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,
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Division of Allergy, La Jolla Institute for Allergy and Immunology, San Diego, CA 92121;
Fred Hutchinson Cancer Research Center, Seattle, WA 98109; and
Department of Biochemistry, University of Washington, Seattle, WA 98195
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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RI) as well as
a variety of stresses induce activation of c-Jun N-terminal protein
kinases (JNKs) stress-activated protein kinases in mast cells. At least
three distinct signaling pathways leading to JNK activation have been
delineated based on the involvements of Bruton’s tyrosine kinase
(Btk), protein kinase C (PKC), and the JNK-activating cascades composed
of multiple protein kinases. The PKC-dependent pathway, which is
inhibited by a PKC inhibitor Ro31-8425 and can be activated by PMA,
functions as a major route in FcRI-stimulated mast cells derived
from btk gene knockout mice. On the other hand,
wild-type mouse-derived mast cells use both PKC-dependent and
PKC-independent pathways for JNK activation. A PKC-independent pathway
is regulated by Btk and SEK1 via the PAK
MEKK1SEK1JNK cascade,
and is sensitive to phosphatidylinositol 3-kinase inhibitors,
wortmannin and LY-294002, while the PKC-dependent pathway is affected
to a lesser extent by both wortmannin treatment and overexpression of
wild-type and dominant negative mutant SEK1 proteins. Another
PKC-independent pathway involves Btk and MKK7, a recently cloned direct
activator of JNK. Among the stresses tested, UV irradiation seems to
activate Btk and JNK via the PKC-independent
pathways. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RI) induces
degranulation, lipid mediator release, and cytokine secretion, leading
to allergic reactions (1, 2, 3). FcRI is a heterotetrameric receptor
composed of one IgE-binding 
subunit, one ß subunit, and two
disulfide-bonded 
subunits (4). Early activation events involve the
activation of receptor-associated protein-tyrosine kinases
(PTKs3): ß subunit-bound
Lyn, a Src family PTK, becomes activated by FcRI cross-linking (5)
and phosphorylates tyrosine residues in the immunoreceptor
tyrosine-based activation motifs (6, 7) of ß and subunits (8, 9).
Phosphorylated ß and subunits in turn recruit and activate Lyn
and Syk, a Syk/ZAP family PTK, respectively (10, 11, 12, 13). Activated Lyn
phosphorylates and activates Btk, a Tec family PTK (14, 15). Another
Tec family PTK, Emt (= Itk/Tsk), is also activated by receptor
cross-linking (16). Syk and Btk are believed to be responsible for
tyrosine phosphorylation and activation of phospholipase C (PLC)-
(17, 18, 19). Diacylglycerol and inositol 1,4,5-trisphosphate, two
metabolites produced by PLC from phosphatidylinositol 4,5-bisphosphate,
activate protein kinase C (PKC) and mobilize Ca2+ from
intracellular storage sites, respectively (20). Both PKC activation
and increased intracellular Ca2+ are required for and
sufficient for maximal degranulatory response (21).
Following activation of the above PTKs, tyrosine phosphorylation of Vav
(22) and Shc and increased association between Shc and Grb2 (23) were
observed. The Shc/Grb2/Sos complex may activate H-Ras, a prototype of a
large family of small m.w. GTPases (24). A recent study showed that
tyrosine-phosphorylated Vav functions as a guanine nucleotide exchange
factor for Rac1, a Rho family GTPase (25, 26). H-Ras activates a
well-characterized protein kinase cascade, i.e.,
c-Raf-1MEK1/2ERK1/2 (reviewed in Refs. 27 and 28). This pathway
is considered to be downstream of Syk and responsible for the
activation of phospholipase A2, an essential enzyme for
arachidonic acid release, in FcRI-stimulated mast cells (29). On the
other hand, Rho family GTPases, Rac1 and Cdc42, may activate another,
less well-characterized cascade, i.e., PAKMEKK1SEK1 (=
MEK4/MKK4/JNKK)JNK1/2) (reviewed in Refs. 30–32). Another
mitogen-activated protein kinase member, p38, is also activated by
FcRI cross-linking (33, 34). These mitogen-activated protein
kinases, i.e., ERK1/2, c-Jun N-terminal protein kinase (JNK)1/2, and
p38, phosphorylate and activate some transcription factors, which have
been demonstrated to play critical roles in the induction of cytokine
genes encoding TNF- and IL-2 (35).
JNK was identified originally as a family of protein kinases that
phosphorylate the critical serine residues, Ser63 and
Ser73, in the activation domain of c-Jun, a component of
AP-1 transcription factor (36, 37). The same kinases were also
identified as stress-activated protein kinases (38). Stresses that
activate this family include physical (-ray, UV light, and heat
shock), chemical (protein synthesis inhibitors and DNA-damaging
genotoxins), and biologic (proinflammatory cytokines, bacterial LPS,
and ischemia) stimuli (39). As mentioned above, a cascade of protein
kinases (PAKMEKK1SEK1JNK) was proposed for the JNK activation
pathway. Although this route for JNK activation may be taken by certain
stress signals (40, 41), there remain uncertainties in the generality
of the usage of this pathway. For example, there are at least four
chromatographically separable activities to activate JNK in rat
fibroblasts, and only one such activity corresponds to SEK1 (42). A
second JNK kinase, termed MKK7, was recently cloned (43, 44). Recent
studies discovered many protein kinases with the ability to activate
SEK1 (45, 46, 47, 48, 49, 50, 51, 52). Null mutation of the sek1 gene in ES cells
affects JNK activation responses differentially dependent on stresses
(53).
Our recent study showed that Btk regulates JNK activity upon FcRI
cross-linking, growth factor stimulation, or growth factor deprivation
(33). Therefore, we investigated JNK activation mechanisms in this cell
type. Using the dependence on Btk, PKC, SEK1, and MKK7, as criteria to
define signaling pathways, multiple pathways were found to operate in
FcRI-stimulated mast cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Culture media and FCS were purchased from Life Technologies (Gaithersburg, MD). B6/129 F2 mice, a wild-type (wt) control, were purchased from The Jackson Laboratory (Bar Harbor, ME), and btk gene knockout mice (54), originally provided by Drs. Wasif N. Kahn and Frederick W. Alt (Children’s Hospital, Boston, MA), were bred in the animal facility of La Jolla Institute for Allergy and Immunology (San Diego, CA). Ro31-8425 is a kind gift from Dr. Koji Yamada (Eisai, Tsukuba, Japan). Recombinant rat stem cell factor was generously donated by Amgen (Thousand Oaks, CA). PMA and Pansorbin were purchased from Calbiochem (La Jolla, CA). LY-294002 was from Alexis (San Diego, CA). Wortmannin and other chemicals of highest grade were obtained from Sigma (St. Louis, MO), unless otherwise mentioned.
Cells
Mast cells were cultured as described previously (55). Briefly,
bone marrow cells derived from femur of B6/129 F2
(wt) or btk knockout (btk null) mice
were cultured in RPMI 1640 medium supplemented with 10% FCS,
nonessential amino acids, 50 µM 2-ME, and 8% conditioned medium of
IL-3 gene-transfected cells. More than 95% of the trypan
blue-excluding viable cells were mast cells (termed BMMC for bone
marrow-derived cultured mast cells) after 4 wk of culture. No
discernible differences in morphology and expression of early signaling
proteins, including FcRIß, FcRI, Lyn, Syk, Grb2,
PLC-1, c-Cbl, Shc, and Sos, were detected between
wt and btk null BMMC (56). BMMC were sensitized
by an overnight incubation with 1 µg/ml anti-DNP IgE mAb, washed once
in Tyrode buffer (112 mM NaCl, 2.7 mM KCl, 0.4 mM
NaH2PO4, 1.6 mM CaCl2, 1 mM
MgCl2, 10 mM HEPES, pH 7.5, 0.05% gelatin, and 0.1%
glucose), resuspended in Tyrode buffer to 2 x 107
cells/ml, and stimulated by polyvalent Ag, 100 ng/ml DNP conjugates of
human serum albumin (DNP-HSA), for the indicated time intervals.
Alternatively, BMMC were stimulated by UV light (0.5- or 5-min exposure
under a bactericidal lamp), heat shock (42°C or 44°C) for 20 min,
cold shock (0°C) for 20 min, 0.5 mM cycloheximide for 20 min, or 0.6
M sorbitol for 15 min. NIH/3T3 cells expressing Btk were cultured in
DMEM supplemented with 10% FCS. These cells were similarly stimulated
by the above stresses.
Plasmids and transfection
pMX-puro-btk and its kinase-dead (K430R) derivative,
pMX-puro-btk (K430R), were described previously (33). Retroviruses
encoding wt or K430R Btk were prepared by transient
transfection of BOSC-23-packaging cells and used for infecting NIH/3T3
cells, as described previously (33). Transfected cells were selected by
puromycin. Glutathione S-transferase (GST) fusion cDNAs
encoding wt or K/R GST-SEK1 proteins (57) were kindly
provided by Dr. John M. Kyriakis. The GST-SEK1 portions were recloned
into pMX-puro vector at the EcoRI and NotI
restriction enzyme recognition sites. Retroviruses were prepared as
above and used for infection of BMMC, which had been cultured in the
presence of IL-3 and stem cell factor for 
2 wk before infection.
Puromycin-resistant mass cultures were expanded for 3 to 6 wk before
use.
Transient transfection of BMMC (2 x 107 cells), which
had been cultured in the presence of IL-3 and stem cell factor, with 3
µg of HA-JNK1 alone or together with 10 µg of Myc-tagged MKK7 (43)
or the constitutively active 
MEKK1 (58) was performed by
electroporation at 400 mV and 950 µm using a Bio-Rad Gene Pulser II
(Richmond, CA). Cells were grown in the presence of IL-3 and stem cell
factor, and started to be overnight sensitized with anti-DNP-IgE
24 h after electroporation. Cells were stimulated with DNP-HSA on
the next day (48 h postelectroporation). One milligram of cell
lysates was subjected to immunoprecipitation with anti-HA mAb
(12CA5; Boehringer Mannheim, Indianapolis, IN), followed by JNK kinase
assays using GST-c-Jun (1–79) as substrate.
Immune complex kinase assays
Cells were lysed in ice-cold 1% Nonidet P-40-containing lysis
buffer (20 mM Tris-HCl, pH 8, 0.15 M NaCl, 1 mM EDTA, 1 mM sodium
orthovanadate, 1 mM PMSF, 10 µg/ml aprotinin, 10 µg/ml leupeptin,
25 µM p-nitrophenyl p'-guanidinobenzoate, and
0.1% sodium azide) immediately after stimulation. Protein
concentrations were measured using Bradford protein assay reagents
(Bio-Rad). Lysates were centrifuged in an Eppendorf microcentrifuge at
4°C for 20 min. For JNK kinase assays, cells were lysed in ice-cold
whole cell extraction buffer (25 mM HEPES, pH 7.5, 0.3 M NaCl, 1.5 mM
MgCl2, 0.2 mM EDTA, 0.1% Triton X-100, 2 µM DTT, 0.5 mM
PMSF, 20 mM ß-glycerophosphate, and 0.5 mM sodium orthovanadate).
Cleared lysates in this buffer were diluted with 3 vol of dilution
buffer (20 mM HEPES, pH 7.5, 0.1 mM EDTA, 2.5 mM MgCl2,
0.05% Triton X-100, 0.5 mM sodium orthovanadate, 0.5 mM PMSF, and 20
mM ß-glycerophosphate). Cleared lysates (0.5, 0.2, 1, 1, or 1 mg
protein) were immunoprecipitated with anti-Btk (M138; Santa Cruz
Biotechnology, Santa Cruz, CA), anti-JNK1 (C-17; Santa Cruz
Biotechnology), MEK4 (K-18, Santa Cruz Biotechnology), anti-PAK65
(N-20 and C-19, Santa Cruz Biotechnology), or anti-HA (12CA5),
respectively. Immune complexes precipitated with an aid of Pansorbin
(Calbiochem) were washed three times with lysis buffer, and once with
kinase buffer without ATP and substrate. Then immunoprecipitates were
incubated with [-32P]ATP without substrate (for Btk
assays in 10-min reactions at 25°C in 50 mM Tris, pH 7.2, 0.1%
Nonidet P-40, 10 mM MnCl2, 2 mM MgCl2, 0.1 µM
ATP, and 10 µCi [-32P]ATP) or with substrate (3 µg
GST-c-Jun (1–79) for JNK assays in 15-min reactions at 30°C in 20 mM
HEPES, pH 7.4, 10 mM MgCl2, 22 mM DTT, 20 mM
ß-glycerophosphate, 50 µM Na3VO4, 20 µM
ATP, and 10 µCi [-32P]ATP; 5 µg myelin basic
protein for PAK65 assays in 20-min reactions at 30°C in 50 mM Tris,
pH 7.4, 100 mM NaCl, 10 mM MgCl2, 1 mM MnCl2,
50 µM ATP, 10 µCi [-32P]ATP; 5 µg myelin basic
protein for SEK1 assays in 20-min reactions at 30°C in 50 mM Tris, pH
7.4, 25 mM MgCl2, 2 mM DTT, 0.1 mM
Na3VO4, 25 mM ß-glycerophosphate, 50 µM
ATP, and 10 µCi [-32P]ATP). Reactions were analyzed
by SDS-PAGE, followed by electroblotting and autoradiography.
Quantitation of phosphorylated substrates was conducted by
densitometry.
Immunoblotting
Cell lysates were analyzed by SDS-PAGE and blotted onto polyvinylidene difluoride membranes (Millipore, Bedford, MA or New England Nuclear, Boston, MA). Blots were blocked and probed with anti-GST (B-14; Santa Cruz Biotechnology), anti-MEK4 (K-18; Santa Cruz Biotechnology), anti-MKK7 (43), anti-HA (12CA5), or anti-Myc (9E10, a gift from Walter Nishioka, Vical, San Diego, CA) in conjunction with enhanced chemiluminescence reagents (Amersham, Arlington Heights, IL, or New England Nuclear).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Btk regulates stress-activated protein kinases, JNK1 and JNK2
(33). Since FcRI cross-linking induces the activation of Btk (14)
and JNK (33, 59), it was interesting to investigate whether various
stresses induce the activation of these kinases and, if so, whether
they share the signaling pathways with those utilized by the FcRI
system. First, we examined whether various stresses induce the
activation of JNK in primary cultured mast cells. As shown in Figure 1
A, stresses such as UV
irradiation, heat shock (44°C), inhibition of protein synthesis, and
hyperosmolarity led to the activation of JNK. UV irradiation induced a
greater JNK response than other stresses. The activation levels of JNK
induced by different stresses were similar between the wt
and btk null BMMC, except for those by UV stimulation. UV
irradiation-induced JNK1 activity was significantly higher in
wt BMMC than in btk null BMMC (Figs. 1A and 2A). The
lack of effects of Btk expression on stress-induced JNK activation was
observed when parental NIH/3T3 fibroblasts and NIH/3T3 cells expressing
Btk ectopically were exposed to heat shock, protein synthesis
inhibitors, or hyperosmolarity (data not shown).
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B (lower panel) shows autophosphorylating
activities of Btk immunoprecipitated from UV-irradiated wt
BMMC. Consistent with differential JNK activation in wt and
btk null BMMC, UV irradiation induced activation of Btk
(Fig. 1B), while none of the other forms of stress activated
Btk (Fig. 1C). IgE/Ag stimulation of the similarly prepared
cells induced the activation of Btk, as previously described (14).
UV-induced activation of Btk and JNK was observed in mast cells
irrespective of the recovery time (10, 30, and 120 min) between UV
irradiation and cell lysis (Fig. 1B). These data demonstrate
that mast cells have the capacity to respond to various stresses by
activating JNK, and suggest that only UV irradiation among them
utilizes Btk to regulate JNK activity. Therefore, in subsequent
studies, we analyzed the involvement of Btk and other signaling
molecules in the FcRI-induced JNK activation and compared it with
the UV-induced JNK activation pathway.
PKC-dependent and PKC-independent pathways for JNK activation
induced by FcRI cross-linking
As a first step toward defining the Btk-mediated JNK activation
pathways, we examined whether more than one JNK-activating pathway
exists in mast cells. Since PKC was extensively shown to be activated
by FcRI cross-linking and to play critical roles in degranulation
(20, 21) and early gene expression in mast cells (60), the PKC
dependence was used as a criterion to distinguish JNK activation
pathways. Mast cells pretreated with or without a potent PKC inhibitor,
Ro31-8425, for 10 min were stimulated by IgE/Ag for 15 min. Cells were
lysed and JNK1 activity was measured after immunoprecipitation.
Ro31-8425 at 2 µM almost completely inhibited IgE/Ag-induced JNK
activation in btk null BMMC, whereas JNK activation was much
more resistant to 2 µM Ro31-8425 in wt BMMC (Fig. 2, A and B). Dose-response analysis (Fig. 2C) demonstrated that JNK activation in the
IgE/Ag-stimulated btk null BMMC is more sensitive to the PKC
inhibitor (IC50 = 1 µM) than that in the
IgE/Ag-stimulated wt BMMC (IC50 > 20 µM).
Without inhibitor, JNK activation that peaked at about 15 min
poststimulation was reduced significantly in btk null BMMC
compared with wt BMMC (Fig. 2, A and
B), as shown previously (33). Ro31-8425 did not change the
time course of JNK1 activation in either wt or btk
null BMMC (Fig. 2B). Interestingly, however, UV-induced
JNK activation was less sensitive to this inhibitor (Fig. 2A). These data suggest that JNK activation to a full extent
by FcRI cross-linking requires PKC, but JNK activation by UV does
not. On the other hand, 100 nM PMA, a potent PKC activator, induced
activation of JNK1, as shown for other types of cells (Fig. 3A). These data suggest that
at least two signaling pathways for FcRI-stimulated JNK activation
exist: PKC-dependent and PKC-independent ones. The PKC-dependent
pathway seems to be a predominant one in btk null BMMC,
since Ro31-8425 almost totally abrogated IgE/Ag-induced JNK activation
in btk null BMMC (Fig. 2, A and B). On
the other hand, Btk seems to control the PKC-independent pathway(s)
(compare IgE/Ag-induced JNK activation between Ro31-8425-treated
wt and btk null BMMC in Fig. 2, A and
B).
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JNK activation was shown to be inhibited by wortmannin in
FcRI-stimulated RBL-2H3 rat basophilic leukemia cells (59).
Therefore, we examined effects of wortmannin on JNK activation in
IgE/Ag-stimulated wt and btk null BMMC (Fig. 3A). JNK activation was reduced significantly by the
treatment of both wt and btk null BMMC with 100
nM wortmannin. Another PI3-K inhibitor, LY-294002, also inhibited
IgE/Ag-stimulated JNK activation in both wt and btk
null BMMC in a dose-dependent manner (Fig. 3B).
However, PMA-induced JNK activation was less sensitive to wortmannin in
both wt and btk null BMMC (Fig. 3A and
data not shown). These results suggest that both the PKC-dependent and
PKC-independent pathways leading to JNK activation involve a
wortmannin/LY-294002-sensitive step, although the PKC-dependent pathway
seems less sensitive to wortmannin. In contrast, UV-induced JNK
activation was resistant to wortmannin (Fig. 3C). In light
of a recent report that Src-mediated Btk activation involves PI3-K in
rat fibroblasts (61), we examined the effects of LY-294002 on the
autophosphorylating activity of Btk in IgE/Ag-stimulated wt
BMMC. The data shown in Figure 3D suggest that the Btk
activity is regulated by PI3-K in mast cells as well.
A PKC-independent, Btk-dependent JNK activation pathway involves SEK1
At least in some cases, JNK activity is regulated by a protein
kinase cascade in the order of PAKMEKKSEKJNK (40, 41). SEK1 is
a well-characterized direct activator of JNK (57). Therefore, possible
involvement of SEK1 in the JNK activation pathways was investigated.
Wt and btk null BMMC were transfected with
wt or dominant negative (K/R) SEK1 expression vectors, and
the puromycin-resistant mass cultures of transfected cells were
analyzed for JNK1 activity. Expression of GST-SEK1 proteins in the
transfectants was confirmed by immunoblotting (Fig. 4C). Wt BMMC
transfectants overexpressing wt SEK1 protein exhibited an
enhanced JNK activation upon FcRI cross-linking, whereas the
dominant negative SEK1(K/R) expression partially (by 20–43%)
suppressed JNK activation in wt BMMC (Fig. 4A),
indicating that SEK1 plays an intermediary role in JNK activation in
these cells. In accordance with this result, we observed 1.5-fold
higher SEK1 activity in IgE/Ag-stimulated wt BMMC over
similarly stimulated btk null BMMC (data not shown). PAK65
activity that increased by IgE/Ag stimulation was two- or
fourfold higher in wt BMMC than in btk null
BMMC, depending on the immunoprecipitating Abs, a C-terminal
peptide-specific or an N-terminal peptide-specific Ab, respectively
(Fig. 4D). However, expression of either wt or
dominant negative SEK1 proteins failed to significantly affect JNK
activation in IgE/Ag-stimulated btk null BMMC (Fig. 4A). Furthermore, expression of a constitutively active
mutant of MEKK1 (MEKK1) induced strong activation of JNK in
btk null BMMC, irrespective of the expression of
wt Btk or kinase-dead Btk (Fig. 4E). These data
suggest that mouse mast cells express functional components of the
PAKMEKK1SEK1JNK cascade and that this pathway is part of the
PKC-independent, Btk-dependent JNK activation pathway.
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In btk null BMMC, Ro31-8425 inhibited IgE/Ag-induced
JNK activity to a very low level (Fig. 2), suggesting a predominant
role of PKC in these cells. This PKC-dependent pathway was insensitive
to overexpression of either wt or K/R mutant SEK1 proteins
when cells were stimulated by IgE and Ag (Fig. 4A). To
further examine the SEK1 involvement in this pathway, we examined
effects of PMA on JNK activation in SEK1-transfected btk
null BMMC. Overexpression of either wt or K/R mutant
SEK1 proteins failed to significantly affect PMA-induced JNK activation
(Fig. 4B), indicating that the PKC-dependent JNK activation
pathway does not involve SEK1. In contrast, JNK activation in response
to UV irradiation is sensitive to the expression of wt SEK1
or SEK1(K/R) in wt BMMC transfectants (Fig. 4A),
similar to FcRI-induced JNK activation in wt BMMC
transfectants.
Another Btk-dependent JNK activation pathway is dependent on MKK7
The enhanced FcRI-induced JNK activation by overexpression of
wt SEK1 (Fig. 4A) shows that SEK1 protein is
limiting. Furthermore, the expression of the dominant negative
SEK1(K/R) protein suppressed JNK activation only partially (Fig. 4A). These findings prompted us to investigate the possible
involvement of another JNK kinase, MKK7, in the FcRI-induced JNK
activation. First, we confirmed the expression of MKK7 in both
wt and btk null BMMC by immunoblotting (Fig. 5A). Then we examined the
effects of MKK7 overexpression on JNK activation. Wt BMMC
were transiently transfected with HA-JNK1 alone or together with
Myc-tagged MKK7, and stimulated via FcRI. JNK1 activity
immunoprecipitated with anti-HA Ab was two- to fourfold greater in
the presence of Myc-MKK7 than that in the absence of Myc-MKK7 (Fig. 5B), suggesting that MKK7 can be activated upon FcRI
cross-linking. When JNK1 activity was compared between wt
and btk null BMMC, which had been transfected with or
without Myc-MKK7, FcRI-induced JNK1 activation was two- to fourfold
higher in wt BMMC expressing Myc-MKK7 than in btk
null BMMC expressing Myc-MKK7 (Fig. 5B). Comparable
expression of Myc-MKK7 and HA-JNK1 in the transfected cells was
confirmed by immunoblotting (Fig. 5C). These data suggest
that FcRI-induced MKK7 activation is at least partly regulated by
Btk. Therefore, Btk appears to regulate the JNK activity through MKK7
as well as SEK1.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RI cross-linking induces the activation of JNK through
PKC-dependent and PKC-independent pathways. In the absence of Btk
expression, mast cells mainly use the PKC-dependent pathway (pathway A
in Fig. 6), while wt mast
cells expressing Btk exhibit a vigorous JNK activation response to
FcRI stimulation via both the PKC-dependent and PKC-independent
pathways (pathways A vs B and C in Fig. 6). It is unlikely that this
difference between wt and btk null mast cells
might be due to differential expression of components of JNK activation
pathways. Thus, no differences were observed in morphology or in the
expression of early signaling proteins between wt and
btk null cells, including SEK1, MKK7 (this study), JNK1 and
JNK2 (33), and c-Jun (62), and importantly, stable and transient
expression of Btk restored IgE/Ag-stimulated JNK activation in
btk null BMMC (33).
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MEKK1SEK1JNK
(40, 41). The downstream portion of JNK activation pathway B seems to
be the same as this cascade. Indeed, our data in support of this notion
include differential activities of PAK65 and SEK1 between wt
and btk null BMMC, MEKK1-induced JNK activation, and more
importantly, the SEK1 involvement in pathway B. As shown in RBL-2H3
cells (59), this pathway is sensitive to PI3-K inhibitors. PI3-K is
physically associated with Ras and works as a Ras effector (63). Our
preliminary data suggest Ras as an intermediary molecule for IL-3
stimulation-induced JNK activation that is dependent on
Btk.4 Therefore, the
Btk-regulated JNK activation pathway, with Ras and PI3-K as intervening
signaling molecules, might be used by the FcRI signaling system. It
is not clear how Btk transduces the activation signal downstream to Ras
and Rho family GTPases. Vav, a hemopoietic cell-specific guanine
nucleotide exchange factor, was recently demonstrated to activate Rac1
in a tyrosine phosphorylation-dependent manner (25), and proposed to
transmit an upstream activation signal to Rac1 in the FcRI system
(64). Therefore, Vav might be involved in Btk-mediated JNK activation
pathways.
MKK7, a recently cloned activator of JNK, is expressed ubiquitously
(43, 44). This JNK kinase is also activated by various stresses.
FcRI-induced JNK activation is enhanced by overexpression of MKK7.
Therefore, these data suggest that MKK7 can be activated upon FcRI
cross-linking in mast cells and that this activation is regulated by
Btk (pathway C). However, we are aware of other possibilities regarding
the involvement of SEK1 and MKK7 in FcRI-induced JNK activation.
Thus, the inhibitory mechanisms of SEK1(K/R) are not known. It might
inhibit the function of SEK1 alone or functions of both SEK1 and MKK7
(and even other unknown JNK activators). Therefore, we do not know
relative contributions of SEK1 and MKK7 to FcRI-induced JNK
activation. Since active forms of Rac1 and MEKK1 induce the activation
of MKK7, pathways B and C might share the signaling components upstream
of SEK1 and MKK7, respectively. This possibility remains to be tested.
On the other hand, SEK1 and MKK7 might preferentially activate distinct
members (or sets of members) of the JNK family (65) and, therefore,
activate distinct downstream pathways to fulfill differential
functions.
PMA was previously shown to modestly activate JNK in some cell types
(37, 38, 66). Similarly, mast cells exhibit this capability (pathway
A). In contrast with pathway B, this PKC-dependent pathway was little
affected by overexpression of SEK1 proteins. Therefore, JNK activation
in this pathway does not seem to involve JNK phosphorylation by SEK1.
Possible involvement of PI3-K in this pathway is not certain at this
point. Thus, wortmannin and LY-294002 inhibited the FcRI-stimulated
JNK activation, whereas JNK activation induced by PMA was more
resistant to wortmannin (Fig. 3). In any event, JNK activation does not
seem to be due to direct phosphorylation of JNK by PKC. Thus, purified
preparations of conventional PKC isoforms from rat brain (a mixture of
, ß, and isoforms) failed to phosphorylate JNK directly in in
vitro kinase assays (data not shown). A study on which PKC isoform is
responsible for JNK activation is underway.
Interestingly, activation of ERKs involves both PKC-dependent and
PKC-independent mechanisms in FcRI-stimulated RBL-2H3 rat basophilic
leukemia cells (67). The PKC-dependent pathway mediates early and
transient activation, while the PKC-independent pathway induces a
longer-lived activation signal. The latter pathway predominates within
5 min of Ag addition. Since tyrosine phosphorylation of Vav and Shc is
unaffected by PKC inhibitor and exhibits a prolonged kinetics, these
proteins remain potential candidates for mediating the long-lived ERK
activation signal. In contrast, the differential kinetics of activation
by PKC-dependent and PKC-independent pathways was not observed for JNK
in mast cells (Fig. 2B). On the other hand, PMA was shown to
increase in GTP-loaded Ras in T cells probably by inhibiting the
activity of GTPase-activating protein (68).
Mast cells were shown to have the machinery to respond to various forms
of stress with JNK activation (Fig. 1). Interestingly, only UV
irradiation among the stresses tested activates Btk. UV
irradiation-induced JNK activation pathway seems to be similar to
pathway B activated by FcRI cross-linking, in that the former
pathway was resistant to PKC inhibitor, and regulated by Btk and
SEK1. Pathway C may also be activated by UV irradiation, since
MKK7 overexpression enhances JNK activation induced by UV irradiation
in COS cells (data not shown). However, UV irradiation-induced JNK
activation was not affected by the presence of ectopically expressed
Btk in NIH/3T3 cells. This observation probably reflects the lack of
hemopoietic cell-specific components (e.g., Vav) in pathway B in the
fibroblasts. Since other stresses seem to use the
Rac1PAKMEKK1SEK1JNK cascade in some cell
types, the lack of effects of Btk expression on JNK activation by these
stresses in mast cells suggests the diversity in upstream signaling
events provoked by these stresses. In this line of study, activation of
Lyn was induced in chicken DT40 B cells by both UV irradiation and
hyperosmolarity, while Syk activation was seen in
hyperosmolarity-shocked cells, but not in UV-irradiated cells (69).
Since Btk can be phosphorylated and activated by Lyn and other Src
family PTKs (15, 70), UV-induced activation of Src family PTKs (71) may
be involved in Btk activation in UV-irradiated mast cells.
In conclusion, mast cells are equipped with the signal-transduction
machinery to activate JNK in response to a variety of stresses. FcRI
cross-linking induces JNK activation through both PKC-dependent and
PKC-independent pathways. A PKC-independent pathway involves steps
sensitive to inhibition by PI3-K inhibitors and dominant negative SEK1,
and therefore dependent on the PAKMEKK1SEK1 cascade to activate
JNK. Another FcRI-induced, Btk-dependent JNK activation pathway
involves MKK7.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Toshiaki Kawakami, La Jolla Institute for Allergy and Immunology, 10355 Science Center Drive, San Diego, CA 92121. E-mail address:
3 Abbreviations used in this paper: PTK, protein-tyrosine kinase; BMMC, bone marrow-derived cultured mouse mast cell; GST, glutathione S-transferase; HA, hemagglutinin; HSA, human serum albumin; PI3-K, phosphatidylinositol 3-kinase; PKC, protein kinase C; PLC, phospholipase C; JNK, c-Jun N-terminal protein kinase; wt, wild-type.
4 J. Deng, Y. Kawakami, S. E. Hartman, T. Satoh, and T. Kawakami. 1998. Involvement of Ras in Bruton’s tyrosine kinase-mediated JNK activation. J. Biol Chem. In press.
Received for publication January 6, 1998. Accepted for publication April 17, 1998.
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