From the
Stimulation of the mast cell line, RBL-2H3, with antigen via the
tetrameric (
Antigen-stimulated mast cells release a variety of inflammatory
mediators through the release of secretory granules and the generation
of arachidonic acid and cytokines
(1) . The multivalent binding
of antigen to receptor-bound IgE and the ensuing aggregation of
receptors for IgE (Fc
Studies in a cultured mast cell
(RBL-2H3) cell line indicate that the phospholipase C
The tyrosine kinases, Lyn (p56
Here we examine
whether or not the Lyn/Syk kinases have an essential role in the
activation of the MAP kinase/phospholipase A
Antibodies were
from the following sources; antibody against phosphotyrosine (PY-20
HRP) from ICN, biotinylated anti-Tac B1.49.9 from Amac, Westbrook, ME;
polyclonal antibody against the carboxyl-terminal peptide of rat MAP
kinase R2 (Erk1-CT) from Upstate Biotechnology Inc., polyclonal
antibody against a synthetic peptide of Vav from Santa Cruz
Biotechnology, Inc., Santa Cruz, CA, and the polyclonal antibody to
synthetic peptide derived from Syk was prepared as described previously
(25) .
As
previously observed
(20) , antigen and carbachol caused an
appreciable increase in MAP kinase activity in intact cells (data not
shown). Further studies, however, were undertaken with permeabilized
cells to avoid the potential interactions between the phospholipase
C/Ca
The
effects of expression of Syk and Syk(T) on release of arachidonic acid
in permeabilized cells are shown in Fig. 7. As was observed with
the activation of MAP kinase, expression of Syk enhanced
antigen-induced release of arachidonic acid, and expression of Syk(T)
blocked this release.
As shown here, Syk not only provides a necessary link between
Fc
Previous
studies have indicated that elevation of Ca
If Syk is the initiator of two divergent
signaling pathways, what can be concluded about events preceding the
tyrosine phosphorylation and activation of Syk? The studies with the
TT
The mechanisms described
here for Fc
It is apparent from the differences between antigen
and carbachol that convergent pathways exist for the activation of the
MAP kinase/phospholipase A
In conclusion, the data in this and previous papers
indicate that for Fc
We thank Drs. S. Chakrabarti and B. Moss, NIAID, for
kindly supplying the pSC-65 vaccinia recombination plasmid.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
) immunoglobulin E receptor
(Fc
R1) leads to the activation of cytosolic phospholipase A
and the release of arachidonic acid. This pathway is dependent on
the activation of the mitogen-activated protein (MAP) kinase. In this
paper, we show that the MAP kinase/cytosolic phospholipase A
pathway is linked to Fc
R1 via the cytosolic tyrosine kinase,
Syk, and that the GDP/GTP exchange factor, Vav, might be one candidate
for accomplishing this link. Cross-linking of transmembrane chimeras
containing the Fc
R1
motif, which is known to activate Syk,
results in the tyrosine phosphorylation of Vav, activation of MAP
kinase, and release of arachidonic acid. Cross-linking of chimeras
containing the Fc
R1
motif does not cause these events.
Furthermore, stimulation of these events by antigen is enhanced by
transient overexpression of a wild-type form of Syk and blocked by
overexpression of a dominant negative form of Syk. By contrast,
stimulation via the transfected, G protein-coupled, muscarinic m1
receptor is not influenced by either form of Syk and does not result in
tyrosine phosphorylation of Vav. These data establish unequivocally
that the two types of receptor are independently linked to the MAP
kinase/cytosolic phospholipase A
pathway and demonstrate
the existence of the Fc
R1-Syk-MAP kinase pathway.
R1) provides the trigger for this release.
Fc
R1 is a multimeric receptor with
,
, and a
homo-dimeric
subunits
(2) . Both the
and
chains contain the motif, ITAM(
)
(3, 4, 5) , which allows recruitment of
tyrosine kinases and the tyrosine phosphorylation of various proteins
(6, 7, 8) , including phospholipase C
1
(9) . The sustained activation of phospholipase C
(10, 11, 12) and D
(13, 14) ,
along with sustained elevation of diglycerides
(14) and
mobilization of Ca
from intracellular and
extracellular sources
(15, 16, 17, 18) ,
results in the activation of protein kinase C. Activation of MAP
kinases is also apparent from an increase in MAP kinase activity
(19, 20) and the tyrosine phosphorylation and shift in
electrophoretic migration of p42
and, much less so, of
p44
(21) .
and the MAP
kinase pathways subserve different functions. For example, elevation of
[Ca
]
and the
activation of certain isozymes of protein kinase C provide sufficient
signals for maximal secretory reponses
(22) , whereas release of
arachidonic acid does not require protein kinase C. This release is
dependent instead on MAP kinase for the phosphorylation and activation
of a cytosolic phospholipase A
and an increase in
[Ca
]
for the binding
of the phospholipase A
with the membrane fraction
(20) .
) and Syk
(p72
), probably link aggregated Fc
R1 to
phospholipase C
, although this has not been unequivocally
established. In unstimulated RBL-2H3 cells, Lyn is normally associated
with the
chain of Fc
R1
(23, 24, 25, 26) . Studies with chimeras
of the extracellular and transmembrane domains of the
subunit of
the IL-2 receptor and the cytosolic, carboxyl-terminal portion of the
(TT
) or the cytosolic domain of the
(TT
) chains
of Fc
R1
(25) , as well as other studies
(26, 27) , indicate that aggregation of Fc
R1 causes
tyrosine phosphorylation of the
and
chains of Fc
R1,
and the recruitment of Syk and additional Lyn. Cross-linking of the
TT
chimeras with biotinylated anti-Tac antibody and avidin suffice
for full expression of responses in RBL-2H3 cells including the
activation of Syk
(25) , mobilization of Ca
,
and release of secretory granules
(25) .
pathway in
RBL-2H3 cells. An early step in this pathway in many types of cells is
the association of Raf-1 with Ras at the plasma membrane
(28, 29, 30) . The serine phosphorylation of
MEK-1 by Raf-1 and, finally, the tyrosine/threonine phosphorylation of
MAP kinases by MEK-1 results in activation of MAP kinase activity and,
in turn, phospholipase A
(reviewed in Ref. 31). This same
pathway is stimulated by a variety of stimulants in RBL-2H3 cells
through different mechanisms
(20) . We have determined that
subunits of Fc
R1 and Syk transduce signals for activation of
this pathway by expression of the aforementioned Tac chimeras, a
porcine Syk, and a truncated Syk, Syk(T), which lacks the kinase
domain, in RBL-2H3 cells. As a control, we have also examined the
activation of the MAP kinase/phospholipase A2 pathway via a G
protein-coupled receptor, the muscarinic m1 receptor, which has been
stably transfected in RBL-2H3 cells
(16) .
Reagents
These were obtained from the following
sources: avidin from Sigma, carbachol from Aldrich, thapsigargin from
LC Services Corp., Woburn, MA; [1-C]arachidonic
acid, from DuPont NEN, reduced streptolysin O from Burroughs Wellcome
Co., phenyl-Sepharose from Pharmacia Biotech, Inc., MAP kinase
substrate (residues 94-102 of myelin basic protein) from Upstate
Biotechnology Inc., Lake Placid, NY, and ECL
detection kit
from Amersham Corp. The antigen, DNP-BSA, and DNP-specific monoclonal
IgE were kindly supplied by Dr. Henry Metzger (NIAMS, National
Institutes of Health). Other reagents and materials were from the
sources listed previously
(22, 32, 33, 34) .
Preparation of Vaccinia Virus/SC-65 Constructs of Syk and
Truncated Syk: Cell Infection
Constructs for expression of
porcine Syk tyrosine kinase and the truncated Syk kinase (Syk(T)) were
prepared by subcloning the respective cDNAs into the pSC-65 vaccinia
recombination plasmid. This plasmid places expression under the control
of a strong synthetic early/late promoter.(
)
Established techniques and the WR wild type vaccinia strain
(American Type Culture Collection) were used to select for recombinant
TK(-)/
-galactosidase-expressing plaques, to amplify the
recombinant plaques into crude stocks, and for establishing titers of
the crude stocks
(35) . The porcine Syk cDNA was prepared as
described previously
(36) . The Syk(T) was constructed from the
porcine Syk cDNA by using polymerase chain reaction to delete an
adenosine at base number 1481, resulting in a frameshift mutation
causing isoleucine 396 to change to serine and leucine 397 to a stop
codon. The sequence of the Syk(T) cDNA construct was confirmed by
dideoxy chain termination sequencing.
Cell Culture and Permeabilization: Measurement of Release
of Arachidonic Acid and Secretion of Hexosaminidase
Experiments
were performed with a subline of RBL-2H3 cells that had been
transfected with the gene for muscarinic m1 receptors (RBL-2H3(m1),
Ref. 16) or the Tac chimeras
(25) , TT (RBL-TT
) and
TT
(RBL-TT
). Cultures were incubated overnight with
DNP-specific IgE (0.5 µg/ml), [
C]arachidonic
acid (0.1 µCi/ml) in complete growth medium in 24-well plates (2
10
cells/400 µl of medium/well)
(22) .
For the experiments with RBL-TT
and RBL-TT
, the IgE or
biotinylated anti-Tac antibody (2.5 µg/ml) was added to the medium
as required 2 h before the experiment
(25) . Cultures were
washed, and experiments were performed with the following buffers: a
glucose/saline/PIPES-buffered medium (pH 7.2) that contained 1
mM Ca
for experiments with intact cells
(37) ; a potassium glutamate/PIPES-buffered medium (pH 7.2) that
contained 5 mM glucose, 7 mM magnesium acetate, 5
mM ATP, 1 mM EGTA, and sufficient Ca
to give the indicated
[Ca
]
(33) for
studies with permeabilized cells. Cells were permeabilized with
streptolysin O
(33) in the presence of stimulant. Stimulants
included DNP-BSA, carbachol, and thapsigargin, at the indicated
concentrations, as well as avidin (15 µg/ml). For measurement of
release of hexosaminidase
(22) and
[
C]arachidonic acid
(34) , reactions were
terminated (on ice) 10 min after the addition of stimulant except where
indicated. For measurement of MAP kinase activity and detection of
proteins by immunoblotting techniques, the reactions were terminated at
2.5 or 5 min as indicated.
Partial Purification and Assay of MAP Kinase
Activity
Procedures were based on those described by Offermanns
et al.(38) . After each experiment, medium was removed
from cultures (in 6-well cluster plates). The cells were harvested in
0.5 ml of a Tris buffer as follows: 25 mM Tris, pH 7.5, 25
mM NaCl, 0.1 mM NaVO
, 2
mM EGTA, 1 mM dithiothreitol, 1 mM p-nitrophenyl phosphate, and 20 µg/ml leupeptin. Cells
were disrupted by freezing and thawing three times before
centrifugation. To the supernatant fraction was added ethylene glycol
(10% final concentration) and then phenyl-Sepharose. The mixture was
centrifuged, and the sedimented phenyl-Sepharose was washed with Tris
buffer that contained 10% (v/v) ethylene glycol. The process was
repeated, but with Tris buffer that contained 30% (v/v) ethylene
glycol. MAP kinase was eluted from the phenyl-Sepharose with 75 µl
of Tris buffer that contained 60% (v/v) ethylene glycol. After
centrifugation, 15 µl of the supernatant fraction was incubated (15
min, 37 °C) in a solution that contained 50 mM Tris, pH
7.5, 10 mM MgCl
,
[
-
P]ATP (10 Ci/mmol, 37 kBq/tube), 25
µg of a MAP kinase substrate (peptide 94-102 of bovine myelin
basic protein). The phosphorylated peptide was isolated by
centrifugation of the incubation mixture through phosphocellulose
membrane (SpinZyme, Pierce), which was then washed twice with 500
µl of 75 mM H
PO
for assay of
radioactivity.
Electrophoretic Separation and Immunoblotting of MAP
Kinase and Other Proteins
After the experiment, cells were lysed
in ice-cold lysis buffer (20 mM HEPES, pH 7.3, 1% Triton
X-100, 1 mM EDTA, 50 mM NaF, 2.5 mM p-nitrophenyl phosphate, 1 mM
NaVO
, 10 µg/ml phenylmethylsulfonyl
fluoride, 10 µg/ml leupeptin, and 10% glycerol).
Immunoprecipitation of Vav was performed with the antibodies listed
under Reagents as recommended by the manufacturer. Proteins in
the cell lysate or immunoprecipitated proteins were separated by
electrophoresis on SDS-polyacrylamide gels and transferred to
nitrocellulose membrane. Proteins were detected by the immunoblotting
technique with the antibodies listed above. The antibodies were used as
recommended by the manufacturer along with alkaline
phosphatase-conjugated antibody to rabbit IgG for the second antibody.
Tyrosine-phosphorylated proteins were detected by the ECL
technique as described previously
(21) .
Expression of Data
Mean values (±S.E. for
three cultures) from a single experiment are presented where these are
representative of at least two experiments. Unless stated otherwise,
values were corrected for spontaneous release of either hexosaminidase
(<1.0 for intact cells and <7% for permeabilized cells) or
arachidonic acid (<1.0% for intact cells and <5.0% for
permeabilized cells) in unstimulated cells. MAP kinase activity was
expressed as a percent of the activity in unstimulated cells (typically
4500-6500 dpm of P incorporated/µg of isolated
protein).
Aggregation of TT
In studies with RBL-TT but Not of TT
Chains in
RBL-2H3 Cells Results in Activation of the MAP Kinase Pathway and
Release of Arachidonic Acid
and
RBL-TT
, cross-linking of TT
with biotinylated anti-Tac
antibody and avidin did not result in release of secretory granules or
arachidonic acid (Fig. 1 A). Both responses were evoked
by cross-linking of TT
(Fig. 1 B) or of
IgE/Fc
R1 with antigen (Fig. 1, A and B).
Anti-Tac and avidin failed to evoke responses in either clone to
indicate that the combination of anti-Tac and avidin were required for
stimulation of RBL-TT
and that dimerization of TT
was an
insufficient stimulus. Similar results were obtained with permeabilized
cells (Fig. 1, C and D).
Figure 1:
Aggregation of TT but not of
TT
chimeras induces release of arachidonic acid as well as
secretion of hexosaminidase. RBL-TT
and RBL-TT
were incubated
for 2 h with optimal concentrations of biotinylated anti-Tac (anti-Tac)
or DNP-specific IgE. Cells were left intact or permeabilized with
streptolysin O in 1 µM [Ca
]
and stimulated with 15 µg/ml avidin ( Avid.) or 20 ng/ml
(intact cells) and 100 ng/ml (permeabilized cells) DNP-BSA
( Ag.) for 10 min. Cells were also treated with biotinylated
anti-Tac or avidin alone for controls as indicated. Values (mean
± S.E. for three cultures for this and subsequent figures) were
corrected for basal release and were from one of two similar
experiments with different clones of RBL-TT
cells.
Release of
arachidonic acid was transient when compared to release of secretory
granules. In this series of experiments, release of arachidonic acid
ceased within 3 to 5 min of addition of stimulants (Fig. 2).
Secretion of granules had essentially ceased by 10 min (Fig. 2),
although in other experiments secretion continued for as long as 15 to
20 min (e.g. Ref. 39). The only difference between anti-Tac/avidin and
antigen was that the responses to antigen were more prompt than those
to the anti-Tac/avidin (compare A with B in
Fig. 2
).
Figure 2:
Time
course of release of arachidonic acid and secretion of hexosaminidase
following aggregation of FcR1 or TT
chimera. RBL-TT
were
incubated for 2 h with optimal concentrations of DNP-specific IgE
( A) or biotinylated anti-Tac ( B) and then stimulated
with 20 ng/ml DNP-BSA ( A) or avidin ( B) for the
indicated times. Values were not corrected for spontaneous release of
mediators.
Cross-linking of TT also resulted in the
tyrosine phosphorylation of various proteins, including p72
(25) , previously identified as Syk
(40) and
p42
(Fig. 3 A). The phosphorylation of
p42
was associated with a shift in electrophoretic
migration of p42
, such that a second retarded band of
p42
was apparent (Fig. 3 A), and an
increase in MAP-kinase activity in cell extracts
(Fig. 3 B). Two additional proteins, possibly
p44
and p46
, were detected by the
antibody
(21, 20) . These proteins also exhibited a
shift in migration but, as in previous studies
(21) , they
exhibited no detectable increase in tyrosine phosphorylation
(Fig. 3 A). Cross-linking of TT
or use of anti-Tac
or avidin failed to evoke these responses ( e.g.Fig. 3
).
Consistent with previous studies
(25) , however, cross-linking
of TT
resulted in tyrosine phosphorylation of a p56 protein, most
probably Lyn. This and concurrent experiments indicated that both
chimeras were consistently expressed in approximately equivalent
amounts (data not shown). These studies indicated collectively that the
MAP kinase/phospholipase A
pathway can be activated by
cross-linking of the
chain alone.
Figure 3:
Aggregation of TT but not of TT
chimeras induces activation of MAP kinase. RBL-TT
and RBL-TT
were incubated for 2 h with optimal concentrations of biotinylated
anti-Tac (anti-Tac) or DNP-specific IgE and then stimulated with avidin
or 20 ng/ml DNP-BSA ( Ag.) for 5 min. Cells were also treated
with biotinylated anti-Tac alone for controls as indicated. For
A, tyrosine-phosphorylated proteins in whole cell extracts
were detected by anti-phosphotyrosine antibody and the ECL-detection
technique ( Anti-PY). The same membrane was used for detection
of p42
by immunoblotting with antibody to MAP kinase
( Anti-MAPK). Note that the protein p40 was previously
identified as unphosphorylated p42
; the identity of the
additional proteins is discussed in the text. For B, cells
were permeabilized with streptolysin O before stimulation in 1
µM [Ca
] and then assayed for
MAP kinase activity. Values indicate percent of the activity in
unstimulated cells.
Overexpression of Syk Enhances Antigen-induced Activation
of MAP Kinase and Expression of Truncated Syk, Syk(T), Blocks This
Activation
Because of the association of Syk with the
chain of Fc
R1 and TT
and the indications that it may
transduce early signals for activation of RBL-2H3 cells
(25) ,
the effects of overexpression of Syk and Syk(T) were examined by use of
a transient expression system. Expression of Syk resulted in enhanced
activation of MAP kinase when cells were stimulated with antigen, as
indicated by the increase in enzyme activity (Fig. 4 A)
and in the amount of protein in the second retarded band of
p42
(Fig. 4 B). This was most evident with
concentrations of antigen that were suboptimal for activation of MAP
kinase (Fig. 4 A). Expression of Syk by itself did not
result in constitutive activation of MAP kinase ( i.e. ``0
ng/ml DNP-BSA'' in Fig. 4, A and B).
Figure 4:
Expression of porcine Syk enhances
antigen-induced activation of MAP kinase. Cultures of RBL-2H3 cells
were incubated for 16 h with 5 plaque-forming units/RBL-2H3 cell of
either wild type (vaccinia/control) or Syk recombinant vaccinia
(vaccinia/Syk). The cultures were permeabilized with streptolysin O
( A) or left intact ( B) before stimulation with the
indicated concentration of DNP-BSA ( Ag.) for 2.5 min. For
A, extracts of the permeabilized cells were assayed for MAP
kinase activity. Values indicate percent of activity of unstimulated
cells that were infected with the vaccinia/Syk virus ( solid
line) or vaccinia/control virus ( dashed line). Similar
results were obtained in another experiment. For B,
immunoblots of intact cells, vaccinia/control (-) or vaccinia/Syk
(+), were probed with antibody to MAP kinase ( Anti-MAPK)
as described in the legend of Fig. 3.
Expression of Syk(T), in contrast, blocked activation of MAP kinase
in antigen-stimulated cells, as indicated by measurement of enzyme
activity (Fig. 5 A), tyrosine phosphorylation of p42
(Fig. 5 B), and the appearance of the second retarded
band of p42 (Fig. 5 C). The expression of
Syk(T) was also associated with marked diminution of all
antigen-induced tyrosine phosphorylated proteins with the notable
exception of a p46 protein (Fig. 5 B). The size of this
protein matched exactly that expected of truncated Syk (estimated size,
46 kDa).
Figure 5:
Expression of truncated Syk (Syk(T))
supresses antigen-induced activation of MAP kinase. Cultures of RBL-2H3
cells were incubated for 16 h with vaccinia/control or Syk(T)
recombinant vaccinia virus ( A, as indicated; B and C,
5 plaque-forming units/RBL-2H3 cell). The cultures were permeabilized
with streptolysin O ( A) or left intact ( B) before
stimulation with the antigen, DNP-BSA (100 ng/ml in A and 20
ng/ml in B), for 5 min. For A, extracts of the
permeabilized cells were assayed for MAP kinase activity, and values
indicate percent of activity of unstimulated cells that were infected
with the vaccinia/Syk(T) virus ( solid line) or
vaccinia/control virus ( dashed line). Similar data were
obtained in two separate experiments. For B and C,
immunoblots of intact cells were probed with anti-phosphotyrosine
antibody ( Anti-PY in Fig. 3) and antibody to MAP kinase
( Anti-MAPK in Fig. 3): Key: wt,
vaccinia/control; Syk(T), vaccinia/Syk(T); (-),
unstimulated; and (+), antigen ( Ag.)-stimulated
cells.
Antigen-induced release of arachidonic acid (data not
shown, but see later experiments) and secretion (data not shown) were
also blocked in Syk(T)-transfected cells and were enhanced in
Syk-transfected cells. These studies indicated, therefore, that Syk
transduced necessary signals for the activation of MAP kinase and
release of arachidonic acid as well as secretion.
Syk Also Regulates Tyrosine Phosphorylation of Vav via
Fc
We next examined the effects of Syk on the
tyrosine phosphorylation of Vav because it is one of several proteins
that are tyrosine-phosphorylated in antigen-stimulated RBL-2H3 cells
(41) and it is known to regulate GTP/GDP exchange activity of
Ras in B cells and T cells
(42, 43, 44) . It may
serve, therefore, as a potential link between Syk and the MAP kinase
pathway via the interaction of Ras with Raf
(28, 29, 30) . Both Syk (Fig. 6, line
1) and Vav (Fig. 6, line 2) showed a marked
increase in tyrosine phosphorylation in antigen-stimulated cells. The
tyrosine phosphorylation of Vav was a specific response in that it was
observed only in cells stimulated via TTR1 and TT
(Fig. 6, line
2) or via Fc
R1 (Fig. 6, line 3). Such
phosphorylation was not observed in cells stimulated with carbachol or
thapsigargin (Fig. 6, line 3). The latter two stimulants
were known to activate the MAP kinase pathway in RBL-2H3 cells, but
probably not through the activation of Syk
(20) . Indeed,
tyrosine phosphorylation of Syk itself was induced by antigen but not
by carbachol (Fig. 6, line 1).
Figure 6:
Vav is
tyrosine-phosphorylated when cells are stimulated via FcR1
and Syk but not when stimulated via carbachol or thapsigargin.
RBL-TT
, RBL-TT
, and RBL-2H3(m1) cells, as well as
vaccinia-infected cells that expressed the porcine Syk or Syk(T) gene,
were exposed to the indicated stimulants for 5 min. Immunoblots of
immunoprecipitated Syk ( blot 1) and Vav ( blots
2-5) were probed with anti-phosphotyrosine antibody. Key:
(-), no stimulant; Ag., 20 ng/ml DNP-BSA; CBC,
1 mM carbachol; Thaps., 150 nM thapsigargin;
T/Av., cells treated with biotinylated anti-Tac and
avidin as described Fig. 1; Syk(T), Syk, or
(-), cells infected with vaccinia/Syk(T), vaccinia/Syk, or
vaccinia/control virus, respectively, as described in Figs. 4 and
5.
Expression of Syk(T)
blocked antigen-induced tyrosine phosphorylation of Vav (Fig. 6,
line 4). Conversely, expression of Syk enhanced this
phosphorylation (Fig. 6, line 5). The lack of response
of Vav to carbachol was not affected by expression of Syk(T) or Syk
(Fig. 6, lines 4 and 5). An incidental
observation was that basal phosphorylation of Vav in unstimulated cells
was reduced, but not blocked, in Syk(T)-transfected cells
(Fig. 6, line 4). Collectively, the results indicated
that Syk and Vav were selectively and specifically
tyrosine-phosphorylated via cross-linking of the chain of
Fc
R1 and that phosphorylation of Vav was downstream to that of
Syk.
A Requirement for Syk for Activation of MAP Kinase and
Release of Arachidonic Acid Is Apparent in Antigen-stimulated Cells but
Not in Carbachol-stimulated Cells
The above studies established
that activation of MAP kinase was dependent on Syk in
antigen-stimulated cells and that Syk was not required for such
activation in carbachol-stimulated cells. Because MAP kinases transduce
necessary signals for release of arachidonic acid through activation of
cytosolic phospholipase A(20) , we next examined
the effect of expression of Syk and Syk(T) on this release.
/protein kinase C and MAP kinase/phospholipase
A
cascades, both of which appear to be activated via Syk in
antigen-stimulated cells. For example, we knew that MAP kinases were
activated by an increase in
[Ca
]
alone and that an
increase in [Ca
]
was
necessary for release of arachidonic acid
(20) . Therefore,
primary and secondary ( i.e. via phospholipase C) effects of
Syk(T) could not be readily distinguished in the intact cell.
(
)
In contrast,
carbachol-induced release was unaffected by these expressions to
indicate that only Fc
R1 was dependent on Syk for activation of the
MAP kinase/phospholipase A
pathway.
Figure 7:
Expression
of porcine Syk or Syk(T) results in, respectively, enhancement or
suppression of antigen-induced release of arachidonic acid without
affecting carbachol-induced release of arachidonic acid. Cultures of
RBL-2H3(m1) cells were incubated for 16 h with 5 plaque-forming
units/RBL-2H3 cell of wild type Syk or Syk(T) recombinant vaccinia. The
cultures were permeabilized with streptolysin O in the presence of 1
µM [Ca] and 100 ng/ml DNP-BSA
( Ag.) or 1 mM carbachol ( CBC). Release of
arachidonic acid was determined 10 min
thereafter.
A further indication
of the differences between FcR1 and the muscarinic m1 receptor was
that activation of MAP kinase, as indicated by increased activity
(Fig. 8) and shift in migration of p42
(data not
shown), by antigen was not dependent on
[Ca
]
, whereas the
activation by carbachol was highly dependent on
[Ca
]
. In these
experiments, [Ca
]
was
buffered at concentrations that mimicked those in unstimulated (75
nM free Ca
) and stimulated (1000 nM
Ca
) intact cells. Although as noted previously
(20) , elevation of
[Ca
]
by itself induced
a modest activation of MAP kinase activity, the data clearly
demonstrated that transduction of signals via Fc
R1 occurred at
basal [Ca
]
.
Figure 8:
Activation of MAP kinase activity by
antigen, in contrast to activation by carbachol, is not dependent on
Ca. Washed-permeabilized cells were stimulated with
100 ng/ml DNP-BSA ( Ag.) or 1 mM carbachol
( CBC) in the presence of 75 nM or 1 µM
[Ca
] for 5 min. Extracts of the cells were
assayed for MAP kinase. Values indicate percent of activity in the
presence of 1 µM [Ca
] alone
and are representative of two similar
experiments.
R1 and phospholipase C
1
(25) , but also between
Fc
R1 and the MAP kinase/cytosolic phospholipase A
pathway. Thus, Syk appears to activate two divergent signaling
pathways; one that leads to the generation of a Ca
signal and release of secretory granules
(25) and another
that results in the activation of MAP kinase and phospholipase A
with release of arachidonic acid (this study).
and
activation of protein kinase C are sufficient signals for secretion and
that secretion is blocked by inhibition or removal of protein kinase C
(22) . Phospholipase A
-mediated release of
arachidonic acid, in contrast, is not dependent on protein kinase C but
rather on the activation of MAP kinase and elevation of
[Ca
]
(20) .
Increased [Ca
]
is not
required, however, for the activation of MAP kinase (this study) and
phospholipase A
(20, 45) when cells are
stimulated via Fc
R1. Rather, the increase in
[Ca
]
is necessary for
the association of phospholipase A
with cell membrane and
the release of arachidonic acid from membrane substrates
(20) (see schema in Fig. 9).
Figure 9:
Pathways for mediating degranulation, via
protein kinase C and Ca, and release of arachidonic
acid, via MAP kinase/phospholipase A
, in RBL-2H3(m1) cells.
The schema is based on present and previous (20, 22) work. It depicts
the simultaneous activation of the phospholipase C
( PLC)/protein kinase C ( PKC) as well as the MAP
kinase ( MAPK)/phospholipase A
( PLA
) pathways through the tyrosine kinase,
Syk, when cells are stimulated via Fc
R1. Also indicated is the
activation of the MAP kinase/phospholipase A
pathway via
the muscarinic m1 receptor by a Syk-independent,
Ca
-dependent mechanism. The stimulatory ligands,
namely, antigen and carbachol, are thought to activate the same
pathway: Ras, Raf1, MEK-1, MAP kinase, and phospholipase A
as shown (20). The connection between Syk and Ras/Raf1 is
undetermined, but the possible participation of Vav and other
components is discussed in the text.
The effects of
overexpression of Syk(T) and porcine Syk demonstrate that Syk is not
only essential for communication between the -subunit of Fc
R1
and the MAP kinase pathway but also for Fc
R1-mediated tyrosine
phosphorylation of Vav which may participate in this communication. Our
data, therefore, do not exclude the possible activation of the MAP
kinase cascade via phospholipase D. Diglycerides are generated through
the activation of the phospholipases C and D in antigen-stimulated
RBL-2H3 cells
(46) and diglycerides are known to activate Vav
(42) which, as previously noted, is thought to regulate Ras in B
cells and T cells
(43, 44) . Nevertheless, if these
reactions occur in RBL-2H3 cells, they must depend on the prior
activation of Syk.
and TT
chimeric receptors revealed that aggregation of the
chain of Fc
R1 is sufficient, and almost as efficient as the
aggregation of intact Fc
R1, for tyrosine phosphorylation of Syk
(25 and this paper), activation of MAP kinase, and release of
arachidonic acid. Expression of the porcine Syk facilitated coupling of
Fc
R1 to the MAP kinase pathway, especially at low suboptimal
concentrations of antigen, whereas expression of Syk(T), which lacks
the kinase domain, abrogated this coupling. These findings collectively
suggest that the SH2 domains of Syk(T) effectively compete with
endogenous Syk for binding to ITAM domains on the Fc
R1
chains. Interestingly, Syk(T) is a substrate for tyrosine
phosphorylation when cells are stimulated through Fc
R1.
Presumably, this phosphorylation is catalyzed by Lyn which is known to
be associated with Fc
R1
(23) , specifically with the
subunit
(25) , in RBL-2H3 cells.
R1 might apply to other immunologically responsive
cells. Activation of the MAP kinase pathway via Fc
R1 or via the
multimeric antigen receptors in T cells and B cells has a common
requirement for cytosolic tyrosine kinases and other signaling
components. The pathway for RBL-2H3 cells appears to be a G protein
(most probably Ras)
Raf-1
MEK-1
p42
(20) as appears to be the case in activated T cells
(47, 48) and B cells
(49) . The Fc
R1
-subunit is functionally analogous to the
-subunit of the
antigen T cell receptor in that the subunits consist of
and
homodimers, and each contain one
or more ITAM domains which when phosphorylated permit association with
Syk-related tyrosine kinases, i.e. Syk with
and ZAP-70
with
(4, 5) . It is unknown whether ZAP-70 is
linked to the MAP kinase pathway in T cells, as Syk appears to be in
RBL-2H3 cells.
in RBL-2H3 cells. For example,
stimulation of the pathway by carbachol is not dependent on Syk but is
dependent on Ca
, whereas the converse is true for
stimulation of the pathway by antigen (see Fig. 9). Our previous
studies
(20) have indicated that all stimulants, including
antigen, carbachol, thapsigargin, and Ca
ionophore,
utilize the same pathway namely; a G protein (Ras?)
Raf-1
MEK-1
MAP kinase
cytosolic phospholipase A
.
If they do so, the point of convergence is likely to be at the level of
Ras for which multiple activation mechanisms have been shown to exist
(30) .
R1-mediated signals, Syk is the point of
divergence for the phospholipase C/protein kinase C and the MAP kinase
signaling pathways. Both pathways, however, have additional stimulatory
inputs from other types of receptors. For the phospholipase C-dependent
cascade of signals, convergent inputs are accommodated by the existence
of different isoforms of phospholipase C to allow activation via
trimeric G proteins, primarily G
, and tyrosine
kinases
(50) . For the MAP kinase pathway, it is apparent that
Syk-dependent and Syk-independent mechanisms exist for the activation
of this pathway.
26-amino acid residue
which was originally recognized by M. Reth (3) in receptors of
immunological importance and since referred to as ARAM, TAM, or Reth
motif (4, 5) among other designations (J. Cambier, personal
communication); [Ca
], concentration of free
cytosolic calcium; IgE, immunoglobulin E; DNP, 2,4-dinitrophenol;
DNP-BSA, antigen consisting of 24 molecules of dinitrophenol conjugated
with 1 molecule of bovine serum albumin; PIPES,
1,4-piperazinediethanesulfonic; MAP kinase, mitogen-activated protein
kinase; Syk(T), the gene for Syk that lacks the kinase domain;
RBL-TT
and RBL-TT
, RBL-2H3 cells that have been stably
transfected and thereby express, respectively, the Tac-Tac-
and
Tac-Tac-
chimeric constructs as discussed in the text.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.