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The Journal of Immunology, 1998, 161: 1795-1802.
Copyright © 1998 by The American Association of Immunologists

Multiple Signaling Pathways for the Activation of JNK in Mast Cells: Involvement of Bruton’s Tyrosine Kinase, Protein Kinase C, and JNK Kinases, SEK1 and MKK71

Yuko Kawakami*, Stephen E. Hartman*, Pamela M. Holland{dagger},{ddagger}, Jonathan A. Cooper{dagger} and Toshiaki Kawakami2,*

* Division of Allergy, La Jolla Institute for Allergy and Immunology, San Diego, CA 92121; {dagger} Fred Hutchinson Cancer Research Center, Seattle, WA 98109; and {ddagger} Department of Biochemistry, University of Washington, Seattle, WA 98195


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Stimulation of the high affinity IgE receptor (Fc{epsilon}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 Fc{epsilon}RI-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->MEKK1->SEK1->JNK 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
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Stimulation of mast cells through the high affinity IgE receptor (Fc{epsilon}RI) induces degranulation, lipid mediator release, and cytokine secretion, leading to allergic reactions (1, 2, 3). Fc{epsilon}RI is a heterotetrameric receptor composed of one IgE-binding {alpha} subunit, one ß subunit, and two disulfide-bonded {gamma} 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 Fc{epsilon}RI cross-linking (5) and phosphorylates tyrosine residues in the immunoreceptor tyrosine-based activation motifs (6, 7) of ß and {gamma} subunits (8, 9). Phosphorylated ß and {gamma} 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)-{gamma} (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-1->MEK1/2->ERK1/2 (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 Fc{epsilon}RI-stimulated mast cells (29). On the other hand, Rho family GTPases, Rac1 and Cdc42, may activate another, less well-characterized cascade, i.e., PAK->MEKK1->SEK1 (= MEK4/MKK4/JNKK)->JNK1/2) (reviewed in Refs. 30–32). Another mitogen-activated protein kinase member, p38, is also activated by Fc{epsilon}RI 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-{alpha} 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 ({gamma}-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 (PAK->MEKK1->SEK1->JNK) 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 Fc{epsilon}RI 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 Fc{epsilon}RI-stimulated mast cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents

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 Fc{epsilon}RIß, Fc{epsilon}RI{gamma}, Lyn, Syk, Grb2, PLC-{gamma}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 {Delta}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 [{gamma}-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 [{gamma}-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 [{gamma}-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 [{gamma}-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 [{gamma}-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).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
UV irradiation activates Btk in mast cells

Btk regulates stress-activated protein kinases, JNK1 and JNK2 (33). Since Fc{epsilon}RI 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 Fc{epsilon}RI system. First, we examined whether various stresses induce the activation of JNK in primary cultured mast cells. As shown in Figure 1GoA, 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. 1GoA 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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  		FIGURE 1. UV irradiation induces the activation of Btk and JNK in mouse mast cells. A, Mast cells from wt or btk null mice were exposed to cold shock (0°C) for 20 min, heat shock (44°C) for 20 min, 0.5 mM cycloheximide for 20 min, 0.6 M sorbitol for 15 min, or UV light (5 min irradiation followed by 10-min incubation in the culture medium at 37°C). Cell lysates were immunoprecipitated with anti-JNK1 Ab, and immune complex kinase assays were performed with GST-c-Jun as substrate, as described in Materials and Methods. Portions of the autoradiogram are shown to indicate the phosphorylated GST-c-Jun bands. B, wt BMMC were left under a room light (-) or irradiated with UV (+) for 5 min, and incubated for 10, 30, or 120 min at 37°C in a CO2 incubator before cell lysis. JNK1 activity was measured as above. Btk-autophosphorylating activity was measured by immune complex kinase assays without substrate. C, The result of Btk immune complex kinase assays without substrate is shown. Molecular masses are indicated on the left. The results shown in A and C are representative of three similar experiments, while those in B are reproduced in another experiment.

 
Next, we examined effects of stresses on Btk kinase activity. Figure 1GoB (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. 1GoB), while none of the other forms of stress activated Btk (Fig. 1GoC). 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. 1GoB). 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 Fc{epsilon}RI-induced JNK activation and compared it with the UV-induced JNK activation pathway.

PKC-dependent and PKC-independent pathways for JNK activation induced by Fc{epsilon}RI 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 Fc{epsilon}RI 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. 2Go, A and B). Dose-response analysis (Fig. 2GoC) 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. 2Go, 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. 2GoB). Interestingly, however, UV-induced JNK activation was less sensitive to this inhibitor (Fig. 2GoA). These data suggest that JNK activation to a full extent by Fc{epsilon}RI 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. 3GoA). These data suggest that at least two signaling pathways for Fc{epsilon}RI-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. 2Go, 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. 2Go, A and B).



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  		FIGURE 2. Effects of a PKC inhibitor on IgE/Ag-induced or UV irradiation-induced JNK activation. IgE-sensitized wt and btk null BMMC were incubated with 2 µM (A) or various concentrations of (C) Ro31-8425 for 10 min. Then the cells were left unstimulated (-) or stimulated with Ag for 15 min or with UV irradiation for 5 min (with 10-min recovery) in the continued presence of Ro31-8425. In B, IgE-sensitized BMMC preincubated with 2 µM Ro31-8425 for 10 min were left unstimulated or stimulated with Ag for the indicated intervals. JNK1 activity was measured as described in Figure 1Go. Relative JNK1 activities determined by densitometry are shown below the gels in A and C. The results shown in A are representative of three similar experiments. Kinetic (B) and dose-response (C) data were repeated in another experiment.

 


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  		FIGURE 3. Effects of wortmannin and LY294002 on IgE/Ag-induced or PMA-induced JNK activation and IgE/Ag-induced Btk activation. A, IgE-sensitized wt and btk null BMMC were pretreated with wortmannin (Wort) or vehicle (DMSO) for 10 min. Then the cells were stimulated with Ag for 15 min or 100 nM PMA (P) for 15 min in the continued presence or absence of 100 nM wortmannin. JNK1 activity was measured as described in Figure 1Go. Representative of two similar experiments. B, IgE-sensitized cells were pretreated with various concentrations of LY294002 for 5 min and stimulated with Ag in the presence of LY294002, and JNK1 activity was measured as above. C, wt BMMC were pretreated with various concentrations of wortmannin for 10 min and irradiated with UV for 5 min. JNK1 activity was measured as described in Figure 1Go. Representative of two similar experiments. D, IgE-sensitized mast cells were pretreated with various concentrations of LY294002 for 5 min and stimulated with Ag for 3 min. Btk-autophosphorylating activity was measured. Representative of two experiments.

 
JNK activation pathways are inhibited by phosphatidylinositol 3-kinase (PI3-K) inhibitors, wortmannin and LY-294002

JNK activation was shown to be inhibited by wortmannin in Fc{epsilon}RI-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. 3GoA). 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. 3GoB). However, PMA-induced JNK activation was less sensitive to wortmannin in both wt and btk null BMMC (Fig. 3GoA 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. 3GoC). 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 3GoD 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 PAK->MEKK->SEK->JNK (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. 4GoC). Wt BMMC transfectants overexpressing wt SEK1 protein exhibited an enhanced JNK activation upon Fc{epsilon}RI cross-linking, whereas the dominant negative SEK1(K/R) expression partially (by 20–43%) suppressed JNK activation in wt BMMC (Fig. 4GoA), 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. 4GoD). However, expression of either wt or dominant negative SEK1 proteins failed to significantly affect JNK activation in IgE/Ag-stimulated btk null BMMC (Fig. 4GoA). Furthermore, expression of a constitutively active mutant of MEKK1 ({Delta}MEKK1) induced strong activation of JNK in btk null BMMC, irrespective of the expression of wt Btk or kinase-dead Btk (Fig. 4GoE). These data suggest that mouse mast cells express functional components of the PAK->MEKK1->SEK1->JNK cascade and that this pathway is part of the PKC-independent, Btk-dependent JNK activation pathway.



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  		FIGURE 4. Involvement of SEK1 and PAK65 in Fc{epsilon}RI-induced JNK activation. A, wt or btk null BMMC transfected with empty (Vec), wt, or K/R SEK1 vectors were sensitized with IgE, and unstimulated (-) or stimulated with Ag for 15 min or with UV irradiation for 5 min with a recovery time of 10 min. B, btk null BMMC transfectants were treated with vehicle (DMSO) for 5 min, with UV for 5 min, or with 100 nM PMA for 5 min. JNK1 activity was measured as described in Figure 1Go. C, Expression of SEK1 protein in transfected and nontransfected BMMC. Whole cell lysates were separated by SDS-PAGE, and the blot was probed with anti-GST (upper). The same blot was stripped and reprobed with anti-MEK4 (lower). Positions of GST, GST-SEK1, and endogenous SEK1 are indicated. Also indicated by an asterisk is a degradation product composed of the GST portion of GST-SEK1 fusion protein. Of note is that more degradation products were found in wt BMMC compared with btk null BMMC. This might indicate the proapoptotic nature of Btk (33, 72) that may activate proteolytic activity toward GST-SEK1 protein. The data shown in A through C were obtained in a set of the transfectants. Similar results were reproduced in another set of the transfectants. D, IgE-sensitized wt and btk null BMMC were stimulated with Ag for the indicated times. Cell lysates were immunoprecipitated with anti-PAK65 (N20) or anti-PAK65 (C-19), and immune complex kinase assays using myelin basic protein (MBP) as substrate were performed as described in Materials and Methods. E, btk null BMMC stably transfected with pMX-puro vector (vec), wt btk cDNA, or kinase-dead (K430R) btk cDNA were transiently transfected with HA-JNK1 together with an empty vector (pCMV5) or {Delta}MEKK1 vector. The cells were sensitized with IgE and stimulated by Ag. JNK1 activity immunoprecipitated with anti-HA was measured.

 
Effects of SEK1 overexpression on PMA-induced JNK activation in btk null mast cells

In btk null BMMC, Ro31-8425 inhibited IgE/Ag-induced JNK activity to a very low level (Fig. 2Go), 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. 4GoA). 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. 4GoB), 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. 4GoA), similar to Fc{epsilon}RI-induced JNK activation in wt BMMC transfectants.

Another Btk-dependent JNK activation pathway is dependent on MKK7

The enhanced Fc{epsilon}RI-induced JNK activation by overexpression of wt SEK1 (Fig. 4GoA) shows that SEK1 protein is limiting. Furthermore, the expression of the dominant negative SEK1(K/R) protein suppressed JNK activation only partially (Fig. 4GoA). These findings prompted us to investigate the possible involvement of another JNK kinase, MKK7, in the Fc{epsilon}RI-induced JNK activation. First, we confirmed the expression of MKK7 in both wt and btk null BMMC by immunoblotting (Fig. 5GoA). 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 Fc{epsilon}RI. 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. 5GoB), suggesting that MKK7 can be activated upon Fc{epsilon}RI cross-linking. When JNK1 activity was compared between wt and btk null BMMC, which had been transfected with or without Myc-MKK7, Fc{epsilon}RI-induced JNK1 activation was two- to fourfold higher in wt BMMC expressing Myc-MKK7 than in btk null BMMC expressing Myc-MKK7 (Fig. 5GoB). Comparable expression of Myc-MKK7 and HA-JNK1 in the transfected cells was confirmed by immunoblotting (Fig. 5GoC). These data suggest that Fc{epsilon}RI-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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  		FIGURE 5. Involvement of MKK7 in Fc{epsilon}RI-induced JNK activation. A, Cell lysates prepared from wt and btk null BMMC were analyzed by immunoblotting with anti-MKK7 Ab. B, Wt and btk null BMMC cultured in the presence of stem cell factor were transfected with HA-JNK1 alone or together with Myc-tagged MKK7. Cells were sensitized with anti-DNP IgE and stimulated by DNP-HSA for the indicated intervals. Cell lysates were immunoprecipitated with anti-HA mAb (12CA5), and immune complexes were subjected to kinase assays using GST-c-Jun (1–79) as substrate. Phosphorylated GST-c-Jun (1–79) bands are indicated. C, Cell lysates were also analyzed by immunoblotting with anti-Myc (left) and anti-HA (right) to check for the expression of Myc-MKK7 and HA-JNK1, respectively. The data shown in A through C are representative of two identical experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Using the PKC dependence as a criterion and comparing the responses in wt and btk null mast cells, we have found that Fc{epsilon}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. 6Go), while wt mast cells expressing Btk exhibit a vigorous JNK activation response to Fc{epsilon}RI stimulation via both the PKC-dependent and PKC-independent pathways (pathways A vs B and C in Fig. 6Go). 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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  		FIGURE 6. Proposed signaling pathways for JNK activation in Fc{epsilon}RI-stimulated mast cells. Fc{epsilon}RIß-associated Lyn becomes activated by receptor cross-linking. Active Lyn phosphorylates ß and {gamma} subunits of Fc{epsilon}RI. Tyrosine-phosphorylated ß and {gamma} subunits recruit and activate Lyn and Syk, respectively. Active Lyn phosphorylates Btk at tyrosine 551 and activates Btk. Btk may phosphorylate PLC-{gamma}1 and PLC-{gamma}2 and other substrates. PLC activation may lead to activation of PKC. Active PKC in turn activates JNK probably indirectly (pathway A). Phosphorylation of another Btk target leads to activation of the protein kinase cascade of PAK65->MEKK1->SEK1->JNK (pathway B), although this important target remains to be identified. Upstream of this cascade may H-Ras and PI3-K be positioned. IP3, inositol 1,4,5-trisphosphate; DAG, diacylglycerol.

 
Rho family GTPases, Rac1 and Cdc42, appear to activate JNK through the phosphorylation cascade PAK->MEKK1->SEK1->JNK (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 Fc{epsilon}RI 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 Fc{epsilon}RI 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. Fc{epsilon}RI-induced JNK activation is enhanced by overexpression of MKK7. Therefore, these data suggest that MKK7 can be activated upon Fc{epsilon}RI 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 Fc{epsilon}RI-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 Fc{epsilon}RI-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 Fc{epsilon}RI-stimulated JNK activation, whereas JNK activation induced by PMA was more resistant to wortmannin (Fig. 3Go). 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 {alpha}, ß, and {gamma} 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 Fc{epsilon}RI-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. 2GoB). 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. 1Go). 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 Fc{epsilon}RI 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 Rac1->PAK->MEKK1->SEK1->JNK 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. Fc{epsilon}RI 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 PAK->MEKK1->SEK1 cascade to activate JNK. Another Fc{epsilon}RI-induced, Btk-dependent JNK activation pathway involves MKK7.


   Acknowledgments
 
We thank Drs. Wasif N. Kahn and Frederick W. Alt for providing breeding pairs of btk knockout mice; John M. Kyriakis for his kind gifts of SEK1 and MEKK1 plasmids; and Koji Yamada for his kind gift of Ro31-8425. Donation of recombinant rat stem cell factor by Amgen is greatly appreciated.


   Footnotes
 
1 This work was supported in part by National Institutes of Health Grants AI33617 and AI38348 (T.K.) and CA54786 (J.A.C.). This article is Publication 198 from La Jolla Institute for Allergy and Immunology. Back

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: Back

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. Back

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. Back

Received for publication January 6, 1998. Accepted for publication April 17, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Beaven, M. A., H. Metzger. 1993. Signal transduction by Fc receptors: the Fc{epsilon}RI case. Immunol. Today 14:222.[Medline]
  2. Galli, S. J.. 1993. New concepts about the mast cell. N. Engl. J. Med. 328:257.[Free Full Text]
  3. Razin, E., I. Pecht, J. Rivera. 1995. Signal transduction in the activation of mast cells and basophils. Immunol. Today 16:370.[Medline]
  4. Ravetch, J. V., J.-P. Kinet. 1991. Fc receptors. Annu. Rev. Immunol. 9:457.[Medline]
  5. Eiseman, E., J. B. Bolen. 1992. Engagement of the high-affinity IgE receptor activates src protein-related tyrosine kinases. Nature 355:78.[Medline]
  6. Reth, M.. 1989. Antigen receptor tail clue. Nature 338:383.[Medline]
  7. Cambier, J. C.. 1995. New nomenclature for the Reth motif (or ARH1/TAM/ARAM/YXXL). Immunol. Today 16:110.[Medline]
  8. Kihara, H., R. P. Siraganian. 1994. Src homology 2 domains of Syk and Lyn bind to tyrosine-phosphorylated subunits of the high affinity IgE receptor. J. Biol. Chem. 269:22427.[Abstract/Free Full Text]
  9. Scharenberg, A. M., S. Lin, B. Cuenod, H. Yamamura, J.-P. Kinet. 1995. Reconstitution of interactions between tyrosine kinases and the high affinity IgE receptor which are controlled by receptor clustering. EMBO J. 14:3385.[Medline]
  10. Jouvin, M.-H., M. Adamczewski, R. Numerof, O. Letourneur, A. Valle, J.-P. Kinet. 1994. Differential control of the tyrosine kinases Lyn and Syk by the two signaling chains of the high affinity immunoglobulin E receptor. J. Biol. Chem. 269:5918.[Abstract/Free Full Text]
  11. Rowley, R. B., A. L. Burkhardt, H.-G. Chao, G. R. Matsueda, J. B. Bolen. 1995. Syk protein-tyrosine kinase is regulated by tyrosine-phosphorylated Ig{alpha}/Igß immunoreceptor tyrosine activation motif binding and autophosphorylation. J. Biol. Chem. 270:11590.[Abstract/Free Full Text]
  12. Shiue, L., M. J. Zoller, J. S. Brugge. 1995. Syk is activated by phosphotyrosine-containing peptides representing the tyrosine-based activation motifs of the high affinity receptor for IgE. J. Biol. Chem. 270:10498.[Abstract/Free Full Text]
  13. Hutchcroft, J. E., R. L. Geahlen, G. G. Deanin, J. M. Oliver. 1992. Fc{epsilon}RI-mediated tyrosine phosphorylation and activation of the 72-kDa protein-tyrosine kinase, PTK72, in RBL-2H3 rat tumor mast cells. Proc. Natl. Acad. Sci. USA 89:9107.[Abstract/Free Full Text]
  14. Kawakami, Y., L. Yao, S. Tsukada, O. N. Witte, T. Kawakami. 1994. Tyrosine phosphorylation and activation of Bruton tyrosine kinase upon Fc{epsilon}RI cross-linking. Mol. Cell. Biol. 14:5108.[Abstract/Free Full Text]
  15. Rawlings, D. J., A. M. Scharenberg, H. Park, M. I. Wahl, S. Lin, R. M. Kato, A.-C. Fluckiger, O. N. Witte, J.-P. Kinet. 1996. Activation of BTK by a phosphorylation mechanism initiated by SRC family kinases. Science 271:822.[Abstract]
  16. Kawakami, Y., L. Yao, M. Tashiro, S. Gibson, G. B. Mills, T. Kawakami. 1995. Activation and interaction with protein kinase C of a cytoplasmic tyrosine kinase, Itk/Tsk/Emt, upon Fc{epsilon}RI cross-linking on mast cells. J. Immunol. 155:3556.[Abstract]
  17. Takata, M., T. Kurosaki. 1996. A role for Bruton’s tyrosine kinase in B cell antigen receptor-mediated activation of phospholipase C-{gamma}2. J. Exp. Med. 184:31.[Abstract/Free Full Text]
  18. Zhang, J., E. H. Berenstein, R. L. Evans, R. P. Siraganian. 1996. Transfection of Syk protein tyrosine kinase reconstitutes high affinity IgE receptor-mediated degranulation in a Syk-negative variant of rat basophilic leukemia RBL-2H3 cells. J. Exp. Med. 184:71.[Abstract/Free Full Text]
  19. Rawlings, D. J., Z. Li, A.-C. Fluckiger, M. I. Wahl, R. M. Kato, O. N. Witte. 1997. Biphasic regulation of B cell receptor mediated Ca2+ flux by Bruton’s tyrosine kinase. J. Allergy Clin. Immunol. 99:S469.
  20. Nishizuka, Y.. 1984. The role of protein kinase C in cell surface signal transduction and tumour promotion. Nature 308:693.[Medline]
  21. Ozawa, K., Z. Szallasi, M. G. Kazanietz, P. M. Blumberg, H. Mischak, J. F. Mushinski, M. A. Beaven. 1993. Ca2+-dependent and Ca2+-independent isozymes of protein kinase C mediate exocytosis in antigen-stimulated rat basophilic RBL-2H3 cells: reconstitution of secretory responses with Ca2+ and purified isozymes in washed permeabilized cells. J. Biol. Chem. 268:1749.[Abstract/Free Full Text]
  22. Margolis, B., P. Hu, S. Katzav, W. Li, J. M. Oliver, A. Ullrich, A. Weiss, J. Schlessinger. 1992. Tyrosine phosphorylation of vav proto-oncogene product containing SH2 domain and transcription factor motifs. Nature 356:71.[Medline]
  23. Jabril-Cuenod, B., C. Zhang, A. M. Scharenberg, R. Paolini, R. Numerof, M. A. Beaven, J.-P. Kinet. 1996. Syk-dependent phosphorylation of Shc: a potential link between Fc{epsilon}RI and the Ras/mitogen-activated protein kinase signaling pathway through SOS and Grb2. J. Biol. Chem. 271:16268.[Abstract/Free Full Text]
  24. Boguski, M. S., F. McCormick. 1993. Proteins regulating Ras and its relatives. Nature 366:643.[Medline]
  25. Crespo, P., K. E. Schuebel, A. A. Ostrom, J. S. Gutkind, X. R. Bustelo. 1997. Phosphotyrosine-dependent activation of Rac-1 GDP/GTP exchange by the vav proto-oncogene product. Nature 385:169.[Medline]
  26. Collins, T. L., M. Deckert, A. Altman. 1997. Views on Vav. Immunol. Today 18:221.[Medline]
  27. Marshall, C. J.. 1995. Specificity of receptor tyrosine kinase signaling: transient vs sustained extracellular signal-regulated kinase activation. Cell 80:179.[Medline]
  28. Herskowitz, I.. 1995. MAP kinase pathways in yeast: for mating and more. Cell 80:187.[Medline]
  29. Hirasawa, N., A. Scharenberg, H. Yamamura, M. A. Beaven, J.-P. Kinet. 1995. A requirement for Syk in the activation of the microtubule-associated protein kinase/phopholipase A2 pathway by Fc{epsilon}RI is not shared by a G protein-coupled receptor. J. Biol. Chem. 270:10960.[Abstract/Free Full Text]
  30. Vojtek, A. B., J. A. Cooper. 1995. Rho family members: activators of MAP kinase cascades. Cell 82:527.[Medline]
  31. Symons, M.. 1996. Rho family GTPases: the cytoskeleton and beyond. Trends Biochem. Sci. 21:178.[Medline]
  32. Machesky, L. M., A. Hall. 1996. Rho: a connection between membrane receptor signalling and the cytoskeleton. Trends Cell Biol. 6:304.[Medline]
  33. Kawakami, Y., T. Miura, R. Bissonnette, D. Hata, W. N. Khan, T. Kitamura, M. Maeda-Yamamoto, S. E. Hartman, L. Yao, F. W. Alt, T. Kawakami. 1997. Bruton’s tyrosine kinase regulates apoptosis and JNK/SAPK kinase activity. Proc. Natl. Acad. Sci. USA 94:3938.[Abstract/Free Full Text]
  34. Zhang, C., R. A. Baumgartner, K. Yamada, M. A. Beaven. 1997. Mitogen-activated protein (MAP) kinase regulates production of TNF-{alpha} and release of arachidonic acid in mast cells: indications of communication between p38 and p42 MAP kinases. J. Biol. Chem. 272:13397.[Abstract/Free Full Text]
  35. Karin, M.. 1995. The regulation of AP-1 activity by mitogen-activated protein kinases. J. Biol. Chem. 270:16483.[Free Full Text]
  36. Hibi, M., A. Lin, T. Smeal, A. Minden, M. Karin. 1993. Identification of an oncoprotein- and UV-responsive protein kinase that binds and potentiates the c-Jun activation domain. Genes Dev. 7:2135.[Abstract/Free Full Text]
  37. Derijard, B., M. Hibi, I.-H. Wu, T. Barret, B. Su, T. Deng, M. Karin, R. J. Davis. 1994. JNK1: a protein kinase stimulated by UV light and Ha-Ras that binds and phosphorylates the c-Jun activation domain. Cell 76:1025.[Medline]
  38. Kyriakis, J. M., P. Banerjee, E. Nikolakaki, T. Dai, E. A. Rubie, M. F. Ahmad, J. Avruch, J. R. Woodgett. 1994. The stress-activated protein kinase subfamily of c-Jun kinases. Nature 369:156.[Medline]
  39. Kyriakis, J. M., J. Avruch. 1996. Sounding the alarm: protein kinase cascades activated by stress and inflammation. J. Biol. Chem. 271:24313.[Free Full Text]
  40. Zhang, S., J. Han, M. A. Sells, J. Chernoff, U. G. Knaus, R. J. Ulevitch, G. M. Bokoch. 1995. Rho family GTPases regulate p38 mitogen-activated protein kinase through the downstream mediator Pak1. J. Biol. Chem. 270:23934.[Abstract/Free Full Text]
  41. Bagrodia, S., B. Derijard, R. J. Davis, R. A. Cerione. 1995. Cdc42 and PAK-mediated signaling leads to Jun kinase and p38 mitogen-activated protein kinase activation. J. Biol. Chem. 270:27995.[Abstract/Free Full Text]
  42. Moriguchi, T., H. Kawasaki, S. Matsuda, Y. Gotoh, E. Nishida. 1995. Evidence for multiple activators for stress-activated protein kinases/c-Jun amino-terminal kinases: existence of novel activators. J. Biol. Chem. 270:12969.[Abstract/Free Full Text]
  43. Holland, P. M., M. Suzanne, J. S. Campbell, S. Noselli, J. A. Cooper. 1997. MKK7 is a stress-activated mitogen-activated protein kinase kinase functionally related to hemipterous. J. Biol. Chem. 272:24992.
  44. Tournier, C., A. J. Whitmarsh, J. Cavanagh, T. Barrett, R. J. Davis. 1997. Mitogen-activated protein kinase kinase 7 is an activator of the c-Jun NH2-terminal kinase. Proc. Natl. Acad. Sci. USA 94:7337.[Abstract/Free Full Text]
  45. Pombo, C. M., J. H. Kehrl, I. Sanchez, P. Katz, J. Avruch, L. I. Zon, J. R. Woodgett, T. Force, J. M. Kyriakis. 1995. Activation of the SAPK pathway by the human STE20 homologue germinal centre kinase. Nature 377:750.[Medline]
  46. Salmeron, A., T. B. Ahmad, G. W. Carlile, D. Pappin, R. P. Narsimhan, S. C. Ley. 1996. Activation of MEK-1 and SEK-1 by Tpl-2 protooncoprotein, a novel MAP kinase kinase kinase. EMBO J. 15:817.[Medline]
  47. Kiefer, F., L. A. Tibbles, M. Anafi, A. Janssen, B. W. Zanke, N. Lassam, T. Pawson, J. R. Woodgett, N. N. Iscove. 1996. HPK1, a hematopoietic protein kinase activating the SAPK/JNK pathway. EMBO J. 15:7013.[Medline]
  48. Hu, M. C.-T., W. R. Qiu, X. Wang, C. F. Meyer, T.-H. Tan. 1996. Human HPK1, a novel human hematopoietic progenitor kinase that activates the JNK/SAPK kinase cascade. Genes Dev. 10:2251.[Abstract/Free Full Text]
  49. Shirakabe, K., K. Yamaguchi, H. Shibuya, K. Irie, S. Matsuda, T. Moriguchi, Y. Gotoh, K. Matsumoto, E. Nishida. 1997. TAK1 mediates the ceramide signaling to stress-activated protein kinase/c-Jun N-terminal kinase. J. Biol. Chem. 272:8141.[Abstract/Free Full Text]
  50. Blank, J. L., P. Gerwins, E. M. Elliott, S. Sather, G. L. Johnson. 1996. Molecular cloning of mitogen-activated protein/ERK kinase kinases (MEKK) 2 and 3: regulation of sequential phosphorylation pathways involving mitogen-activated protein kinase and c-Jun kinase. J. Biol. Chem. 271:5361.[Abstract/Free Full Text]
  51. Ellinger-Ziegelbauer, H., K. Brown, K. Kelly, U. Siebenlist. 1997. Direct activation of the stress-activated protein kinase (SAPK) and extracellular signal-regulated protein kinase (ERK) pathways by an inducible mitogen-activated protein kinase/ERK kinase kinase 3 (MEKK) derivative. J. Biol. Chem. 272:2668.[Abstract/Free Full Text]
  52. Ichijo, H., E. Nishida, K. Irie, P. ten Dijke, M. Saitoh, T. Moriguchi, M. Takagi, K. Matsumoto, K. Miyazono, Y. Gotoh. 1997. Induction of apoptosis by ASK1, a mammalian MAPKKK that activates SAPK/JNK and p38 signaling pathways. Science 275:90.[Abstract/Free Full Text]
  53. Nishina, H., K. D. Fischer, L. Radvanyl, A. Shahinian, R. Hakem, E. A. Rubie, A. Bernstein, T. W. Mak, J. R. Woodgett, J. M. Penninger. 1997. Stress-signalling kinase Sek1 protects thymocytes from apoptosis mediated by CD95 and CD3. Nature 385:350.[Medline]
  54. Khan, W. N., F. W. Alt, R. M. Gerstein, B. A. Malynn, I. Larsson, G. Rathbun, L. Davidson, S. Mueller, A. B. Kantor, L. A. Herzenberg, F. S. Rosen, P. Sideras. 1995. Defective B cell development and function in Btk-deficient mice. Immunity 3:283.[Medline]
  55. Kawakami, T., N. Inagaki, M. Takei, H. Fukamachi, K. M. Coggeshall, K. Ishizaka, T. Ishizaka. 1992. Tyrosine phosphorylation is required for mast cell activation by Fc{epsilon}RI cross-linking. J. Immunol. 148:3513.[Abstract]
  56. Hata, D., Y. Kawakami, N. Inagaki, C. S. Lantz, T. Kitamura, W. N. Khan, M. Maeda-Yamamoto, T. Miura, W. Han, S. E. Hartman, L. Yao, H. Nagai, A. E. Goldfeld, F. W. Alt, S. J. Galli, O. N. Witte, T. Kawakami. 1998. Involvement of Bruton’s tyrosine kinase in Fc{epsilon}RI-dependent mast cell degranulation and cytokine production. J. Exp. Med. 187:1235.[Abstract/Free Full Text]
  57. Sanchez, I., R. T. Hughes, B. J. Mayer, K. Yee, J. R. Woodgett, J. Avruch, J. M. Kyriakis, L. I. Zon. 1994. Role of SAPK/ERK kinase-1 in the stress-activated pathway regulating transcription factor c-Jun. Nature 372:794.[Medline]
  58. Yan, M., T. Dai, J. C. Deak, J. M. Kyriakis, L. I. Zon, J. R. Woodgett, D. J. Templeton. 1994. Activation of stress-activated protein kinase by MEKK1 phosphorylation of its activator SEK1. Nature 372:798.[Medline]
  59. Ishizuka, T., A. Oshiba, N. Sakata, N. Terada, G. L. Johnson, E. W. Gelfand. 1996. Aggregation of the Fc{epsilon}RI on mast cells stimulates c-Jun amino-terminal kinase activity: a response inhibited by wortmannin. J. Biol. Chem. 271:12762.[Abstract/Free Full Text]
  60. Baranes, D., E. Razin. 1991. PKC regulates proliferation of mast cells and the expression of Fos and Jun proto-oncogene mRNAs during activation by IgE-Ag or calcium ionophore A23187. Blood 78:2354.[Abstract/Free Full Text]
  61. Li, Z., M. I. Wahl, A. Eguinoa, L. R. Stephens, P. T. Hawkins, O. N. Witte. 1997. Phosphatidylinositol 3-kinase-{gamma} activates Bruton’s tyrosine kinase in concert with Src family kinases. Proc. Natl. Acad. Sci. USA 94:13820.[Abstract/Free Full Text]
  62. Hata, D., J. Kitaura, S. E. Hartman, Y. Kawakami, T. Yokota, T. Kawakami. 1998. Bruton’s tyrosine kinase-mediated IL-2 gene activation in mast cells: dependence on the JNK activation pathway. J. Biol. Chem. 273:10979.[Abstract/Free Full Text]
  63. Rodriguez-Viciana, P., P. H. Warne, R. Dhand, B. Vanhaesebroeck, I. Gout, M. J. Fry, M. D. Waterfield, J. Downward. 1994. Phosphatidylinositol-3-OH kinase as a direct target of Ras. Nature 370:527.[Medline]
  64. Teramoto, H., P. Salem, K. C. Robbins, X. R. Bustelo, J. S. Gutkind. 1997. Tyrosine phosphorylation of the vav proto-oncogene product links Fc{epsilon}RI to the Rac1-JNK pathway. J. Biol. Chem. 272:10751.[Abstract/Free Full Text]
  65. Gupta, S., T. Barrett, A. J. Whitmarsh, J. Cavanagh, H. K. Sluss, B. Derijard, R. J. Davis. 1996. Selective interaction of JNK protein kinase isoforms with transcription factors. EMBO J. 15:2760.[Medline]
  66. Su, B., E. Jacinto, M. Hibi, T. Kallunki, M. Karin, Y. Ben-Neriah. 1994. JNK is involved in signal integration during costimulation of T lymphocytes. Cell 77:727.[Medline]
  67. Zhang, C., N. Hirasawa, M. A. Beaven. 1997. Antigen activation of mitogen-activated protein kinase in mast cells through protein kinase C-dependent and independent pathways. J. Immunol. 158:4968.[Abstract]
  68. Downward, J., J. D. Graves, P. H. Warne, S. Rayter, D. A. Cantrell. 1990. Stimulation of p21ras upon T-cell activation. Nature 346:719.[Medline]
  69. Qin, S., Y. Minami, T. Kurosaki, H. Yamamura. 1997. Distinctive functions of Syk and Lyn in mediating osmotic stress- and ultraviolet C irradiation-induced apoptosis in chicken B cells. J. Biol. Chem. 272:17994.[Abstract/Free Full Text]
  70. Mahajan, S., J. Fargnoli, A. L. Burkhardt, S. A. Kut, S. J. Saouaf, J. B. Bolen. 1995. Src family protein tyrosine kinases induce autoactivation of Bruton’s tyrosine kinase. Mol. Cell. Biol. 15:5304.[Abstract]
  71. Devary, Y., R. A. Gottlieb, T. Smeal, M. Karin. 1992. The mammalian ultraviolet response is triggered by activation of Src tyrosine kinases. Cell 71:1081.[Medline]
  72. Uckun, F. M., K. G. Waddick, S. Mahajan, X. Jun, M. Takata, J. Bolen, T. Kurosaki. 1996. BTK as a mediator of radiation-induced apoptosis in DT-40 lymphoma B cells. Science 273:1096.[Abstract]



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