From the Department of Molecular Genetics, Chiba
University Graduate School of Medicine, 1-8-1 Inohana, Chuo-ku, Chiba
260-8790, the
Department of
Neurophysiology, Tokyo Metropolitan Institute of Gerontology, 35-2 Sakae-cho, Itabashi-ku, Tokyo 173-0015, the § Department of
Internal Medicine, Institute of Pulmonary Cancer Research, School of
Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8790, Japan, the §§ Lymphocyte Cell Biology Section,
NIAMS, National Institutes of Health,
Bethesda, Maryland 20892-1820
Received for publication, December 18, 2000, and in revised form, May 10, 2001
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ABSTRACT |
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Jak3 is responsible for growth signals by various
cytokines such as interleukin (IL)-2, IL-4, and IL-7 through
association with the common The function and fate of T cells after antigen recognition are
determined by the quality and quantity of the combination of antigen-recognition signals through the T cell receptor
(TCR)1 complex,
co-stimulation signals through co-stimulation receptors such as CD28,
and growth signals through cytokine receptors such as IL-2 receptor
(R). The TCR complex is composed of clonotypic TCR On the other hand, growth signals in T cells are mediated through
cytokine receptors such as IL-2R, IL-4R, or IL-7R. These cytokine
receptor complexes contain a common During functional characterization of these peripheral T cells in
Jak3-deficient mice, we found that these T cells from the knockout mice
failed to exhibit not only the response to cytokines, which was
expected, but also the early activation signals such as
Ca2+ mobilization upon TCR stimulation (18). These analyses
led us to assume the possible involvement of Jak3 in TCR signaling. Indeed, the present study demonstrates that Jak3 is directly associated with the TCR complex, particularly with the CD3 Cells and Abs--
23-1-8 is a KLH-specific T cell clone and is
maintained by periodic antigen stimulation as previously described
(19). 2B4 and DO11.10 were widely used murine T hybridoma cells
specific for cytochrome c and ovalbumin, respectively. E6.1
is a wild-type Jurkat cell line, and J.CaM1 (20, 21) and P116 (22) are Jurkat mutant cell lines deficient for Lck and ZAP-70, respectively (kindly provided by Dr. A. Weiss, Univ. California San Francisco, CA
and Dr. R. Abraham, Mayo Clinic, MN, respectively). Anti-Jak3 Abs
(anti-peptide Asp169-Gly182 for Western
blotting and anti-Jak3Pro498-Pro794 Ab for precipitation) were raised
in rabbit as described (15). Rabbit antisera against the C and N
termini of human Jak3 were described (23). Anti-TCR Plasmid Construction and Transfection--
To analyze the
association of Jak3 and CD3 Immunoprecipitation, Blotting, and Surface
Biotinylation--
For biochemical analysis, T cells were stimulated
by cross-linking with anti-CD3 In Vitro Binding Assay--
A construct of GST fusion protein
containing the cytoplasmic domain of CD3 Measurement of Intracellula Ca2+ Response--
1 × 106 purified splenic T cells were loaded with Indo-1
(Indo-1 AM, Molecular Probes, Eugene, OR) in the presence of F127 (Molecular Probes), washed, and incubated with
anti-CD4-phycoerythrin and anti-CD8-fluorescein
isothiocyanate(PharMingen, San Diego, CA) in the presence or absence of
anti-CD3 Impaired Early T Cell Activation Signal in Jak3-deficient T
Cells--
We reported previously that splenic T cells from
Jak3-deficient mice exhibited impaired early activation signals such as
Ca2+ response upon cross-linking of the TCR complex with
anti-CD3 Induction of Jak3 Phosphorylation upon TCR Stimulation--
Jak3
is assembled with IL-2 receptor through the association with
Although our results showed that IL-2 signals were not involved in
TCR-induced Jak3 phosphorylation (Fig.
3), we found that T cell hybridoma cells
do not express IL-2R Jak3 Phosphorylation Is Mediated by Lck and ZAP-70--
We next
addressed the question of how Jak3 is activated upon TCR stimulation.
To determine the tyrosine kinase responsible for phosphorylating Jak3,
we first analyzed Jak3 phosphorylation in Jurkat variant cell lines
lacking either Lck (J.CaM1) (20) or ZAP-70 (P116) (22). The wild-type
Jurkat cells induced Jak3 phosphorylation upon TCR cross-linking, but
the Jurkat cell line lacking either Lck or ZAP-70 failed to induce the
phosphorylation of Jak3, suggesting that Lck and ZAP-70 are involved in
phosphorylation of Jak3 as upstream kinases in TCR activation (Fig.
4A).
Next, to elucidate how Lck and ZAP-70 contribute to the phosphorylation
of Jak3, we analyzed by transfection with Jak3 and these kinases. We
used a mutant Jak3 (K855A) bearing a point mutation at the ATP binding
site as a substrate to avoid auto-phosphorylation. Transfection of
either Lck or ZAP-70 induced Jak3 phosphorylation (Fig. 4B,
lanes 2 and 3), and enhanced phosphorylation was
observed when Lck and ZAP-70 were transfected together with Jak3 (Fig. 4B, lane 4). Collectively, these data
demonstrated that Lck and ZAP-70 cooperatively phosphorylate Jak3.
Direct Association of Jak3 with the TCR Complex--
The
phosphorylation data suggested the possibility that Jak3 might directly
associate with the TCR complex. To test this possibility, cell lysates
of normal T cell clones were immunoprecipitated with anti-CD3 Ab and
blotted with anti-Jak3 Ab. As shown in Fig.
5A (top panel),
both anti-CD3 Association of Jak3 with CD3 In the present study, we demonstrated that Jak3 is activated upon
TCR stimulation. Although Beadling et al. (32) reported that
cytokine but not TCR stimulation induced the activation of Jak and
STATs, they analyzed Jak1, Jak2, and various STATs but not Jak3
activation. On the other hand, Welte et al. (33) recently reported that STAT5 is activated upon TCR stimulation and assembles with the TCR complex. We have not observed significant induction of
STAT5 phosphorylation upon TCR stimulation even by using Abs specific
for phosphotyrosine of STAT5 in our
system.2 A recent
paper on STAT5-deficient mice (34) as well as the paper by Beadling
et al. (32) also concluded that STAT5 is not involved in TCR
activation signals. Considering these data that TCR stimulation does
not induce STAT activation despite the fact that a minor population of
STAT5 might be activated upon TCR stimulation, the activation of Jak3
and subsequent signaling through activated Jak3 may dominate the
contribution to T cell activation. The STAT5 activation observed by
Welte et al. (33) might be induced through TCR-activated
Jak3, or alternatively, there may be another pathway to activate STAT5
independently of Jak3 activation. The recent report by Malaviya
et al. demonstrating that Jak3 is phosphorylated in mast
cells by Fc We started to analyze the involvement of Jak3 in TCR signaling from our
previous analysis of Jak3-deficient mice (15, 18). We demonstrated that
peripheral CD4+ T cells in Jak3 It is important to determine how TCR-stimulated Jak3 and
IL-2R-stimulated Jak3 discriminate the downstream signaling pathway in
the same cells without mixing the two activation signals.
Physiologically, the association of Jak3 with both TCR complex and
IL-2R complex indicates that Jak3 plays pivotal roles in regulating T
cell activation/growth at two distinct time points in the sequence of T
cell activation events. Jak3 contributes to initial TCR activation
signals (on day 0), then T cells start to express cytokine receptors
and produce cytokines, and thereafter Jak3 mediates growth signals upon
cytokine binding on the receptors (on days 2-3). Thus, the
constitutive associations of Jak3 to TCR and IL-2R induce T cell
activation and cell growth, respectively. A possible mechanism for the
differential activation of Jak3 by TCR and IL-2 signals may be based on
the physical dissection of these two signaling complexes by membrane compartmentalization. Recent findings revealed that the
glycolipid-enriched membrane compartment (GEM)/raft contains most of
the phosphorylated signaling molecules such as phospho-CD3 The association of Jak3 with chain (
c) in lymphocytes. We found
that T cells from Jak3-deficient mice exhibit impairment of not only
cytokine signaling but also early activation signals and that Jak3 is
phosphorylated upon T cell receptor (TCR) stimulation. TCR-mediated
phosphorylation of Jak3 is independent of IL-2 receptor/
c but is
dependent on Lck and ZAP-70. Jak3 was found to be assembled with the
TCR complex, particularly through direct association with CD3
via
its JH4 region, which is a different region from that for
c
association. These results suggest that Jak3 plays a role not only in
cell growth but also in T cell activation and represents cross-talk of
a signaling molecule between TCR and growth signals.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
dimers
recognizing the antigen-major histocompatibility complex as well
as the CD3 complex responsible for signal transduction and is comprised
of three dimers:
,
, and
homodimers (1, 2). All CD3
chains contain a common signaling motif, ITAM (immunoreceptor tyrosine-based activation motif), within the cytoplasmic region. The
CD3
,
, and
chains each contains one ITAM whereas CD3
possesses three. Antigen recognition by TCR induces tyrosine
phosphorylation of ITAM of the CD3 chains, particularly the CD3
chain, by src family tyrosine kinases, Lck or Fyn, followed by
recruitment of ZAP-70 to the phosphorylated ITAM (3). Subsequently,
activated ZAP-70 induces downstream phosphorylation of various adaptor
proteins including LAT, SLP-76, Vav, and PLC
(4).
chain (
c) as a component
that has been shown to be crucial for signal transduction of cell
growth (5). Functional mutation of
c was reported to result in
X-linked severe combined immunodeficiency (6). Jak3 kinase is
associated with
c (7, 8) and is responsible for transducing growth
signals through the activation of STATs (signal transducers and
activators of transcription) (9-11) and STAM (signal transducing
adaptor molecule) (12). It has been shown in mouse and man that
functional mutation of Jak3 also results in autosomal recessive severe
combined immunodeficiency (13). We and others showed that
Jak3-deficient mice revealed a similar severe combined
immunodeficiency phenotype, characterized by a lack of B cells, NK
cells, and by reduced numbers of most T cells (13-18). Nevertheless,
despite the number of T cells being strongly reduced in these mice, the
pattern of thymocyte development was almost normal, and T cells
recovered with age in the periphery (17, 18).
chain, and is phosphorylated upon TCR stimulation. We found that Jak3 utilizes a
region for assembling with CD3
that is distinct from that with
c.
These data suggest that Jak3 may be involved in TCR activation signals
independently of
c that is involved in growth signals, and they
imply the occurrence of cross-talk by a signaling molecule between
pathways for antigen-recognition signals and growth signals.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
mAb (H57-597)
and anti-CD3
mAb (H146-698A) were kind gifts from Dr. R. Kubo (La
Jolla Institute for Allergy and Immunology, San Diego, CA).
Anti-
c mAbs (TUGm2, TUGm3) were previously described (5, 12) and
provided by Dr. K. Sugamura (Tohoku Univ. Sendai). Anti-CD3
mAb
(145-2C11) was a gift from Dr. Jeff Bluestone (Chicago Univ., Chicago,
IL). Anti-phosphotyrosine mAb (4G10) and anti-FLAG(M2) were purchased
from Upstate Biotechnologies Inc., Lake Placid, NY and SIGMA,
respectively, and goat anti-hamster Ig Ab (GAH) and sheep anti-mouse Ig
Ab (SAM) were from Organon Teknica Corp.
and the phosphorylation of Jak3,
1-3 × 106 293T cells were transiently transfected
with the expressible constructs of Jak3 (pME-Jak3), CD3
(pME-
) as
well as with Lck and ZAP-70 using the
LipofectAMINETMReagent (Life Technologies, Inc.) according
to the manufacturer's protocol. The C-and N-terminal deletion mutants
and the kinase-dead mutant Jak3 (K855A) were previously generated (23).
JH4 deletion mutant of Jak3 lacking
Phe289-Ser432 was constructed. Wild type and
JH4 Jak3 were attached with a FLAG tag and subcloned into pME18S.
All the constructs were confirmed by DNA expressive vector sequencing
using Applied Biosystems PRISM Dye Terminator Cycle Sequencing kit
(PerkinElmer Life Sciences).
mAb (2C11) and GAH as described
previously (24, 25). Stimulated T cells were lysed with a lysis buffer (1% digitonin, 1% Brij 97 or 1% Nonidet P-40/0.1% SDS (radioimmune precipitation buffer), 150 mM NaCl, 5 mM EDTA,
10 mM NaF, 1 mM phenylmethylsulfonyl fluoride,
leupeptin (10 µg/ml), aprotinin (10 µg/ml), antipain (25 µg/ml),
chymostatin (25 µg/ml), pepstatin (10 µg/ml), and 10 mM
iodoacetamide) at 4 °C for 40 min, and the cell lysates were
immunoprecipitated with the indicated Abs followed by analysis on
SDS-PAGE. For Western blotting the proteins were transferred onto a
polyvinylidene difluoride membrane (Millipore), and the membrane was
blotted with Abs and visualized using the ECL detection system
(Amersham Pharmacia Biotech). For analysis of cell-surface labeling,
cells were labeled by surface-biotinylation as previously described
(24, 25).
(GST-
) has been
described (26). 35S-labeled, in vitro translated
Jak3 probe containing the JH2-JH4 region (amino acids 281-783) was
constructed by subcloning of SacI fragment of Jak3 into
pCITE vector and was in vitro transfected using
[35S]methionine. In vitro binding assay using
the 35S-labeled Jak3 probe was performed as described
previously (26, 27). Briefly, purified GST-
was adsorbed onto
glutathione-Sepharose® 4B-beads and incubated with in vitro
translated Jak3 protein. Subsequently, Jak3 attached to the beads was
eluted by 10-50 bead volumes of 20 mM glutathione and
analyzed on SDS-PAGE.
-biotin plus anti-CD4-biotin (RM4-5-biotin). The binding of
RM4-5 to CD4 molecules was not blocked by another anti-CD4 mAb (PL-172)
(data not shown). After washing, T cells were stimulated by
cross-linking with anti-CD3
mAb (2C11) alone or with a mixture of
anti-CD3 and anti-CD4 mAbs followed by co-cross-linking with
streptavidin. Labeled and stimulated cells were subjected to
Ca2+ analysis by FACS Vantage (Becton Dickenson, Mountain
View, CA). The Ca2+ flux was monitored for 512 s, and
the results were analyzed with MULTITIME software (Phoenix Flow
Systems, San Diego, CA).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
mAb (18). In contrast, Thomis et al. (28)
described a similar analysis with a different conclusion, namely, that
Jak3
/
peripheral T cells exhibited Ca2+ flux comparable
with normal T cells upon TCR stimulation. The different results appear
to be explainable by the different stimuli used in the two systems. We
stimulated T cells by cross-linking with anti-CD3
mAb alone, whereas
Thomis et al. utilized stimulation by cross-linking of both
CD3 and CD4 with biotin-conjugated anti-CD3 and anti-CD4 mAbs together
with avidin. It is known that cross-linking of CD4 induces a
severalfold increase of Lck activity (29), and further co-cross-linking
of CD3 and CD4 with biotinylated Abs and avidin induced extremely
strong activation signals for Lck activation and Ca2+ flux.
We assumed that such strong signals might bypass the involvement of
Jak3 for T cell activation. To examine this possibility,
CD4+ T cells from Jak3
/
mice were stimulated by
cross-linking with either CD3 alone or CD3+CD4 in the biotin-avidin
system, and Ca2+ responses were compared (Fig.
1). We found that Jak3
/
T cells failed to elicit Ca2+ flux upon CD3 cross-linking alone as
we previously described (18), but these cells did respond at a level
fairly similar to wild-type T cells upon cross-linking of CD3+CD4 in
the biotin-avidin system. These results suggest that Jak3 is involved
in early TCR activation signals. Therefore, we next tested whether Jak3
is phosphorylated upon TCR stimulation in normal T cells. Because T
cells from Jak3-deficient mice did not proliferate at all and the
cellularity was small, we could not use them for biochemical analysis,
so we used T cell clones and hybridoma cells as described below.
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Fig. 1.
Splenic T cells from Jak3 exhibited
impaired intracellular Ca2+ mobilization. Splenic T
cells from Jak3+/ (left panels) and Jak3
/
mice
(right panels) were loaded with Indo-1 and F127 and then
stained with anti-CD4-phycoerythrin and anti-CD8-fluorescein
isothiocyanate. T cells were stimulated with anti-CD3
mAb (2C11)
alone or anti-CD3
-biotin plus anti-CD4-biotin (RM4-5-biotin)
followed by streptavidin at the time point which resulted in a
white interval in the figures. The binding of RM4-5 to CD4
did not interfere with the binding of another anti-CD4 mAb (PL-172)
(data not shown). Labeled and stimulated cells were subjected to
Ca2+ analysis by FACS Vantage and the results were analyzed
with MULTITIME software.
c and
is known to be phosphorylated upon IL-2 stimulation. Therefore, to
avoid the involvement of IL-2 signaling in testing Jak3 phosphorylation
upon TCR stimulation, a normal T cell clone 23-1-8 was cultured in the
absence of exogenous IL-2 for 2 days, and then T cells in the resting
stage were stimulated with immobilized anti-CD3
mAb for 2-5 min.
The cell lysates of stimulated T cells were immunoprecipitated with
anti-Jak3 Ab and blotted with anti-phosphotyrosine mAb (4G10) and
anti-Jak3 Ab. Tyrosine phosphorylation of Jak3 was induced as early as
2 min (Fig. 2A).
Immunoprecipitation of Jak3 from IL-2 dependent T cell line CTLL-2 was
used as a positive control for phosphorylated Jak3 (Fig. 2A,
lane 6). It is unlikely that IL-2 secreted from the T cells
upon TCR stimulation induced Jak3 phosphorylation during such a short
period. When T cells were also stimulated in the presence of inhibitory
mAbs against IL-2R
and
chains to avoid the contribution of
growth signals through IL-2R, Jak3 phosphorylation was similarly
induced under such condition (data not shown). Furthermore, because T
cell hybridoma cells lack the functional expression of IL-2R, we
stimulated two widely used T cell hybridoma cell lines, 2B4 specific
for cytochrome c (Fig. 2B) and DO11.10 specific
for ovalbumin (data not shown). Jak3 phosphorylation was also observed
upon TCR stimulation in both cell lines. To confirm whether TCR-induced
Jak3 phosphorylation is also observed in normal T cells, purified
splenic T cells were stimulated similarly with anti-CD3
mAb and
immunoprecipitated with anti-Jak3 Ab. As with T cell clones and T cell
hybridoma cells, Jak3 was phosphorylated upon TCR cross-linking in
normal splenic T cells within 2 min (Fig. 2C). These data
demonstrate that Jak3 is phosphorylated upon TCR stimulation
independently of IL-2R signaling.
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Fig. 2.
Phosphorylation of Jak3 upon TCR
stimulation. A, tyrosine phosphorylation of Jak3 upon
TCR stimulation in a normal T cell clone. A Th1 clone 23-1-8 (5 × 107) was stimulated by cross-linking with anti-CD3 mAb
(2C11) and goat anti-hamster (GAH) Ab for the indicated periods. The
cell lysates prepared in 1% digitonin were immunoprecipitated with
anti-Jak3 Ab or rabbit IgG as a control Ab and analyzed on 8%
SDS-PAGE, followed by blotting with anti-PY mAb (4G10) (upper
panel) or anti-Jak3 (lower panel). The result of
blocking IL-2R function was the same in the presence of anti-IL-2R
mAb (3C7) or anti-IL2R
mAb (TM
1) (40 µg/ml each) (data not
shown). The arrow indicates the position of Jak3.
B, Jak3 phosphorylation upon TCR stimulation in T cell
hybridoma cells. 5 × 107 of 2B4 hybridoma cells
specific for cytochrome c/H-2k were stimulated
with 2C11 and GAH for 2 or 5 min, and the cell lysates were
precipitated with anti-Jak3 Ab or rabbit IgG as a control Ab followed
by blotting with 4G10 (upper panel) or anti-Jak3
(lower panel) as described in A. The arrow
indicates Jak3. C, Jak3 phosphorylation in purified splenic
T cells upon TCR stimulation. 5 × 107 B cell-depleted
splenic T cells from C57BL/6 mice were stimulated with 2C11 and GAH.
The cell lysates were precipitated with anti-Jak3 Ab or rabbit IgG as a
control Ab followed by blotting with 4G10 (upper panel) or
anti-Jak3 (lower panel). The cell lysate of
IL-2-unstimulated CTLL-2 was used as a positive control of
phosphorylated Jak3. IP, immunoprecipitation; WB,
Western blotting.
and
chains but do express
c on the cell
surface (data not shown). Then, to avoid the possible involvement of
c in TCR-mediated Jak3 phosphorylation, T hybridoma cells were
stimulated for 2 min in the presence of an anti-
c mAb that is known
to inhibit
c-mediated signals. Despite the fact that all growth
signals through IL-2, IL-4, and IL-7 were completely blocked under this
condition (30, 31), Jak3 was similarly phosphorylated upon TCR
stimulation (Fig. 3A). Furthermore, cross-linking of
c
with anti-
c mAb and anti-rat Ig Ab did not induce Jak3
phosphorylation (Fig. 3A, lane 4), ruling out the possibility that
c cross-linking with Abs induced Jak3
phosphorylation. One other possible mechanism of the involvement of
c in inducing Jak3 phosphorylation upon TCR stimulation is that
c
might be physically assembled in the TCR complex and that Jak3 is
phosphorylated through
c upon T cell activation. To investigate this
possibility, T hybridoma cells were stimulated by TCR cross-linking
after surface biotinylation, and the cell lysates were
immunoprecipitated with anti-CD3
mAb to analyze the TCR complexes on
the cell surface. Although
c was clearly detected with anti-
c Ab,
even if only one-tenth of the lysate was used (Fig. 3B,
lanes 5 and 6), the band corresponding to
c
was not detected by immunoprecipitation with anti-CD3
mAb (Fig.
3B, lanes 1-4), suggesting that
c was not
involved in the TCR complex upon T cell activation. Collectively, these
results indicate that TCR stimulation induces tyrosine phosphorylation of Jak3 independently of
c.
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Fig. 3.
c-independent phosphorylation
of Jak3 upon TCR stimulation. A, Jak3 phosphorylation
in the presence of blocking anti-
c mAb. DO11.10 cells were
stimulated for 2 min with 2C11 and GAH in the presence of 10 µg/ml of
anti-
c mAb (TUGm2) (lane 3). T cells were cross-linked
with anti-
c mAb and anti-Ig Ab (lane 4). The cell lysates
were precipitated with anti-Jak3 Ab and blotted with 4G10 (upper
panel) or anti-Jak3 (lower panel). The arrow
indicates Jak3. B,
c is not involved within the TCR
complex upon TCR stimulation. 5 × 107 DO11.10
hybridoma cells were surface-biotinylated (25) and then stimulated with
2C11 and GAH. The indicated amounts of cell lysates (1.0 corresponds to
5 × 107 cell lysates) were immunoprecipitated with
anti-CD3
mAb, 2C11 (lanes 1-4), anti-
c mAb (TUGm3)
(lanes 5 and 6), and control hamster and mouse
IgG (lanes 7 and 8, respectively). The proteins
were analyzed on 10% SDS-PAGE and detected by the ECL system for
biotinylated proteins. The position of
c is indicated by an
arrow. Molecular size markers are indicated at the
left margin in all figures. IP,
immunoprecipitation; WB, Western blotting.
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Fig. 4.
Lck and ZAP-70 are upstream signaling
pathways for TCR-stimulated Jak3. A, mutant Jurkat
cells failed to phosphorylate Jak3. Lck-deficient (J.CaM1) and
ZAP-70-deficient (P116) mutant Jurkat cell lines and Jurkat cells
(5 × 107 each) were stimulated by cross-linking with
anti-CD3 mAb (OKT3) and anti-mouse Ig, and the cell lysates were
immunoprecipitated with anti-Jak3 Ab followed by blotting with anti-PY
mAb (4G10) (upper panel) or anti-Jak3 (lower
panel). Phosphorylated Jak3 from CTLL-2 under
IL-2-dependent growth was used as a positive control of
Jak3. The arrow indicates Jak3. Note that an ~70-kDa band in the lane
of stimulated Jurkat was reproducibly observed and appears to be STAM.
B, Lck and ZAP-70 cooperatively phosphorylate Jak3. A mutant
Jak3 lacking the kinase activity with the mutation of the ATP binding
site (K855A) was used as the substrate for kinases to avoid
auto-phosphorylation. 293T cells were co-transfected with 4 µg of Lck
and/or ZAP-70. Two days later, cells were lysed, immunoprecipitated
with anti-Jak3, and immunoblotted with mAb 4G10. IP,
immunoprecipitation; WB, Western blotting. The
arrow indicates Jak3.
and CD3
mAbs, which precipitate the TCR-CD3
complex, immunoprecipitated Jak3 whereas a control hamster mAb did not
indicating that Jak3 is physically and constitutively associated with
the TCR complex in normal T cells. On the other hand, Jak1 and Jak2
were not detected in the immunoprecipitation of the TCR complexes (Fig.
5A, middle and bottom panels,
respectively). To examine which CD3 chain interacts with Jak3, we first
tested the CD3
chain because anti-CD3
Ab clearly co-precipitated
Jak3. The expressible cDNAs encoding CD3
and Jak3 were
co-transfected into 293T cells and the cell lysates were
immunoprecipitated with either anti-CD3
(Fig. 5B,
left panel) or anti-Jak3 (Fig. 5B, right
panel) Abs, followed by blotting with anti-Jak3 or CD3
mAbs,
respectively. Jak3 was co-precipitated with CD3
in both experiments
(Fig. 5B, lanes 1, 4), suggesting that
Jak3 is directly associated with CD3
. The direct association between
Jak3 and CD3
was further confirmed by in vitro binding
analysis, where the binding capacity of in vitro translated
35S-labeled Jak3 protein corresponding to JH2-JH4 to a GST
fusion protein containing the cytoplasmic tail of CD3
(GST-
) was
analyzed. As shown in Fig. 5C, Jak3 specifically bound to
GST-
but not to GST, whereas luciferase as a control did not bind to
GST-
. These in vivo and in vitro
results strongly suggest that Jak3 directly associates with the TCR-CD3
complex, particularly with the CD3
, though the possibility of a
similar association with CD3
remains.
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Fig. 5.
Direct association of Jak3 with the TCR
complex. A, co-precipitation of Jak3 with the TCR-CD3
complex in normal T cell clone. T cell clones 23-1-8 (5 × 107) were lysed in 1% digitonin, and the lysate was
immunoprecipitated with either anti-CD3 mAb (2C11), anti-CD3
mAb
(H146), or hamster Ig as a control. The precipitates were analyzed on
8% SDS-PAGE followed by blotting with anti-Jak3 Ab (upper
panel), anti-Jak1Ab (middle panel), and anti-Jak2 Ab
(lower panel). IP, immunoprecipitation. Note that
the amount of Jak3 coimmunoprecipitated with the TCR complex was
approximately equivalent to 1.5 × 104 cell lysate of
23-1-8 clones. B, association between Jak3 and CD3
by
co-transfection into 293T cells. 1 × 106 293Tcells
were transfected with expressible constructs (4 µg each) of Jak3
(pME-Jak3) and/or CD3
(pME-
). The cell lysates in 1% digitonin
were immunoprecipitated with either anti-CD3
mAb (H146) (upper
panel, lanes 1-3) or anti-Jak3 Ab (upper
panel, lanes 4-6), which was followed by blotting with
anti-Jak3 Ab (upper left panel) or H146 (upper right
panel). As controls, each cell lysate was blotted with anti-Jak3
Ab (middle panels) or H146 (lower panel). Note
that a significant band of CD3
, though weak, was detected in the
upper right panel. C, direct binding of Jak3 with
CD3
in in vitro binding assay. 35S-labeled
in vitro translated Jak3 protein containing the JH2
4
region (lanes 3, 4, and 6) and
luciferase as a control protein (lanes 1, 2, and
5) were assessed for interaction with GST fusion product
with the cytoplasmic tail of CD3
(lanes 2 and
4) or GST alone (lanes 1 and 3) as a
control. One fiftieth of each probe used for the binding assay was
applied in lanes 5 and 6. A fraction (~1/150)
of Jak3 probe bound to CD3
(lane 4). The
arrows indicate the position of Jak3.
via Jak3-JH4 Region--
We next
attempted to determine the binding region of Jak3 to CD3
and, in
particular, to discover whether
c and CD3
bind to the same region
of Jak3. For this purpose, we prepared a series of C-terminal (J1-J5)
and N-terminal (
J7 and
J7-5) deletion mutants of Jak3 (Fig.
6, A and B,
upper panels). cDNAs corresponding to various Jak3
mutants were co-transfected with CD3
into 293T cells. The
association between CD3
and the Jak3 mutants was then assessed by
immunoprecipitation with anti-CD3
mAbs, followed by blotting with
Abs against the N or C terminus of Jak3. As shown in Fig.
6A, because deletion of JH1-JH3 did not affect the
association between Jak3 and CD3
, the JH1-3 domains were not
required for the Jak3-CD3
interaction (Fig. 6A,
lanes 1-4). However, the J4 mutant lacking JH1-JH4 as well
as J5 lacking JH1-JH5 failed to bind to CD3
(Fig. 6A,
lane 5), suggesting that the JH4 domain is a crucial region
for Jak3-CD3
association. This conclusion was further supported by
the analysis using N-terminal deletion mutants (Fig. 6B).
Even
J7-5 lacking JH7-JH5 was still co-precipitated with CD3
(Fig. 6B, lanes 1-3), indicating that the
JH7-JH5 domains were not necessary for the CD3
binding. Finally, to
confirm that the JH4 region is required for the association with
CD3
, FLAG-tagged wild-type Jak3 and JH4-deletion mutant,
JH4,
which lacks only the JH4 region, were constructed. As shown in Fig.
6C,
JH4 failed to associate with CD3
. Collectively,
these data demonstrate that JH4 of Jak3 is the crucial domain of Jak3
for the association with CD3
.
View larger version (21K):
[in a new window]
Fig. 6.
Jak3 associates with CD3
through its JH4 region. A, JH4 of Jak3 is
required for CD3
binding. A schematic representation of the
wild-type and C-terminal deletion mutants of Jak3 (J1-J5) is shown in
the upper panel. 3 × 106 293T cells were
transfected with 12 µg of the indicated cDNAs together with
CD3
. Lysates were immunoprecipitated with H146 and blotted with
anti-Jak3 C-terminal Ab (middle left panel).
Expression levels of various mutant Jak3 were analyzed by
immunoblotting with anti-JAK3 C-terminal (middle right
panel) and H146 (lower panel). B, the JAK3 N
terminus is not necessary for CD3
binding. A schematic
representation of the Jak3 N-terminal deletion mutants is shown. 1 × 106 293T cells were transfected with 4 µg of wild-type
or mutant Jak3 together with CD3
cDNAs. Lysates were
immunoprecipitated with H146 and blotted with anti-Jak3 C-terminal Ab
(upper panel). Expression levels of various mutants were
analyzed by immunoblotting with anti-Jak3 C-terminal Ab (middle
panel) and H146 (lower panel). C, JH4 is
responsible for the binding of Jak3 with CD3
. FLAG-tagged wild-type
or JH4-deletion mutants of Jak3 were transfected together with CD3
into 293T cells. Lysates were immunoprecipitated with H146 and blotted
with anti-FLAG mAb (upper panel). Expression levels of wild
type and the mutant Jak3 were analyzed by blotting with anti-FLAG mAb
(middle panel) and H146 (lower panel). The
arrow indicates FLAG wild-type Jak3.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RI cross-linking (35) is consistent with our results in
terms of
c-independent and ITAM-containing receptor-mediated activation of Jak3.
/
mice showed impaired
early activation signals such as Ca2+ mobilization upon T
cell activation. In the present study, we solved the discrepancy from
Thomis et al. (28), which described that Jak3
/
peripheral T cells exhibited Ca2+ flux comparable with
normal T cells, by analyzing stimulation conditions. When Jak3
/
T
cells exhibited impaired Ca2+ response by CD3 cross-linking
alone, strong aggregation of CD3+CD4 in the biotin-avidin system
resulted in the induction of Ca2+ flux in Jak3
/
T cells
comparable with normal T cells (Fig. 1). This result suggests that such
strong signals may bypass some function of Jak3 for early T cell
activation. A similar requirement has been described for CD4/CD8
co-receptors (36) in which strong activation such as super antigen
skips the requirement of co-receptors but the same receptor needs a
co-receptor for stimulation with a weak agonist. Such strong
cross-linking may induce superactivation of Lck, which then bypasses
the requirement of some signaling molecules such as Jak3 and induces
strong activation of ZAP-70 and the downstream signaling pathway.
Therefore, it is possible that under the physiological condition
in vivo of stimulation with antigen/major histocompatibility
complex or a suboptimal activation condition Jak3 may play a role in
augmenting T cell activation signals. Because Jak3 is involved in both
T cell activation and growth, these results provide evidence of the
cross-talk of a signaling molecule between TCR signal and growth signal
in the same T cells. Recently, similar cross-talk between cytokine
signal and TCR signal has been reported in which IFN signals in T cells for growth arrest utilize TCR signal machinery including ZAP-70 and
CD45 (37), whereas IFN signals in cells other than T cells utilize
Jak-STAT pathways. In this case, the choice of TCR machinery versus Jak-STAT pathway by IFN-R appears to be determined in
a cell type-specific manner. In our case, in contrast, Jak3 can be used
in both TCR- and cytokine-signal pathways by assembling with distinct
receptor components, CD3
and
c, in the same cells.
, ZAP-70,
LAT, Lck, and PLC
and is crucial for T cell activation (38-41). It
is possible to assume that TCR-activated Jak3 is localized within
GEM/raft and may be involved in the activation of these TCR signaling
molecules, whereas IL-2-activated Jak3 may not be located within
GEM/raft.
c is mediated by the consensus sequence
box1 (7, 8). CD40 has been reported to associate with Jak3 as a
non-cytokine receptor (42). However, because CD40 contains a box1-like
motif, the association appears to follow a similar rule to
c-associated cytokine receptors. In contrast, the CD3
chain does
not contain such a box1-like motif. However, considering that the
association of STAM with Jak3 has been shown to be mediated through the
ITAM region within STAM (12) in the absence of its phosphorylation, it
might be possible that Jak3 associates with CD3
through the
non-phosphorylated ITAM. In this study, we determined the JH4 region of
Jak3 to be the responsible region for the association with CD3
. The
failure of CD3
binding by the specific JH4-deletion mutant of Jak3
supports this conclusion, although the possibility of an indirect
structural alteration by JH4 deletion is not excluded. In addition,
although we focused on CD3
as a responsible CD3 chain of
Jak3-association, the possibility of a similar association with CD3
still remains because anti-CD3
Ab immunoprecipitated Jak3. In
contrast, it has been demonstrated that
c associates with Jak3
through the N terminus region including JH7-6 (23). Therefore, the
distinct association of Jak3 with CD3
and
c appears to be
regulated through distinct regions of Jak3 as the binding sites (JH4
for TCR, JH7 for
c) as well as through different kinetics during T
cell activation (day 0 for TCR stimulation, days 2-3 for IL-2R
signaling). The precise mechanism and maps of the association between
Jak3 and the TCR-CD3 complex may enable us to discriminate the in
vivo contributions of Jak3 to activation and growth of T cells.
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ACKNOWLEDGEMENTS |
---|
We thank S. Miyatake, S. Taki, and K. Sugamura for discussion; K. Sugamura, J. Bluestone, T. Hirano, G. Koretzky, R. Kubo, H. Wakao, M. Miyasaka, and L. Samelson for Abs; R. Abraham and A. Weiss for Jurkat variant cells; J. Ihle, A. Miyajima, K. Sugamura, and T. Taniguchi for cDNAs; Y. Hamano, K. Sugaya, M. Sakuma, and R. Shiina for experimental help; and H. Yamaguchi for secretarial assistance.
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FOOTNOTES |
---|
* This work was supported by a grant-in-aid for scientific research from the Ministry of Education of Japan and a grant from the Human Frontier Scientific Program.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.
¶ Present address: Laboratory for Lymphocyte Signaling, Rockefeller Univ., 1230 York Ave., New York, NY 10021.
Present address: Dept. of Microbiology and Immunology,
Univ. of California, San Francisco, 152 Parnassus Ave., San
Francisco, CA 94143.
** Present address: Div. of Molecular Membrane Biology, Cancer Research Institute, Kanazawa University, 13-1 Takaramachi, Kanazawa 920-0934, Japan.
¶¶ To whom correspondence should be addressed. Tel.: 81-43-226-2198; Fax: 81-43-222-1791; E-mail: saito@med.m.chiba-u.ac.jp.
Published, JBC Papers in Press, May 10, 2001, DOI 10.1074/jbc.M011363200
2 K. Tomita, K. Saijo, and T. Saito, unpublished observation.
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ABBREVIATIONS |
---|
The abbreviations used are:
TCR, T cell
receptor;
R, receptor;
ITAM, immunoreceptor tyrosine-based activation
motif;
IL, interleukin;
c,
chain;
STATs, signal transducers and
activators of transcription;
STAM, signal transducing adaptor molecule;
Ab(s), antibody(ies);
mAb(s), monoclonal antibody(ies);
GAH, goat
anti-hamster Ig Ab;
PAGE, polyacrylamide gel electrophoresis;
GST, glutathione S-transferase;
IFN, interferon.
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