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Department of Cell Biology and Histology, Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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RI-mediated signaling and release
of inflammatory mediators from mast cells. We now show that activation
of rat peritoneal mast cells by the nonimmunological,
Gi-mediated pathway also results in the activation of Syk.
We show that compound 48/80 (c48/80), a receptor analogue that
activates directly G proteins, activates Syk in a pertussis
toxin-sensitive fashion. We further show that Syk activation by c48/80
is blocked by the protein kinase C inhibitor GF109203X, by the
phosphatidylinositol 3-kinase inhibitors, wortmannin and LY294002, by
EGTA, and by the selective src-like kinase inhibitor
PP1. These results suggest that in the nonimmunological,
Gi-mediated pathway, Syk is located downstream from
phospholipase C and phosphatidylinositol 3-kinase. However, in common
with the FcRI-mediated pathway, activation of Syk by c48/80 is
dependent on a src-like protein tyrosine kinase.
Finally, we show that in the nonimmunological pathway, Syk plays a
central role in the release of arachidonic acid/eicosanoid metabolites,
but not in the release of prestored mediators such as
histamine. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The major pathway of activating mast cells is the immunological
trigger brought about by the aggregation of high affinity receptors
(FcRI) for IgE, by corresponding Ags (3). The earliest
event in this pathway is the activation of src-like
cytosolic protein tyrosine kinases (PTKs), p53/56lyn or
p62c-yes, leading to the rapid tyrosine phosphorylation of
the FcRI 
and 
subunits (4). These
phosphorylations then enable the recruitment and activation of
additional cytosolic PTKs, such as the Syk kinase (5, 6, 7)
and the tyrosine phosphorylation of a number of protein substrates,
including phospholipase C (PLC), Vav, Nck, and paxillin (reviewed
in Ref. 8). Syk activation is essential for
FcRI-induced activation of mitogen-activated protein kinase (MAPK)
(9) probably through phosphorylation of shc and
triggering of the Grb2/Sos/Ras cascade (10).
An alternative way of activating mast cells is by a large number of polycationic molecules collectively known as the basic secretagogues of mast cells (11). The latter include positively charged neuropeptides such as substance P and bradykinin, various amines such as the synthetic compound 48/80 (c48/80), and naturally occurring polyamines (11). Basic secretagogues activate connective tissue type, but not mucosal mast cells, in an IgE-independent manner. These agonists act as receptor mimetic agents, which trigger mast cell exocytosis by activating directly pertussis toxin (Ptx)-sensitive Gi proteins that control exocytosis (12, 13, 14, 15). The mechanism by which basic secretagogues activate exocytosis and release of the preformed mediators appears to be distinct from that of the immunological trigger. Unlike the immunological evoked response, release by basic secretagogues is inhibited by Ptx, does not require the presence of external Ca2+, and is completed within <1 min (12, 16). However, despite these marked differences, we have recently demonstrated similarities in the mechanisms by which the immunological or the G protein-mediated trigger stimulates the production and release of class II mediators. We have shown that c48/80 stimulates the activity of PTKs, leading to the enhanced tyrosine phosphorylation of a number of cellular proteins and to the activation of both the p42 and p44 MAPKs (17, 18). Activation of the PTK pathway occurred via a mechanism that involves protein kinase C (PKC), phosphatidylinositol 3-kinase (PI-3K), and Ca2+ as intermediates (17, 18). We have further demonstrated that this signaling pathway importantly contributes to the production and release of class II mediators, while it is dispensable for histamine release. In the present work, we have identified the Syk kinase as one of the PTKs, which are activated by c48/80. We also demonstrate that Syk activation occurs via a mechanism, which involves PKC, Ca2+, and PI-3K as well as a src-like PTK. Finally, we show that in the nonimmunological pathway, Syk mediates the release of AA/metabolites, but not degranulation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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GF109203X, Go 6976, wortmannin, and PD98059 were purchased from
Calbiochem (La Jolla, CA). Piceatannol was obtained from Sigma (St.
Louis, MO). The protease inhibitor cocktail complete was obtained from
Boehringer Mannheim (Indianapolis, IN), and the src
inhibitor PP1 was obtained from Alexis (San Diego, CA).
[-32P]ATP (3000 Ci/mmol) and
[3H]AA (60–100 Ci/mmol) were obtained from
DuPont-NEN (Boston, MA).
The plasmid encoding the hemopoietic lineage cell-specific protein (HS1) fused with GST (GST-HS1) was kindly provided by U. Blank (Immuno-Allergie, Institute Pasteur, Paris, France), and the protein was expressed in Escherichia coli and was affinity purified on GST beads.
Antibodies
mAbs against phosphotyrosine (P-Tyr; PY20) were obtained from Transduction Laboratories (Lexington, KY); anti-active MAPK Abs were purchased from Promega (Madison, WI); Syk Ab directed against a peptide comprising the 11 C-terminal aa of Syk was kindly provided by U. Blank. Peroxidase-conjugated Affinipure goat anti-mouse or anti-rabbit IgGs were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA).
Isolation and purification of mast cells
Rat peritoneal mast cells were obtained from Wistar rats by a peritoneal lavage and purified as previously described (12). Briefly, a suspension of washed peritoneal cells was layered over a cushion of 30% Ficoll 400 (Pharmacia Biotech, Uppsala, Sweden) in buffered saline supplemented with 0.1% BSA, and centrifuged at 150 x g for 15 min. The purity of mast cells recovered from the bottom of the tube was 90% as assessed by toluidine blue staining.
Triggering of intact cells
Purified mast cells (5 x 105 cells/ml in a final volume of 0.5 ml) were incubated in Tyrode’s buffer (137 mM NaCl, 2.7 mM KCl, 20 mM HEPES, pH 7.4, 1 mM CaCl2, 5.6 mM glucose, 1 mg/ml BSA) with buffer or with the indicated stimuli for 20 min. Reactions were terminated by placing the tubes in ice, followed by a brief spin (12,000 x g, 20 s) at 4°C.
In vitro kinase assay
Cells were washed three times with cold PBS and subsequently
lysed in lysis buffer (50 mM HEPES, pH 7.4, 100 mM NaF, 10 mM EDTA,
10% glycerol, 1% Triton X-100, 1 mM sodium orthovanadate, 50 mM
-glycerophosphate, and 40 µg/ml protease inhibitor mixture) for 20
min on ice. Cell lysates were cleared by centrifugation at 12,000
x g for 15 min at 4°C. For immunoprecipitation, cell
lysates were incubated for 2 h at 4°C with the primary
anti-Syk Ab, which was preconjugated to protein A-Sepharose beads.
The immune complexes were washed once with 0.5 M LiCl/0.1 M Tris-HCl,
at pH 8, three times with lysis buffer, once with lysis buffer without
Triton X-100, and once with buffer containing 50 mM HEPES, pH
7.4, 150 mM NaCl, 1 mM sodium orthovanadate, and 1 mM PMSF. The immune
complexes were subsequently incubated for 10 min at 30°C with 2 µg
GST-HS1 in 40 µl kinase buffer containing 10 mM
MgCl2, 2 mM MnCl2, 30 mM
HEPES, pH 7.4, 1 µM ATP, 1 mM sodium orthovanadate, and 10 µCi
[-32P]ATP. Reactions were terminated by the
addition of 5-fold concentrated Laemmli sample buffer
(19). Samples were boiled for 5 min, centrifuged for 1 min
at 14,000 x g, and resolved by SDS/12% PAGE under
reducing conditions.
Determination of protein tyrosine phosphorylation
Cell lysates prepared in lysis buffer comprising 150 mM sucrose,
80 mM -glycerophosphate, 2 mM EDTA, 2 mM EGTA, 2 mM sodium
orthovanadate, 10 mM sodium pyrophosphate, 1% Triton X-100, 1 mM PMSF,
and 40 µg/ml protease inhibitor cocktail were centrifuged for 15 min
at 12,000 x g, and the supernatants were mixed with
5x concentrated Laemmli sample buffer. Samples were boiled, resolved
by SDS/10% PAGE under reducing conditions, and transferred to
polyvinylidene difluoride membranes. The membranes were incubated
overnight at 4°C with mAbs directed against P-Tyr (PY20, 1 µg/ml).
Bound Abs were visualized by ECL detection with the use of goat
antiserum to mouse coupled to HRP (Jackson ImmunoResearch
Laboratories).
Determination of MAPK activation
Cell lysates, prepared as described above for the determination of protein tyrosine phosphorylation, were resolved by SDS/10% PAGE under reducing conditions and transferred to polyvinylidene difluoride membranes. The membranes were incubated overnight at 4°C with polyclonal Abs directed against the active phosphorylated form of p42/p44 MAPKs (1/20,000 dilution). Bound Abs were visualized by ECL detection with the use of goat antiserum to rabbit coupled to HRP (Jackson ImmunoResearch Laboratories).
Determination of AA release
Purified mast cells (1 x 106 cells/ml) were incubated with 2–5 µCi/ml [3H]AA for 2 h at 37°C. The cells were subsequently washed three times in Tyrode’s buffer, resuspended in Tyrode’s buffer at 5 x 105 cells/ml, and triggered for 20 min. Reactions were terminated by placing the tubes in ice, followed by a brief centrifugation (12,000 x g, 20 s at 4°C). Supernatants were collected and used to determine the amount of radiolabeled AA released by liquid scintillation.
Determination of histamine release
The amount of histamine released was determined, as previously described (12), using the O-phthaldialdehyde fluorometric method (20).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To investigate whether c48/80 can activate the Syk kinase, rat
peritoneal mast cells were incubated for 20 min in the absence or
presence of c48/80 (5 µg/ml), and Syk was subsequently
immunoprecipitated. Initially, we attempted to determine whether c48/80
stimulated tyrosine phosphorylation of Syk. However, the low cell
number did not allow detection of P-Tyr or Syk protein by Western blot
analyses. Therefore, we used instead an in vitro kinase assay, using
the Syk substrate GST-HS1 (21). As shown in Fig. 1
, the activity of Syk derived from
c48/80-treated cells was markedly higher, as evidenced by its increased
potency to phosphorylate the exogenous substrate (lane
2 vs lane 1). This ability to phosphorylate GST-HS1 was
completely abolished when piceatannol (50 µg/ml), a specific
inhibitor of Syk (22), was included in the kinase assay
(lane 3 vs lane 2). Therefore, these
results have indicated that c48/80 was able to activate Syk in intact
rat peritoneal mast cells.
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To determine whether the activation of Syk by c48/80 was dependent
on Gi proteins, we examined the effect of Ptx on
the state of Syk activation. As shown in Fig. 2, pretreatment of the cells with Ptx
(300 ng/ml for 2 h) has completely eliminated the ability of
c48/80 to activate Syk. Therefore, c48/80-induced activation of Syk is
indeed mediated by Ptx-sensitive Gi
protein(s).
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We have shown previously that the enhancement in protein tyrosine
phosphorylation and MAPK activation, effected by c48/80, is dependent
on the presence of external Ca2+ and on the
activation of PI-3K and PKC (17, 18). Therefore, we
investigated whether Syk activation also requires these intermediates.
As shown in Fig. 3A, in cells
treated with c48/80 in the presence of EGTA, activation of Syk was
abolished. Similarly, exposing the cells to GF109203X (100 nM), which
is considered a specific inhibitor for the PKC isozymes 
, , ,
, and 
, has completely abolished Syk activation (Fig. 3B). In contrast, Go 6976, which blocks specifically the
activity of the PKC and isoforms, failed to affect Syk (Fig. 3B). Hence, although not proven, these findings strongly
implicate the Ca2+-independent PKC isozymes
PKC and in mediating Syk activation by c48/80.
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, A and B). In
contrast, an ionophore for Ca2+ (A23187, 1 µM)
failed to induce such activation (Fig. 4). Notably, the combined
addition of a subdose of TPA (5 nM) with the Ca2+
ionophore did not increase Syk activation beyond activation achieved by
TPA alone (Fig. 4, A and B). This was in marked
contrast to the synergistic effect produced on histamine release (Fig. 4C). Therefore, these results suggest that
Ca2+ is required at a step upstream to PKC, in
the cascade of events leading to Syk activation by c48/80.
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B) and LY294002 (data not shown), two
structurally unrelated inhibitors of PI-3K. As shown in Fig. 3B, in cells triggered with c48/80, in the presence of
wortmannin (100 nM), activation of Syk was eliminated. To determine
whether PI-3K was located up or downstream of PKC, the effect of
wortmannin on TPA-induced Syk activation was tested as well. As shown
in Fig. 4, wortmannin (100 nM) has completely abolished the ability of
TPA to activate Syk, suggesting that the PI-3K was located downstream
from PKC. Activation of Syk is dependent on a src-like PTK, but not on MAPK activation
Recently, we have shown that c48/80 activates the MAPKs by a
mechanism, which involves PTKs, Ca2+, PKC, and
PI-3K as intermediates (18). Because activation of Syk
included similar intermediates, we investigated whether Syk was
localized downstream from MAPKs. For this purpose, the effect of
PD98059, a specific inhibitor of mitogen-activated
protein/extracellular signal-related kinase kinase, was evaluated. As
shown in Fig. 5, PD98059 had no effect on
Syk activation at concentrations that we have previously shown to block
MAPK activation (18). These results have indicated that
MAPKs were not involved in the pathway leading to Syk activation.
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RI by the src kinase family member
lyn (5, 23). To investigate whether Syk
activation by c48/80 also requires activation of a src-like
PTK, we used PP1, an inhibitor of src-like PTKs
(24). As shown in Fig. 5, incubation of the cells with PP1
(5 µM) diminished the activation of Syk by 2-fold. Inhibition by PP1
was dose dependent, with half-maximal inhibition achieved at a
concentration of 3 µM and maximal inhibition at 10 µM (Fig. 6A). This finding suggested
that in the nonimmunological activation pathway, Syk activation is
dependent on the activation of a src-like PTK.
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B).
However, PP1 failed to inhibit the activation of the MAPKs (Fig. 6C). Therefore, these results have indicated that the
signaling pathways leading to Syk or MAPK activation bifurcate at or
upstream to a src-like PTK.
Next we examined whether activation of Syk by TPA was also dependent on
a src-like PTK. Indeed, as shown in Fig. 6D, PP1
also inhibited TPA-induced activation of Syk. Taken together, our
results indicate that both src-like and Syk kinases are
located downstream from PKC in the Gi-mediated
signaling pathway.
Syk activation is not dependent on trans activation of the receptor for epidermal growth factor (EGFR)
Trans activation of the EGFR is one pathway by which G
protein-coupled receptors activate protein tyrosine and MAP kinases
(25, 26). Although the mechanism of trans
activation remains obscure, it involves activation of the
src kinase (27). Therefore, we explored the
possibility that activation of Syk by c48/80 was mediated by the EGFR.
For this purpose, we used AG1478, a specific inhibitor of the EGFR
tyrosine kinase. As shown in Fig. 7, incubation with AG1478 had no effect on c48/80-induced activation of
Syk, therefore, ruling out the possibility of trans
activation of the EGFR. As a matter of fact, incubation of mast cells
with EGF (10 ng/ml) failed to evoke Syk activation, excluding further
involvement of the EGFR (Fig. 7).
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We have previously shown that c48/80-induced release of
AA/metabolites is dependent on PTKs, but not on the activation of the
MAPKs (17, 18). Because activation of Syk was also
dependent on PTKs, but not on MAPKs, we investigated whether Syk played
a role in the stimulation of AA/metabolites release. For this purpose,
we made use of piceatannol, an inhibitor of the Syk kinase. As shown in
Fig. 8, incubation of the cells with
piceatannol inhibited the release of AA/metabolites in a dose-dependent
manner. Half-maximal inhibition was achieved at 20 µg/ml and maximal
at 50 µg/ml. In contrast, histamine release induced by c48/80 under
the identical experimental conditions was unaffected by piceatannol
(Fig. 8). Therefore, these results have implicated Syk in mediating
c48/80-induced release of class II, but not class I, mediators.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RI-mediated signaling. Indeed,
in Syk-deficient mast cells, aggregation of the FcRI fails to induce
mast cell activation (9, 28). In a similar fashion,
inhibitors, such as piceatannol (22) or ER-27319
(29), which inhibit the Syk kinase, inhibit Ag-induced
release of allergic mediators. In this study, we show that activation
of mast cells by the nonimmunological,
Gi-dependent pathway also results in the
activation of Syk (Fig. 1). As expected, activation of Syk by c48/80, a
receptor analogue that activates directly Gi
proteins (12, 13, 14, 15), is Ptx sensitive (Fig. 2). It is
presently unknown which G protein, or which subunits, or ,
mediate the activation of the Syk kinase by c48/80. However, based upon
our previous results (30), rat peritoneal mast cells
express only two Ptx-sensitive G proteins. These include
Gi2 and Gi3, whereby
Gi3 serves as the principal mediator of
exocytosis (30). Therefore, the finding that Ptx treatment
prevents Syk activation limits the G proteins, which are possibly
involved, to Gi2 or
Gi3.
Activation of the Syk kinase by c48/80 implicates Syk as one of the
signaling molecules that are activated by either the immune receptor
FcRI or upon activation of Ptx-sensitive Gi
proteins by receptor analogues. However, the mechanism by which c48/80
activates Syk is different from its mode of activation by the FcRI.
In the latter case, tyrosine phosphorylation of the activated FcRI
by the lyn kinase creates the protein modules, which allow
the activated receptor to bind and activate Syk. This interaction
between the tyrosine-phosphorylated receptor and Syk is mediated by
tandem SH2 groups located in the N-terminal region of Syk
(31). Therefore, activation of Syk by FcRI reflects one
of the early events following receptor aggregation. As such, activation
of Syk precedes the phosphorylation of PLC and the subsequent
mobilization of Ca2+ and activation of PKC. In
contrast, activation of Syk by Gi occurs at a
later step. We show in this study that in this signaling pathway, Syk
is localized downstream from PKC. Two observations support this
conclusion. First, GF109203X, an inhibitor that blocks specifically
PKC, 1, 2, , , and , prevents activation of Syk by
c48/80 (Fig. 3B). Second, the phorbol ester TPA, which
activates directly PKC, also promotes Syk activation (Fig. 4).
Interestingly, Go 6976, an inhibitor that selectively blocks the
and , Ca2+-dependent PKC isozymes (reviewed in
Ref. 32), has no effect (Fig. 3B), therefore
implicating the Ca2+-independent PKC or PKC
isozymes in mediating this response. Indeed, we have previously shown
that stimulation of protein tyrosine phosphorylation and activation of
the MAPKs by c48/80 are mediated by PKC isozymes sensitive to
GF109203X, but resistant to Go 6976 (17, 18).
Nevertheless, Ca2+ is required for activation of
either the MAPKs (18) or Syk, as EGTA abolishes
c48/80-induced activation of both (Fig. 3A). It is presently
unknown at which step Ca2+ is required.
Ca2+ does not interfere with the ability of
c48/80 to activate Gi, as indicated by the fact
that external Ca2+ is not required for the
trigger of histamine release by c48/80 (33). The finding
that Ca2+ ionophore neither activates Syk, nor
does it synergize with a suboptimal dose of TPA may suggest that
Ca2+ is involved at a step downstream to
Gi activation by c48/80, but upstream to PKC
activation. For example, Ca2+ may be required to
activate phospholipase D (PLD) and produce diacylglycerol, which in
turn activates PKC (34). Nevertheless, taken together our
results clearly demonstrate that whereas in the immunological pathway
Syk is located upstream to PLC and therefore also upstream to
Ca2+ mobilization and PKC activation, in the
Gi-mediated pathway, Syk is located downstream
from PKC. These results are consistent with the finding that in a
Syk-deficient mast cell line, G protein-coupled receptors (adenosine
and thrombin), but not FcRI aggregation, could still induce tyrosine
phosphorylation of Pyk2, a member of the focal adhesion kinase family,
whose phosphorylation requires Ca2+ and PKC
(35).
We have previously shown that two mechanistically different inhibitors
of PI-3K, wortmannin and LY294002, prevent the stimulation of protein
tyrosine phosphorylation and activation of the MAPKs by c48/80
(18). These findings indicated the involvement of PI-3K in
mediating this signaling pathway. We now show that wortmannin (Fig. 3B) and LY294002 (data not shown) also prevent Syk
activation, indicating that Syk also resides downstream from PI-3K.
Moreover, the fact that wortmannin also prevents TPA-induced activation
of Syk (Fig. 4) suggests that PI-3K is located downstream from PKC in
the cascade leading to Syk activation (see model, Fig. 9). However, it should be noted that
heterotrimeric G proteins, belonging to the Gi
family, can activate PI-3K via their subunits (36).
Therefore, we cannot exclude the possibility that activation of Syk by
c48/80 may be mediated by PI-3K(s) activated simultaneously by
subunits and by PKC (see model, Fig. 9).
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RI
by the src family member lyn is a prerequisite
for Syk activation. Similarly, in the nonimmunological,
Gi pathway, activation of Syk is prevented if the
cells are treated with PP1, a selective inhibitor of
src-like tyrosine kinases (Figs. 5 and 6A).
However, it is presently unknown what member of the src-like
PTKs is involved and whether activation results from a direct
interaction between the upstream src-like kinase and Syk, or
whether like in the case of the FcRI, an intermediate
tyrosine-phosphorylated substrate is involved in the binding and
activation of Syk.
In contrast to its marked effect on Syk, PP1 exerted only a moderate
inhibitory effect on the total cell protein tyrosine phosphorylation
(Fig. 6B) and had no effect on MAPK activation (Fig. 6C). Therefore, these results indicate that the signaling
pathway initiated by c48/80-activated Gi diverges
downstream from PKC, Ca2+, and PI-3K to a MAPK
cascade and to a src-like PTK/Syk pathway (see model, Fig. 9). Activation of neither of these pathways is mediated by
trans activation of the EGFR (Fig. 7).
A clue to what might possibly be the function of Syk was obtained from
the observation that like Syk also, release of AA/eicosanoid
metabolites from c48/80-activated cells is dependent on PTKs, but not
on MAPKs (18). Therefore, in the present study, we
explored the possibility that Syk may mediate release of class II
mediators. Indeed, the Syk inhibitor piceatannol inhibits AA/eicosanoid
metabolites release in a dose-dependent manner (Fig. 8). Surprisingly,
however, PP1 was ineffective and could not reproduce the inhibitory
action of piceatannol (data not shown). Whereas the reason for this
discrepancy is presently unknown, we would like to propose a model
(Fig. 9), according to which two distinct pathways can stimulate
AA/eicosanoid metabolite release. One pathway is mediated by Syk,
whereas the other is Syk independent, but negatively regulated by a
src-like PTK. Therefore, under conditions in which the
src-like PTK is blocked (e.g., by PP1), the alternative,
Syk-independent pathway dominates. Both phospholipase
A2 and PLD contribute to AA release in mast cells
(37) and, therefore, may represent the two distinct
pathways. Consistent with this model are the findings that in the
mucosal mast cell line (rat basophilic leukemia, RBL-2H3), activation
of phospholipase A2 by transfected, G
protein-coupled muscarinic m1 receptor is not influenced by
overexpression of a dominant-negative form of Syk (38),
whereas activation of PLD was recently shown to depend on Syk
(39).
When triggered via the FcRI, Syk mediates release of both
AA/eicosanoid metabolites and class I mediators stored in the secretory
granules (9). However, when triggered by c48/80, Syk is
dispensable to histamine release (Fig. 8). Therefore, these results
indicate that the immune and the Gi
protein-mediated signaling pathways converge to stimulate production
and release of class II and possibly class III mediators. In contrast,
the signaling pathways evoking degranulation are essentially
different.
In summary, in the present study we have identified src-like and Syk kinases as two of the PTKs, which are activated by the nonimmunological, Gi-mediated pathway. Although the mechanism underlying this activation remains obscure, we have shown that it includes PKC, Ca2+, and PI-3K as intermediates, and that it does not involve trans activation of the receptor for EGF. Finally, we show that although the mechanisms by which the immunological and nonimmunological triggers elicit degranulation and release of the prestored class I mediators are tremendously different, the signaling pathways responsible for release of class II mediators are similar or at least share common signaling molecules.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Ronit Sagi-Eisenberg, Department of Cell Biology and Histology, Sackler School of Medicine, Tel Aviv University, Tel Aviv, 69978 Israel. E-mail address: histol3{at}post.tau.ac.il
3 Abbreviations used in this paper: AA, arachidonic acid; EGF, epidermal growth factor; MAPK, mitogen-activated protein kinase; PI-3K, phosphatidylinositol 3-kinase; PKC, protein kinase C; PLC, phospholipase C; PLD, phospholipase D; PTK, protein tyrosine kinase; Ptx, pertussis toxin; P-Tyr, phosphotyrosine; TPA, 12-O-tetradecanoylphorbol-13-acetate.
Received for publication November 6, 2000. Accepted for publication April 25, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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/cachectin. Nature 346:274.[Medline]
chain after receptor aggregation. J. Biol. Chem. 268:23318.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.RI signal transduction. J. Allergy Clin. Immunol. 94:1214.[Medline]
RI and the Ras/mitogen-activated protein kinase signaling pathway through SOS and Grb2. J. Biol. Chem. 271:16268.R1-mediated signaling and effector function by the Syk-selective inhibitor, piceatannol. J. Biol. Chem. 269:29697. receptors. Eur. J. Immunol. 27:321.[Medline]
subunit-mediated activation of mitogen-activated protein kinases. J. Biol. Chem. 271:19443. receptor I-mediated activation of Syk. Proc. Natl. Acad. Sci. USA . 94:12539. and subunits of the high-affinity receptor for immunoglobulin E, FcRI. Mol. Cell. Biol. 15:272.[Abstract]
RI signaling. J. Biol. Chem. 272:32443.52 is a highly selective activator of phospholipid-dependent enzymes. J. Biol. Chem. 275:13746.R1 is not shared by a G protein-coupled receptor. J. Biol. Chem. 270:10960.2. FEBS Lett. 445:371.[Medline]
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