(Received for publication, September 24, 1996, and in revised form, November 5, 1996)
From the Department of Biochemistry, Kobe University
School of Medicine, Kobe 650, Japan, § Fukui Medical School,
Fukui 910-11, Japan, ¶ Biomedical Research Center, Osaka
University Medical School, Osaka 565, Japan, and
Kansai
Medical University, Moriguchi 570, Japan
It was found that Syk protein-tyrosine kinase is
rapidly activated in B cells after H2O2
treatment (oxidative stress) or increased extracellular NaCl
concentration (osmotic stress) as well as in response to B cell
receptor activation. In this study we examined the involvement of Syk
in responses elicited by these types of extracellular stress,
particularly Ca2+ responses and c-Jun amino-terminal kinase
(JNK) activation, using a chicken B cell line, DT40, as well as the
DT40-derived mutant DT40/Syk(), which does not express Syk. Osmotic
stress evokes increases in
[Ca2+]i by stimulating an
extracellular Ca2+ influx in both DT40 and DT40/Syk(
)
cells. In comparison, oxidative stress elicits an increase in
[Ca2+]i by stimulating both an
extracellular Ca2+ influx and Ca2+ release from
internal stores in DT40 cells, but this Ca2+ response is
partially abolished in DT40/Syk(
) cells, indicating that the
oxidative stress-induced Ca2+ response is at least partly
dependent on Syk. Interestingly, the depletion of Ca2+
results in a significantly decreased level of Syk activation in DT40
cells stimulated by oxidative but not osmotic stress. Furthermore, JNK
is activated to different extents by these two types of stress. The
extent of JNK activation in DT40/Syk(
) cells in response to osmotic
stress is comparable to that observed in DT40 cells. Intriguingly,
oxidative stress-induced JNK activation is significantly compromised in
DT40/Syk(
) cells. Collectively, these results indicate that both the
Ca2+ response and JNK activity induced by oxidative stress
are partly dependent on Syk, whereas those induced by osmotic stress
are independent of Syk.
Protein-tyrosine kinases (PTKs)1 play crucial roles in a wide variety of cellular responses, including cellular activation, proliferation, and differentiation (1). The binding of growth factors and cytokines to their cognate receptors stimulates intrinsic tyrosine kinase activities associated with these receptors or nonreceptor PTKs that couple to these receptors, thereby triggering downstream signaling events (2). It has also been reported that extracellular stress, ionizing radiation, UV irradiation, and H2O2 or genotoxic agents activate the nonreceptor PTKs of the Src- and/or Syk/ZAP-70 family (3-7), although the exact roles of these nonreceptor PTKs in stress-activated signaling pathways remain to be elucidated. It has been well established that extracellular stimuli promoting cellular activation and proliferation induce the activation of receptor PTKs or nonreceptor PTKs, leading to the subsequent activation of the Ras-Raf-Mek-extracellular signal-regulated kinase (ERK) signaling cascade (8). Activated ERKs of the mitogen-activated protein kinase superfamily, in turn, regulate gene expression by phosphorylating transcription factors such as Elk1 (9).
Recently, novel members of the mitogen-activated protein kinase superfamily, now referred to as stress-activated protein kinases or the c-Jun amino-terminal kinases (JNKs), have been identified in yeast and mammalian cells. JNKs exhibit an extraordinarily high affinity for their substrate, c-Jun, and phosphorylate it on specific amino-terminal serine residues (Ser-63 and Ser-73), thereby augmenting the function of c-Jun as a transcriptional activator (10). Stimuli that primarily activate Raf-Mek-ERK can only poorly activate the JNK cascade (11). Unlike ERKs, JNKs are strongly activated by a variety of extracellular stress (12-16). It has been recently reported that Rac/Cdc42-MEKK1-SEK protein kinases act upstream of JNK (17, 18). Although the participation of PTKs in stress signaling has been indicated (19), the identities and exact roles of such PTKs are largely unknown. Interestingly, recent studies reveal that c-Abl, a nonreceptor PTK localized in both the cytoplasm and the nucleus (20, 21), positively regulates the activities of JNKs in response to extracellular stress that selectively induces DNA damage (22, 23).
It has been shown that extracellular stress induces the activation of
the Syk/ZAP-70 family of PTKs and Ca2+ mobilization in T
and B lymphocytes, identical to those observed after antigen receptor
activation (24, 25). Unlike the Src family of PTKs, the Syk/ZAP-70
family of PTKs, Syk and ZAP-70, contain two SH2 domains, no
myristylation site, and no carboxyl-terminal negative regulatory
tyrosine residues (26, 27). Syk is expressed in a wide range of
hematopoietic cells, including T cells, B cells, myeloid cells, and
platelets, whereas ZAP-70 expression is restricted to T cells and
natural killer cells (28). Previous studies have demonstrated that Syk
plays a crucial role in B cell receptor (BCR)-mediated signaling (29,
30). Syk-negative mutants of the chicken B cell line DT40 could be
readily obtained due to the high frequency of homologous recombination
(31). Thus, DT40 cells and the DT40-derived mutant lacking Syk
[DT40/Syk()] have provided a powerful tool to examine the role of
Syk in BCR-mediated signaling (32). BCR activation in wild-type DT40
cells resulted in a rapid activation of Syk as well as a rapid increase
in [Ca2+]i. It was found that the
BCR-mediated Ca2+ response is almost completely abolished
in DT40/Syk(
) cells (32). There is growing evidence that strongly
suggests that upon BCR-activation, Syk mediates the activation of
phospholipase C
, resulting in the subsequent production of inositol
1,4,5-trisphosphate, thereby inducing an elevation in
[Ca2+]i (32). Interestingly,
H2O2 treatment (oxidative stress) also induces
a rapid activation of Syk as well as a rapid increase in
[Ca2+]i in DT40 cells, and this
Ca2+ response is drastically reduced in DT40/Syk(
) cells
(33).
Here we report that osmotic as well as oxidative stress induces Syk
activation, increases intracellular calcium concentration, and
activates JNK in a chicken B cell line, DT40. In this study we examined
the roles of Syk in the observed Ca2+ response and JNK
activity induced by the respective stress, using DT40 and DT40/Syk()
cells. Interestingly, it was found that both the Ca2+
response and JNK activation induced by oxidative stress were partly
dependent on Syk, yet those induced by osmotic stress were independent
of Syk. We will discuss the significance of our findings in respect to
the role of Syk in different cellular stress responses.
Acetoxymethyl esters of
bis-(o-aminophenoxy) ethane-N,N,N,N
-tetraacetic
acid (BAPTA-AM) were purchased from Life Technologies, Inc. Protein A
was from Calbiochem. Hydrogen peroxide and Fura 2-AM were from Wako
Pure Chemicals. Anti-phosphotyrosine antibody (4G10), mouse anti-human
JNK1 monoclonal antibody, and polyclonal anti-ERK/mitogen-activated
protein kinase antiserum were from Upstate Biotechnology Inc.,
Pharmingen (San Diego, CA), and Santa Cruz Biotechnology, respectively.
Enhanced chemiluminescence reagents were from DuPont.
Glutathione-Sepharose 4B was from Pharmacia Biotech Inc.
Establishment of DT40/Syk() and
DT40/Syk(
) expressing porcine Syk was performed as described
previously (32). DT40 and DT40-derived cells as well as the human B
cell line Raji were maintained in RPMI 1640 medium supplemented with
10% (v/v) fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml
streptomycin in a humidified 95% air, 5% CO2 atmosphere.
For experiments, cells were collected by centrifugation as described
previously (33). For experiments requiring the depletion of calcium,
cells were resuspended in calcium-free Hanks' balanced salt solution
buffer, and EGTA (final concentration, 1 mM) was added 2 min before stimulation. Cells were stimulated by hydrogen peroxide
(oxidative stress) or sodium chloride (osmotic stress) at 37 °C.
pGEX3X-c-Jun (amino acids 1-79) glutathione S-transferase fusion protein expression vector was transfected into Escherichia coli XL1Blue. Proteins were purified following the protocol recommended by the manufacturer (Pharmacia).
Measurement of [Ca2+]iCalcium mobilization was measured using fluorescent indicator Fura-2 as described previously (33).
Preparation of Cell ExtractsStimulated cells (1 × 107 cells/ml) were lysed in ice-cold lysis buffer (5 mM EDTA, 150 mM NaCl, 2% Triton X-100, 100 µM Na3VO4, 2 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and 50 mM Tris, pH 7.4) after a short centrifugation step. Lysates were clarified by centrifugation at 16,000 × g for 15 min at 4 °C.
Immunoblot AnalysisCell extracts or immunoprecipitates were resolved by SDS-polyacrylamide gel electrophoresis, transferred electrophoretically onto polyvinylidene difluoride membranes, and then immunoblotted with the indicated antibodies. Immunoreactive proteins were visualized by enhanced chemiluminescence.
Immunoprecipitation and Kinase AssaysThe
immunoprecipitated kinase activity of Syk was measured in a 30-µl
reaction mixture that included 10 µg of H2B histone as the exogenous
substrate (27). Immunoprecipitates by anti-JNK1 antibody (1 µg) with
40 µl of protein A-Sepharose 4B were washed three times with lysis
buffer, once with washing buffer (50 mM HEPES, pH 7.6, and
10 mM MgCl2), and once with kinase assay buffer (10 mM HEPES, pH 7.6, 10 mM MgCl2,
10 µM of cold ATP, and 10 µM vanadate).
Immune complex kinase assays were performed in a 30-µl kinase assay
buffer containing 1 µCi of [-32P]ATP (3,000 Ci/mmol)
and 5 µg of glutathione S-transferase-c-Jun as the
substrate. After a 20-min incubation at 30 °C, reactions were
terminated by the addition of SDS sample buffer and 5 min of boiling.
Autoradiography was carried out and quantitated using a phosphoimager
(Fuji BAS 2000).
In our previous study we showed that
H2O2 treatment (oxidative stress) as well as
BCR activation induced rapid tyrosine phosphorylation and activation of
Syk in chicken B cell line DT40 (33, 34) (Fig.
1A). To investigate whether or not high
osmolar exposure (osmotic stress), a distinct extracellular stress, can
induce tyrosine phosphorylation of Syk in DT40 cells, anti-Syk
immunoprecipitates of cell lysates from DT40 cells treated with or
without sodium chloride were subjected to immunoblotting with
anti-phosphotyrosine antibody. As shown in Fig. 1B
(top), the exposure of DT40 cells to sodium chloride
triggered a rapid and sustained tyrosine phosphorylation of Syk. This
increase in tyrosine phosphorylation was almost maximal within 1 min
and remained elevated throughout the 15-min incubation (Fig.
1B, top). This observed tyrosine phosphorylation
of Syk was dependent on the concentration of sodium chloride used. When exposed to low concentrations of sodium chloride, such as 0.2 M, significant tyrosine phosphorylation of Syk was observed
after a 5-min incubation (Fig. 1B). In parallel with Syk
tyrosine phosphorylation, sodium chloride stimulated a rapid and
transient increase in Syk activity (Fig. 1C). The ability of
Syk to phosphorylate the exogenous substrate, H2B histone, was
increased 2-3-fold upon treatment. Immunoblot analysis with an
anti-Syk antibody revealed that the amounts of Syk immunoprecipitated
from treated or untreated DT40 cells were comparable (Fig. 1,
A and B, bottom), indicating that the
result obtained reflects changes in the specific activity of Syk.
Furthermore, it was shown that Syk activation was also induced by other
high-osmolarity producers, such as potassium chloride, lithium
chloride, and sorbitol (Fig. 1D). Like DT40 cells, tyrosine
phosphorylation of Syk was observed in a human B cell line, Raji, upon
exposure to the respective high-osmolarity producers (data not shown).
These observations indicate that Syk activation by osmotic stress is
not restricted to DT40 cells and may be a common biochemical event for
cells of a B cell lineage.
Oxidative and Osmotic Stress Elicit Differential Ca2+ Responses via Syk-dependent and -independent Mechanisms
An increase in
[Ca2+]i has been appreciated as
one of the earliest events during lymphocyte activation. To address whether oxidative and osmotic stress can elicit a Ca2+
response, intracellular Ca2+ mobilization in DT40 cells was
examined using a fluorescent indicator, Fura-2. A representative
profile is shown in Fig. 2. Both oxidative and osmotic
stress induced increases in
[Ca2+]i, however, the kinetics of
the response differed. Oxidative stress triggered a rapid and sustained
increase in [Ca2+]i that was
maximal within 1 min and decreased only slightly over the duration of
the experiment. In contrast, osmotic stress induced a relatively slow
and sustained increase in [Ca2+]i.
In the absence of calcium influx after EGTA chelated the extracellular
calcium, oxidative stress was still capable of inducing an increase in
[Ca2+]i, but to a lesser extent,
whereas osmotic stress failed to trigger an increase in
[Ca2+]i under the same conditions.
Hence, it became apparent that an increase in
[Ca2+]i by oxidative stress was
mediated by both release from intracellular calcium stores and
extracellular calcium influx, but that the increase in
[Ca2+]i observed for osmotic
stress was solely dependent on extracellular calcium influx.
Syk plays a critical role in regulating an increase in
[Ca2+]i during anti-IgM-induced B
cell activation (32). To investigate whether the Ca2+
responses induced by extracellular stress were dependent on Syk, [Ca2+]i was monitored in the
Syk-deficient DT40/Syk() cell line. As shown in Fig. 2, A
and E, oxidative stress induced a delayed increase in
[Ca2+]i in DT40/Syk(
) cells
compared to that observed in DT40 cells. The impairment of the
oxidative stress-induced calcium response in DT40/Syk(
) cells was
largely restored by an ectopic expression of porcine Syk (data not
shown), excluding the possibility that the differing calcium responses
observed in DT40 and DT40/Syk(
) cells were due to clonal alteration.
In contrast, the osmotic stress-induced increase in
[Ca2+]i in DT40/Syk(
) cells was
comparable to that observed in DT40 cells (Fig. 2, B and
F). These results indicate that Syk plays a crucial role in
oxidative stress-induced but not osmotic stress-induced
Ca2+ responses in DT40 cells.
The result showing a partially impaired Ca2+
response in DT40/Syk() cells subjected to oxidative stress suggests
that Syk is one of the upstream signaling molecules required for
mediating the Ca2+ response in oxidative stress signaling.
Next we examined whether an increase in
[Ca2+]i could, in turn, modulate
the activity of Syk in oxidative stress- and osmotic stress-induced
signaling. For this purpose, tyrosine phosphorylation of Syk induced by
these types of stress was examined in the absence of intracellular free
calcium, utilizing the calcium chelators EGTA and BAPTA-AM. As shown in
Fig. 3A, osmotic stress-induced tyrosine
phosphorylation of Syk was slightly affected by the depletion of
intracellular/extracellular free calcium. Consistent with this result,
the tyrosine phosphorylation of cellular proteins by osmotic stress was
marginally affected under the same conditions (data not shown).
Interestingly, the depletion of intracellular/extracellular free
calcium resulted in a drastic decrease in tyrosine phosphorylation of
Syk, as well as other cellular proteins, in response to oxidative
stress (Fig. 3B and data not shown). Collectively, these
observations revealed that an increase in
[Ca2+]i was required for
full-scale activation of Syk by oxidative but not osmotic stress.
Differential Regulation of JNKs and ERKs by Oxidative and Osmotic Stress
Previous studies demonstrate that a variety of
extracellular stimuli activate members of the mitogen-activated protein
kinase superfamily, such as JNKs and ERKs, to different extents
(35-37). Hence, we address the question of whether JNKs as well as
ERKs are activated in DT40 cells by oxidative and osmotic stress. JNK activity was drastically increased (~9-fold increase compared to
basal) by oxidative stress (1 mM
H2O2) as measured by the phosphorylation of
glutathione S-transferase-c-Jun (Fig.
4A). JNKs were also activated (approximately
33-fold compared to basal) by osmotic stress (0.3 M NaCl).
As shown in Fig. 4A, JNK activation by both oxidative and
osmotic stress occurred in a dose-dependent manner.
ERKs have been extensively studied in the context of growth factor signal transmission (38). However, it has been recently reported that in addition to JNKs, ERKs are also activated by some extracellular stress (39-41). Accordingly, we examined whether ERKs were also activated in DT40 cells by oxidative and osmotic stress. Elevated ERK activity correlates well with the phosphorylation state of ERKs, which can be exemplified by the extent of the shift in their electrophoretic mobilities (42). As shown in Fig. 4B, oxidative stress resulted in a retarded migration of p44 ERK, which is the only ERK isoform expressed in DT40 cells (43), in a dose-dependent manner. However, the exposure of cells to osmotic stress resulted in marginal, if any, activation of ERK. Thus, these results demonstrate that oxidative but not osmotic stress can activate ERK in DT40 cells.
Differential Requirement of Syk for Activation of JNKs and ERKs by Oxidative and Osmotic StressERKs are well characterized
downstream effectors of receptor or nonreceptor PTKs. JNKs are shown to
be regulated by distinct protein kinases in a similar cascade, however,
the roles of PTKs involved in the regulation of JNKs remain largely
unknown. Recently, c-Abl was shown to regulate the activation of JNKs
in response to cellular stress that selectively damages DNA (22, 23). This finding led us to analyze whether Syk plays a similar role in
regulating JNK activation in response to oxidative and osmotic stress.
As shown in Fig. 5A, JNKs were activated by
osmotic stress to comparable levels in both DT40 and DT40/Syk()
cells. The increase in JNK activity was 2.4- and 18.0-fold in DT40
cells and 4.6- and 16.0-fold in DT40/Syk(
) cells upon stimulation by
0.1 and 0.3 M sodium chloride, respectively (the 2-fold
difference in JNK activity between DT40/Syk(
) and DT40 cells was not
a consistent observation when stimulated by 0.1 M NaCl).
However, in the case of oxidative stress, JNK activation was
compromised in DT40/Syk(
) cells (Fig. 5A). An
approximately 3- and 4-fold reduction in JNK activities was observed in
response to 0.5 and 1 mM hydrogen peroxide stimulation,
respectively, in DT40/Syk(
) cells when compared to that measured in
DT40 cells. Immunoblot analysis revealed that the amounts of JNK
immunoprecipitated from DT40 and DT40/Syk(
) cell lysates were
comparable (data not shown). These results strongly indicate that Syk
is involved in JNK activation by oxidative but not osmotic stress in
DT40 cells.
It has been demonstrated that by overexpressing a dominant-negative Syk
mutant, Syk is required for the activation of ERKs after immunoglobulin
E receptor activation (44). Therefore, we addressed the involvement of
Syk in oxidative stress-induced ERK activation. As assessed by a gel
mobility shift assay, oxidative stress-induced activation of ERK in
DT40/Syk() cells was essentially identical to that observed in DT40
cells (Fig. 5B). Therefore, it became evident that Syk was
not required for ERK activation stimulated by oxidative stress.
Recent studies have indicated that different types of extracellular stress, including ionizing radiation, UV irradiation, and H2O2, activate a set of nonreceptor PTKs, such as the Src- and Syk/ZAP-70 family of PTKs (3-7), although the functional roles of these PTKs in stress-induced signaling pathways remain unclear. It has also been well documented that such stress can elicit other signaling events, including the activation of JNKs (and ERKs), and increase intracellular Ca2+ concentrations (12, 13, 24, 25, 39-41, 45). Here, we show that Syk is activated in a B cell line (DT40 cells) after treatment with H2O2 (oxidative stress) and with NaCl (osmotic stress), although the kinetics of Syk activation are different (Fig. 1). Furthermore, it was found that both oxidative and osmotic stress induce a Ca2+ response and activation of JNKs (and ERKs) (Figs. 2 and 4). In this study, we have focused on the possible role(s) of Syk in regulating the Ca2+ response as well as the activation of JNKs (and ERKs) induced by oxidative and osmotic stress.
Previous studies have demonstrated that Syk plays a crucial role in
platelet activation, B cell development, and B cell activation (29, 30,
46-48). An important role of Syk in BCR-mediated signaling was further
elucidated by utilizing a chicken B cell line, DT40, and the
DT40-derived mutant DT40/Syk(), which does not express Syk (32). In
fact, the BCR-mediated Ca2+ response was almost completely
abolished in DT40/Syk(
) cells, indicating the crucial role of Syk in
the Ca2+ response upon BCR activation (43).
DT40 as well as DT40/Syk() cells have provided a powerful tool to
dissect signaling cascades triggered by oxidative and osmotic stress.
We have shown that both oxidative and osmotic stress elicit a
Ca2+ response in DT40 cells, although the oxidative stress-
but not osmotic stress-induced Ca2+ response was delayed
and lower in magnitude in DT40/Syk(
) cells (Fig. 2). Thus, it became
evident that the osmotic stress-induced Ca2+ response is
independent of Syk, whereas that induced by oxidative stress is partly
dependent on Syk. The dependency of the oxidative stress-induced
Ca2+ response on Syk was further emphasized when the
expression of porcine Syk in DT40/Syk(
)
cells2 resulted in a calcium response very
similar to that observed in DT40 cells. In addition, experiments
utilizing calcium chelators highlighted that the requirement for
calcium exhibited by Syk differed between oxidative and osmotic stress
(Fig. 3). Oxidative but not osmotic stress-induced tyrosine
phosphorylation of Syk (a surrogate marker for Syk activation) was
significantly affected by the depletion of intracellular/extracellular
free calcium (Fig. 3). Thus, it became obvious that an increase in
[Ca2+]i was required for maximum
oxidative stress-induced activation of Syk. Furthermore, our results
revealed that the Ca2+ response induced by oxidative stress
involved both release from intracellular calcium stores and
extracellular calcium influx, whereas the osmotic stress-induced
calcium response is solely from extracellular calcium influx (Fig. 2).
Considering our previous observation that oxidative stress-induced
inositol 1,4,5-trisphosphate production was impaired in DT40/Syk(
)
cells (33) and that oxidative stress induced tyrosine phosphorylation
of phospholipase C
1 as well as promoting the association of Syk with
phospholipase C
1 in porcine peripheral blood lymphocytes (49), it is
likely that oxidative stress-induced Syk activity results in the
tyrosine phosphorylation of phospholipase C
and the subsequent
production of inositol 1,4,5-trisphosphate, thereby inducing the
release of Ca2+ from intracellular stores. However, it
remains unclear how osmotic as well as oxidative stress induces
extracellular calcium influx. In contrast to hyperosmolar stress, it
was reported that the Ca2+ response induced by hypoosmolar
stress was mediated by a release from intracellular calcium stores
(50).
Recently, much attention has been paid to the role(s) of JNKs (and ERKs) in stress signaling pathways (51). In fact, a variety of extracellular stress have been shown to induce activation of JNKs (and ERKs) (12, 13, 39-41). To date, the role(s) of PTKs in stress-induced activation of JNKs has not been elucidated, with the exception of c-Abl. c-Abl is a nonreceptor PTK localized in both the cytoplasm and nucleus and is activated upon the exposure of cells (U-937 and NIH3T3 cells) to DNA-damaging agents (22, 23). Interestingly, it has been shown that cells deficient in c-Abl fail to activate JNKs after exposure to DNA-damaging agents and that activation of JNKs can be restored by an ectopic expression of c-Abl in these cells (22, 23). Collectively, these results suggest that c-Abl plays a crucial role in mediating the activation of JNKs upon exposure to DNA-damaging agents.
It was found that osmotic stress induced the activation of JNKs,
whereas oxidative stress induced the activation of both JNKs and ERKs
in DT40 cells as assessed by an in vitro kinase assay (for
JNKs) and by SDS-polyacrylamide gel electrophoresis mobility shift
analysis (for ERKs) (Fig. 4). Interestingly, oxidative but not osmotic
stress-induced activation of JNKs was compromised in DT40/Syk()
cells, indicating that Syk was required for oxidative stress-induced
activation of JNKs (Fig. 5). In contrast, oxidative stress-induced
activation of ERKs was not affected by the absence of Syk (Fig. 5).
Accordingly, it became obvious that Syk played an important role in the
oxidative stress-induced signaling cascades, particularly in the case
of the Ca2+ response and the activation of JNKs in the B
cell line DT40. However, Syk alone is not sufficient to mediate the
signaling cascades elicited by oxidative stress because both
Ca2+ response and JNK activation are observed in
DT40/Syk(
) cells, although to a lesser degree. It is of importance to
identify the additional upstream signaling molecule(s) that mediates
the oxidative stress-induced Ca2+ response as well as the
activation of JNKs. Because it was reported that Rac/Cdc42-MEKK1-SEK
protein kinases work upstream of JNKs under certain conditions, it is
possible that oxidative stress may also activate this cascade. Further
study will be required to clarify such a possibility. On the other
hand, Syk is not required for the osmotic stress-induced
Ca2+ response or the activation of JNKs, although Syk is
apparently activated in response to osmotic stress. What is the exact
role(s) of Syk in osmotic stress-induced signaling? Is the activation of Syk induced by osmotic stress an epiphenomenon? At present, the
functional role(s) of Syk in osmotic stress-induced signaling remains
unclear. However, it is likely that Syk plays an important role in
osmotic stress-induced signaling cascades other than the Ca2+ response and JNK activation. In fact, Syk-negative
mutants of DT40 cells are more susceptible to osmotic stress-induced
apoptosis than the wild-type cells, and ectopic expression of porcine
Syk in Syk-negative mutants renders cells resistant to osmotic
stress-induced apoptosis.2
An important finding made in this study is that the functional role(s) of Syk differs between oxidative and osmotic stress-induced signaling pathways. Although both stresses can activate Syk, this activation is qualitatively different. At the present time, we do not know the molecular basis that could explain the qualitative difference observed in Syk activation. Further study will be required to clarify this issue. The results presented in this study also reveal that the mechanism for activating (or regulating) Syk by different stresses may be distinct. As shown, there are differences in the kinetics of Syk activation as well as the calcium requirement for Syk activation stimulated by oxidative and osmotic stress.
Syk is a 72-kDa nonreceptor PTK that is widely expressed in immunohematopoietic cells (27). Our results clearly demonstrate an important role for Syk in stress-induced signaling cascades in B cells. ZAP-70, another member of the Syk/ZAP-70 family of PTKs, is mainly expressed in mature T cells and natural killer cells (26) and is also activated by oxidative stress (25). Therefore, it will be of interest to examine whether or not ZAP-70 plays a role similar to that of Syk in stress-induced signaling cascades in both T cells and natural killer cells.
We thank Dr. Allison Stewart for her help in manuscript preparation.