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* Department of Immunology and Signal Transduction, Tokyo Metropolitan Institute for Neuroscience, Tokyo Metropolitan Organization for Medical Science, and
Graduate School of Science, Tokyo Metropolitan University, Tokyo, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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N) lacking the
NH2-terminal region, which contains four tyrosine residues,
was introduced in SHP-1-C/S-expressing WEHI-231 cells, the enhanced JNK
activation was inhibited. Among candidate proteins likely to regulate
JNK activation through BLNK, Nck adaptor protein was found to associate
with tyrosine-phosphorylated BLNK and this association was more
pronounced in SHP-1-C/S-expressing cells. Furthermore, expression of
dominant-negative forms of Nck inhibited BCR-induced JNK activation.
Finally, BCR-induced apoptosis was suppressed in SHP-1-C/S-expressing
cells and coexpression of Nck SH2 mutants or a dominant-negative form
of SEK1 reversed this phenotype. Collectively, these results suggest
that SHP-1 acts on BLNK, modulating its association with Nck, which in
turn negatively regulates JNK activation but exerts a positive effect
on apoptosis. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and Ig
(1, 2). Phosphorylated ITAMs of Ig and Ig then
recruit and activate Syk PTK, which in turn phosphorylates multiple
cellular substrates, and transduces a variety of downstream signaling
events (3, 4), including activation of Ras, activation of
mitogen-activated protein kinases (MAPKs), phosphoinositide turnover,
increases in intracellular calcium, and other intermediary events,
which ultimately leads to cell proliferation, differentiation,
activation, or cell death. Transmission of BCR signals to intracellular
signaling machinery is highly dependent on reversible protein tyrosine
phosphorylation, which is precisely balanced between PTKs and protein
tyrosine phosphatases (PTPs) (5, 6).
Src homology region 2 (SH2) domain-containing phosphatase 1
(SHP-1) is a cytoplasmic PTP containing two SH2 domains at the
NH2 terminus, which is implicated in negative
regulation of several receptors, including BCR (6, 7). In
the resting state, SHP-1 interacts with the BCR complex, most likely
maintaining its dephosphorylated state (8). Upon BCR
ligation, SHP-1 dissociates from the BCR complex and binds to
BCR-inducible tyrosine-phosphorylated CD22, CD72, and paired Ig-like
receptor B (PIR-B), all of which contain immunoreceptor
tyrosine-based inhibition motifs (ITIMs) in their cytoplasmic portions
(9, 10, 11, 12, 13). Since SHP-1 is activated when its SH2 domains
interact with tyrosine-phosphorylated peptides (14), SHP-1
bound to CD22, CD72, or PIR-B might efficiently dephosphorylate
signaling molecules such as Ig/Ig and Syk, thereby acting as a
negative regulator (15).
In addition, our earlier study (16) showed that SHP-1
dephosphorylates B cell linker protein (BLNK) (17), also
named SH2 domain leukocyte protein of 65 kDa (SLP-65) (18)
or B cell adaptor containing SH2 domain (19). Upon BCR
ligation, BLNK is tyrosine-phosphorylated by Syk and translocates to
the membrane fraction (17), where it acts as a scaffold
for recruiting various signaling proteins, such as phospholipase C
,
Vav, Grb2, Nck, and Btk and is therefore capable of mediating an array
of distinct cellular outcomes (20). For example, B cell
development is blocked at the transition from pro-B to pre-B cells in
BLNK-deficient mice (21, 22). Moreover, BLNK-deficient B
cells fail to enter the cell cycle upon BCR ligation due to the
inability to induce the expression of cell cycle regulatory proteins
and exhibit impaired activation of NF-
B (23). Our
finding that BLNK is a physiological substrate of SHP-1 in B cells
suggests that SHP-1 can regulate multiple signaling pathways by
dephosphorylating BLNK tyrosine residues to which multiple signaling
molecules are recruited (16).
By introducing a form of SHP-1 lacking PTP activity (SHP-1-C/S) into WEHI-231 cells, we were also able to show that BCR-induced c-Jun NH2-terminal kinase (JNK) is significantly enhanced (16). JNK is a member of the MAPK family and is known to play an important role in a variety of downstream signaling pathways (for review, see Ref. 24). When cells are treated with cytokines or exposed to environmental stress, JNK is phosphorylated at tyrosine and threonine residues by SEK1 (also known as MKK4) or MKK7 and activated (24). Several lines of evidence indicate that JNK is activated by Rho family small GTPases (25, 26), Nck adaptor protein and Ste20-like protein kinase, Nck-interacting kinase (NIK) (27), TNFR-associated factor 2 (TRAF2) (28), or Ste20-like kinase, HPK1 (29, 30). However, it is not yet clear how these upstream molecules are interrelated or how JNK modulates final cellular responses. For example, one group demonstrated that activation of JNK and p38 correlates with cell death in one human B cell line (31), whereas no correlation was found between JNK activation and BCR-mediated cell death in WEHI-231 cells (32, 33). In the present study, we investigated molecular mechanisms whereby SHP-1 regulates BCR-induced JNK activation and apoptosis in B cells. Our findings indicate that SHP-1 negatively regulates JNK activation by modulating the association of BLNK with Nck, while exerting a positive effect on BCR-induced apoptosis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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WEHI-231 cells were obtained from the American Type Culture Collection (Manassas, VA) and maintained in RPMI 1640 (Sigma-Aldrich, St. Louis, MO) supplemented with 10% FBS (PAA Laboratories, Linz, Austria), 50 µM 2-ME, 100 µg/ml streptomycin, and 100 U/ml penicillin (complete medium).
Motheaten mice and preparation of splenic B cells
Mice homozygous for the motheaten (me) mutation were obtained by mating C3HeBFeJ-me/+ breeding pairs, which were originally purchased from The Jackson Laboratory (Bar Harbor, ME) and were maintained in our animal facility in accordance with institutional policies for animal care. All mice were genotyped by using PCR amplification of tail DNA as described previously (34) and used at 2 wk of age. Splenic B cells were prepared by treating spleen cells from C3HeBFeJ-me/me and C3HeBFeJ-+/+ mice with anti-Thy-1.2 mAb and rabbit C. The resultant cells were >95% positive for surface IgM expression.
Antibodies
Goat anti-mouse IgM Ab was purchased from Cappel, Organon Teknika (Durham, NC). Anti-phosphotyrosine (PY) mAb (PY20) and anti-HPK1 Ab were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-Vav, anti-Nck, and anti-SHP-1 Abs were purchased from Upstate Biotechnology (Lake Placid, NY). Anti-flag epitope M2 mAb was obtained from Sigma-Aldrich. Anti-BLNK Ab was described previously (16). Anti-hemagglutinin (HA) mAb (12CA5) was purchased from Roche Diagnostics (Mannheim, Germany). Rabbit anti-mouse phospho-specific p38 and phospho-specific JNK Abs were purchased from New England Biolabs (Beverly, MA) and rabbit anti-mouse phospho-specific extracellular signal-regulated kinase (ERK) Ab was obtained from Promega (Madison, WI). Rabbit anti-mouse ERK-2, anti-JNK-2, and anti-p38 Abs were purchased from Santa Cruz Biotechnology. Alkaline phosphatase (AP)-conjugated goat anti-mouse IgG and AP-conjugated mouse anti-rabbit IgG were obtained from Bio-Rad (Richmond, CA) and Jackson ImmunoResearch Laboratories (West Grove, PA), respectively. HRP-conjugated anti-rabbit and anti-mouse IgGs were purchased from Santa Cruz Biotechnology.
Immunoprecipitation and Western blot analysis
Immunoprecipitation and Western blot analysis were performed as described previously (16). Briefly, cells (1 x 107) were suspended in 1 ml of complete medium and stimulated for the indicated times with 20 µg/ml anti-IgM Ab. The reactions were stopped with 6 ml ice-cold PBS containing 1 mM Na3VO4 and 2 mM EDTA (PBS-VE). After washing twice with PBS-VE, cells were lysed in 500 µl of TNE buffer (1% Nonidet P-40, 10 mM Tris-HCl (pH 7.5), 150 mM NaCl, 2 mM Na3VO4, and 2 mM EDTA) and centrifuged. The supernatants were immunoprecipitated with protein G-Sepharose coupled with anti-flag mAb or Abs against BLNK and Nck. Immunoprecipitates were boiled in reducing Laemmli SDS sample buffer and subjected to 10% SDS-PAGE, followed by transfer to nitrocellulose membranes. The membranes were incubated with anti-PY mAb, anti-flag mAb, anti-BLNK Ab, or anti-Nck Ab, visualized by incubating with AP-conjugated goat anti-mouse IgG or AP-conjugated mouse anti-rabbit IgG, and developed with nitroblue tetrazolium and 5-brono-4-chloro-3-indoyl phosphate. Alternatively, the membranes were incubated with HRP-conjugated anti-mouse IgG or anti-rabbit IgG and then visualized using an ECL Western blot detection kit (Amersham Pharmacia Biotech, Piscataway, NJ).
Expression constructs and transfection
Expression constructs for flag-tagged SHP-1-wild type
(wt), SHP-1-C/S, and BLNK-wt have been described elsewhere
(16). A flag-BLNK cDNA in which aa 1–190 and aa 130–312
were deleted was generated by PCR using BLNK-wt cDNA as a template and
subcloned into pEF-flag/stop vector (a gift from Dr. G. Koretzky,
University of Pennsylvania, Philadelphia, PA), yielding
pEF-flag-BLNKN and pEF-flag-BLNKPro, respectively. To generate an
expression construct for Nck-wt, a PCR fragment containing the entire
open reading frame of mouse Nck was cloned downstream of the EF
promoter in pEF-flag/stop vector (pEF-flag-Nck-wt). A Nck cDNA with a
deletion of the SH2 domain was generated by PCR, and the amplified
product, encompassing aa 1–281 of Nck, was cloned in pEF-flag/stop
vector, yielding pEF-flag-NckSH2. To introduce an arginine to lysine
substitution at position 373 in the SH2 domain of BLNK and position 289
in the SH2 domain of Nck, in vitro mutagenesis was respectively
performed on full-length BLNK and Nck cDNAs cloned in pBluescript using
a Gene Editor in vitro Site-directed Mutagenesis System (Promega).
Mutation was verified by sequencing and the mutated cDNA fragments were
then cloned in pEF-flag/stop vector, yielding pEF-flag-BLNK-RK and
pEF-flag-Nck-RK. The expression plasmid encoding a HA-tagged
dominant-negative SEK1 (SEK-DN) has been described (35).
Transient transfection was performed as described previously
(16). The expression level of transfected genes was
20–22% as revealed by flow cytometric analysis with cells transfected
with pEF-flag-green fluorescence protein.
Assays for MAPKs
TNE-soluble supernatants from cells, either left unstimulated or stimulated with anti-IgM for 5 min, were subjected to 10% SDS-PAGE and transferred to nitrocellulose membranes. The blots were incubated first with Abs against ERK, JNK, p38, and their phosphorylated forms, then with HRP-conjugated anti-rabbit IgG, after which they were visualized using an ECL Western blot detection kit. The intensity of each band was measured with a Bio-Rad imaging densitometer. Under the conditions examined, the intensity of bands was linearly correlated to the amount of samples. The results were expressed as fold activation, with the intensity of unstimulated cells being assigned a value of 1.
Assays for apoptosis
Cells (1.5 x 106) were suspended in 5 ml of complete medium and either left untreated or stimulated with 5 µg/ml anti-IgM Ab for 48 h. The cells were then harvested, washed twice with PBS, and subjected to DNA fragmentation assays (36) and TUNEL assays, as previously described (37).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our previous work demonstrated that BCR-induced JNK activation is
selectively enhanced in SHP-1-C/S transfectants, suggesting a negative
regulatory role for SHP-1 in JNK activation (16). To
confirm the physiological relevance of SHP-1 regulation of JNK
activation, we used splenic B cells from SHP-1-deficient
C3HeBFeJ-me/me and control C3HeBFeJ-+/+
mice. B cells were cultured with or without anti-IgM Ab for 5 min
and then phosphorylation of total cellular proteins were examined by
Western blot with anti-PY mAb. As shown in Fig. 1
A, tyrosine phosphorylation
was constitutively enhanced in me/me mice, suggesting a
negative regulatory role of SHP-1 in B cells. Activation of MAPK family
members was assayed by immunoblotting with Abs against activated forms
of ERK, JNK, and p38. As in WEHI-231 cells, SHP-1 regulates activation
of JNK, but not ERK or p38 (Fig. 1B). Furthermore, in
me/me B cells, JNK was constitutively activated and further
enhanced upon BCR ligation. The level of JNK activation, either without
or with BCR ligation, was higher than that of control B cells. These
results corroborate the physiological significance of negative,
selective regulation of JNK by SHP-1.
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Having confirmed negative regulation of JNK activation by SHP-1 in
B cells, we assessed the mechanisms of SHP-1 action using WEHI-231
cells. Initially, we asked whether increased tyrosine phosphorylation
of BLNK (16) was responsible for the enhanced JNK
activation. WEHI-231 cells, transiently transfected with
pEF-flag-tagged SHP-1-wt or SHP-1-C/S along with pEF-flag-tagged
wild-type or mutant forms of BLNK, were stimulated with anti-IgM Ab
for 5 min, after which activation of MAPK family members was measured
by immunoblotting with anti-phospho-ERK, -JNK, and -p38 Abs (Fig. 2). The mutant forms of BLNK used in this
study lacked the NH2-terminal acidic region
(BLNKN) or proline-rich region (BLNKPro) or contained an arginine
to lysine substitution at the position 373 in the SH2 domain (BLNK-RK;
Fig. 2A). Since arginine 373 is the predicted phosphate
binding site, BLNK-RK would be expected to lack the ability to bind
phosphotyrosine-containing proteins. As shown in Fig. 2B,
activation of ERK and p38 was not significantly different among
transfectants. By contrast, JNK activation was affected by various
transfections. In our system, JNK2 was predominantly activated. As
shown in Fig. 2B (top panel, sample 1 vs
sample 2), JNK2 was more strongly activated in transfectants with
BLNK-wt and this enhancement was inhibited by cotransfection with
SHP-1-wt (sample 2 vs sample 3). However, transfection with BLNK-wt and
SHP-1-C/S reversed this inhibition (Fig. 2B, sample 3
vs sample 4) and the level of JNK2 activation was higher than that
observed in transfectants with BLNK-wt only (Fig. 2B, sample
2 vs sample 4). This enhanced JNK2 activation was reduced in cells
expressing SHP-1-C/S and BLNKN (Fig. 2B, sample 4 vs
sample 5), whereas the expression of BLNKPro or BLNK-RK along with
SHP-1-C/S exhibited little effects on BCR-induced JNK2 activation. This
result suggests that the NH2-terminal region of
BLNK containing tyrosine residues plays an essential role in JNK
activation and that the enhanced JNK activation seen in
SHP-1-C/S-expressing cells might be due to phosphotyrosine-based
binding of signaling molecules to BLNK.
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Several upstream regulators for JNK have been identified in
various cellular systems, including Vav, Nck, HPK1, and TRAF2. Because
tyrosine phosphorylation of BLNK is required for JNK activation, we
first attempted to identify such upstream regulators in our system by
identifying molecules whose association with BLNK was enhanced in
SHP-1-C/S-expressing cells upon BCR ligation. WEHI-231 cells,
transfected with BLNK-wt along with SHP-1-wt or SHP-1-C/S, were either
left untreated or stimulated with anti-IgM Ab for 2 or 7 min, after
which immunoprecipitated BLNK was blotted with Abs against candidate
proteins. Although we could not detect an association between BLNK and
TRAF2 (data not shown), BLNK was found to bind Nck within 2 min of BCR
ligation, and this binding was significantly enhanced in
SHP-1-C/S-expressing cells (Fig. 3A). Reciprocal experiments in
which anti-Nck immunoprecipitates were blotted with anti-BLNK
Ab again showed the association between BLNK and Nck to be increased in
SHP-1-C/S-expressing cells than in SHP-1-wt-expressing cells (Fig. 3B).
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C). We found that
association of Nck-wt with endogenous BLNK was higher in SHP-1-C/S
transfectants than in SHP-1-wt transfectants. In this experiment,
association between Nck and BLNK was constitutively observed. One of
the possibilities is that BLNK was phosphorylated, albeit at a low
level, before BCR ligation. Additionally, overexpression of Nck-wt may
contribute to the detectable level of association between BLNK and Nck.
Significantly, even under these conditions, association between BLNK
and Nck was completely abrogated when Nck-RK was introduced. It is thus
very likely that Nck binds to BLNK via its SH2 domain and this
association is dependent on tyrosine phosphorylation of BLNK. Nck is required for SHP-1-mediated JNK regulation
The finding that Nck associates with BLNK prompted us to examine
how Nck regulates JNK activation following BCR ligation. To address
this question, we investigated the effect of Nck expression on
BCR-induced JNK activation (Fig. 4A). WEHI-231 cells were
transiently transfected with a combination of SHP-1-wt or SHP-1-C/S and
Nck-wt, Nck-RK, or a Nck mutant lacking its SH2 domain (NckSH2),
after which BCR-induced JNK activation was compared. Both Nck-RK and
NckSH2 were expected to act as dominant-negatives by attenuating the
association of Nck with tyrosine-phosphorylated BLNK (38).
We found that Nck-wt transfection strongly enhanced BCR-induced JNK2
activation (Fig. 4B, top, sample 1 vs sample 2)
and this enhancement was diminished by transfection with Nck-RK or
NckSH2 (Fig. 4B, sample 2 vs samples 3 and 4).
Similarly in transfectants with SHP-C/S, enhanced JNK2 activation was
significantly attenuated in cells expressing NckSH2 and Nck-RK (Fig. 4B, sample 7 vs samples 8 and 9). The fact that inhibition
was more pronounced in NckSH2-expressing cells than in
Nck-RK-expressing cells may reflect higher expression of NckSH2.
Again, there was no significant difference in the level of ERK and p38
activation among these transfectants (Fig. 4B). Taken
together, these results suggest that Nck recruited to
tyrosine-phosphorylated BLNK via its SH2 domain acts as an upstream
mediator of BCR-induced JNK activation.
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WEHI-231 cells have characteristics of immature B cells and
therefore are susceptible to growth arrest and apoptotic cell death
upon BCR stimulation (36, 39). To clarify how SHP-1
regulates the final outcome of BCR signaling through dephosphorylation
of BLNK, we examined the effect of transient SHP-1-C/S expression on
BCR-induced apoptosis. WEHI-231 cells were transfected with vector
alone, SHP-1-wt, or SHP-1-C/S, after which they were left unstimulated
or stimulated with anti-IgM Ab for 48 h and then subjected to
DNA fragmentation and TUNEL assays. As shown in Fig. 5, in contrast to augmented BCR-induced
apoptosis in SHP-1-wt-expressing cells, SHP-1-C/S expression
significantly inhibited BCR-induced apoptosis, implying a positive
regulation of BCR-induced apoptosis by SHP-1.
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The results thus far suggest that BCR-induced JNK activation is
enhanced in SHP-1-C/S transfectants, while BCR-induced apoptosis is
suppressed. If JNK activation is directly involved in the inhibition
BCR-induced apoptosis, specific inactivation of JNK should increase
cell death following BCR ligation. To test this possibility, we
transfected WEHI-231 cells with SEK-DN (40) to inhibit an
upstream activator of JNK (Fig. 6A). As shown in Fig. 6B, transfection of SEK-DN significantly reduced JNK
activation, but had little effect on ERK and p38 activities (data not
shown). Significantly, it partially reversed the suppression of
BCR-induced apoptosis in cells overexpressing SHP-1-C/S (Fig. 6, C and D, samples 4 vs 5), suggesting that JNK
activation directly regulates apoptosis in WEHI-231 cells in a negative
fashion.
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Finally, to assess the involvement of Nck adaptor protein in the
regulation of BCR-induced apoptosis, we transiently transfected
WEHI-231 cells with the aforementioned forms of Nck (Fig. 3) and
performed DNA fragmentation and TUNEL assays. As shown in Fig. 7, BCR-induced apoptosis was
significantly diminished in cells expressing Nck-wt, whereas the
expression of Nck-RK and NckSH2 reversed this effect. These data,
along with those in Fig. 5, suggest that there is an inverse
correlation between JNK activation and apoptosis following BCR
ligation, and that the SH2 domain of Nck is directly involved in
SHP-1-mediated regulation of apoptosis through its interaction
with BLNK.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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/Ig, Lyn (42), Syk
(15), and BLNK (16), thus acting as an
inhibitor of BCR-driven activation events. However, the precise
molecular mechanisms by which SHP-1 regulates downstream signaling
pathways are still largely unknown. We previously demonstrated that cells expressing a form of SHP-1 lacking PTP activity (SHP-1-C/S) exhibited increased tyrosine phosphorylation of BLNK and selectively enhanced JNK activation upon BCR ligation. In this study, we first assessed the physiological relevance of the findings in a B cell line using splenic B cells from SHP-1-deficient me/me mice. In the event, JNK, but not ERK or p38, was constitutively activated and further induced upon BCR ligation in me/me B cells, corroborating a selective, negative role of SHP-1 in B cells. Phosphorylation of BLNK, however, could not be examined in splenic B cells, because BLNK was not effectively immunoprecipitated in our hands. We therefore focused, in this study, on WEHI-231 cells to investigate molecular mechanisms linking BLNK tyrosine phosphorylation to JNK activation and to apoptotic processes. We found that expression of a BLNK mutant lacking the NH2-terminal region inhibited the enhanced JNK activation seen in SHP-1-C/S-expressing cells, indicating that this region of BLNK containing six tyrosine residues is required for the enhanced JNK activation seen in SHP-1-C/S-expressing cells. In addition, Nck adaptor protein was clearly shown to associate with BLNK in a tyrosine phosphorylation-dependent manner. Cells expressing Nck SH2 mutants that do not interact with phosphotyrosine-containing proteins did not show enhanced JNK activation upon BCR ligation, even when cotransfected with SHP-1-C/S. It is thus concluded that among several upstream activators of JNK, Nck is a candidate protein linking BLNK with JNK activation.
Nck is a ubiquitously expressed adaptor protein possessing one SH2 and two SH3 domains (43). Since it has no catalytic activity, Nck might regulate signaling processes by coupling a catalytic component, which binds to its SH3 domains, to tyrosine-phosphorylated proteins. For example, NIK is a Ste20-family serine/threonine kinase that is constitutively associated with Nck (44). As in the case of other members of the Ste20 protein kinase family, overexpression of NIK itself activates the MAPK kinase kinase 1/SEK1/JNK pathway but fails to activate ERK or p38 cascade (44). Thus, SHP-1 may regulate the amount of the Nck-NIK complex recruited to phosphorylated tyrosine residues on BLNK by controlling the level of tyrosine phosphorylation of BLNK during BCR signaling.
The MAPK module consists of three protein kinases that sequentially phosphorylate and activate downstream kinase cascade, MAP kinase kinase kinase (or MAPK kinase kinase), MAP kinase kinase (MAPKK or MEK), and MAPK (45). MAPK is phosphorylated at threonine and tyrosine residues by MAPKK to be activated. In the case of JNK, 13 MAPKKKs, 2 MAPKKs, and 3 JNKs have been identified in the mammalian cells (24). Although the kinase cascade leading to JNK activation is highly conserved, the upstream components that regulate the JNK pathway are diverse, depending on cell types and types of stimulation. Accumulating evidence indicates that the Rho family small GTPases (25, 26) and the TRAF group of adaptor proteins (28), respectively, mediate the activation of JNK in receptor-type PTK and cytokine receptor signaling pathways. Rho family GTPase is of particular interest to us because Vav, a Rho family guanine nucleotide exchange factor, is activated by tyrosine phosphorylation and is demonstrated to interact with tyrosine-phosphorylated BLNK through its SH2 domain (20). However, we could not detect differences in the amount of Vav1 recruited to BLNK in cells expressing SHP-1-wt or SHP-1-C/S (data not shown). Nevertheless, the observation that expression of the dominant-negative form of Vav1, possessing the RK mutation in its SH2 domain, inhibited enhanced JNK activation in SHP-1-C/S-expressing cells suggests that Vav1 might be involved in the pathways leading to JNK activation. The contribution of Vav to this signaling pathway is under study.
Recently, HPK1, a Ste20 family protein kinase (29, 30), was shown to be involved in BCR- and TCR-mediated signaling (46). Although HPK1 associates with BLNK and SLP-76 upon AgR stimulation in B and T cells, respectively, their association is quite different from that between BLNK and Nck or Vav. Following AgR ligation, HPK1 is phosphorylated at tyrosine 379 and subsequently interacts with the SH2 domains of BLNK and SLP-76, which in turn induces full activation (47, 48). Consistent with this model, association between BLNK and HPK1 was observed after BCR stimulation in our system, but the amount of HPK1 in the anti-BLNK immunoprecipitates was similar in lysates from SHP-1-C/S- and SHP-1-wt-expressing cells (data not shown). Interaction of BLNK with HPK1 may thus be independent of SHP-1 action.
Finally, we focused on the potential role of SHP-1 as a regulator of BCR-induced apoptotic signals. Using WEHI-231 cells, we clearly demonstrated that BCR-induced apoptosis is enhanced in SHP-1-wt transfectants but inhibited in SHP-1-C/S-expressing cells, suggesting a positive role for SHP-1 in this process. Interestingly, the inhibition of apoptosis seen in SHP-1-C/S transfectants was reversed by the expression of SEK-DN, implying that JNK plays a positive role in this pathway. Despite extensive study, the precise roles of the MAPK family members in apoptosis or cell survival remains controversial, possibly because of differences in the experimental procedures, cell types, and ligands used. The JNK pathway has been implicated in both apoptosis and cell survival signaling (49). Given that JNK is activated by exposing cells to stress, it is likely that JNK may mediate some effects of stress itself which in turn results in the induction of apoptosis. Alternatively, JNK activation may serve as a protective response to stress, facilitating antiapoptotic processes. Evidence favoring the latter comes from the observation that apoptosis is enhanced in the developing forebrain of jnk1/jnk2 double null mouse embryos (50, 51) and that integrin-mediated survival signaling is mediated by the JNK signaling pathway (52). Other studies have demonstrated that there is a positive correlation between ERK activation and BCR-induced apoptosis (32, 33) and that BCR-induced activation of JNK and p38 correlates apoptosis (31). The definitive reasons for these discrepancies are unclear at present.
In summary, we have demonstrated that SHP-1 negatively regulates BCR-induced JNK activation by dephosphorylating its physiological substrate, BLNK, thereby attenuating the association of BLNK with Nck. This negative regulation of JNK activation by SHP-1 contributes, at least in part, to the enhancement of BCR-induced apoptotic cell death. The molecular events linking JNK activation to apoptosis remains to be elucidated.
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Acknowledgments |
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Footnotes |
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2 Current address: Toyama Medical and Pharmaceutical University, Toyama 930-0194, Japan.
3 Address correspondence and reprint requests to Dr. Hidetaka Yakura, Tokyo Metropolitan Institute for Neuroscience, Fuchu, Tokyo 183-8526, Japan. E-mail address: yakura{at}tmin.ac.jp
4 Abbreviations used in this paper: BCR, B cell Ag receptor; BLNK, B cell linker protein; JNK, c-Jun NH2-terminal kinase; MAPK, mitogen-activated protein kinase; SH2, Src homology 2; SHP-1, SH2 domain-containing phosphatase 1; wt, wild type; PTK, protein tyrosine kinase; ITAM, immunoreceptor tyrosine-based activation motif; PTP, protein tyrosine phosphatase; ITIM, immunoreceptor tyrosine-based inhibition motif; SLP-65, -76, SH2 domain leukocyte protein of 65 and 76 kDa, respectively; NIK, Nck-interacting kinase; TRAF, TNFR-associated factor; PY, phosphotyrosine: HA, hemagglutinin; ERK, extracellular signal-regulated kinase; AP, alkaline phosphatase; DN, dominant negative; MAPKK, MAPK kinase.
Received for publication February 1, 2002. Accepted for publication May 9, 2002.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B and cell cycle entry and survival of B lymphocytes. J. Biol. Chem. 276:20055.B activation, and increased sensitivity to TNF-induced cell death in TRAF2-deficient mice. Immunity 7:715.[Medline]
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) or without
(
) 5 µg/ml anti-IgM Ab for 48 h. The cells were then
subjected to TUNEL assays and the percentage of apoptotic cells was
measured with a flow cytometer. The results are expressed as means of
the percent apoptotic cells ± SEM (error bars) from three
independent experiments.
