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RI-Mediated Signal Transduction in Mast Cells
* Receptors and Signal Transduction Section, Oral Infection and Immunity Branch, and
Secretory Physiology Section, Gene Therapy and Therapeutics Branch, Department of Health and Human Services, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD 20892
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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RI
aggregation induced the tyrosine phosphorylation of Gab2 and
translocation of a significant fraction of it from the cytosol to the
plasma membrane. As in other cells, Gab2 was found to associate with
several signaling molecules including Src homology 2-containing protein
tyrosine phosphatase 2, Grb2, Lyn, and phospholipase C
(PLC). The association of Gab2 with Lyn and PLC were enhanced
after receptor aggregation. Overexpression of Gab2 in rat basophilic
leukemia 2H3 cell line cells inhibited the FcRI-induced tyrosine
phosphorylation of the subunits of the receptor, and the
phosphorylation and/or activation of Syk and mitogen-activated protein
kinase. Downstream events such as calcium mobilization, degranulation,
and induction of TNF-
and IL-6 gene transcripts were decreased in
Gab2 overexpressing cells, although Akt phosphorylation as a measure of
PI3-kinase activation was unaffected. These results suggest that in
addition to the positive effects mediated by PI3-kinase that are
apparent in Gab2-/- mast cells, Gab2 by interacting with
Lyn and PLC may have negative regulatory effects on FcRI-induced
mast cell signaling and functions. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Gab2 is a newly identified 97-kDa molecule that is a member of the
Dos/Gab adapter family which also includes Gab1, the insulin receptor
substrates, and Drosophilia daughter of sevenless Dos
(3, 4, 5). Gab2 contains an N-terminal Pleckstrin homology
domain followed by a long region with multiple tyrosine-containing
motifs and two proline-rich domains. The tyrosine-containing motifs,
once phosphorylated, provide binding sites for Src homology
(SH)2 2 domain-containing
signaling molecules including p85 subunit of phosphatidylinositol-3
kinase (PI3-kinase), phospholipase C (PLC), and SH2-containing
protein tyrosine phosphate 2 (SHP-2). The proline-rich regions of Gab2
are potential binding sites for SH3-domain-containing proteins such as
Src family protein tyrosine kinases (4).
Gab2 has been shown to function in various signaling pathways activated
by the binding of cytokine, Ag, and growth factor receptors
(5, 6, 7). The importance of Gab2 has been demonstrated by
its ability to assemble multiple proteins, which then couple receptors
to downstream pathways. The IL-3R activates Akt via a
Shc/Grb2/Gab2/PI3-kinase pathway to result in cell proliferation and
survival (8). Gab2 is also involved in the regulation of
Grb2/Ras/extracellular regulated kinase (ERK) mitogen-activated protein
kinase (MAPK) pathway (3, 7). Recent studies with bone
marrow-derived mast cells (BMMC) from Gab2-deficient mice demonstrates
an essential role for Gab2 in the regulation of mast cell functions
through its coupling to PI3-kinase (9). However, because
Gab2 is a scaffolding molecule that can couple to different signaling
molecules including not only PI3-kinase, but also SHP-2, PLC, Grb2,
Shc, Src, and SH2-containing inositol phosphatase (3, 4, 10, 11), it could have complex roles in multiple signaling
pathways.
Aggregation of the FcRI on mast cells initiates a biochemical
cascade that ultimately results in the release of inflammatory
mediators and generation of cytokines (12, 13, 14, 15). Because
FcRI itself has no intrinsic tyrosine enzymatic activity,
nonreceptor protein tyrosine kinases such as Lyn and Syk are essential
in this signaling pathway (16, 17, 18, 19, 20, 21, 22, 23). Several adapter
molecules such as LAT and Vav are also critical for FcRI-mediated
mast cell signal transduction (24, 25). The purpose of the
present experiments was to study the role of another adaptor molecule,
Gab2, in FcRI signaling, using the rat basophilic leukemia 2H3 mast
cell line (RBL-2H3). FcRI aggregation induced the tyrosine
phosphorylation of Gab2 and translocation of a significant fraction of
Gab2 from the cytosol to the plasma membrane. Overexpression of Gab2 in
RBL-2H3 cells inhibited the FcRI-induced signal transduction. These
results suggest that Gab2, in addition to the positive effects mediated
by PI3-kinase that are apparent in the BMMC from
Gab-/- mice, can regulate FcRI-induced mast
cell signaling by interacting with Lyn and PLC negatively.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Aprotinin, Triton X-100, and protein A-conjugated agarose were obtained from Sigma Aldrich (St. Louis, MO). Polyvinylidene difluoride transfer membrane was purchased from Millipore (Bedford, MA) and the ECL reagent was from NEN (Boston, MA). The materials for electrophoresis were purchased from NOVEX (San Diego, CA). The mouse Gab2 cDNA in pEBB vector was kindly provided by Dr. B. G. Neel (Beth Israel Hospital, Boston, MA).
Antibodies
Rabbit polyclonal anti-Gab2 and anti-c-Jun N-terminal
kinase (JNK) Abs, HRP-conjugated anti-phospho-Tyr (pTyr) mAb, 4G10,
and mixed monoclonal anti-PLC1 Abs were obtained from Upstate
Biotechnology (Lake Placid, NY). Rabbit polyclonal anti-phospho-ERK
(phospho-Thr (pThr)202 and
pTyr204), anti-phospho-p38
(pThr180 and pTry182),
anti-phospho-Akt (phospho-Ser473),
anti-Akt, anti-ERK, and anti-p38 Abs were purchased from
New England Biolabs (Beverly, MA). Goat polyclonal anti-Gab2,
rabbit polyclonal anti-PLC2, and monoclonal anti-phospho-JNK
(pThr183 and pTyr185) Abs
were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
Stable transfection
A total of 20 µg of linearized Gab2 cDNA in pEBB vector or
empty vector together with 2 µg of pSV2-neo plasmid were
cotransfected into 5 x 106 RBL-2H3 cells by
electroporation (960 µF, 310 V) as described previously
(26). Clones were selected with 350 µg/ml of active G418
(Life Technologies, Rockville, MD). Cell lines were screened for the
level of Gab2 expression by immunoblotting of total cell lysates with
anti-Gab2 Ab, using blotting with anti-FcRI
Ab as an
internal control. Cell lines were selected for additional experiments
that expressed high levels of the Gab2 molecule.
Cell culture and activation
RBL-2H3 cells and transfectants were cultured as monolayers in Eagle’s MEM supplemented with 15% heat-inactivated FBS, penicillin, streptomycin, amphotericin, and glutamine (27). For activation, cells were cultured overnight with or without anti-trinitrophenyl-specific IgE. For histamine release assays, the cell monolayers were washed twice with MEM containing 0.1% BSA and 10 mM Tris (pH 7.4). The cells incubated with IgE were stimulated with Ag (DNP coupled to human serum albumin, 35:1 molar ratio), or with calcium ionophore A23187 in the same medium. After incubation for 45 min at 37°C, the medium was removed for histamine analysis (28). For RNA protection assays, cells were stimulated in culture medium for 1 or 2 h, and total RNA was isolated using the RNeasy kit (Qiagen, Santa Clarita, CA). In the Akt experiments, cells were cultured in MEM medium without FBS for 24 h before stimulation with Ag.
Immunoprecipitation and immunoblotting
After stimulation, the cell monolayers were rinsed once with ice-cold PBS containing Na3VO4 (1 mM) and protease inhibitors (2 mM aminoethyl-benzenesulfonyl-fluoride hydrochloride, 10 µg/ml leupeptin, 5 µg/ml pepstatin A, and 0.2 U/ml aprotinin). Cells were then solubilized in Triton lysis buffer (1% Triton X-100, 50 mM Tris (pH 7.5), 150 mM NaCl, 1 mM Na3VO4, and protease inhibitors). The supernatants after a 15 min 20,000 x g centrifugation were mixed with protein A-coupled agarose beads and then proteins were immunoprecipitated with Abs prebound to protein A-agarose beads. Rabbit anti-mouse IgG Ab was used to couple mouse mAb with protein A-agarose. After gentle rotation for 1 h at 4°C, the beads were washed four times with ice-cold Triton lysis buffer, and the precipitated proteins were eluted by boiling for 15 min with SDS-PAGE sample buffer containing 1% 2-ME. For the preparation of total cell lysates, monolayers were rinsed once with PBS as described above, and directly lysed by the addition of SDS-PAGE sample buffer containing 2-ME.
Immunoprecipitated proteins or whole-cell lysates were separated by SDS-PAGE under reducing conditions and electrotransferred to polyvinylidene difluoride membranes. The membrane was incubated with 4% BSA blocking buffer (10 mM Tris (pH 7.4), 150 mM NaCl, and 0.1% Tween 20) for 1 h at room temperature and the tyrosine-phosphorylated proteins were detected by HRP-conjugated anti-pTyr Ab, 4G10. The membranes were then stripped and reprobed with primary Abs. In all these blots, the proteins were visualized by the ECL reagent (NEN).
RNA protection assay
Cytokine mRNA was measured by using a multiprobe rat cytokine
RNA protection kit (RiboQuant kit; BD PharMingen, San Diego, CA) as
recommended by the manufacturer. Briefly, by in vitro transcription,
32P-labeled RNA probes were synthesized using the
set of cytokine cDNA templates. The synthesized probes were purified
using SELECT-D (RF) spin chromatography column (5 Prime
3 Prime,
Boulder, CO) and hybridized overnight at 56°C with 20 µg of RNA.
After digestion with RNase, the protected RNA were purified, resolved
by QuickPoint PAGE (Invitrogen, Carlsbad, CA), and visualized by
autoradiography.
Intracellular free calcium concentration ([Ca2+]i) measurements
Fura 2 fluorescence in single cells was measured using an SLM Aminco 8000/DMX 100 spectrofluorometer (Jobin Yvon Horiba, Edison, NJ) attached to an inverted Nikon Diaphot microscope (Nikon, Melville, NY) with a Fluor x40 oil-immersion objective. Images were acquired using an enhanced charge-coupled device camera (CCD-72; Maryland Technologies, Michigan City, IN) and the Image-1 software (Universal Imaging, Downingtown, PA) at excitation wavelengths of 340 and 380 nm, with emission at 510 nm. Analog plots of the fluorescence ratio (340:380) in single cells are shown. Cells were grown overnight in culture medium on coverslips, and then washed twice with loading medium (medium 199; Biofluid, Rockville, MD) supplemented with 2 mM CaCl2 and 0.1% BSA, and loaded with 2 µM fura 2 for 45 min at 37°C. After loading, cells were washed four times with working medium (medium 199 containing 2 mM CaCl2, 10 mM Tris (pH 7.4), and 0.01% BSA). All other details are given in the text and figure legends.
Subcellular fractionation
For the preparation of cytosolic and membrane fractions, 5 x 106 cells were washed with ice-cold PBS containing 1 mM NaVO4, 0.5 mM PMSF, 5 µg/ml leupeptin, and resuspended on ice in hypotonic buffer (42 mM KCl, 10 mM HEPES (pH 7.4), 5 mM MgCl2, and protease inhibitors). Cell lysates were centrifuged (10 min at 200 x g), and the supernatants were centrifuged for 30 min at 100,000 x g. Supernatants of the second centrifugation were collected as the cytosolic fraction. The pellet was washed once with hypotonic buffer, then directly solubilized in sample buffer as the membrane fraction.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Gab2 is widely expressed in many tissues and cells including
heart, lung, kidney, testis, spinal cord, blood leukocytes, T cells,
some B cell lines, and recently shown to be present in mouse mast cells
(4, 5, 9). Immunoblotting demonstrated the presence of
Gab2 in the RBL-2H3 mast cell line, which is a useful model for
defining FcRI-mediated signaling pathways. To investigate the role
of Gab2 in FcRI-mediated signaling, we first examined the tyrosine
phosphorylation status of this molecule in RBL-2H3 cells. There was
some basal constitutive tyrosine phosphorylation of Gab2, which rapidly
increased after FcRI aggregation (Fig. 1
A). This FcRI-induced
phosphorylation was clearly apparent at 3 min, was stronger at 10 min,
and then gradually decreased to baseline by 45 min. These results,
together with the data with the BMMC (9), indicate that
Gab2 may play an important role in FcRI-mediated signaling.
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B and data not shown). We
next examined whether the increased Gab2 protein in the transfected
cells was still tyrosine phosphorylated after FcRI aggregation
(Fig. 1C). As in the control vector-transfected cells,
receptor stimulation induced a rapid increase in phosphorylations that
returned to baseline at 30 min. As there was much more Gab2 in these
transfected cells, we would assume that there was actually an increase
in the concentration of this molecule that was tyrosine-phosphorylated.
Therefore, Gab2 is tyrosine phosphorylated in these transfected cells
in the same time course as in the controls, and participates in the
FcRI signaling pathway.
FcRI-stimulated histamine release and cytokine gene
transcription are inhibited in Gab2-overexpressing cells
FcRI-mediated activation of mast cells results in the release
of histamine from cytoplasmic granules. Therefore, we examined the
effect of Gab2 overexpression on Ag-stimulated histamine release. In
transfected cells that expressed high levels of Gab2, the Ag-induced
histamine release was significantly reduced to <50% of that in the
RBL-2H3 parental cells (Fig. 2A). In contrast, there were
no changes in the calcium ionophore-induced release (data not shown),
suggesting that inhibition was at an early step after receptor
activation, and that the cells were still capable of degranulation.
There was no inhibition of Ag-induced histamine release in the control
vector-transfected cells and in Gab2-transfected cell lines where the
expression level of this protein was increased by only 2- to 7-fold
(clone nos. 3 and 4 and data not shown). Further evidence that the
inhibition of histamine release was related to the expression level of
Gab2 were studies in a third cloned line that after transfection
initially expressed high levels of Gab2 (43-fold increase) and had 45%
inhibition of Ag-induced histamine release. When maintained in culture,
there was a gradual decrease in the expression level of Gab2 (to
27-fold) and the histamine release reverted to that of the parental
cells. These results indicate that overexpression of Gab2 negatively
regulates FcRI-mediated degranulation.
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RI not only causes the release of pre-formed
mediators such as histamine, but also stimulates the synthesis of
various cytokines including TNF-, IL-6, and IL-2
(29, 30, 31, 32). In RBL-2H3 cells, a multiprobe RNA protection
assay demonstrates the FcRI-induced increase in the mRNA for
multiple cytokines, the most prominent of which are IL-4, IL-6, and
TNF- (33). Using this assay, there was significant
suppression of the FcRI aggregation-induced production of mRNA for
TNF- and IL-6 in the cells that overexpressed Gab2 (Fig. 2B). The inhibition of TNF- and IL-6 mRNA production was
more dramatic (73–94%, Fig. 2, C and D) than
the inhibition of histamine release (50–66%, Fig. 2A).
However, the changes in the mRNA for IL-4 were variable. These results,
taken together with the inhibition of histamine release, suggested a
negative regulatory role of Gab2 overexpression on FcRI-mediated
mast cell function.
Gab2 overexpression inhibits the FcRI-induced increase
in intracellular calcium
Aggregation of FcRI results in the generation of inositol
1,4,5-triphosphate (IP3) that next results in the
release of calcium from intracellular stores, and calcium influx
through calcium release-activated calcium channels in the plasma
membrane (34, 35). The inhibition of FcRI-induced, but
not ionophore-induced, histamine release by overexpression of Gab2
suggested that these effects of Gab2 were at an early step after
FcRI aggregation. Therefore, intracellular measurements in
individual cells were used to monitor the effects of Gab2 expression on
receptor-mediated [Ca2+]i
(Fig. 3). Both the rapid and the
sustained response to Ag stimulation were suppressed by 
50% in the
Gab2-transfected cells. In the absence of extracellular calcium, there
was again an inhibition of the calcium response. RBL-2H3 cells have a G
protein-coupled seven-transmembrane receptor for thrombin that when
activated, results in calcium influx. The thrombin-induced increase in
intracellular calcium was similar in all the different cell lines.
Furthermore, there were similar responses with thapsigargin (data not
shown) and ionomycin in all the cell lines, indicating that the
expression of Gab2 had no effects on intracellular calcium storage.
These controls confirmed that the changes in calcium influx were
limited to the FcRI-activated pathway. The results indicate that the
major inhibitory effects of Gab2 overexpression were upstream of the
rise in intracellular calcium.
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Cellular protein tyrosine phosphorylation is an early event after
FcRI stimulation, and is critical for the propagation of downstream
signal transduction (16, 36, 37). The inhibition of the
calcium release suggested that the overexpression of Gab2 had effects
at an early stage of cell activation. As compared with the control
vector-transfected cells, the Ag-induced total cellular protein
tyrosine phosphorylation was decreased in Gab2-overexpressing cells
(Fig. 4A). However, the
general pattern was similar except for the phosphorylated band at about
p97 kDa in the cells overexpressing Gab2, which was recognized by
anti-Gab2 Ab (data not shown). In both the control and transfected
cells, Gab2 was tyrosine phosphorylated under quiescent conditions and
after receptor aggregation (Fig. 1C). The suppression of
cellular protein tyrosine phosphorylation by Gab2 overexpression
further suggests that Gab2 was regulating signal transduction at an
early step after FcRI activation.
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RI results in the rapid tyrosine phosphorylation of
the and receptor subunits (37). The
phosphorylation of the tyrosines in the immunoreceptor tyrosine-based
activation motif of these subunits then recruits signaling molecules
such as Syk to transduce downstream activation events (18, 38, 39). The decreases in total cellular tyrosine phosphorylations
suggest that Gab2 may interfere at a very early stage in the signaling
cascade after receptor aggregation. Indeed, tyrosine phosphorylation of
both the - and -chains of FcRI were decreased in
Gab2-overexpressing cells compared with those of controls (Fig. 4B). Tyrosine phosphorylation of Syk was also clearly
reduced in these cells (Fig. 4C). Phosphorylation of the
activation loop tyrosines is a measure of Syk activation and critical
for downstream propagation of signals (40, 41). In these
cells, the phosphorylation of the tyrosines in the activation loop of
Syk was also decreased as detected by an anti-phosphoactivation
loop tyrosine-specific Ab (data not shown). The recruitment and
activation of Syk by FcRI is mainly by the phosphorylated
immunoreceptor tyrosine-based activation motif of the
subunit (28, 42), suggesting that the decreased Syk
activation is probably due to the changes in FcRI tyrosine
phosphorylation. Therefore, overexpression of Gab2 by interfering with
receptor phosphorylation, results in decreased Syk
activation.
Gab2 associates with Lyn protein tyrosine kinase and translocates
to the plasma membrane after FcRI activation
The protein tyrosine kinase Lyn, a Src kinase family member,
associates with FcRI, and this association is increased after
receptor aggregation (17, 21, 43). After FcRI
aggregation, Lyn is thought to phosphorylate the and subunits
of the receptor, initiating the intracellular signaling cascades
(44). The SH3 domain of Src tyrosine kinase has also been
shown to bind to Gab2 in vitro (4). Therefore, we
investigated whether there was an interaction between Lyn and Gab2.
Indeed, Gab2 was detected in Lyn immunoprecipitates from the lysates of
Gab2-overexpressing cells, and this association increased after FcRI
aggregation, suggesting that Gab2 might be recruited to Lyn after
receptor aggregation (Fig. 5A). However, this association
was below the detection threshold in control cells that have much less
Gab2. Lyn was constitutively tyrosine phosphorylated, and there were no
changes in this phosphorylation after FcRI stimulation in both the
control and Gab2-transfected cells (data not shown). Therefore, Gab2,
by interacting with Lyn, could interfere with the phosphorylation of
downstream substrates such as the subunits of the receptor.
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RI, as expected,
was only in the membrane fraction (Fig. 5, B and
C). After receptor aggregation, there was rapid
translocation of Gab2 from the cytosol to the plasma membrane, which
was apparent 3 min after stimulation, the earliest time point examined.
However, at all time points examined, 70% of the Gab2 remained in
the cytosol fraction after cell activation. A similar fraction of the
total Gab2 was present in the plasma membrane of stimulated cells in
both the control (Fig. 5B) and Gab2-transfected cells (Fig. 5C). Therefore, after FcRI aggregation there was
translocation of Gab2 to the plasma membrane, presumably due to its
Pleckstrin homology domain, and interaction with Lyn. The interaction
of Gab2 with Lyn kinase might play a role in FcRI signaling.
Gab2 binds PLC2 after FcRI stimulation, and inhibits its
tyrosine phosphorylation
The aggregation of FcRI results in tyrosine phosphorylation and
activation of PLC, which generates IP3 that in
turn mediates the increase in intracellular calcium. The dramatic
decrease in the FcRI-induced intracellular calcium response in the
Gab2-transfected cells suggested that there could be changes in the
activation of PLC. The extent of the tyrosine phosphorylation of
PLC1 was similar in the control and the Gab2-overexpressing cells
(Fig. 6A). However, the amount
of PLC1 protein precipitated in Gab2-overexpressing cells was much
greater than that in vector control cells, suggesting that Gab2 may
up-regulate the expression of PLC1. Anti-PLC1 Ab analysis of
total cell lysates confirmed that the expression level of PLC1 in
Gab2-transfected cells was indeed higher than that in the controls
(data not shown). Therefore, in the Gab2-transfected cells, there is a
decrease in the fraction of the PLC1 that is tyrosine phosphorylated
after receptor activation. Similarly, Ag induced a rapid tyrosine
phosphorylation of PLC2 in control cells; however, this
phosphorylation was dramatically decreased in Gab2-transfected cells
(Fig. 6B). Therefore, there is decreased tyrosine
phosphorylation of PLC that could account for the decrease in the
reduction in the signals that result in the intracellular calcium
response.
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on the Gab2 molecule,
phosphorylation of tyrosines at these sites allows binding by the SH2
domain of PLC (3). Indeed, Gab2 was coprecipitated with
PLC2, and this association was increased after activation (Fig. 6B), suggesting that PLC2 is recruited to Gab2 after Ag
stimulation. The association of Gab2 with PLC2 was clearly evident
in the Gab2-transfected cells, but was below the detection threshold in
the control vector-transfected cells. There was also association of
PLC1, Grb2, and SHP-2 with Gab2 (data not shown). However, there was
no significant change in the association of Gab2 with PLC1 after
receptor activation (data not shown). Therefore, these data suggest
that interactions of PLC with Gab2 may play a role in the inhibition
of the receptor-induced increase in
[Ca2+]i. Overexpression of Gab2 inhibits MAPK pathways
Gab2, and its structurally related molecule Gab1, by interacting
with Grb2/Sos/Shc plays a role in the activation of the ERK MAPK
pathway (7, 45, 46), thereby regulating downstream
transcription factors (3, 10). The suppression of
Ag-induced TNF- and IL-6 mRNA production in Gab2-overexpressing
cells suggested that there could be changes in the MAPK pathways.
Therefore, phospho-specific Abs were used to investigate the ERK, JNK,
and p38 MAPK pathways. These Abs recognize phosphorylated Thr and Tyr
residues that are critical for their activation (47, 48).
The phosphorylation of ERK at 3 min after FcRI activation was
similar in the different cell lines; however, at 10 min it was
decreased in the Gab2-transfected compared with the control cells (Fig. 7A). A very similar pattern
was observed with p38 MAPK phosphorylation; strong signals that were
similar to controls at 3 min in the Gab2-transfected cells, but a
faster return to baseline (Fig. 7B). Therefore, there was a
more rapid rate of dephosphorylation and inactivation of ERK and p38 in
Gab2-overexpressing cells. However, the pattern was different with the
JNK proteins; there was some inhibition of the FcRI-induced
phosphorylation in the Gab2-transfected cells, although the changes
were not dramatic (Fig. 7C). Therefore, FcRI stimulation
resulted in a more transient activation of the ERK and p38 MAPK
pathways in the Gab2-transfected cells.
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Gab2 has multiple binding sites for the p85 subunit of PI3-kinase
and this interaction is involved in the activation of the PI3-kinase in
response to growth factors, cytokines, and Ag receptors
(7). The activation of PI3-kinase results in the
production of phosphatidylinositol 3,4,5-triphosphate, which plays an
important role in the sustained influx of calcium and also recruits Akt
to the plasma membrane where Akt is Thr/Ser-phosphorylated and
activated (49, 50, 51). The decrease in the calcium response
in the Gab2-transfected cells could be due to inhibition of
receptor-mediated PI3-kinase stimulation. Therefore, we used an Ab
specific for phosphorylated Ser473 that is
critical for the activation of Akt as a measure of the simulation of
the PI3-kinase pathway (50). There was significant Akt
phosphorylation in cells not stimulated by receptor aggregation;
however, this disappeared when cultures were maintained for 24 h
under serum-free conditions (data not shown). FcRI-induced
phosphorylation of Akt was similar in the control and Gab2-transfected
cells cultured for 24 h under serum-free conditions (Fig. 8). Similar results were observed in
cells that had been cultured in regular media. Because there was
neither suppression nor enhancement of Akt activation in Gab2
transfectants, it can be assumed that PI3-kinase activation is not
affected in these cells. Therefore, these results suggest that the
PI3-kinase/Akt pathway is not involved in the Gab2-mediated inhibition
of mast cell histamine release and cytokine production.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RI aggregation results in the
tyrosine phosphorylation of Gab2 and translocation of a significant
fraction of it from the cytosol to the plasma membrane. Gab2 associates
with several signaling molecules including protein tyrosine phosphatase
SHP-2, p85 subunit of PI3-kinase, Lyn, and PLC. The coprecipitation
of Gab2 with Lyn and PLC were enhanced after receptor aggregation.
The association of Gab2 with Lyn might be through proline-rich domains
of Gab2 interacting with the SH3 domain of Lyn (4).
Overexpression of Gab2 in RBL-2H3 cells inhibited the FcRI-induced
tyrosine phosphorylation of the receptor subunits, and the
phosphorylation and activation of Syk and MAPK. Downstream events such
as PLC tyrosine phosphorylation, calcium mobilization,
degranulation, and induction of TNF- and IL-6 gene transcripts were
similarly decreased in these cells. These results suggest that in
addition to the positive effects mediated by PI3-kinase that are
apparent in Gab2-/- mast cells, Gab2 by
interacting with Lyn and PLC may have negative regulatory effects on
FcRI-induced mast cell signaling and functions.
The results of the present experiments have to be explained in light of
the recent description of defects in FcRI-mediated signaling in BMMC
from Gab2-/- mice (9). In these
cells, there is a decrease in the receptor-induced activation of the
PI3-kinase pathway with a decrease in the generation of
phosphatidylinositol 3,4,5-triphosphate, the tyrosine phosphorylation
of PLC1, and the formation of IP3. There is
also a decrease in the receptor-induced increase in
[Ca2+]i, especially of
the delayed response with parallel decreases in degranulation and
cytokine generation. These data suggest that Gab2 recruits and
activates the PI3-kinase, thus playing an important role in
degranulation. Multiple signaling pathways are activated in cells in
response to a particular stimulus. A scaffolding molecule such as Gab2
binds different signaling proteins, thereby coupling to several
pathways, some of which could have positive or negative effects. The
absence of Gab2 as a scaffolding molecule would affect the related
signaling pathways such as PI3-kinase, PLC, Lyn, SHP-2, and Grb2.
The observed functional changes would represent the major positive role
of the molecule. In BMMC from Gab2-/- mice, the
FcRI-induced PI3-kinase activation was decreased and mast cell
functions were defective, suggesting that PI3-kinase is a major
positive effector of Gab2 in FcRI signaling. In contrast,
overexpression of Gab2 in cells would amplify additional pathways
dependent on other interactions, such as PLC and Lyn. These
inhibitory effects would not be apparent in the deficient cells if they
act downstream of the positive effects of Gab2.
Although the expression level of Gab2 was quite high, the effects of
Gab2 were specific for FcRI signaling. The localization of Gab2 in
transfected cells was similar to that in the controls both in the
quiescent state and after Ag stimulation. This suggests that there are
no changes in subcelluar distribution of Gab2 that could account for
the inhibition of signal transduction in transfected cells. Similarly,
thrombin, which binds to a seven-transmembrane G-protein-coupled
receptor, induced calcium mobilization that was not inhibited in these
cells suggesting a specific regulatory role of Gab2 in IgE receptor
signaling. Calcium ionophore-induced calcium mobilization and histamine
release were not affected in these cells, indicating a normal
degranulation function of the cells and a specific action of Gab2
upstream of calcium mobilization in FcRI signal transduction.
The present experiments strongly suggest that overexpression of Gab2
interferes at a very early stage in FcRI signal transduction. The
reduction in the tyrosine phosphorylation of subunits of FcRI and
Syk kinase would implicate the step at the Lyn-mediated phosphorylation
of the receptor subunits. Because Lyn is localized to the plasma
membrane, it is possible that binding of Gab2 with Lyn may directly
interfere with its capacity to phosphorylate the receptor subunits. It
is also possible that Gab2 may indirectly interfere with receptor
subunit phosphorylation by recruiting multiple other proteins such as
SHP-2, PI3-kinase, PLC, or Shc and keep these away from
FcRI-organized signaling complexes (42, 52). Although
Gab2 and SHP-2 were associated in RBL-2H3 cells, we could not detect a
significant change in membrane translocation of SHP-2 and
overexpression of SHP-2 in RBL-2H3 cells did not inhibit histamine
release (data not shown). Therefore, the decrease in the tyrosine
phosphorylation of subunits of FcRI and PLC2 do not appear to be
due to Gab2-mediated membrane recruitment of SHP-2. Nevertheless,
whatever the mechanism, the decrease in receptor phosphorylation would
then be a major contributor to the inhibition of the subsequent
signaling cascade.
The tyrosine phosphorylation and activation of PLC1 and PLC2 for
the generation of IP3 are downstream of Syk
(26). The FcRI-induced tyrosine phosphorylation of
PLC2 was dramatically decreased by over-expression of Gab2. In
contrast, although the extent of the total tyrosine phosphorylation of
PLC1 was similar in control and Gab2-transfected cells, there was a
decrease in the fraction of the total PLC1 that was phosphorylated.
These results suggest that the interaction of Gab2 with PLC
inhibited the phosphorylation, and therefore, the activation of PLC.
The binding of Gab2 to PLC might prevent its tyrosine
phosphorylation by mechanisms such as blocking tyrosine residues,
sequestering the molecule away from kinases, or by promoting
dephosphorylation by phosphatases. The difference in the extent of the
decrease in the phosphorylation of PLC1 and PLC2 may be due to
variation in their interaction with Gab2 or their subcellular
locations. By electron microscopy, PLC1 and PLC2 isoforms are
differentially distributed in the RBL-2H3 cells with the PLC2
inherently associated with the membrane, whereas PLC1 is recruited
to membrane ruffles only after receptor aggregation (53, 54). Studies with inhibitors suggest that the FcRI-induced
tyrosine phosphorylation and activation of PLC1, but not of PLC2,
depends on PI3-kinase (54). Therefore, the minimal changes
in the phosphorylation of PLC1 compared with PLC2 would further
indicate that in the Gab2-expressing cells the PI3-kinase pathway is
still active.
The overexpression of Gab2 also had dramatic effects on the
FcRI-induced rise in intracellular calcium. There was 50%
inhibition in both the initial and sustained increase in intracellular
calcium, which would indicate a decrease in release from intracellular
stores as well as in influx from the extracellular medium. The
PI3-kinase-generated lipid products play an important role in the rise
in intracellular calcium by recruiting PLC and Tec protein tyrosine
kinases to the membrane (55, 56, 57). The Tec protein kinase
Btk then regulates the protein tyrosine phosphorylation and activation
of PLC (57). Similarly, PI3-kinase inhibitors block the
rise in intracellular calcium (53). The normal
FcRI-induced activation of Akt in the Gab2-transfected cells
suggests that PI3-kinase was not inhibited. Therefore, the decreased
calcium response to Ag in these cells is probably due to the reduced
activation of PLC, and would contribute to the subsequent inhibition
of mast cell functions.
Similar to its role in signaling from cytokine and growth factor
receptors, Gab2 is important for FcRI-induced activation of the
PI3-kinase pathway (9). Therefore, it was surprising that
PI3-kinase pathway appeared to be unchanged in Gab2-overexpressing
cells, as demonstrated by the phosphorylation of Akt. Because tyrosine
phosphorylation of the and subunits of the receptor (as well as
Syk) was inhibited by Gab2 overexpression, it would be assumed that
receptor-induced PI3-kinase/Akt pathway should also be suppressed.
However, the normal Akt phosphorylation might reflect two opposing
effects on the PI3-kinase pathway: 1) reduced activation on the one
hand, due to the decrease in Syk activity; and 2) increased activation
due to overexpression of Gab2. PI3-kinase could also be both an
upstream regulator and downstream effector of Gab2, as has been shown
with the closely related molecule of Gab1 (58). Therefore,
our data supports a positive role of Gab2 in regulating PI3-kinase
activation.
In summary, these experiments demonstrate that FcRI induced the
tyrosine phosphorylation of Gab2 and its translocation to the membrane.
Gab2 interacted with several signaling molecules the binding to two of
which (PLC2 and Lyn) was increased after Ag stimulation. These
interactions appear to play a negative regulatory role in FcRI
signaling. Overexpression of Gab2 dramatically suppressed
FcRI-induced signal transduction, except for the activation of the
PI3-kinase pathway as evidenced by Akt phosphorylation. These results
demonstrate complex positive and negative regulatory effects of Gab2 in
FcRI signaling in mast cells.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: SH, Src homology; BMMC, bone marrow-derived mast cells; JNK, c-Jun N-terminal kinase; ERK, extracellular regulated kinase; MAPK, mitogen-activated protein kinase; pTyr, phosphotyrosine; pThr, phosphothreonine; RBL-2H3, rat basophilic leukemia 2H3 cell line; SHP-2, SH2-containing protein tyrosine phosphotase 2; PLC, phospholipase C; PI3-kinase, phosphatidylinositol-3 kinase; [Ca2+]i, intracellular free calcium concentration; IP3, inositol 1,4,5-trisphosphate.
Received for publication September 13, 2001. Accepted for publication February 15, 2002.
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