZAP-70 and Syk play overlapping roles in mediating
pre-TCR function. Whereas zap-70/ mice
accumulate CD4+ CD8+ thymocytes, mice deficient
in both zap-70 and syk are blocked at an earlier
developmental stage and accumulate CD4 CD8
thymocytes (12, 48). These data indicate that either ZAP-70 or Syk is sufficient to mediate the transition of CD4
CD8 to CD4+ CD8+ thymocytes.
Moreover, mice deficient in both zap-70 and syk
demonstrate a developmental block similar to that of mice deficient in
both lck and fyn. Hence, Syk alone is unable to
regulate the pre-TCR transition in the absence of Src PTKs. Together,
these observations also support a requirement of Src PTKs in Syk
function.
Once activated, however, Syk, but not ZAP-70, appears to be able to
more efficiently amplify its own autophosphorylation.
Hence, while
ZAP-70 and Syk may have similar upstream signaling
requirements (i.e.,
CD45 and Lck) for their initiation of activation,
the biochemical
differences in the modulation of downstream effector
functions by
activated ZAP-70 and Syk may alter the thresholds
for signaling
mediated by these two PTKs. Latour et al. have recently
analyzed a
similar panel of chimeric PTKs in COS cells and described
a 100-fold
difference in the enzymatic activities of the ZAP-70
and Syk kinase
domains (
42). While these regulatory differences
between
ZAP-70 and Syk are reflected in differences in the efficiencies
of
proximal signaling events, such as calcium responses and cytolysis,
they do not appear to cause significant differences in ITAM-based
-T-cell development or in antibody- or mitogen-induced
proliferative
responses (
12,
28,
37). However, it is
intriguing to speculate
that differential expression of ZAP-70 and Syk
may be able to
alter the strength of the proximal signaling events and
convert
a low-affinity ligand or an altered peptide ligand response
(reviewed
in references
33 and
59) into a high-affinity or wild-type
peptide
response. In addition, the selection of a TCR repertoire
may be altered
depending on whether the ZAP-70 or Syk PTK is activated
in a given
developmental stage.
Finally, in contrast to
-T-cell function, ZAP-70 and Syk play
distinct roles in the development and function of
T cells,
in
the activation of the high-affinity immunoglobulin G and E
receptors,
in natural killer cell function, and in integrin-mediated
signaling
(
17,
23,
28,
45,
64). Hence, it is likely
that in these and
other cellular systems, less-constrained signaling
motifs may result in
selective activation of Syk but not ZAP-70.
Additional in vivo studies
to translate these observed potential
mechanistic differences between
ZAP-70 and Syk into functional
differences in biological outcomes are
ongoing.
| |
ACKNOWLEDGMENTS |
We thank Eric Brown, Matt Thomas, Andrey Shaw, and Julie Bubeck
Wardenburg for critical reading of the manuscript.
This work was supported in part by grants from the National Institutes
of Health (RO1CA71516 and 5T32DK07126). A.C.C. is a Pew Scholar in the
Biomedical Sciences.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Box 8022, Washington University School of Medicine, 660 S. Euclid Ave., St.
Louis, MO 63110. Phone: (314) 362-9012. Fax: (314) 454-0175. E-mail: chan{at}im.wustl.edu.
| |
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Mol Cell Biol, May 1998, p. 2855-2866, Vol. 18, No. 5
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Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
