From the Division of Allergy, La Jolla Institute for Allergy and
Immunology, San Diego, California 92121 and the Faculty
of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan
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ABSTRACT |
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Defects in Bruton's tyrosine kinase (Btk) result in B cell immunodeficiencies in humans and mice. Recent studies showed that Btk is required for maximal activation of JNK, a family of stress-activated protein kinases, induced by several extracellular stimuli including interleukin (IL)-3. On the other hand, IL-3-induced JNK activation is dependent on Ras. In the present study we have investigated whether Ras is involved in Btk-mediated JNK activation in BaF3 mouse pro-B cells. Overexpression of wild-type Btk protein in these cells enhanced JNK activation upon IL-3 stimulation, whereas expression of kinase-dead Btk partially suppressed JNK activation. Induced expression of the dominant negative Ras(N17) in the cells overexpressing wild-type Btk suppressed JNK activation. Importantly, overexpression of Btk enhanced the level of the GTP-bound, active form of Ras in response to IL-3 stimulation. Btk overexpression also increased the Shc-Grb2 association induced by IL-3 stimulation. Expression of either N17Ras or V12Ras did not impose any effects on Btk kinase activity. These data collectively indicate that Ras plays a role of an intermediary signaling protein in Btk-mediated JNK activation induced by the IL-3 signaling pathway.
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INTRODUCTION |
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Defects in Bruton's tyrosine kinase
(Btk)1 lead to X-linked
agammaglobulinemia in humans (1, 2) and X-linked immunodeficiency (xid)
in mice (3, 4). In addition to its crucial role in B lymphocyte
development, Btk is implicated in signal transduction triggered by
several immune cell receptors and cytokine receptors (for review, see
Refs. 5-7). For example, we showed recently that Btk plays an
important role in cytokine production induced by cross-linking of the
high affinity IgE receptor (FcRI) in murine mast cells (8, 9). This
protein-tyrosine kinase (PTK), a representative member of the Tec PTK
family, is also required for apoptosis induced by anti-IgM stimulation,
-irradiation, or growth factor deprivation (10-12).
A biochemical function of Btk is to mobilize a signaling pathway leading to the activation of stress-activated protein kinases, JNK/SAPK (JNK), in response to various stimuli including interleukin (IL)-3 (12). JNK is a subfamily of mitogen-activated protein kinases (MAPKs) involved in cell growth, differentiation, and transformation (for review, see Refs.13 and 14). MAPK, in general, is activated by phosphorylation of critical threonine and tyrosine residues in the region between subdomains VII and VIII by a dual specificity kinase, generically termed MAPK kinase. MAPK kinases are activated by MAPK kinase kinases. In the case of JNK, two MAPK kinases, MKK4 (=SEK1 and JNKK) and MKK7, are known to activate JNK, and numerous MAPK kinase kinases for JNK have been described recently (15-22). Furthermore, transfection studies demonstrated that Rho family GTPases, Rac1 and Cdc42, regulate JNK activity (23, 24). PAK65, a mammalian homolog of Saccharomyces cerevisiae Ste20, is activated directly by a GTP-bound form of Cdc42 or Rac1 (25-28) and activates MEKK1, a MAPK kinase kinase, under some conditions (29). In some situations these Rho family proteins are regulated by Ras, a prototypic GTPase (for review, see Refs. 30 and 31). Terada et al. (32, 33) showed recently that Ras regulates the catalytic activities of JNK as well as ERK, a prototypic MAPK, upon IL-3 stimulation.
IL-3 binding to the IL-3 receptor complex induces the activation of several nonreceptor PTKs: Jak1, Jak2, Lyn, Fes, and Tec (for a review, see Ref. 34). Therefore, several proteins are tyrosine phosphorylated in the IL-3-simulated cells (35-37). Because Lyn and other Src family PTKs can phosphorylate Btk at the tyrosine residue (Tyr-551) in the activation loop and activate the enzymatic activity of the latter PTK (38), Tec may also be activated by the same mechanism. Similar to other cytokines, IL-3 stimulation leads to the activation of Ras (39, 40). Tyrosine phosphorylation of Shc, an adaptor molecule with a phosphotyrosine binding domain at its amino terminus and a Src homology 2 (SH2) domain at its carboxyl terminus, and its association with Grb2, another adaptor protein composed of an SH2 domain flanked by two SH3 domains, are also induced by IL-3 (41-44). Although the precise mechanisms for linking these observations have not been elaborated in the IL-3 signaling system, many insights were obtained in growth factor receptor signaling systems. Some growth factor receptors with intrinsic PTK activity activate Ras through the direct or indirect recruitment of Grb2 adaptor protein (for a review, see Ref. 45). For example, ligand-bound and thereby autophosphorylated epidermal growth factor receptor binds Grb2 as well as Shc. The SH2 domain of Grb2 binds directly to the tyrosine-phosphorylated receptor or through interactions with Shc. Sos, a guanine nucleotide exchange factor for Ras, constitutively bound to Grb2 through SH3-proline-rich sequence interactions is then mobilized to the plasma membrane where Sos interacts with Ras. GDP-bound inactive Ras is then turned on to the active GTP-bound form (for review, see Ref. 44).
In the present report, transfection studies of IL-3 responsive pro-B cells with an inducible Ras expression system demonstrated that Ras plays a role in mediating the activation signal from Btk to JNK activation. Btk also enhanced IL-3-induced association between Shc and Grb2, therefore accounting for the involvement of Ras in this pathway.
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EXPERIMENTAL PROCEDURES |
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Materials-- Recombinant mouse IL-3 was a generous gift from Kirin Brewery Co. BaF3-N6 and BaF3-V2 cell lines were described previously (32). Anti-JNK1(C17), anti-Btk(M138), and anti-Grb2(C23) antibodies were obtained from Santa Cruz Biotechnology. Anti-phospho-MAPK antibody was purchased from New England Biolabs, and anti-Shc and anti-phosphotyrosine (4G10) antibodies from Upstate Biotechnology Inc. Retroviral expression vectors, pMXpuro-btk and pMXpuro-btk(K430R), were described previously (12). Pansorbin and hygromycin B were purchased from Calbiochem and enhanced chemiluminescence reagents from NEN Life Science Products. G418 was from Alexis Corporation. Other chemicals of the highest grade were obtained from Sigma Chemical Company unless otherwise mentioned.
Cell Culture and Transfection-- BaF3-N6, BaF3-V2 cell lines and their transfectants were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum, 50 µM 2-mercaptoethanol, 2 mM glutamine, 1 mg/ml G418, 1 mg/ml hygromycin B, and mouse IL-3 (culture supernatants of mouse IL-3 gene-transfected cells). For the btk transfectants, 1.2 µg/ml puromycin was included in the culture medium. Bone marrow cells from femurs of B6/129 F2 (wild type) 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. After 4 weeks of culture, more than 95% of the cells are immature mast cells. These mast cells were incubated overnight with IgE anti-dinitrophenyl monoclonal antibody and stimulated with antigen, dinitrophenyl conjugates of human serum albumin, for the indicated periods of time.
Transfection of Bosc-23 packaging cells (47) with pMXpuro, pMXpuro-btk, pMXpuro-btk(K430R) was performed with a LipofectAMINE reagent (Life Technologies, Inc.). Retroviruses (5-6 × 105 colony-forming units/ml) in the culture supernatants were used to infect BaF3-N6 and BaF3-V2 cells. Transfected cells were selected in the presence of 1.2 µg/ml puromycin for 2 weeks and cloned by limiting dilutions.Induction of Ras Expression and IL-3 Stimulation-- Expression of a dominant negative ras (N17Ras) gene in BaF3-N6 transfectants and a constitutively active ras (V12Ras) gene in BaF3-V2 transfectants was induced by culturing the cells in the presence of 5 mM IPTG for 18 h at 37 °C (32). During the last 6 h of IPTG treatment, the cells were cultured in RPMI 1640 medium containing 1 mg/ml bovine serum albumin for growth factor starvation. Then, the cells were stimulated with recombinant mouse IL-3 (100 ng/ml) in RPMI 1640 medium containing 1 mg/ml bovine serum albumin and 25 mM HEPES (pH 7.25) at 37 °C for the indicated periods.
Immunoprecipitation and Immunoblotting--
IL-3-stimulated
cells were lysed in 1% Nonidet P-40 lysis buffer (1% Nonidet P-40, 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, 0.1 mM sodium orthovanadate, 1 mM phenymethylsulfonyl fluoride, 16.5 µg/ml aprotinin, 10 µg/ml leupeptin, 25 µM p-nitrophenyl p'-guanidinobenzoate, 1 µM pepstatin, and
0.1% NaN3) on ice for 30 min, followed by centrifugation
at 15,000 × g for 10 min at 4 °C. For JNK kinase
assays, the cells were lysed in WCE lysis buffer (25 mM
HEPES (pH 7.5), 300 mM NaCl, 1.5 mM
MgCl2, 0.2 mM EDTA, 0.1% Triton, 2 µM dithiothreitol, 0.5 mM phenymethylsulfonyl fluoride, 20 mM -glycerophosphate, 0.5 mM
sodium orthovanadate). Cell lysates were incubated with the indicated
antibodies for 90 min on ice. Immune complexes were precipitated by a
brief centrifugation after incubation with Pansorbin for another 30 min
at 4 °C. Precipitated proteins were subjected to immune complex
kinase assays or solubilized directly with SDS sample buffer and
resolved by SDS-PAGE followed by blotting onto Immobilon-P membranes
(Millipore). Blots were blocked and incubated sequentially with a
primary antibody, with a secondary antibody conjugated with horseradish
peroxidase, and with enhanced chemiluminescence reagents before
exposure to ReflectionTM autoradiography films (NEN Life
Science Products). Some of the detected bands were quantified by
densitometry.
In Vitro Kinase Assays--
Btk activity was measured by
autophosphorylation. Btk immune complexes were washed with lysis buffer
and then with kinase buffer (minus ATP) before incubation with kinase
buffer (50 mM Tris-HCl (pH 7.2), 2 mM
MgCl2, 10 mM MnCl2, 0.1% Nonidet
P-40, 10 µM ATP, and 10 µCi of
[-32P]ATP) at 25 °C for 10 min. Reaction products
were analyzed by SDS-PAGE and blotted onto Immobilon-P membranes
followed by autoradiography. On the other hand, JNK1 immunoprecipitates
after being washed as described above were incubated with 2.7 µg of
GST-c-Jun(1-79) in JNK kinase buffer (20 mM HEPES (pH
7.5), 10 mM MgCl2, 22 mM dithiothreitol, 20 mM
-glycerophosphate, 50 µM sodium orthovanadate, 20 µM ATP, and 10 µCi [
-32P]ATP) at 30 °C for 10 min.
Analysis of Ras-bound Guanine Nucleotide-- Analyses of Ras-bound guanine nucleotide in BaF3-N6 transfectants were performed as described previously (48).
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RESULTS AND DISCUSSION |
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Btk Is Activated by IL-3 Stimulation--
Our previous study
demonstrated that JNK activation in response to IL-3 stimulation is
up-regulated by Btk in mouse bone marrow-derived mast cells. Thus,
IL-3-induced JNK activity at its peak in btk null mast cells
was less than half of that in wild-type mast cells (12). These data
suggest that Btk is involved in IL-3 signaling pathways, although a
previous study reported that Btk is not activated by IL-3 (49). To
reevaluate this possibility, we deprived BaF3 pro-B cells of serum and
IL-3 for 6 h and then restimulated the cells with IL-3. In
vitro kinase assays were performed on Btk immune complexes. As
shown previously (49), no apparent changes in Btk autophosphorylating
activity were observed during IL-3 stimulation for 30 min (Fig.
1, top panel). Equal amounts
of Btk protein in the immunoprecipitates were detected by reprobing the same blot with anti-Btk antibody (Fig. 1, bottom panel).
However, Btk is known to be associated with serine/threonine kinases,
one of which was identified to be protein kinase C (50). To
eliminate the effects of contaminating serine/threonine kinases, the
blot containing the Btk autophosphorylation products was probed with anti-phosphotyrosine antibody. The result clearly showed that tyrosine
phosphorylation of Btk is increased by IL-3 stimulation (Fig. 1,
middle panel), suggesting that Btk is involved in IL-3 receptor signal transduction. Because Tec, a prototype PTK of this
family, was shown previously to be activated by IL-3 stimulation in the
same cell line (49), IL-3 receptor occupancy leads to the activation of
these Tec family PTKs.
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Btk Overexpression Enhances JNK Activity in BaF3 Cells--
Terada
et al. showed that IL-3-induced JNK activation is dependent
on Ras in BaF3 cells (33). They described BaF3-N6 and BaF3-V2 cell
lines equipped with the inducible expression vectors for dominant
negative Ras(S17N) and constitutively active Ras(G12V) proteins,
respectively. To investigate whether Ras is involved in the
Btk-mediated JNK activation pathway induced by IL-3 stimulation, we
transfected the BaF3-N6 cell line with the wild-type btk
cDNA. JNK catalytic activity and ERK phosphorylation were examined
upon IL-3 stimulation in two representative transfected cell clones, BaF3-N-wt8 and BaF3-N-wt9. Expression of 2-3-fold more Btk protein over the endogenous protein was observed in these transfectants by
immunoblotting (Fig. 2A).
Serum- and IL-3-starved BaF3-N6 transfectants were stimulated with 100 ng/ml mouse IL-3 for 30 min. JNK1 kinase activity was measured in an
in vitro immune complex kinase assay using GST-c-Jun(1-79)
as a substrate. As shown in Fig. 2B (IPTG), increases in
JNK kinase activity were observed in N-wt8 and N-wt9 cell clones
compared with those in vector-transfected cells. Clone N-wt9 exhibited
a stronger JNK activity than clone N-wt8. This finding correlates with
the higher level of Btk expression in N-wt9. Expression of JNK1 protein
detected by immunoblotting cell lysates with anti-JNK1 antibody
revealed no significant change among the transfectants (Fig.
2B, lower panel). Kinetic studies showed that
overexpression of Btk does not change the time course of IL-3-induced
JNK activation, and it peaks around 20-40 min (data not shown) as
shown previously (33). These results suggest that overexpressed Btk
enhances JNK activation in a mouse pro-B cell line and also confirmed
our previous data with mouse bone marrow-derived mast cells (12). ERK
activity was also evaluated by immunoblotting with anti-phospho-MAPK
antibody. After normalized against ERK amounts, ERK phosphorylation at
the critical tyrosine residue (Tyr-204) induced by IL-3 stimulation was
not significantly changed in either Btk-overexpressing cells (Fig.
3C,
IPTG and data not
shown). Although BaF3-N-wt9 cells had approximately 50% more ERK
amounts for an unknown reason, the other clone, BaF3-N-wt8, had amounts
of ERK proteins similar to those in the vector-transfected cells or
nontransfected cells (data not shown). These results are consistent
with our previous data that Btk did not affect phosphorylation and
enzymatic activity of ERKs in Fc
RI-stimulated mast cells (12).
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Effects of N17Ras and V12Ras on JNK Catalytic Activity in Wild-type
btk and Kinase-dead btk Transfected BaF3 Cells, Respectively--
To
investigate the effect of Ras on Btk-mediated JNK activation pathway,
we induced the expression of the dominant negative Ras (N17Ras) in
BaF3-N6 transfectants and the constitutively active Ras (V12Ras) in
BaF3-V2 transfectants in the presence of 5 mM IPTG. As
shown in Fig. 2B, the enhancement in JNK activation induced by Btk overexpression was almost totally abrogated in both N-wt8 and
N-wt9 cells (compare the lanes between IPTG and +IPTG). On the other
hand, JNK activation was enhanced slightly (by 20-60% at its peak
activity) by expression of V12Ras in both vector- and
btk(K430R)-transfected BaF3-V2 cells (Fig. 3B).
These results suggest that Ras is involved in the Btk-mediated JNK
activation pathway.
Btk Overexpression Enhances Ras Activation upon IL-3 Stimulation-- Because Btk overexpression induced a remarkable enhancement in JNK activation in IL-3-stimulated BaF3-N-wt9 cells, we measured the GTP/GDP ratio of Ras in these cells before and after IL-3 stimulation. As shown in Fig. 4, the active GTP-bound form of Ras in a control cell line, BaF3-N-vec, increased upon IL-3 stimulation as described previously (32). The GTP/GDP ratios in BaF3-N-wt9 cells were twice as high as those in BaF3-N-vec. Therefore, this experiment clearly demonstrates that Btk regulates the Ras activity.
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Neither N17Ras nor V12Ras Affects Btk Activity in BaF3 Cells-- We next examined the effects of the dominant negative and constitutively active Ras mutants on Btk kinase activity. Btk autophosphorylating activity was not affected by the IPTG-induced expression of either N17Ras or V12Ras (data not shown). Because Btk kinase activity is correlated with its tyrosine phosphorylation (51), the blot containing the Btk autophosphorylation products was probed with anti-phosphotyrosine antibody. This latter experiment also confirmed the lack of effects on Btk kinase activity by mutant Ras expression (data not shown). Further, IPTG treatment of the parental BaF3 cells did not change Btk expression or Btk kinase activity. These results are consistent with the notion that Btk works upstream of Ras, and active Ras in turn leads to the activation of JNK.
Btk Overexpression Enhances the Shc-Grb2 Association in BaF3
Cells--
To gain insight into how Btk activates Ras, we analyzed the
effects of IL-3 stimulation on two Ras-regulatory adaptor proteins, Shc
and Grb2, in vector- or wild-type btk-transfected BaF3-N6 cells. Anti-Shc immunoprecipitates were analyzed by immunoblotting with
anti-phosphotyrosine antibody as well as anti-Grb2 antibody. As shown
in Fig. 5A, Shc-Grb2
association was increased markedly in the Btk-overexpressing cells
(BaF3-N-wt9), although tyrosine phosphorylation levels of Shc were
indistinguishable between vector- and btk-transfected BaF-N6
cells. After being normalized for the amount of Shc as revealed by
reprobing the blot, the coimmunoprecipitated Grb2 in BaF3-N-wt9 cells
at 5 min after IL-3 stimulation was severalfold (5.9- and 8.7-fold in
two experiments) more than those in BaF3-N-vec cells. Similar
observations were made on FcRI-stimulated cultured mast cells
derived from bone marrow cells of wild-type and btk null
mice. Thus, the amount of Grb2 associated with Shc upon Fc
RI stimulation was slightly higher (22% and 28% in two experiments) in
wild-type mast cells than in btk null mast cells (Fig.
5B). Therefore, it seems that the amount of Grb2 associated
with Shc is controlled by expression levels of Btk. Because the
Shc-Grb2 association is an early signaling event preceding Ras
activation, Btk might up-regulate Ras activation through its effects on
the Shc-Grb2 association. In contrast, neither Shc tyrosine
phosphorylation nor Shc-Grb2 association changed significantly in the
BaF3-V-K/R1 transfectants (data not shown). This might reflect the weak
effect by K430R Btk. This also suggests that Btk-enhanced Shc-Grb2
association might require the kinase activity of Btk. Although the
precise mechanisms remain to be explored, these results demonstrate
collectively that Btk operates upstream of Ras in the IL-3 receptor
signaling pathway.
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Concluding Remarks--
Both Btk and JNK are activated by a
variety of extracellular stimuli. Some stimuli, e.g. FcRI
and B cell receptor stimulation, induce the activation of both Btk and
JNK (12, 51-55). Although the current study deals mostly with IL-3
receptor-activated signaling pathways, Ras may mediate the Btk
activation signal to JNK activation not only in the IL-3 system but
also in other signal transduction systems such as the Fc
RI system.
Indeed, Ras activation was shown by this and the aforementioned stimuli
(56-59). Increased association of Shc and Grb2 seems to be part of the
Ras activation mechanisms in these signaling processes.
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ACKNOWLEDGEMENT |
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We thank Dr. Yoshito Kaziro for enthusiasm and encouragement during this study and for a critical reading of the manuscript.
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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 211 from the 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. Fax: 619-558-3526; E-mail: toshi_kawakami{at}liai.org.
1
The abbreviations used are: Btk, Bruton's
tyrosine kinase; FcRI, high-affinity IgE receptor; GST, glutathione
S-transferase; IL, interleukin; IPTG, isopropyl
-D-thiogalactopyranoside; MAPK, mitogen-activated
protein kinase; PTK, protein-tyrosine kinase; PAGE, polyacrylamide gel
electrophoresis; SH domain, Src homology domain.
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REFERENCES |
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