From the Division of Allergy, La Jolla Institute for Allergy and
Immunology, San Diego, California 92121 and the
Department of Stem Cell Regulation, The Institute of
Medical Science, The University of Tokyo, 4-6-1 Shirokanedai,
Minato-ku, Tokyo 108, Japan
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ABSTRACT |
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Cross-linking of the high affinity IgE receptor
(FcRI) on mast cells induces secretion of cytokines, including
interleukin (IL)-2, through transcriptional activation of cytokine
genes. Previously, defects in the gene coding for Bruton's tyrosine
kinase (Btk) were shown to result in defective cytokine production in mast cells, and thereby mice carrying btk mutations
exhibited diminished anaphylactic reactions in response to IgE and
antigen. In this study, we provide evidence that the transcription
factors involved in the IL-2 gene expression in T cells are also
required for maximal activation of the IL-2 gene in Fc
RI-stimulated
mast cells. Among them, AP-1 (Jun/Fos) and NF-AT were identified as candidate transcription factors that are regulated by Btk. Consistent with our previous data indicating that Btk regulates stress-activated protein kinases, c-Jun N-terminal kinase (JNK), c-Jun and other JNK-regulatable transcription factors are activated by Fc
RI
cross-linking in a Btk-dependent manner. Further,
Fc
RI-induced IL-2 gene activation is dependent on c-Jun and a
component, SEK1, of its upstream activation pathway. Collectively,
these data demonstrate that Btk regulates the transcription of the IL-2
gene through the JNK-regulatable transcription factors in
Fc
RI-stimulated mast cells.
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INTRODUCTION |
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Cross-linking of antibody-bound high affinity IgE receptor
(FcRI)1 with multivalent
antigen initiates allergic reactions by inducing degranulation, lipid
mediator release, and cytokine secretion in mast cells (1, 2). Fc
RI
is a heterotetrameric receptor composed of an IgE-binding
-subunit,
a
-subunit with four transmembrane domains, and two disulfide-bonded
-subunits (3). Several protein-tyrosine kinases were shown to be
activated following receptor cross-linking; Lyn associated with Fc
RI
-subunit is believed to phosphorylate
- and
-subunits (4, 5).
Phosphorylated immunoreceptor tyrosine-based activation motifs on
-and
-subunits recruit more Lyn and Syk, respectively (5-8).
These receptor-bound protein-tyrosine kinases are activated and
phosphorylate target proteins such as phospholipase C-
. Activated
phospholipase C-
hydrolyzes phosphatidylinositol 4,5-bisphosphate
into two second messengers; inositol 1,4,5-trisphosphate mobilizes
Ca2+ from intracellular storage sites, and
diacylglycerol activates protein kinase C (9). Both protein kinase
C and Ca2+ are required for degranulation (10).
Bruton's tyrosine kinase (Btk) plays an indispensable function in B
cell development, as exemplified in human (X-linked agammaglobulinemia) and mouse (xid) immunodeficiencies (11-14). Btk is also
implicated in signal transduction for several immune cell and cytokine
receptors (15, 16). In contrast with Lyn and Syk, Btk and Emt (also known as Itk and Tsk) do not physically associate with FcRI. However, these Tec family protein-tyrosine kinases are activated by
Fc
RI cross-linking (17, 18). Recently, Btk was shown to play an
important role in the Fc
RI signaling
system.2 Thus, xid
(with a substitution of Arg-28 in Btk with Cys) and btk null
mice exhibited diminished anaphylactic reactions. Correspondingly, Fc
RI-induced cytokine production was severely impaired in mast cells
derived from these mutant mice. Defects in the Fc
RI-induced production of interleukin (IL)-2 and tumor necrosis factor
(TNF-
) in the mutant cells were found in the transcription of these
cytokine genes.2
FcRI cross-linking induces activation of three major
mitogen-activated protein (MAP) kinases, i.e. ERK1/2,
JNK1/2, and p38 (20-25). Activities of MAP kinases are regulated
through a unique set of protein kinases in a cascade, e.g.
Raf-1
MEK1/2
ERK1/2 and MEKK1
SEK1 (also known as MKK4,
MEK4, or JNKK)
JNK1/2 (26-28). Phosphorylation targets of MAP
kinases include transcription factors. Typically, JNK phosphorylates
c-Jun, a component of the AP-1 transcription factor, at the critical
serine residues (Ser-63 and Ser-73) in the activation domain of c-Jun
(29, 30), causing increased c-Jun transcriptional activity (31-33).
More recently, similar phosphorylation and transcriptional activation
by JNK were shown for other transcription factors such as ATF2 (34, 35), Elk-1 (36), and Sap-1a (37). xid and btk
null mutations affect JNK activities severely and, to a lesser
extent, p38 in Fc
RI-stimulated mast cells, while ERK activities are
not significantly changed (25).
Transcriptional regulation of IL-2 and TNF- genes has been
extensively characterized in T cells. IL-2 promoter activity depends on
the 300-base pair region upstream of the transcription initiation site
(38-40). Several cis-acting elements that are binding sites for the nuclear factor of activated T cells (NF-AT), nuclear
factor-
B (NF-
B), CD28RC, AP-1, and Oct have been identified (41,
42). Cooperation of these transcription factors is required for maximal activation of the IL-2 promoter. NF-AT, a family of related proteins, exists in the cytoplasm before activation and translocates to the
nucleus in response to an increase in intracellular concentrations of
Ca2+ (43). This translocation depends on dephosphorylation
of NF-AT by a Ca2+- and calmodulin-dependent
phosphatase, calcineurin, which is a major target of the
immunosuppressive drugs cyclosporin A and FK506. Translocated NF-AT
forms a complex with a ubiquitous nuclear factor AP-1 to exert its
transcriptional action (reviewed in Ref. 44). NF-
B is composed of
Rel family members of p50 and p65 (reviewed in Ref. 45). p50/p65
heterodimers seem to stimulate the transcription of the IL-2 gene,
while p50/p50 homodimers inhibit IL-2 gene transcription (46). The IL-2
promoter region contains two conventional and two noncanonical AP-1
binding sites. The latter AP-1 sites exist in a close apposition to the
distal NF-AT and proximal Oct sites. AP-1 sites are bound by Jun/Fos or
Jun/Jun dimers. The binding activity for the proximal Oct (Octp) site is constitutive, although the Octp site-dependent
transcription requires T cell stimulation (40). Similar to the IL-2
gene, transcriptional activation of the TNF-
gene induced by T cell receptor stimulation involves NF-ATp and ATF-2/Jun heterodimer proteins
bound to the
3 site and a cyclic AMP response element, respectively,
in the 200-base pair TNF-
promoter (47-49).
In this study, we determined which cis-regulatory elements
in the IL-2 gene promoter are involved in FcRI-induced activation of
the IL-2 gene. Among the transcription factors involved in activation
of the IL-2 gene, c-Jun and other JNK-activable transcription factors
are shown to be regulated by Btk. Since Btk regulates JNK activity, we
investigated the possible involvement of the MEKK1
SEK1
JNK
pathway in the IL-2 gene activation in mast cells.
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EXPERIMENTAL PROCEDURES |
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Reagents-- B6/129 F2 mice were purchased from Jackson Laboratory. btk null mice (50) originally provided by Drs. Wasif N. Khan and Frederick W. Alt were bred in an animal facility in La Jolla Institute for Allergy and Immunology. Recombinant rat stem cell factor was kindly donated by Amgen. Anti-Btk antibody was described previously (51). Antibodies against MEK-4 (SEK1) and MEKK1 were from Santa Cruz Biotechnology, Inc. Anti-phospho-c-Jun(Ser-63) and anti-c-Jun antibodies were purchased from New England Biolabs, and anti-EE antibody was a kind gift of Dr. Arpad Molnar.
Cells-- Bone marrow cells from femurs of B6/129 F2 (wild-type (WT)) and btk null mice with the same genetic background were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum, 50 µM 2-mercaptoethanol, 2 mM glutamine, and IL-3 (culture supernatants of mouse IL-3 gene transfected cells). For transient transfection experiments, mast cells (BMMC for bone marrow-derived mast cells) after 4 weeks of culture were used. At this point, more than 95% of the live cells are immature mast cells. The lack of Btk expression in btk null mice was routinely monitored by immunoblotting of spleen cell lysates with anti-Btk.
DNA Constructs--
WT and K430R btk cDNAs in
expression vectors, pMX-puro (a retroviral vector) and pME18S, have
been described (25, 52). An IL-2 gene promoter construct (IL-2Luc,
encompassing nucleotides 321 to +46) fused with the firefly
luciferase gene and some mutated versions of this construct,
i.e. mut NF-ATdLuc, mut NF-
BLuc, mut AP-1pLuc, and mut
OctpLuc, were described previously (53). Several more mutants of the
IL-2 promoter were generated by two-step polymerase chain reactions in
this study (Fig. 1A) and cloned into the
KpnI/HindIII site of a luciferase reporter
vector, pUC00Luc (54). Mutations were confirmed by nucleotide
sequencing. Luciferase reporter constructs in the pGL2-promoter vector
(Promega), containing multiple copies of the NF-ATd/AP-1 (
290 to
261, seven times), NF-
B (
211 to
192, eight times), or
AP-1/Octp (
97 to
64, seven times) sites, were described (54).
pG5E1bLuc (55), a reporter plasmid containing five copies of GAL4
binding sites fused with the firefly luciferase gene, and GAL4 DNA
binding domain expression vectors (56) fused with activation domains of
transcription factors, c-Jun (Ref. 57), c-Jun(S63/73A), ATF-2 (Ref.
34), ATF-2(T69/71A), Elk-1 (Ref. 59), Elk-1(S383A), Sap-1a (Ref. 37),
and Sap-1a(A361/366/381/387/420/425) were kindly provided by Drs.
Michael Karin, Richard Treisman, and Ralf Janknecht. Jun in 282 expression vector (Ref. 61) and its empty vector (pSR
MSVtkneo), SEK1(K/R) (Ref. 62) and its empty vector (pEBG), and
MEKK1 tagged
with an EE epitope (63) and its empty vector (pCMV5) were donated by
Drs. Charles L. Sawyers, John M. Kyriakis, and Arpad Molnar.
Stable Transfectants--
Mast cells derived from btk
null mice after 2 weeks of culture in IL-3 were incubated in the
presence of rat stem cell factor as well as IL-3 for another 2 weeks to
accelerate cell proliferation before retroviral infection. pMX-puro
vectors containing no cDNAs, WT btk cDNA, or K430R
btk cDNA were transduced into BOSC-23 packaging cells (64) with LipofectAMINE (Life Technologies, Inc.) according to
the manufacturer's instructions. Culture supernatants were recovered
48 h after transfection. Virus titers (1-5 × 105 colony-forming units/ml) in the supernatants were
measured by infecting NIH/3T3 cells and selecting in 1 µg/ml
puromycin. Mast cells cultured in the presence of stem cell factor were
infected with the retroviruses in the presence of 10 µg/ml polybrene.
Forty-eight h after infection, selection in puromycin (1.5 µg/ml) was
started to obtain sufficient numbers of drug-resistant cells. Drug was omitted from the culture 2 days before transient transfection. Successful transfection by WT btk cDNA was routinely
confirmed by restored secretion of IL-2 and TNF- from
immunologically stimulated transfectants.2
Transient Transfection and Luciferase Assays-- BMMC or stably transfected mast cells (1-1.5 × 107 cells) prepared as described above were transfected with 5-10-µg reporter plasmids together with or without the indicated combinations of expression plasmids by electroporation at 400 V and 950 microfarads using a Bio-Rad Gene Pulser II system. Twenty-four h after transfection, cells were started to be sensitized overnight with anti-dinitrophenyl monoclonal IgE antibody. Forty-one h after transfection, cells were left unstimulated or stimulated with antigen, 30 ng/ml dinitrophenyl conjugates of human serum albumin for 7 h before cell harvest. Cells were lysed in 0.2% Triton X-100 in 100 mM potassium phosphate (pH 7.8)/1 mM dithiothreitol. Luminescence of cleared lysates after the addition of ATP and luciferin solutions was measured using a Monolight 2010 luminometer (Analytical Luminescence Laboratory).
Immunoblotting-- Cells were lysed in 1% Nonidet P-40-containing lysis buffer (65), and cleared lysates were analyzed by SDS-polyacrylamide gel electrophoresis followed by electroblotting. Blots were blocked and incubated consecutively with primary antibody, horseradish peroxidase-conjugated secondary antibody, and enhanced chemiluminescence reagents (Amersham Pharmacia Biotech or NEN Life Science Products). Fluorescent bands were visualized by exposure to ReflectionTM autoradiography films (NEN Life Science Products).
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RESULTS |
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AP-1 and NF-AT Elements Are Crucial for IL-2 Gene Transcription in
FcRI-stimulated Mast Cells--
We and others recently showed that
rodent mast cells, including BMMC, secrete IL-2 in addition to numerous
other cytokines upon Fc
RI cross-linking (66, 67). Transcriptional
activation of the IL-2 gene turned out to be a major mechanism for this
phenomenon.2 In the murine IL-2 promoter region, several
cis-regulatory elements have been identified (41, 42).
Therefore, it is interesting to determine which element is important
for IL-2 gene transcription in mast cells. To this end, expression of
IL-2 promoter-luciferase constructs with mutations at each
cis-element was measured in transiently transfected BMMC
derived from B6/129 F2 or CBA/J mice (Fig.
1). Mutations in the distal NF-AT
(NF-ATd, positions
285 to
281), AP-1 next to the NF-ATd site
(NF-ATd/AP-1,
277 to
274), distal Oct (Octd,
253 to
251),
NF-
B (
206 to
202), CD28RE (
160 to
157), proximal AP-1
(AP-1p,
151 to
149), proximal NF-AT (NF-ATp,
137 to
133), AP-1
next to the proximal Oct site (AP-1/Octp,
86 to
83), or proximal
Oct (Octp,
79 to
77) sites drastically reduced Fc
RI-induced
transcriptional activation of the IL-2 promoter, while the effect of
mutations at the distal AP-1 site (positions
185 to
183) was
relatively small (
(B6/129 F2) or
(CBA/J) of the
activity of the WT promoter). These results indicate that most of the
known cis-regulatory elements are required for maximal
activation of the IL-2 promoter in mast cells as well as in T cells.
The most severe impairments in Fc
RI-mediated IL-2 promoter
activation were observed with the mutant promoters containing mutated
NF-ATd, NF-ATd/AP-1, NF-ATp, or AP-1/Octp sites in BMMC from both mouse strains (Figs. 1, B and C), suggesting that these
AP-1 and NF-AT elements are crucial for Fc
RI-mediated IL-2 gene
transcription in mast cells. These data are consistent with the
accumulated observations on T cells that NF-AT family and AP-1 proteins
play essential roles in the transcription of the IL-2 gene (44).
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Btk Regulates IL-2 Gene Transcription through AP-1 and/or NF-AT
Sites--
We recently showed that FcRI-induced transcriptional
activation of the IL-2 as well as TNF-
genes is severely compromised in xid and btk null mast cells.2 To
further confirm this observation, we measured transcriptional activities of the IL-2 promoter in btk null BMMC that were
stably transfected with the empty vector, WT btk, or
kinase-dead (K430R) btk cDNAs. Comparable expression of
WT and K430R Btk in the transfectants was confirmed by immunoblotting
(Fig. 2A). Upon Fc
RI
cross-linking, the transcriptional activity was strongly induced in
WT btk transfected cells compared with those in vector or
kinase-dead btk transfected cells (Fig. 2A). This
result demonstrates that Btk regulates expression of the IL-2 gene and
that the intact enzymatic activity of Btk is required for this
activity. This Btk function could be ascribed to its effects on mast
cell differentiation. However, this possibility is unlikely for two
reasons. First, btk null BMMC is phenotypically indistinguishable from WT BMMC.2 Second, transient
expression of WT Btk, but not vector or K430R Btk, in btk
null BMMC restored the Fc
RI-induced transcriptional activation
of the IL-2 gene (Fig. 2B). In these experiments, cells were
harvested for luciferase assays only 48 h after transfection. This
short period of time would not be long enough for cells to differentiate. These data together with the results shown in Fig. 1
raise the possibility that Btk regulates IL-2 gene transcription by
regulating the transcription factors that target the critical AP-1
and/or NF-AT sites. Therefore, we examined the transcriptional activity
of individual cis-elements of the IL-2 promoter in btk null BMMC. For these transient transfection experiments, we used the basic enhancerless SV40 promoter-luciferase fusion vectors containing multiple copies of either the NF-ATd/AP-1 or AP-1/Octp sequences. Robust transcriptional activation was seen of
NF-ATd/AP-1-Luc and AP-1/Octp-Luc constructs before Fc
RI stimulation
of WT btk-transfected cells, and Fc
RI cross-linking
further enhanced their transcriptional activities (Fig. 2C).
In contrast, a similar luciferase construct containing eight copies of
the NF-
B site exhibited a high basal transcriptional activity, but
no further enhancement by Fc
RI cross-linking was observed.
Transcriptional activities of these reporter constructs were much
smaller in vector or kinase-dead btk transfected cells.
These data suggest that Btk-regulated IL-2 gene transcription is
mediated by AP-1- and/or NF-AT-dependent mechanisms.
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Btk Regulates c-Jun and Other Transcription Factors That Are
Activated by JNK--
Previous studies demonstrated that FcRI
cross-linking induces activation of all three major subfamilies of MAP
kinases, i.e. ERK1/2, JNK1/2, and p38 (20-25). Btk was
shown to critically regulate JNK1/2 and, to a lesser extent, p38, while
it did not affect ERKs significantly (25). Targets of JNK include the
transcription factors, such as c-Jun, Elk-1, and Sap-1a. Therefore, we
tested whether activities of these transcription factors are under the control of Btk in mast cells. To this end, btk null-BMMC
stably transfected with the empty vector, WT btk, or K430R
btk cDNAs were transiently co-transfected with a
luciferase reporter plasmid containing five repeats of GAL4 binding
sites (pG5E1bLuc) and GAL4 DNA binding domain expression vectors fused
with activation domains of these transcription factors. Fc
RI-induced
luciferase expression by a GAL4-c-Jun construct in WT btk
transfectants was at least 2.5-5-fold higher than those in vector or
K430R btk transfectants (Fig.
3A). Substitutions of serine
residues at positions 63 and 73, the critical phosphorylation sites by
JNK (29, 30), with alanine in a GAL4-c-Jun construct
(GAL4-c-Jun(S63/73A)) abolished the activation of c-Jun, suggesting
that this c-Jun activation is mediated by phosphorylation at these
serine residues by JNK. Therefore, we analyzed the phosphorylation
status of c-Jun in WT and btk null BMMC by immunoblotting
with antibody specific for the c-Jun peptide with phosphorylation at
Ser-63. As shown in Fig. 3B, phosphorylation at Ser-63 was
induced in Fc
RI-stimulated WT BMMC in a time-dependent
manner, whereas the same phosphorylation was drastically reduced in
btk null BMMC. The presence of comparable amounts of c-Jun
protein in both BMMC was confirmed by immunoblotting with anti-c-Jun
antibody. These data demonstrate that Btk regulates the activity of JNK
and thereby the phosphorylation and activity of c-Jun.
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The SEK1-JNK-c-Jun Pathway Is Crucial for FcRI-induced,
Btk-dependent IL-2 Gene Transcription in Mast
Cells--
Upstream of c-Jun, various stimuli lead to JNK activation
through a cascade of protein kinases: MEKK1
SEK1
JNK (26-28). Therefore, we examined whether c-Jun and this cascade are used to
activate the IL-2 gene. First, the effects of a c-Jun dominant negative
mutant were tested on IL-2 promoter activity. A c-Jun mutant, Jun in
282 protein, which lacks the DNA binding ability and acts as a dominant
negative protein in Bcr-Abl-induced transformation (61), was
transiently expressed in WT BMMC. Jun in 282 protein was expressed
~2-fold more than the endogenous c-Jun protein in the transfected WT
BMMC (Fig. 4A,
inset). Fc
RI-induced transcriptional activation of the
IL-2Luc reporter in WT BMMC was suppressed by ~60% by Jun in 282 protein compared with the vector transfected cells under our conditions
(Fig. 4A). btk null BMMC were also transfected
with control or Jun in 282 expression vectors together with
btk expression and IL-2Luc plasmids. Jun in 282 protein
almost completely abolished the enhancing effect of Btk on
Fc
RI-induced IL-2 promoter activity (Fig. 4B). These data
clearly indicate that c-Jun plays an important role in IL-2 gene
transcription in mast cells and that Btk regulates the activity of
c-Jun.
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DISCUSSION |
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Production of cytokines is regulated at various levels,
e.g. mRNA stability, posttranslational modification, and
processing of precursor proteins. However, transcriptional activation
is a major regulatory mechanism generally adopted by numerous cytokine genes. Similar to T cells, mast cells seem to use this strategy in
response to FcRI cross-linking. As shown in Fig. 1, most of the
known cis-elements that are binding sites for transcription factors are required for maximal activation of the IL-2 gene. Since
mutations in any one of these elements lead to drastic decreases in
IL-2 gene expression, individual transcription factors cooperate for
maximal gene activation, a finding also consistent with previous studies on T cells. Among the cis-elements tested, mutations
in the two AP-1 (NF-ATd/AP-1 and AP-1/Octp) and two NFAT (NF-ATd and
NF-ATp) sites affected the gene expression most severely in mast cells.
On the other hand, transcriptional activities of the cis-elements containing the NF-ATd/AP-1 and AP-1/Octp
regions were shown to be regulated by Fc
RI cross-linking in a
Btk-dependent manner. These results suggest that Btk
regulates activities of AP-1 and/or NF-AT transcription factors. AP-1
is a complex of Jun and Fos family members. Our present studies have
demonstrated that activities of c-Jun and other JNK-activable
transcription factors, i.e. JunB, JunD, Elk-1, and Sap-1a,
are regulated by Btk. Jun and Fos family members associate in various
combinations and with other transcription factors, including NF-AT
(reviewed in Ref. 77). Therefore, one possible explanation for the Btk dependence of transcriptional activities of NF-ATd/AP-1-Luc and AP-1/Octp-Luc in mast cells is that the activity of the transcription factors bound to the AP-1 sites, but not those of the factors bound to
the NF-ATd or Octp sites, is dependent on Btk. Although we have not
studied NF-AT or Oct as a candidate Btk-regulated transcription factor
in further detail, this possibility certainly warrants further
investigation.
Btk regulates the enzymatic activities of JNK and p38, but not ERK
(25). At least some stimuli use the cascade of protein kinases, MEKK1
SEK1
JNK1/2 (28). Mast cells seem to use this pathway to
transcriptionally activate the IL-2 gene upon Fc
RI stimulation.
Thus, dominant negative mutants of SEK1 and c-Jun inhibited the
Fc
RI-induced IL-2 gene expression, while a constitutively active
MEKK1 mutant activated the transcription of the IL-2 gene. However, our
study does not rule out the possibility that a protein kinase(s) other
than MEKK1 might be activated by Fc
RI cross-linking to activate SEK1
and JNK. There are several reports on other protein kinases with
SEK1-activating ability (69-76). Furthermore, there are several
chromatographically separable activities that activate JNK (78). One
such kinase, MKK7, was recently cloned (79, 80). This and other JNK
kinases might be activated by Fc
RI cross-linking and regulated by
Btk. These possibilities should be explored in future work.
Btk was shown to be activated through phosphorylation of Tyr-551 by Lyn
and other Src family protein-tyrosine kinases (81, 82). Activated Btk
then autophosphorylates at Tyr-231 in heterologous (83) and mast cells
(84) and phosphorylates target proteins such as phospholipase C-
(85). On the other hand, the enzymatic activity of Btk is negatively
regulated by phosphorylation by protein kinase C (51). However, it is
not known how Btk activation leads to JNK activation. One possible
scenario is that phospholipase C-
activated by Btk results in an
increase in [Ca2+]i, which then leads to
activation of Pyk2, a Ca2+-sensitive protein-tyrosine
kinase that activates JNK in response to stresses (86). Another
possible route to JNK activation is through Ras activation. Indeed,
this pathway is shown to be used when pro-B cells were stimulated by
IL-3.3 Alternatively, Btk may
phosphoarylate and activate Vav, a tyrosine phosphorylation-dependent guanine nucleotide exchange
factor for Rac1 (87), and activated GTP-bound Rac1 leads to the
activation of JNK (19). Experiments to test these possibilities are
under way.
In conclusion, the current data demonstrate that Btk regulates the
transcription of the IL-2 gene via the JNK activation pathway upon
FcRI cross-linking. Since btk mutant mast cells have
defective secretion of several other cytokines, i.e.
TNF-
, IL-6, and granulocyte-macrophage colony-stimulating factor,
c-Jun and other JNK-regulated transcription factors also seem to
activate the transcription of these cytokine genes in a
Btk-dependent manner. These proinflammatory cytokines are
implicated in allergic and other inflammatory diseases.
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ACKNOWLEDGEMENTS |
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We are grateful to Drs. Frederick W. Alt, Ken-ichi Arai, Ralf Janknecht, Michael Karin, Wasif N. Khan, John M. Kyriakis, Arpad Molnar, Charles L. Sawyers, and Richard Treisman for kind gifts of animals and reagents. We acknowledge Anne E. Goldfeld for kind advice.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grants AI33617 and AI38348 (to T. K.). This article is Publication 208 from La Jolla Institute for Allergy and Immunology.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.
§ To whom correspondence should be addressed: La Jolla Institute for Allergy and Immunology, 10355 Science Center Dr., San Diego, CA 92121. Tel.: 619-558-3538; Fax: 619-558-3526; E-mail: toshi_kawakami{at}liai.org.
1
The abbreviations used are: FcRI, high
affinity IgE receptor; Btk, Bruton's tyrosine kinase; BMMC, bone
marrow-derived mouse cultured mast cell(s); IL, interleukin; MAP,
mitogen-activated protein; NF-AT, nuclear factor of activated T cells;
NF-ATd, distal NF-AT; NF-ATp, proximal NF-AT; Octp, proximal Oct; Octd,
distal Oct; NF-
B, nuclear factor-
B; TNF, tumor necrosis factor;
WT, wild-type; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase.
2 Hata, D., Kawakami, Y., Inagaki, N., Kitamura, T., Lantz, C., Khan, W. N., Maeda-Yamamoto, M., Miura, T., Han, W., Hartman, S. E., Yao, L., Nagai, H., Goldfeld, A. E., Alt, F. W., Galli, S. J., Witte, O. N., and Kawakami, T. (1998) J. Exp. Med. 187, 1-13.
3 J. Deng, Y. Kawakami, and T. Kawakami, unpublished data.
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REFERENCES |
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