From the Division of Biochemistry and Cellular
Biology, National Institute of Neuroscience, National Center of
Neurology and Psychiatry, Kodaira, Tokyo 187-8502, Japan, the
§ Department of Cell Biology, University of Virginia,
Charlottesville, Virginia 22908, and the
Division of Cellular
Proteomics, Institute of Medical Science, University of Tokyo,
Shirokanedai, Minato-ku, Tokyo 108-8639, Japan
Received for publication, August 5, 2002, and in revised form, December 2, 2002
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ABSTRACT |
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Chat
(Cas/HEF1-associated signal
transducer) is a novel adaptor protein with an N-terminal
Src homology-2 domain and C-terminal Cas/HEF1 association
domain. We report here the molecular cloning of Chat-H, the
hematopoietic isoform of Chat. Chat-H has an extended N-terminal domain
besides the known Chat domain structures, suggesting a unique function
of Chat-H in hematopoietic cells. Jurkat transfectants overexpressing
Chat-H show a marked increase in interleukin-2 production after
costimulation of T cell receptor and CD28. The degree of JNK activation
is enhanced substantially in the Chat-H transfectants upon
costimulation. The Src homology-2 domain mutant of Chat-H loses this
signal modulating activity. Expression of the Cas/HEF1 association
domain mutant exhibits a dominant negative effect on both JNK
activation and interleukin-2 production. We further found that Chat-H
forms a complex with Pyk2H and enhances its tyrosine 402 phosphorylation, an up-regulator of the JNK pathway. These results
suggest that Chat-H positively controls T cell function via
integrating the costimulatory signals.
The antigen-induced activation of T cell receptor
(TCR)1 triggers a variety of
T cell responses such as cytokine production and cell proliferation,
differentiation, adhesion, and migration (1-3). It has been well
characterized that nonreceptor type tyrosine kinases including Lck,
Fyn, and ZAP-70 play pivotal roles in initiating the TCR signals (4,
5). Several adaptor proteins that comprise various signaling modules
mediating protein-protein or protein-lipid interactions relay the
TCR-elicited signals to induce downstream immune responses (6-8).
In addition to integrating extracellular signals, cell type-specific
expression of the signaling molecules may be critical in determining
the cell type-specific response to extracellular stimuli. SLP-76 and
LAT are expressed exclusively in T cells and function as essential
adaptor proteins in the TCR signaling. Besides the TCR-derived signals,
costimulation signals elicited by engagement of coreceptors such as
CD28 are crucial to induce sufficient immune responses (9). When TCR
activation occurs in the absence of the costimulation signals, T cells
become anergic, and these T cells never produce interleukin-2 (IL-2) upon restimulation of the TCR (10).
TCR-mediated signals activate members of the mitogen-activated protein
kinase (MAPK) family (11-15). These kinases phosphorylate various
transcription factors inducing the expression of several immune
response-related genes. The MAPK subgroups include the extracellular
signal-regulated kinases (ERK), c-Jun N-terminal kinases (JNK), and p38
MAPKs, which differ in their response and substrate specificities. In T
cells, JNK and p38 MAPK are synergistically activated by costimulation
of TCR and CD28 auxiliary receptors. By contrast, no synergy is
observed in the ERK activation. Efficient induction of IL-2 synthesis
appears to require the activation of these three distinct MAPKs.
Anergic T cells exhibit defective activation of ERK and JNK (16).
Recently, we proposed the nonreceptor tyrosine kinase, Pyk2H, as a key
regulator of the JNK pathway leading to IL-2 production by Jurkat cells
(17). However, the molecular mechanisms underlying the differential
regulation of MAPKs, which determine the T cell fate, still remain elusive.
Cas-L/HEF1 functions in TCR and In our previous study, we identified Chat as a Cas/HEF1-associated
adaptor protein (28). Chat consists of an N-terminal SH2 domain, a
C-terminal Cas/HEF1 association domain, and a central serine/proline-rich region containing four potential ERK
phosphorylation sites. Indeed, Chat is phosphorylated by ERK when
stimulated with epidermal growth factor or nerve growth factor.
Overexpression of Chat induces up-regulation of JNK in COS7 cells.
Another group has reported the cDNA cloning of NSP3, the human
ortholog of Chat (29). Association of the NSP3 relative, NSP1, with
tyrosine kinase receptors of the epidermal growth factor or insulin has been demonstrated (29). Chat also shares the most C-terminal sequence
with SHEP1, which binds through its SH2 domain to a
tyrosine-phosphorylated motif of Eph receptors (30). These data
indicate that Chat family proteins are implicated in the signaling
pathways of tyrosine kinases and MAPKs.
We have also described a 115-kDa Chat isoform, which is expressed
primarily in hematopoietic cells and forms a complex with HEF1 (28). In
this study, we report the molecular cloning of the hematopoietic Chat
isoform, Chat-H. Jurkat transfectant cells overexpressing Chat-H show a
marked up-regulation of IL-2 production when stimulated with a
concurrent ligation of TCR and CD28. IL-2 synthesis is suppressed
significantly in cells overexpressing the C-terminal deletion mutant of
Chat-H. The activation of JNK in these transfectants is well
synchronized with the IL-2 production. Chat-H also promotes the
phosphorylation of its tyrosine 402, an intermediate of the JNK
activation. Taken together, we propose a role for Chat-H as a positive
regulator of TCR signaling in Jurkat cells.
Antibodies--
Anti-Chat SH and anti-Chat CT polyclonal
antibodies that recognize both Chat and Chat-H were raised as described
in our previous work (28). Anti-human CD3 monoclonal antibody (OKT3)
was obtained from the American Type Culture Collection, and anti-human
CD28 (CD28.2) was from Immunotech. Anti-Pyk2 (N-19) and anti-Pyk2
(C-19) were purchased from Santa Cruz. Anti-phosphotyrosine 402 Pyk2 was from BIOSOURCE. Anti-FLAG (M2) was purchased from Sigma.
Anti-phosphotyrosine (4G10) was from Upstate Biotechnology Inc.
Antibodies detecting the active form of ERK, JNK, or p38 MAPK were
obtained from Promega Corp., and anti-ERK, anti-JNK, and anti-p38 MAPK
were from Santa Cruz.
cDNA Cloning and Establishment of Stable
Transfectants--
Mouse Chat-H cDNA was isolated from a mouse
spleen Cell Stimulation and Preparation of Cell Lysates--
Jurkat
cells (5 × 106-107 cells) were serum
starved for 4-6 h to become "resting" cells and then were treated
on ice for 15 min with a saturating concentration (10 µg/ml) of OKT3
and/or CD28.2 monoclonal antibodies. Goat anti-mouse IgG (10 µg/ml,
Cappel) was added to the cell suspensions to cross-link CD3 and/or CD28 on the cells. The cells were then immediately placed at 37 °C for
the indicated time periods. After the desired time point, the cells
were lysed in lysis buffer (1% Triton X-100, 25 mM Hepes, pH 7.4, 150 mM NaCl, 5 mM EDTA, 1 mM Na3VO4, 10 mM
Immunoprecipitation and Immunoblotting--
Immunoprecipitation
was carried out by using protein G-Sepharose 4B (Amersham Biosciences)
as described above (17). Alkaline phosphatase treatment of
immunoprecipitates was described previously (31). For in
vitro phosphorylation by active ERK, the immunoprecipitates were
washed three times with ERK buffer (50 mM Hepes, pH 7.5, 1 mM MgCl2, 1 mM dithiothreitol)
followed by resuspension in ERK buffer containing 100 units of
recombinant active ERK (Calbiochem) and 50 µM ATP. After
a 1-h incubation at 30 °C, samples were treated with or without
alkaline phosphatase and eluted by SDS sample buffer. Immunobotting was
performed by the Renaissance enhanced chemiluminescence detection
system (PerkinElmer Life Sciences) with a polyvinylidene difluoride
membrane (Millipore) as described (17).
Determination of ERK, JNK, and p38 MAP Kinase
Activities--
The activated state of MAPKs (ERK, JNK, and p38 MAPK)
was determined by immunoblotting using polyclonal antibodies that
recognize the dually phosphorylated, active form of ERK, JNK, or p38
MAPK. To normalize the amount of loaded samples, the blots were
reprobed with anti-ERK, anti-JNK, or anti-p38 MAPK antibodies. JNK
activity was also determined by a solid phase kinase assay using
glutathione S-transferase (GST)-c-Jun1-79
(Santa Cruz) and glutathione-Sepharose 4B (Amersham Biosciences) as
described (17).
IL-2 Assay--
Jurkat transfectants (2 × 106
cells) were stimulated with either monoclonal antibodies OKT3 and
CD28.2 cross-linked by goat anti-mouse IgG or with 10 ng/ml phorbol
12-myristate 13-acetate (Sigma) and 1 µg/ml calcium ionophore
(A23187; Calbiochem), as described above (17). After a 24-h incubation,
the concentration of IL-2 secreted in the culture supernatant was
measured by using human IL-2 enzyme-linked immunosorbent assay kit
(Endogen Inc.) according to the manufacturer's instructions.
Isolation of Hematopoietic Cell-specific Chat-H
cDNA--
Recently, we identified a novel adaptor protein,
Cas/HEF1-associated signal transducer, Chat (28). Chat is expressed in a wide range of tissues with an apparent molecular mass of 78 kDa. Interestingly, a 115-kDa Chat isoform is detected in hematopoietic tissues by using anti-Chat antibodies. Because it is expressed exclusively in hematopoietic cells, we tentatively named this protein Chat-H, a hematopoietic cell-specific Chat. In this study, we
isolated a cDNA encoding Chat-H from a mouse spleen cDNA
library. Chat-H is encoded by an mRNA isoform that shares most of
the 3'-sequence with Chat. The unique 5'-sequence implies differential
initiation and splicing of the Chat-H transcript in hematopoietic
cells. The Chat-H cDNA sequence is identical to that of SHEP1,
reported as an activated Eph receptor-binding protein (30). Chat-H
consists of 854 amino acid residues; the molecular mass ~94 kDa (Fig.
1). However, when the Chat-H mRNA was
expressed ectopically in 293T cells, the translated product showed an
apparent molecular mass of around 115 kDa on SDS-PAGE as observed in
hematopoietic tissues (data not shown). The N-terminal amino acid
sequence specific for Chat-H (amino acid residues 1-166) does not show
any significant similarity to other known protein sequences.
Establishment of Jurkat Clones Expressing Chat-H Variants--
To
investigate the role of Chat-H in T cell activation, we established
Jurkat transfectants overexpressing Chat-H or its mutants (Fig.
2A). An SH2 domain mutant of
Chat-H, Chat-H-R238K, was engineered by replacing a conserved arginine
with lysine. The loss of function was verified by its abrogated binding
to tyrosine-phosphorylated EphA4 in 293T cell expression system (data
not shown). In Chat-H- TCR Engagement Induces ERK-dependent Phosphorylation of
Chat-H--
First, we examined whether Chat-H becomes tyrosine
phosphorylated in response to TCR stimulation because Chat-H has some
potential target motifs for tyrosine kinases. However, we did not
detect phosphorylation of these tyrosine residues upon TCR engagement (data not shown). Instead, we found that Chat-H in TCR-stimulated Jurkat cells and mouse splenic T cells exhibits reduced mobility on
SDS-PAGE (data not shown). As shown in Fig.
3A, this mobility shift was
also observed in TCR-stimulated Chat-H-overexpressing Jurkat cells
(JT-ChatH, +Stim.). Pretreatment with a MAPK/ERK kinase inhibitor PD98059 prior to TCR ligation suppressed this mobility
change (JT-ChatH, +Stim. +PD). As expected, Chat-H- Chat-H-overexpressing Jurkat Cells Show Enhanced IL-2 Production
after Costimulation of TCR and CD28--
Next, we assessed the IL-2
production level by Jurkat transfectants after costimulation of TCR and
CD28 (Fig. 4). The IL-2 concentration in
the culture supernatant of Chat-H-overexpressing transfectants
(JT-ChatH) was increased up to 4-fold compared with that of
control transfectants (JT-Vector). Chat-H- Chat-H Is Not Involved in the Activation of ERK and p38
MAPK--
The MAPK family comprises three distinct kinases, ERK, JNK,
and p38 MAPK. The activation of all three kinases is required for the
full activation of T cells (11-15). Therefore, we studied the
activation of MAPKs in the Jurkat transfectants upon costimulation of
TCR and CD28 and examined the role of Chat-H in IL-2 production by
Jurkat cells. We first compared the activation kinetics of ERK and p38
MAPK between Jurkat and Chat-H transfectants upon costimulation (Fig.
5A); the detection was carried
out by the antibodies specific for their activated forms. The
activation of ERK and p38 MAPK reached its maximum at 5 min and
decreased to less than 50% at 20 min. Several independent experiments
(data not shown) gave a consensus that there was virtually no
difference in the activation kinetics between these transfectants.
Next, we examined whether ERK and p38 activation is influenced by the presence of various Chat-H mutants. All of the transfectants were found
to be similar in the TCR-induced ERK activation and
costimulation-elicited p38 MAPK activation at 5 min (Fig. 5,
B and C) and at 20 min (data not shown). Here we
show ERK activation data from TCR-stimulated transfectants because
TCR-stimulated cells (Fig. 5B) and TCR/CD28-costimulated cells (data not shown) gave essentially the same results. Thus, the
activation of ERK and p38 MAPK downstream of the TCR is not affected by
the function of Chat-H.
Chat-H Is Essential for the Activation of JNK upon Costimulation of
TCR and CD28--
We next addressed the activation of another MAPK,
JNK, in these transfectants. After concurrent ligation of TCR and CD28, JNK activation estimated by activated JNK-specific antibody was promoted significantly in Chat-H-overexpressing cells compared with the
control Jurkat cells (data not shown). To quantify the JNK activation
level in various transfectants, we employed a solid phase JNK assay.
The JNK activity in JT-Chat-H cells was 5-8-fold higher at 10-40 min
after stimulation than those in the Jurkat control (Fig.
6A). As shown in Fig.
6B, the Jurkat transfectants overexpressing Chat-H and
Chat-H- Chat-H Forms a Complex with Pyk2H and Enhances Phosphorylation of
Its Tyrosine 402--
To reveal the mechanism underlying the JNK
activation by Chat-H, we examined the effect of Chat-H expression on
the tyrosine phosphorylation state of several TCR signal-related
molecules. Vav, SLP-76, LAT, and protein kinase C
Nonreceptor tyrosine kinase Pyk2, highly expressed in cells of the
hematopoietic lineage and in the central nervous system, is proposed as
a key regulator of the JNK signaling pathway in various cellular
responses (32-34). We demonstrated recently that Pyk2H, an
alternatively spliced isoform of Pyk2, is involved in JNK activation in
T cells (17). We further found that the phosphorylation of Pyk2H
tyrosine 402 is crucial for the JNK activation; this is also verified
in a different system, i.e. angiotensin II signaling in
smooth muscle cells (35). As shown in Fig.
7, A and B,
anti-Pyk2H immunoprecipitates from the lysates of JT-Pyk2H included a
significant amount of Chat-H, and anti-Chat-H-precipitates from
JT-Chat-H contained Pyk2H. This coprecipitating activity was abolished
in JT- Recently, we identified a novel adaptor protein, Chat, which forms
a complex with Cas family docking proteins. We have also described a
Chat isoform, Chat-H, abundantly expressed in hematopoietic cells. In
the present study, we report the molecular cloning of the Chat-H
cDNA. Chat-H is encoded by a mRNA isoform that shares most of
the 3'-sequence with Chat. In other words, Chat-H has an extra
N-terminal domain absent in Chat. Chat-H is associated with
tyrosine-phosphorylated HEF1 in both splenic T cells and thymocytes
(Ref. 28 and data not shown), suggesting a possible interplay between
Chat-H and HEF1 in T cell development. By using various Jurkat
transfectants overexpressing Chat-H or its mutants, we provide evidence
that Chat-H is involved in the positive regulation of T cell signaling,
which activates IL-2 gene expression: 1) Chat-H overexpression induces
a marked enhancement of IL-2 production under costimulation with TCR
and CD28; 2) the function of the Chat-H SH2 domain is essential for
this up-regulation; and 3) Chat-H- The activation of ERK, JNK, and p38 MAPK is required for adequate
induction of IL-2 synthesis in T cells (11-15). Indeed, the enzymatic
activities of ERK and JNK are reduced after costimulation of TCR and
CD28 in murine anergic T cells (16). These data suggest the close
correlation between the activities of the MAPK superfamily and T cell
activation. The identification of the signaling components, which
integrate costimulatory signals for the differential activation of
MAPKs, is a key for clarifying T cell fate determination. Intriguingly, we found that the activation level of JNK, but not of ERK or p38, is
greatly augmented in response to the costimulation in Jurkat transfectants overexpressing Chat-H. These cells also exhibit a
dramatic increase in their IL-2 production level. In addition, we
demonstrated the importance of Chat-H-HEF1-interaction in both the
up-regulation of JNK pathway and IL-2 production. Because the members
of CrkL-DOCK2-Rac pathway, downstream of HEF1 in T cells, are
implicated in JNK activation and IL-2 gene expression (24, 25, 27), it
is plausible that Chat-H·HEF1 complexes participate in the positive
regulation of IL-2 synthesis via activation of the JNK pathway.
Pyk2H has been shown to up-regulate JNK activation and subsequent IL-2
production after costimulation of TCR and CD28 (17). This process is
mediated by phosphorylation of Pyk2H tyrosine 402. Interestingly,
Chat-H was found to interact with Pyk2H in a
phosphorylation-independent manner, and overexpression of Chat-H further enhances the tyrosine 402 phosphorylation of Pyk2H upon costimulation. Both activities of Chat-H require the functional Cas/HEF1 association domain. This implicates HEF1 in the connection because of its capability to interact with Chat-H and Pyk2H discretely (Refs. 28 and 34 and data not shown). These findings suggest that
molecular networks involving Chat-H, HEF1, and Pyk2H may function as a
modulator of costimulatory signals to induce efficient T cell activation.
Tyrosine kinases and adaptor proteins play crucial roles in diverse
immune cell signaling pathways (4-8). Our data clearly indicate the
functional requirement of the Chat-H SH2 domain in the Chat-H-mediated
up-regulation of JNK activation and IL-2 synthesis after costimulation.
However, Chat-H SH2 domain target(s) responsible for the augmentation
of these T cell responses have not yet been identified. Fascinatingly,
Chat-H is identical to SHEP1, which binds to activated Eph receptors
via its SH2 domain (30). Eph receptor tyrosine kinases function as
receptors for ephrins, and the ephrin-Eph system is implicated in a
wide range of intercellular communications (36, 37). However, little is
known about the role of the ephrin-Eph system in hematopoietic cells.
It is very intriguing to assess whether the ephrin-Eph system is
involved in T cell activation and differentiation.
Chat-H is phosphorylated by ERK upon TCR stimulation. This raises the
possibility that ERK regulates Chat-H function. Chat-H promotes both
JNK activation and IL-2 production in the absence of four ERK
phosphorylation sites, although the latter activity shows an apparent
reduction. It is possible that there is a subordinate route besides the
JNK pathway for the enhancement of the IL-2 gene expression downstream
of Chat-H. Identification of effector protein(s) whose function is
controlled by ERK-mediated Chat-H phosphorylation will provide some
insights into its significance in the TCR signaling.
In this report, we illuminated the positive regulatory role of a novel
hematopoietic adaptor protein Chat-H in the T cell activation. We also
revealed that interactions via its domain structures are crucial for
the up-regulation of costimulatory signals. Further
investigations on the molecular mechanism of signal integration by
Chat-H and in vivo studies using transgenic mice will be the
next approaches to understanding the physiological function of
Chat-H in the T cell fate determination.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1-integrin
costimulation-mediated induction of cell migration and activation (18,
19). HEF1 belongs to the Cas family of docking proteins consisting of
an SH3 domain, substrate domain, and Src family kinase-binding domain
(20, 21). The substrate domain includes numerous tyrosine phosphorylation motifs that serve as binding sites for CrkL when phosphorylated by Src family kinase(s) in TCR-mediated signaling (22).
Binding of CrkL to HEF1 induces the recruitment of downstream effectors
to the HEF1 sites. Two potent CrkL effectors in T cells, C3G and DOCK2,
activate the small GTPases Rap1 and Rac, respectively (23-25). This
activation further controls cell adhesion and gene expression (26,
27).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ZAP II cDNA library (Stratagene) by hybridization
screening using the mouse Chat cDNA (GenBank AB030442) probe,
followed by nucleotide sequencing. N-terminally FLAG-tagged expression
plasmids of Chat-H and its variants, Chat-H-R238K (the SH2 domain
arginine 238 was mutated to lysine), Chat-H-
MAPK (amino acid
residues 324-456 including four MAPK phosphorylation sites were
deleted), or Chat-H-
CT (C-terminal 83 amino acid residues essential
for Cas/HEF1 association were deleted) were constructed as described
(28); a schematic representation is depicted in Fig. 2A. The
human acute T cell leukemia Jurkat clone E6-1 was obtained from the
American Type Culture Collection. Jurkat cells were transfected with
the expression plasmid together with pcDNA3.1-Hygro by
electroporation (17). The transfectants were selected by 250 µg/ml
hygromycin B (Wako Chemicals). The expressed protein level of each
hygromycin-resistant clone was examined by immunoblot analysis against
the FLAG tag. The expression levels of CD3 and CD28 were determined by
flow cytometry as described previously (17). The clones expressing FLAG-tagged Chat-H mutants with amounts of CD3 and CD28 equivalent to
those of parental Jurkat cells transfected with pcDNA3.1-Hygro vector alone were selected for the following analyses. Established clones were maintained in 250 µg/ml hygromycin B-containing medium.
-glycerophosphate, 10 mM pyrophosphate, 100 units/ml
aprotinin, 10 µg/ml leupeptin, 25 µM
p-nitrophenyl p'-guanidinobenzoate, 1 mM phenylmethylsulfonyl fluoride, 10 mM
iodoacetamide) for 30 min at 4 °C. For treatment with a MAPK
inhibitor, Jurkat cells were incubated with 50 µM PD98059
or a control solvent (dimethyl sulfoxide) for 30 min at 37 °C,
washed, and then stimulated with anti-CD3.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Primary structure of Chat-H. The amino
acid sequence of Chat-H deduced from the nucleotide sequence of mouse
Chat-H cDNA is shown. The SH2 domain sequence is
underlined. MAPK phosphorylation consensus sites are shown
by double underlines. The region that corresponds to the
Cas/HEF1 association domain is indicated on the left side of
the sequence. The junction site between the N-terminal Chat-H-specific
sequence and Chat/Chat-H common region is marked with an
arrowhead.
MAPK, the central region of Chat-H consisting
of four ERK phosphorylation consensus sites was deleted. Based on our
previous observation, the deletion of the Chat C-terminal 83 amino acid
residues is enough to abolish its Cas binding activity (28). Therefore, we constructed Chat-H-
CT; the analogous region was deleted. Impaired interaction between Chat-H-
CT and HEF1 was confirmed by a
coimmunoprecipitation assay (data not shown). The Jurkat clones
expressing these Chat-H mutants were screened by immunoblotting as
shown in Fig. 2B. The transfectants, showing an 8-10-fold
expression level of Chat-H variants over the endogenous level, were
selected for the following functional analyses. We also chose the
clones with similar expression levels of both CD3 and CD28 to the
control cells and established three to four independent clones
expressing each Chat-H-related protein (data not shown).
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Fig. 2.
Establishment of Jurkat transfectants
overexpressing Chat-H variants. A, schematic representation
of Chat-H and its mutants used for establishment of Jurkat
transfectants. Full-length Chat-H (ChatH), the SH2 domain
point mutant (R238K), deletion mutant of four ERK
phosphorylation sites ( MAPK), and the C-terminal Cas/HEF1
association domain mutant (
CT) are shown. B,
expression level of Chat-H variant in each transfectant analyzed by
anti- Chat-H immunoblotting.
MAPK, lacking four potential ERK phosphorylation sites, did not show the
mobility shift. In addition, alkaline phosphatase treatment of Chat-H
reverted the mobility, whereas active ERK induced the mobility change
in vitro (Fig. 3B). These results suggest that Chat-H is phosphorylated by ERK upon TCR engagement. This agrees well
with our previous observation that ERK mediates Chat phosphorylation in
EGF-stimulated PC12 cells (28). There is no difference in HEF1 binding
activity between the nonphosphorylated and phosphorylated states of
Chat-H (data not shown).
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Fig. 3.
ERK-mediated phosphorylation of Chat-H
induced by TCR activation. A, Jurkat transfectants
expressing Chat-H (JT-ChatH) or Chat-H- MAPK
(JT-
MAPK) were incubated with (+Stim.) or
without (
Stim.) anti-CD3 for 3 min. Anti-Chat
immunoprecipitates of each stimulated sample were immunoblotted with
anti-Chat antibody. To evaluate the effect of a MEK inhibitor on Chat-H
phosphorylation, the cells were incubated with 50 µM
PD98059 (+PD) for 30 min before stimulation. B,
JT-Chat-H cells were incubated with (
CD3) or without
(None) anti-CD3 for 3 min. For in vitro
phosphorylation and dephosphorylation experiments, anti-Chat
immunoprecipitates were treated with alkaline phosphatase
(+AP), alkaline phosphatase with phosphatase inhibitors
(+AP+PI), active ERK (+ERK), or active ERK and
alkaline phosphatase (+ERK+AP). The mobility of each Chat-H
band was detected by anti-Chat immunoblotting.
MAPK also
enhanced IL-2 production although the impact was significantly weaker
than that of Chat-H (p < 0.05). This enhancement was
not observed in the transfectant overexpressing the SH2 domain mutant
of Chat-H (JT-R238K). Overexpression of Chat-H-
CT significantly
decreased the IL-2 level (p < 0.01). All of these
transfectants produced essentially the same level of IL-2 in response
to the combined stimulation with phorbol 12-myristate 13-acetate and
A23187, suggesting their equivalent potential in IL-2 synthesis (Fig. 4). Taken together, Chat-H is likely to play a positive role in the
coupling signals from TCR and CD28 to IL-2 production, and the
interactions via both the SH2 and Cas/HEF1 association domains of
Chat-H are essential for the process.
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Fig. 4.
Overexpression of Chat-H promotes IL-2
synthesis after costimulation of TCR and CD28. Jurkat
transfectants (2 × 106 cells/well) expressing vector
alone (JT-Vector), Chat-H (JT-ChatH),
Chat-H-R238K (JT-R238K), Chat-H- MAPK
(JT-
MAPK), and Chat-H-
CT (JT-
CT) were
incubated with (gray columns) or without (black
columns) anti-CD3 and anti-CD28, or phorbol 12-myristate
13-acetate and A23187 (open columns) for 24 h. The
concentration of IL-2 in the supernatants was measured by enzyme-linked
immunosorbent assay. The values shown are mean values ± S.D.
(n = 3).
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Fig. 5.
Chat-H is not involved in the activation of
ERK and p38 MAPK downstream of TCR signaling. A, Jurkat
transfectants expressing vector alone (JT-Vector) or Chat-H
(JT-ChatH) were incubated with anti-CD3 and anti-CD28 for
the indicated times. The whole cell lysates were immunoblotted with
anti-active ERKs (phospho-ERK) or anti-active p38 MAPK
(phospho-p38). B, Jurkat transfectants expressing
vector alone (JT-Vector), Chat-H (JT-ChatH),
Chat-H-R238K (JT-R238K), Chat-H- MAPK
(JT-
MAPK), and Chat-H-
CT (JT-
CT) were
incubated with (+Stim.) or without (
Stim.)
anti-CD3 for 5 min followed by immunoblotting the cell lysates with
anti-active ERKs (upper panel; Phospho-ERK). The
expression level of ERKs in these cells is shown (lower
panel; ERK1, ERK2). C, the same
transfectants used in B were costimulated with
(+Stim.) or without (
Stim.) anti-CD3 and
anti-CD28 for 5 min, then immunoblotted with anti-active p38 MAPK
(upper panel; Phospho-p38). These transfectants
express similar amounts of p38 MAPK (lower panel; p38
MAPK).
MAPK exhibited 3-7-fold higher levels of JNK activity over
that of the control cells. In contrast, the JNK activation level of
JT-
CT was reduced to 30-40% of the controls. The JT-R238K showed
almost the control level of JNK activity. The degrees of JNK activation
in different Jurkat transfectants agree well with their IL-2 production
levels (Figs. 4 and 6B). This strongly suggests the possible
augmentation of IL-2 production by Chat-H via up-regulation of the JNK
activity.
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Fig. 6.
Chat-H overexpression enhances JNK activation
upon costimulation of TCR and CD28. A, Jurkat transfectants
expressing vector alone (JT-Vector) and Chat-H
(JT-ChatH) were stimulated with anti-CD3 and anti-CD28 for
the indicated times, and the lysates were subjected to a solid phase
JNK assay. The activity of endogenous JNK pulled down with
GST-c-Jun1-79 was evaluated by an autoradiogram of
32P-labeled GST-c-Jun (Phospho-GST-c-Jun).
B, Jurkat transfectants expressing vector alone
(JT-Vector), Chat-H (JT-ChatH), Chat-H-R238K
(JT-R238K), Chat-H- MAPK (JT-
MAPK), and
Chat-H-
CT (JT-
CT) were incubated with
(+Stim.) or without (
Stim.) anti-CD3 and
anti-CD28 for 5 min followed by the JNK assay described above. Relative
JNK activities are indicated below.
are tyrosine
phosphorylated upon TCR stimulation and implicated in the activation of
MAPKs (4-8, 11). However, we did not detect significant alterations in
their phosphorylation levels between the control and
Chat-H-overexpressing cells (data not shown). Tyrosine phosphorylation
levels of Lck, ZAP-70, Fyn, phospholipase C-
, and
phosphatidylinositol 3-kinase also did not show any significant
changes (data not shown).
CT, suggesting a HEF1-mediated interaction between Chat-H and
Pyk2H. Neither tyrosine 402 phosphorylation of Pyk2H nor
ERK-dependent Chat-H phosphorylation induced by TCR
activation influenced the extent of the Chat-H-Pyk2H interaction.
Intriguingly, overexpression of Chat-H increased the phosphorylation
level of Pyk2H tyrosine 402 at about 5-fold over the control upon
costimulation of TCR and CD28, whereas Chat-H-
CT overexpression
decreased it (Fig. 7C). These data suggest that Chat-H and
Pyk2H may function together in the integration of costimulatory signals
for JNK activation.
View larger version (34K):
[in a new window]
Fig. 7.
Chat-H functionally interacts with Pyk2H in T
cells. A, Jurkat transfectants expressing Chat-H
(JT-ChatH) or Chat-H- CT (JT-
CT) were
incubated with (+Stim.) or without (
Stim.)
anti-CD3. Cell lysates were immunoprecipitated with anti-Chat-H
(ChatH) or control rabbit IgG (Rab.IgG) followed
by immunoblotting with anti-Pyk2H (left panel) or antibody
specific for phosphotyrosine 402 of Pyk2H (right panel).
B, upper panel, Pyk2H-expressing Jurkat
transfectant (JT-Pyk2H) was stimulated with anti-CD3. The
amount of Chat-H in anti-Pyk2H immunocomplex (Pyk2H) or its
control (Rab.IgG) was examined as in A. Lower panel, the same lysates were immunoblotted with
anti-Chat-H. C, Jurkat transfectants expressing vector alone
(JT-Vector), Chat-H (JT-ChatH), or Chat-H-
CT
(JT-
CT) were incubated with (+Stim.) or
without (
Stim.) anti-CD3 and anti-CD28 followed by
anti-Pyk2H immunoprecipitation. The immunoprecipitates were
immunoblotted with anti-phosphotyrosine 402 Pyk2H. The
numbers under the panel represent the relative
phosphorylation level of Pyk2H tyrosine 402.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
CT, the Cas/HEF1 association
domain mutant, shows a dominant negative effect on the IL-2 production.
Thus, Chat-H is likely to play a key role in coupling costimulatory
signals to IL-2 gene expression collaborating with HEF1 and unknown
target(s) of its SH2 domain.
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ACKNOWLEDGEMENTS |
---|
We thank D. J. Webb, K. Momotani, R. B. Riggins, and A. H. Bouton for critical reading of the manuscript. We also thank the members of Division of Biochemistry and Cellular Biology, National Institute of Neuroscience for helpful discussions and technical advice.
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FOOTNOTES |
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* This work was supported in part by a grant for cancer cell biology from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AB043953.
¶ Recipients of a domestic research fellowship from the Japan Science and Technology Corporation.
** To whom correspondence may be addressed: Division of Cellular Proteomics, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan. Tel.: 81-3-5449-5314; Fax: 81-3-5449-5314; E-mail: katagiri@ims.u-tokyo.ac.jp or hattoris{at}ims.u-tokyo.ac.jp.
Published, JBC Papers in Press, December 14, 2002, DOI 10.1074/jbc.M207942200
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ABBREVIATIONS |
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The abbreviations used are: TCR, T cell receptor; Chat, Cas/HEF1-associated signal transducer; Chat-H, hematopoietic isoform of Chat; ERK, extracellular signal-regulated kinase; GST, glutathione S-transferase; IL-2, interleukin-2; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; SH domain, Src homology domain.
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REFERENCES |
---|
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---|
1. | Paul, W. E., and Seder, R. A. (1994) Cell 76, 241-251[Medline] [Order article via Infotrieve] |
2. |
Woods, M. L.,
and Shimizu, Y.
(2001)
J. Leukocyte Biol.
69,
874-880 |
3. | Serrador, J. M., Nieto, M., and Sánchez-Madrid, F. (1999) Trends Cell Biol. 9, 228-232[CrossRef][Medline] [Order article via Infotrieve] |
4. | Qian, D., and Weiss, A. (1997) Curr. Opin. Cell Biol. 9, 205-212[CrossRef][Medline] [Order article via Infotrieve] |
5. | Werlen, G., and Palmer, E. (2002) Curr. Opin. Immunol. 14, 299-305[CrossRef][Medline] [Order article via Infotrieve] |
6. | Rudd, C. E. (1999) Cell 96, 5-8[Medline] [Order article via Infotrieve] |
7. |
Leo, A.,
Wienands, J.,
Baier, G.,
Horejsi, V.,
and Schraven, B.
(2002)
J. Clin. Invest.
109,
301-309 |
8. | Samelson, L. E. (2002) Annu. Rev. Immunol. 20, 371-394[CrossRef][Medline] [Order article via Infotrieve] |
9. |
Frauwirth, K. A.,
and Thompson, C. B.
(2002)
J. Clin. Invest.
109,
295-299 |
10. | Powell, J., Ragheb, J. A., Kitagawa-Sakakida, S., and Schwartz, R. H. (1998) Immunol. Rev. 165, 287-300[Medline] [Order article via Infotrieve] |
11. | Rincón, M., Flavell, R. A., and Davis, R. J. (2001) Oncogene 20, 2490-2497[CrossRef][Medline] [Order article via Infotrieve] |
12. | Franklin, R. A., Tordai, A., Patel, H., Gardner, A. M., Johnson, G. L., and Gelfand, E. W. (1994) J. Clin. Invest. 93, 2134-2140[Medline] [Order article via Infotrieve] |
13. | Su, B., Jacinto, E., Hibi, M., Kallunki, T., Karin, M., and Ben-Neriah, Y. (1994) Cell 77, 727-736[Medline] [Order article via Infotrieve] |
14. |
Nishina, H.,
Bachmann, M.,
Oliveira-dos-Santos, A. J.,
Kozieradzki, I.,
Fischer, K. D.,
Odermatt, B.,
Wakeham, A.,
Shahinian, A.,
Takimoto, H.,
Bernstein, A.,
Mak, T. W.,
Woodgett, J. R.,
Ohashi, P. S.,
and Penninger, J. M.
(1997)
J. Exp. Med.
186,
941-953 |
15. |
Matsuda, S.,
Moriguchi, T.,
Koyasu, S.,
and Nishida, E.
(1998)
J. Biol. Chem.
273,
12378-12382 |
16. | Li, W., Whaley, C. D., Mondine, A., and Mueller, D. L. (1996) Science 271, 1272-1276[Abstract] |
17. |
Katagiri, T.,
Takahashi, T.,
Sasaki, T.,
Nakamura, S.,
and Hattori, S.
(2000)
J. Biol. Chem.
275,
19645-19652 |
18. |
Ohashi, Y.,
Iwata, S.,
Kamiguchi, K.,
and Morimoto, C.
(1999)
J. Immunol.
163,
3727-3734 |
19. |
Kamiguchi, K.,
Tachibana, K.,
Iwata, S.,
Ohashi, Y.,
and Morimoto, C.
(1999)
J. Immunol.
163,
563-568 |
20. | Minegishi, M., Tachibana, K., Sato, T., Iwata, S., Nojima, Y., and Morimoto, C. (1996) J. Exp. Med. 184, 1365-1375[Abstract] |
21. | Bouton, A. H., Riggins, R. B., and Bruce-Staskal, P. J. (2001) Oncogene 20, 6448-6458[CrossRef][Medline] [Order article via Infotrieve] |
22. | Kanda, H., Mimura, T., Hamasaki, K., Yamamoto, K., Yazaki, Y., Hirai, H., and Nojima, Y. (1999) Immunology 97, 56-61[CrossRef][Medline] [Order article via Infotrieve] |
23. |
Ohashi, Y.,
Tachibana, K.,
Kamiguchi, K.,
Fujita, H.,
and Morimoto, C.
(1998)
J. Biol. Chem.
273,
6446-6451 |
24. |
Nishihara, H.,
Maeda, M.,
Oda, A.,
Tsuda, M.,
Sawa, H.,
Nagashima, K.,
and Tanaka, S.
(2002)
Blood
100,
3968-3974 |
25. | Nishihara, H., Maeda, M., Tsuda, M., Makino, Y., Sawa, H., Nagashima, K., and Tanaka, S. (2002) Biochem. Biophys. Res. Commun. 296, 716-720[CrossRef][Medline] [Order article via Infotrieve] |
26. | Bos, J. L., de Rooij, J., and Reedquist, K. A. (2001) Nat. Rev. Mol. Cell. Biol. 2, 369-377[CrossRef][Medline] [Order article via Infotrieve] |
27. |
Takai, Y.,
Sasaki, T.,
and Matozaki, T.
(2001)
Physiol. Rev.
81,
153-208 |
28. |
Sakakibara, A.,
and Hattori, S.
(2000)
J. Biol. Chem.
275,
6404-6410 |
29. |
Lu, Y.,
Brush, J.,
and Stewart, T. A.
(1999)
J. Biol. Chem.
274,
10047-10052 |
30. |
Dodelet, V. C.,
Pazzagli, C.,
Zisch, A. H.,
Hauser, C. A.,
and Pasquale, E. B.
(1999)
J. Biol. Chem.
274,
31941-31946 |
31. |
Sakakibara, A.,
Furuse, M.,
Saitou, M.,
Ando-Akatsuka, Y.,
and Tsukita, S.
(1997)
J. Cell Biol.
137,
1393-1401 |
32. | Tokiwa, G., Dikic, I., Lev, S., and Schlessinger, J. (1996) Science 273, 792-794[Abstract] |
33. |
Yu, H., Li, X.,
Marchetto, G. S., Dy, R.,
Hunter, D.,
Calvo, B.,
Dawson, T. L.,
Wilm, M.,
Anderegg, R. J.,
Graves, L. M.,
and Earp, H. S.
(1996)
J. Biol. Chem.
271,
29993-29998 |
34. | Avraham, H., Park, S.-Y., Schinkmann, K., and Avraham, S. (2000) Cell. Signal. 12, 123-133[CrossRef][Medline] [Order article via Infotrieve] |
35. | Frank, G. D., Eguchi, S., Motley, E. D., Sasaki, T., and Inagami, T. (2001) Biochem. Biophys. Res. Commun. 286, 692-696[CrossRef][Medline] [Order article via Infotrieve] |
36. |
Holder, N.,
and Klein, R.
(1999)
Development
126,
2033-2044 |
37. | Schmucker, D., and Zipursky, S. L. (2001) Cell 105, 701-704[CrossRef][Medline] [Order article via Infotrieve] |