From the Department of Medicine and Pathology,
University of Chicago, Chicago, Illinois 60637 and the
Department of Medicine and Howard Hughes Medical Institute,
Washington University School of Medicine,
St. Louis, Missouri 63110
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ABSTRACT |
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Initiation of T-cell antigen receptor (TCR)
signaling is dependent upon the activity of protein tyrosine kinases.
The Src family kinase Lck is required for the initial events in TCR
signaling, such as the phosphorylation of the TCR complex and the
activation of ZAP-70, but little is known of its role in downstream
signaling. Expression of a mutated form of Lck lacking SH3 domain
function (LckW97A) in the Lck-deficient T-cell line JCaM1 revealed a
requirement for Lck beyond the initiation of TCR signaling. In cells
expressing LckW97A, stimulation of the TCR failed to activate the
mitogen-activated protein kinase (MAPK) pathway, despite normal TCR Activation of T-lymphocytes is initiated by engagement of the
T-cell antigen receptor
(TCR)1 (1-3). Signaling
through the TCR is dependent upon the activity of protein-tyrosine
kinases which induce the phosphorylation of a number of proteins,
including the subunits of the TCR complex itself. Tyrosine
phosphorylation of the TCR complex occurs within amino acid sequences
known as immunoreceptor-based tyrosine activation motifs (ITAMs) and is
required for receptor signaling function (4-6). Activation of
downstream signaling pathways, including the Ras/mitogen-activated
protein kinase (MAPK) pathway, the phosphatidylinositol (PIP2) pathway, and the phosphatidylinositol 3-kinase
(PI-3-K) pathway, is dependent upon protein-tyrosine kinase function
(7-10). However, the mechanisms by which the induction of tyrosine
kinase activity initiates these diverse signaling processes is unknown.
Unlike receptor tyrosine kinases, the cytoplasmic portion of the TCR
lacks intrinsic catalytic activity. Instead, the induction of tyrosine
phosphorylation following engagement of the TCR requires the expression
of non-receptor kinases. Both the Src family and the Syk/ZAP-70 family
of tyrosine kinases are required for normal TCR signal transduction
(11-18). A role for Lck has been identified in the initiation of TCR
signaling; T-cells lacking functional Lck fail to initiate ITAM
phosphorylation or induce ZAP-70 recruitment and activation (12, 17,
19). ZAP-70 has been implicated in downstream signaling events such as
activation of the PIP2 and the MAPK pathways (18, 20-22).
A current model for the initiation of TCR signaling proposes that Lck
and ZAP-70 function sequentially (1-3). Heterologous cell systems
expressing ZAP-70 and the cytoplasmic domain of the TCR Like other Src family kinases, Lck contains a C-terminal kinase domain,
a single Src homology 2 (SH2) and SH3 domain, and an N-terminal region
that is distinct from other family members (26). SH3 domains are able
to mediate protein interactions by binding certain proline-rich amino
acid sequences (27, 28), and a number of proteins have been reported to
bind the Lck SH3 domain including c-Cbl, PI-3-K, Ras-GAP, HS1, and CD2
(29-33). The SH3 domain of Lck has also been proposed to stabilize the formation of Lck homodimers which may potentiate TCR signaling following co-ligation of the receptor and CD4 (34, 35). Previous work
indicated that deletion of the Lck SH3 domain interfered with the
ability of an oncogenic form of Lck to enhance interleukin-2 production, supporting a role for the Lck SH3 domain in regulating T-cell activation (36).
In the present study, we have examined the involvement of the Lck SH3
domain in TCR signaling processes by expressing an altered form of Lck
which contained an inactive SH3 domain in the Lck-deficient T-cell line
JCaM1 (12). TCR signaling pathways displayed a differential sensitivity
to loss of Lck SH3 domain function. The induction of tyrosine
phosphorylation and activation of the PIP2 pathway was
independent of the Lck SH3 domain, whereas activation of the MAPK
pathway was strictly SH3 domain-dependent. The inability of
this altered form of Lck to activate the MAPK pathway despite ZAP-70
activation suggests that Lck participates directly in the stimulation
of downstream signaling pathways following TCR ligation.
Cells and Plasmids--
Derivatives of the Lck-deficient Jurkat
cell line JCaM1 were maintained at 37 °C and 5% CO2 in
RPMI 1640 supplemented with 10% fetal bovine serum, glutamine,
penicillin, and streptomycin. The LckW97A mutant cDNA was generated
from wild type human Lck cDNA using base pair mismatched primers
and polymerase chain reaction amplification and was confirmed by
sequencing. The Lck cDNA was subcloned into pBP1, a plasmid derived
from pUCH-13 (37) which contains a cytomegalovirus promoter sequence
regulated by tetracycline operators, and a gene conferring resistance
to the antibiotic hygromycin. Clones that express wild type Lck or
LckW97A were generated by electroporation of a G418-resistant JCaM1
derivative, which expresses a VP16-tetracycline repressor fusion
protein (37), and were isolated by plating at limiting dilution in the
presence of hygromycin and G418. Clones that expressed TCR and wild
type Lck, or LckW97A, at levels equivalent to the parental Jurkat cell line were maintained for further analysis.
Analysis of TCR and CD69 Expression--
TCR expression was
measured by staining cells with a mouse antibody recognizing CD3
(Leu-4), followed by a FITC-conjugated goat anti-mouse secondary
antibody, and analyzed by fluorescence flow cytometry. To assess the
induction of CD69, cells expressing wild type Lck or LckW97A, or
Lck-deficient control cells, were incubated with media alone, PHA (0.3 µg/ml), or PMA (50 ng/ml) for 16-20 h at 37 °C. Cells were
stained with a mouse anti-CD69 antibody (PharMingen) and a
FITC-conjugated goat anti-mouse secondary antibody then evaluated by
flow cytometry.
Immunoprecipitations and Immunoblotting--
Cell suspensions
(40 × 106 per ml in phosphate-buffered saline) were
stimulated for 2 min at 37 °C with the anti-Jurkat TCR monoclonal
antibody C305 (38). Cells were lysed in 1% (v/v) Nonidet P-40 solution
containing (in mM) 10 Tris (pH 7.8), 150 NaCl, 1 phenylmethylsulfonyl fluoride, 0.4 sodium orthovanadate, 10 NaF, as
well as 1 µg/ml leupeptin. Particulate matter was removed by
centrifugation at 12,500 × g for 10 min at 4 °C,
and lysates were precleared with fixed Staphylococcus aureus
(Pansorbin, Calbiochem). Lck, ZAP-70, and PLC In Vitro Kinase Assays of Lck and ZAP-70--
Lck
immunoprecipitates were washed twice in Nonidet P-40 lysis buffer
followed by two washes in 20 mM Tris (pH 7.4), 0.5 M LiCl, 0.4 mM sodium vanadate, and a single
wash in distilled H2O. The immobilized Lck was resuspended
at 37 °C in Lck kinase buffer containing (in mM) 20 Tris
(pH 7.4), 10 MnCl2, 0.01 ATP, as well as 1 µg of GST-
ZAP-70 immunoprecipitates were prepared from unstimulated and
TCR-stimulated cells using Nonidet P-40 lysis buffer containing 2 mM EDTA, washed twice in the same buffer, then twice 10 mM Tris (pH 7.4), 0.5 M LiCl, and once with
ZAP-70 kinase buffer containing (in mM) 10 Tris (pH 7.4),
10 MnCl2, 10 MgCl2. The ZAP-70 kinase assay was
conducted at room temperature for 10 min by resuspending in ZAP-70
kinase buffer with 20 µM ATP, 1 µg of GST-Band III
fusion protein, and 15 µCi of [
Following SDS-PAGE and staining with Coomassie Blue, the gels were
dried to filter paper and exposed to x-ray-sensitive film (X-OMat,
Eastman Kodak Co.). The regions corresponding to phospho-GST- Calcium Measurement--
Cells were loaded with the fluorescent
calcium indicator indo-1, washed extensively in Hepes-buffered saline
(HeBS, pH 7.4) solution containing (in mM) 25 Hepes, 125 NaCl, 5 KCl, 1 CaCl2, 0.5 MgCl2, 1 Na2HPO4, 0.1% (w/v) bovine serum albumin and
0.1% (w/v) D-glucose, and kept on ice. Prior to use, the
aliquots were warmed to 37 °C for 10 min and then placed in a
spectrofluorometer equipped with a thermally jacketed cuvette holder
maintained at 37 °C. The fluorescence intensity was monitored
continuously at the emission wavelength of 400 nm following excitation
at 334 nm. Fluorescence intensity values were corrected for cell
autofluorescence and then converted to [Ca2+]i
using a Kd value of indo-1 for Ca2+ of
250 nM (39).
To examine the role of Lck in TCR signaling, we have utilized the
Lck-deficient JCaM1 T-cell line, derived from the Jurkat cell line (12,
40). The advantage of using JCaM1 is that TCR signaling can be studied
in clones expressing an altered form of Lck independent of the
contributions of endogenous wild type Lck (41). As such, the importance
of the SH3 domain of Lck in TCR signaling was examined by transfecting
JCaM1 cells with an Lck cDNA that encoded a protein containing an
inactive SH3 domain. A conserved tryptophan in SH3 domains stabilizes
their association with proline-rich target peptides (28); mutation of
this tryptophan residue abolishes ligand binding (42, 43). In this
study, the SH3 domain of Lck was inactivated by mutating this essential tryptophan, located at position 97, to alanine using site-directed mutagenesis (LckW97A). In preliminary experiments, we observed that the
SH3 domain of LckW97A, when expressed as a fusion protein with
glutathione S-transferase (GST), was unable to bind the
known substrate c-Cbl (not shown). Thus, the LckW97A mutation
represents a loss of function mutation for the Lck SH3 domain. Stable
transfection of JCaM1 cells generated several independent clones
expressing similar levels of TCR, and levels of wild type Lck or
LckW97A, equivalent to those in the parental Jurkat cell line (Fig.
1).
chain phosphorylation, ZAP-70 recruitment, and ZAP-70 activation.
Activation of extracellular signal-regulated kinase (ERK) and MAPK
kinase (MEK), as well as the induction of CD69 expression, was greatly
impaired in JCaM1/LckW97A cells. In contrast, the phosphorylation of
phospholipase C
1 (PLC
1) and corresponding elevations in
intracellular calcium concentration ([Ca2+]i) were intact. Thus, cells
expressing LckW97A exhibit a selective defect in the activation of the
MAPK pathway. These results demonstrate that Lck has a role in the
activation of signaling pathways beyond the initiation of TCR signaling
and suggest that the MAPK pathway may be selectively controlled by
regulating the function of Lck.
INTRODUCTION
Top
Abstract
Introduction
References
chain
displayed enhanced ITAM phosphorylation and ZAP-70 activation when Lck
is co-transfected with ZAP-70 (19, 23, 24), suggesting Lck mediates
both the initial ITAM phosphorylation and the subsequent
phosphorylation and activation of ZAP-70. However, this does not
preclude a role for Lck in TCR signaling which is independent of the
activation of ZAP-70 (25).
EXPERIMENTAL PROCEDURES
1 were
immunoprecipitated using rabbit antisera (Upstate Biotechnology), and
TCR
chain was immunoprecipitated using the 6B10.2 monoclonal
antibody (). Immunoprecipitates
were collected on protein A-Sepharose, washed twice each in Nonidet
P-40 lysis buffer and lysis buffer containing 0.5 M NaCl,
analyzed by SDS-PAGE, and transferred to polyvinylidene difluoride
membranes. Monoclonal antibodies were used to detect Lck (1F6; A. Burkhardt and J. Bolen), phosphotyrosine (4G10; Upstate Biotechnology,
Inc.), ZAP-70 (2F3.2; Upstate Biotechnology, Inc.), PLC
1 (mixed
monoclonal, Upstate Biotechnology, Inc.), and TCR
chain (6B10.2;
). The activated forms of ERK1
and ERK2 or MEK1 and MEK2 were detected using rabbit antibodies which
recognize phospho-ERK or phospho-MEK (New England Biolabs). Proteins
were detected using horseradish peroxidase-conjugated goat anti-mouse
or anti-rabbit IgG secondary antibody and enhanced chemiluminescence.
fusion protein and 10 µCi of [
-32P]ATP. Aliquots
were withdrawn and transferred to microcentrifuge tubes containing
ice-cold Nonidet P-40 lysis buffer, 2 mM EDTA, 10 mM ATP, and glutathione-agarose; after 30 min at 4 °C
the samples were washed twice with Nonidet P-40 lysis buffer and
resuspended in SDS sample buffer.
-32P]ATP. The
reactions were terminated by addition of 2× SDS sample buffer.
and
phospho-Lck or phospho-GST-Band III were excised, and the incorporation
of 32P was measured by counting Cerenkov radiation.
RESULTS
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Fig. 1.
JCaM1 cells transfected with wild type Lck,
or LckW97A, express levels of TCR and Lck that are similar to the
parental Jurkat cell line. A, TCR expression;
B, Lck levels in Jurkat, JCaM1, and JCaM1 cells transfected
with wild type Lck or LckW97A. TCR expression was analyzed by
fluorescence flow cytometry of cells stained with anti-TCR antibody
(Leu-4), followed by a FITC-conjugated secondary antibody. Lck levels
were analyzed by immunoblotting Nonidet P-40-soluble cell lysates
prepared from equivalent numbers of cells.
Lck kinase activity is essential for the initiation of TCR signaling,
and it is possible that disruption of the SH3 domain of Lck affects its
catalytic activity. To determine if the SH3 domain of Lck influenced
its catalytic activity in vitro, we incubated immunoprecipitates of LckW97A or wild type Lck with
[-32P]ATP and an exogenous substrate consisting of the
cytosolic domain of the
chain of the TCR fused to GST (Fig.
2A). Both wild type Lck and
LckW97A displayed autophosphorylation and GST-
phosphorylation, indicating that the SH3 domain of Lck was not required for kinase activity. In fact, LckW97A displayed enhanced catalytic activity relative to wild type Lck, consistent with an autoinhibitory role for
the Lck SH3 domain (Fig. 2B). Thus, any disturbances in TCR signaling in cells expressing LckW97A cannot be attributed to a general
loss of catalytic activity.
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Although LckW97A was active in vitro, mutation of the SH3 domain may disrupt TCR-induced substrate phosphorylation within the cell, possibly by altering Lck distribution or by interfering with the binding of potential substrates. Therefore, we examined the levels of cellular tyrosine phosphoproteins in unstimulated and TCR-stimulated JCaM1 transfectants expressing wild type Lck, or LckW97A, by immunoblotting lysates with an anti-phosphotyrosine antibody (Fig. 3A). The profiles of tyrosine phosphoproteins in unstimulated and TCR-stimulated cells expressing LckW97A closely resembled those of cells expressing wild type Lck, whereas TCR stimulation of plasmid vector-transfected JCaM1 cells failed to induce tyrosine phosphorylation (not shown). Although cells expressing LckW97A displayed a slight, but reproducible, elevation in the basal level of tyrosine phosphorylation, it is apparent that mutation of SH3 domain of Lck did not substantially alter the tyrosine phosphorylation of cellular phosphoproteins prior to or following TCR stimulation. As such, LckW97A was capable of inducing tyrosine phosphorylation upon TCR stimulation.
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Although the induction of tyrosine phosphorylation appeared to be
intact, we examined in greater detail the effects of mutation of the
Lck SH3 domain on the initiation of TCR signaling. Specifically, we
focused on the induction of ITAM and ZAP-70 phosphorylation in cells
expressing LckW97A since these events are mediated directly by Lck.
Immunoprecipitates of TCR chain were immunoblotted for phosphotyrosine to evaluate ITAM phosphorylation and associated ZAP-70
phosphorylation (Fig. 3B). TCR stimulation of cells
expressing either wild type Lck or LckW97A induced the phosphorylation
of TCR
chain and association of tyrosine-phosphorylated ZAP-70 (Fig. 3B), but vector-transfected control cells failed to
induce detectable levels of TCR
chain or ZAP-70 phosphorylation
(not shown). To confirm that TCR stimulation of cells expressing
LckW97A elicited ZAP-70 activation, we examined ZAP-70 phosphorylation in the JCaM1 transfectants and evaluated ZAP-70 kinase activity in vitro. Anti-phosphotyrosine blotting of ZAP-70
immunoprecipitates revealed that TCR stimulation induced similar levels
of ZAP-70 phosphorylation in cells expressing either LckW97A or wild
type Lck but not vector-transfected cells (Fig.
4A). The induction of ZAP-70
phosphorylation in TCR-stimulated cells corresponded to an elevation in
ZAP-70 kinase activity in vitro, demonstrating that the SH3
domain of Lck is not required for the activation of ZAP-70 (Fig.
4B). Although a slight reduction in TCR-induced ZAP-70
activation was observed in JCaM1 cells expressing LckW97A, this
difference was not statistically significant (p > 0.05). Thus, the previously identified roles for Lck in the initiation of TCR signaling, namely ITAM phosphorylation and the subsequent phosphorylation and activation of ZAP-70, can occur independently of
Lck SH3 domain function.
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Since mutation of the SH3 domain did not alter the initial TCR
signaling events that are known to require Lck, we investigated the
possibility that the SH3 domain of Lck participates in the activation
of downstream signaling events. Specifically, we examined the ability
of this altered form of Lck to activate the PIP2 and the
MAPK signaling pathways following TCR stimulation. Activation of the
PIP2 pathway upon TCR stimulation was assessed by measuring elevations in [Ca2+]i and the induction of
PLC1 phosphorylation. Elevations in [Ca2+]i
elicited by TCR stimulation were examined using cells loaded with the
Ca2+-sensitive fluorescent dye indo-1. We analyzed a range
of anti-TCR antibody concentrations since subtle disturbances in the
activation of the PIP2 pathway would be more apparent at
submaximal levels of TCR stimulation. A deficit in the activation of
the PIP2 pathway would be manifested as a shift in the
concentration-response curve of TCR-stimulated elevations in
[Ca2+]i. However, TCR stimulation of cells
expressing wild type Lck or LckW97A induced similar elevations in
[Ca2+]i at every concentration of anti-TCR
antibody tested (Fig. 5A).
Similarly, the kinetics of these TCR-stimulated elevations in
[Ca2+]i were identical in cells expressing wild
type Lck or LckW97A (Fig. 5B). The time-to-onset, peak
elevation, decline phase, and plateau were all unaltered by
inactivation of the Lck SH3 domain. We confirmed that the Lck SH3
domain was not required for the activation of the PIP2
pathway by measuring PLC
1 phosphorylation in cells expressing
LckW97A. Anti-phosphotyrosine immunoblots of PLC
1 immunoprecipitates
from cells expressing LckW97A showed that PLC
1 tyrosine
phosphorylation was induced upon TCR stimulation (Fig. 5C).
Although lower levels of inducible PLC
1 phosphorylation were
observed in JCaM1/LckW97A cells, it did not alter the ability of these
cells to elicit elevations in [Ca2+]i following
TCR stimulation. Thus, the activation of the PIP2 pathway
following TCR stimulation is not dependent upon the SH3 domain of Lck.
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We also examined the activation of the MAPK pathway following TCR stimulation of cells expressing LckW97A. In contrast to the initiation of TCR signaling and the activation of the PIP2 pathway, the activation of the MAPK pathway was strictly dependent upon the SH3 domain of Lck. MAPK pathway activity was assessed initially by immunoblotting cell lysates with antibodies that specifically recognize the activated forms of the MAP kinases ERK1 and ERK2 and the MAPK kinases MEK1 and MEK2. TCR stimulation of cells expressing LckW97A failed to induce ERK or MEK phosphorylation, whereas stimulation of cells expressing wild type Lck elicited substantial ERK and MEK phosphorylation (Fig. 6A). A kinetic analysis demonstrated that extending the period of TCR stimulation as much as 20 min did not enhance ERK phosphorylation in JCaM1 cells expressing LckW97A (not shown). TCR-independent activation of the MAPK pathway using PMA resulted in phosphorylation of both ERK and MEK irrespective of Lck expression (Fig. 6A). The selective disruption of the MAPK pathway in cells expressing LckW97A demonstrates that an additional requirement for Lck exists in the activation of downstream signaling events, which is distinct from its previously identified role in mediating the initiation of TCR signaling.
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Engagement of the TCR initiates intracellular signaling processes that
culminate in characteristic changes in gene expression, including
expression of the cell-surface marker CD69 (44). In light of the
pronounced deficits in MAPK pathway activation, we suspected that TCR
stimulation of JCaM1 cells expressing LckW97A would be incapable of
inducing CD69 expression (45). Treatment of cells expressing LckW97A
with phytohemagglutinin (PHA) resulted in little or no induction of
CD69 expression, whereas PHA treatment of cells expressing wild type
Lck elicited a 10-fold elevation in median fluorescence intensity (Fig.
6B). TCR-independent stimulation with PMA elicited similar
levels of CD69 expression in all cell lines, regardless of Lck
expression. Thus, the SH3 domain of Lck is required for
TCR-dependent CD69 expression. The inability of LckW97A to
activate the MAPK pathway or induce CD69 expression demonstrates a
critical role for the SH3 domain of Lck in TCR signaling and T-cell activation.
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DISCUSSION |
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We have identified a crucial role for the SH3 domain of Lck in
activation of the MAPK pathway following TCR stimulation. The Lck-deficient cell line JCaM1 was transfected with an altered form of
Lck in which a conserved tryptophan located within the SH3 domain was
mutated to alanine, thereby abolishing Lck SH3 domain function.
Substantial biochemical and genetic evidence has demonstrated that this
conserved tryptophan is essential for ligand binding (28, 42, 43, 46);
thus, it is very unlikely that LckW97A has any residual SH3 domain
function. JCaM1 clones expressing LckW97A failed to induce CD69
expression and MAPK activation upon TCR stimulation, revealing a defect
in TCR signaling. Since LckW97A possessed kinase activity in
vitro, the block in T-cell activation in cells expressing LckW97A
could not be attributed to loss of catalytic function. Furthermore,
cells expressing LckW97A had only minor alterations in the tyrosine
phosphorylation of cellular proteins and exhibited inducible TCR chain phosphorylation and ZAP-70 activation upon TCR stimulation. Thus,
the Lck SH3 domain is not required for the initiation of TCR signaling
but is essential for events that are independent of, or subsequent to,
the recruitment and activation of ZAP-70. Additional analysis of
downstream signaling pathways demonstrated that cells expressing LckW97A were capable of activating the PIP2 pathway
following TCR stimulation; both PLC
1 phosphorylation and subsequent
elevations in [Ca2+]i were independent of Lck SH3
domain function. However, unlike the PIP2 pathway, TCR
stimulation of cells expressing LckW97A failed to induce activation of
ERK or MEK, indicating that there was a selective loss of this TCR
signaling pathway. Thus, the Lck SH3 domain is required specifically
for the activation of the MAPK pathway but not the initiation of TCR
signaling or the activation of the PIP2 pathway.
A current model of TCR signal transduction proposes that Lck and ZAP-70 function sequentially to initiate signaling (1-3). Lck phosphorylates the TCR which allows ZAP-70 recruitment, followed by ZAP-70 phosphorylation and activation, which is also mediated by Lck. The subsequent stimulation of the PIP2 and MAPK pathways is dependent upon the activation of ZAP-70. In the simplest model, the sole role of Lck in TCR signaling would be to recruit and activate ZAP-70. However, our data demonstrate that the involvement of Lck in TCR signaling is more complex and establish that a selective requirement for Lck exists in the activation of downstream signaling pathways. TCR stimulation of JCaM1/LckW97A cells failed to activate the MAPK pathway, despite activation of ZAP-70 and the PIP2 pathway. Recent studies with a constitutively active form of ZAP-70 support the notion that Lck is required for downstream signaling (25). Constitutively active ZAP-70 is able to elicit interleukin-2 gene expression independently of TCR stimulation but only when co-expressed with Lck. Taken together, these studies indicate an additional role for Lck in TCR signaling beyond the recruitment and activation of ZAP-70. Several models could explain the requirement for both activated ZAP-70 and Lck in the stimulation of the MAPK pathway. Lck and activated ZAP-70 may provide separate signals, both of which are needed for activation of the downstream pathway. Alternatively, ZAP-70 and Lck may act together on a common substrate that is essential for the stimulation of the MAPK pathway.
The Lck SH3 domain has been shown to bind several signaling molecules implicated in the regulation of the MAPK pathway. The binding of these target proteins by the Lck SH3 domain may modify their activity as a result of conformational changes, subsequent tyrosine phosphorylation, or recruitment to the membrane. PI-3-K binds to the Lck SH3 domain (30) and is capable of activating the MAPK pathway in some cell types (47). However, it is unlikely that the loss of MAPK activation in JCaM1/LckW97A cells is due to alterations in the function of PI-3-K since we did not observe any alteration in PI-3-K localization or activity following TCR stimulation of JCaM1 cells expressing either wild type Lck or LckW97A (not shown). The proto-oncogene product c-Cbl also associates with the SH3 domain of Lck (29). c-Cbl acts as a negative regulator of MAPK activation (48), although this appears to be the result of a block in the activation of Syk/ZAP-70 making it unlikely that c-Cbl would selectively regulate the MAPK pathway (49). Lck has also been shown to interact directly with the kinases comprising the MAPK signaling pathway, including Raf-1, MEK, and MAPK (50-53) as well as the inhibitor of Ras activity, Ras-GAP (31). Future studies will be required to understand the specific function of the Lck SH3 domain in the activation of the MAPK pathway following stimulation of the TCR.
The SH3 domains of Src family kinases have been proposed to be autoinhibitory. X-ray crystallographic studies examining the structure of Src and Hck revealed that the SH3 domain mediates an intramolecular interaction with an atypical binding site located in the region linking the SH2 and kinase domains (54, 55). Inactivating the SH3 domain of Src induces an 8-10-fold elevation in its kinase activity (42). However, in the present study, mutation of the SH3 domain of Lck caused a comparatively modest 2-fold elevation in its kinase activity. Moreover, deleting the SH3 domain of Lyn reduces its kinase activity (56). These findings suggest that despite their significant structural similarity, the SH3 domains of Src family kinases may not be functionally identical, although it is possible that other regulatory mechanisms may compensate for loss of SH3 domain function in Lck and Lyn.
In summary, we have identified an additional role for Lck in TCR
signaling which is distinct from its ability to mediate ITAM phosphorylation and ZAP-70 activation. Our results demonstrate that
TCR-induced activation of the MAPK pathway, but not the
PIP2 pathway, requires the SH3 domain of Lck. The selective
requirement for Lck SH3 domain function in the MAPK pathway suggests
that the activation of specific downstream signaling events could be controlled by regulating the function of Lck. Such a mechanism may be
involved in thymocyte development where activation of the MAPK pathway
is required for positive selection but not negative selection (57, 58).
Future studies will address the molecular basis of the role of the Lck
SH3 domain in activation of the MAPK pathway.
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ACKNOWLEDGEMENTS |
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We thank Anne Burkhardt, Joe Bolen, Nicolai van Oers, and Arthur Weiss for providing reagents; Jean Maguire and Angus MacNicol for critical review of the manuscript; and Barbara Patai and Judith Austin for technical assistance.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Recipient of an Arthritis Foundation Postdoctoral Fellowship.
¶ Current address: Dept. of Medicine, New York University, New York, NY.
** Supported in part by National Institutes of Health Grant CA71516-01.
Supported in part by a research award from the Arthritis
Foundation. To whom correspondence should be addressed: Dept.
Medicine/MC6084, University of Chicago, 5841 S. Maryland Ave., Chicago,
IL 60637. Tel.: 773-702-4708; Fax: 773-702-2281; E-mail:
dstraus{at}midway.uchicago.edu.
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ABBREVIATIONS |
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The abbreviations used are:
TCR, T-cell antigen
receptor;
ITAMs, immunoreceptor-based tyrosine activation motifs;
MAPK, mitogen-activated protein kinase;
MEK, mitogen-activated protein kinase
kinase;
ERK, extracellular-signal regulated kinase;
PIP2, phosphatidylinositol;
PI-3-K, phosphatidylinositol 3-kinase;
SH2, Src
homology 2;
SH3, Src homology 3;
PAGE, polyacrylamide gel
electrophoresis;
FITC, fluorescein isothiocyanate;
PHA, phytohemagglutinin;
PMA, phorbol 12-myristate 13-acetate;
GST, glutathione S-transferase;
[Ca2+]i, intracellular calcium concentration;
PLC1, phospholipase
C
1.
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REFERENCES |
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