(Received for publication, December 23, 1996, and in revised form, March 11, 1997)
From the The SH2 domain-containing
inositol-polyphosphate 5-phosphatase, SHIP, associates with Fc Aggregation of the high affinity IgE receptors
(Fc The high affinity IgE receptor (Fc We report here evidence that SHIP is tyrosine-phosphorylated after
IgE stimulation in rat basophilic leukemia cell line (RBL-2H3). This
phosphorylation was an early event after receptor aggregation but was
not secondary to the increase of intracellular calcium or to the
activation of protein kinase C. Furthermore the phosphorylation of SHIP
did not depend on protein kinase Syk. SHIP was precipitated by
immobilized tyrosine-phosphorylated synthetic peptides based on the
ITAM of the Protein A, aprotinin, and Triton X-100 were from
Sigma. GammaBind plus Sepharose 4B was from Pharmacia Biotech Inc.
Streptavidin coupled to agarose beads was from Pierce. The materials
for electrophoresis were purchased from Novex (San Diego, CA), and the
source of other materials was as described previously (28). The
non-phosphorylated and phosphorylated peptides based on the sequences
of the Mouse monoclonal anti-Grb2 antibody, mouse
monoclonal anti-Shc antibody (S14620), and rabbit polyclonal anti-Shc
antibody (S14630) were from Transduction Laboratories (Lexington, KY). Rabbit polyclonal anti-Shc antibody (06-203) was from Upstate Biotechnology Inc. (Lake Placid, NY). Rabbit polyclonal anti-SHIP was
kindly provided by Dr. Gerald Krystal (Terry Fox Laboratory, Vancouver,
Canada) (13). The affinity-purified polyclonal rabbit anti-phosphotyrosine antibodies were coupled to Sepharose 4B beads as
recommended by the manufacturer. All other antibodies have been
described previously (28-31).
The RBL-2H3 cells were maintained as monolayer
cultures in Eagle's minimum essential medium (BioWhittaker,
Walkersville, MD) supplemented with 15% heat-inactivated fetal bovine
serum (Life Technologies, Inc.), penicillin, and streptomycin (1, 32). The Syk negative variant of the RBL-2H3 was described previously (33).
RBL-2H3
cells were stimulated essentially as described previously (1, 32).
Briefly, for Fc For immunoprecipitation, lysates from 107
cells in 1.0 ml were precleared by mixing for 90 min at 4 °C with
protein A-agarose beads or GammaBind plus Sepharose 4B beads. The
lysates were then incubated with each antibody that had been
preincubated with 20 µl of beads. After gentle rotation at 4 °C
for 90 min, the beads were washed three times with wash buffer (lysis
buffer with Triton X-100 concentration decreased to 0.5%), once with
150 mM NaCl, 50 mM Tris, pH 7.4, and the
proteins eluted by boiling for 5 min with Laemmli's sample buffer as
described previously (28). For precipitation with ITAM peptides,
lysates from 1.5 × 107 cells in 1.0 ml were
precleared by mixing for 90 min at 4 °C with streptavidin coupled to
agarose beads and then were incubated with biotinylated ITAM peptides
that had been preincubated with 20 ml of streptavidin beads. The beads
were then washed as described above.
Samples from the precipitates were separated
by SDS-PAGE under reducing conditions and electrotransferred to
polyvinylidine difluoride membranes (Millipore, Bedford, MA).
Tyrosine-phosphorylated proteins were detected with the monoclonal
antibody PY-20 conjugated to horseradish peroxidase as described
previously (36). Other proteins were immunoblotted with specific
antibodies and then detected using horseradish peroxidase-conjugated
protein A, donkey anti-rabbit IgG, or donkey anti-mouse IgG antibodies.
In all blots, proteins were visualized using the enhanced
chemiluminescence. In some experiments antibodies were stripped from
the membranes according to the protocol of the manufacturer and then
the membranes were reprobed with other antibodies.
Cross-linking the high affinity IgE receptor on mast
cells results in activation of protein-tyrosine kinases and rapid
changes in inositol phosphates (1-3). We therefore investigated
whether the recently described SHIP protein was tyrosine-phosphorylated after receptor aggregation. By immunoblot and immunoprecipitation analysis, SHIP was present in RBL-2H3 cells (Fig. 1).
There were proteins of 145, 140, and 105 kDa recognized by the
anti-SHIP antibody. The low level tyrosine phosphorylation of the
145-kDa SHIP protein was dramatically enhanced after receptor
aggregation. There was also the tyrosine phosphorylation of the 140-kDa
protein. Similar data were obtained when Fc
To further define the tyrosine phosphorylation of SHIP and its
interaction with other molecules, Shc was immunoprecipitated with
anti-Shc antibody from non-stimulated or stimulated cells (Fig.
2). As previously reported, Shc was already
tyrosine-phosphorylated before stimulation, and there were no changes
in its tyrosine phosphorylation after receptor aggregation (37). As
expected, SHIP was associated with Shc in these anti-Shc
immunoprecipitates. Similar to the previous results, the 145-kDa SHIP
protein was tyrosine-phosphorylated in non-stimulated cells, and after
receptor aggregation the increase in this phosphorylation was
detectable at 3 min with further gradual increase to reach a maximum at
30 min. With stimulation there was also the coprecipitation of the 140- and 105-kDa SHIP proteins. Grb2 was also coimmunoprecipitated with Shc
from both non-stimulated and stimulated cells. However, Grb2 did not
get tyrosine-phosphorylated, and there was no detectable change in the
extent of its association with Shc after receptor aggregation. The SHIP
associated with Shc was already tyrosine-phosphorylated in
non-stimulated cells (Figs. 2 and 3). In the absence of
the tyrosine phosphatase inhibitor, vanadate, there was no
coprecipitation of SHIP with Shc (Fig. 3). However, there was still
association of Grb2 with Shc. Therefore, SHIP in this mast cell line
was tyrosine-phosphorylated in non-stimulated cells, and its
tyrosine phosphorylation was enhanced by receptor aggregation. This
phosphorylation was important for its association with Shc.
Some proteins are
tyrosine-phosphorylated very early after Fc
The
protein-tyrosine kinase Syk is essential for Fc In previous studies we observed the association of Shc with
tyrosine-phosphorylated peptides based on the ITAM of the
These experiments identified SHIP as one of the substrates that is
tyrosine-phosphorylated after Fc SHIP hydrolyzes inositol 1,3,4,5-tetraphosphate with the formation of
inositol 1,3,4-trisphosphate by a reaction that requires Mg2+ (13). It also catalyzes the hydrolysis of
phosphatidylinositol 3,4,5-trisphosphate to
phosphatidylinositol-3,4-bisphosphate. Inositol 1,3,4,5-tetraphosphate
stimulates the release of intracellular Ca2+, which results
in activation of the calcium release-activated Ca2+
channel, Icrac, and the influx of Ca2+ into the
cell (38, 39). In mast cells, receptor aggregation activates
phospholipase C The tyrosine phosphorylation of SHIP does not enhance its enzymatic
activity (13). However, tyrosine phosphorylation of SHIP and other
proteins could result in SH2-mediated interactions and changes in the
cellular localization of SHIP to sites where it could play important
physiological roles. Thus, the association of SHIP with Shc and with
the tyrosine-phosphorylated The Ras-signaling pathway after Fc In the present model for mast cell signaling, the earliest event
after aggregation of Fc While these experiments were in progress two reports appeared that
suggest that SHIP may play a role in the negative regulation of
signaling in B cells or in mast cells (43, 44). The optimal tyrosine
phosphorylation of SHIP occurred when the B-cell receptor was
coclustered with Fc We thank Drs. Mark Swieter and
Nicholas Ryba for helpful discussions and for reviewing this
manuscript. We also thank Greta Bader and Elsa Berenstein for excellent
technical help.
Laboratory of Immunology,
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
RIIB
and negatively regulates both B-cell and mast cell function. We report
here that SHIP was tyrosine-phosphorylated after high affinity IgE
receptor (Fc
RI) aggregation in rat basophilic leukemia RBL-2H3
cells. The tyrosine phosphorylation of SHIP was an early event after
receptor aggregation and was present in cells deficient in the
protein-tyrosine kinase Syk. Furthermore it was not secondary to the
increase of intracellular calcium or the activation of protein kinase
C. SHIP was precipitated by immobilized phosphorylated synthetic
peptides based on the immunoreceptor tyrosine-based activation motif
(ITAM) of the
but not the
subunit of the high affinity IgE
receptor. Tyrosine phosphorylation of SHIP and its association with the
tyrosine-phosphorylated
subunit of Fc
RI could play an important
role in down-regulating receptor-mediated signal transduction in mast
cells. Thus, whereas the activation molecule Syk associates with the
subunit ITAM, the
subunit ITAM binds the negative signaling
molecule SHIP. Therefore, unlike B cells where the antigen receptor and
coreceptors such as Fc
RIIB or CD22 each recruits molecules with
opposite effects, the Fc
RI contains subunits which recruit molecules
that activate and inhibit signal transduction.
RI)1 on basophils and mast cells
initiates a cascade of events that results in the release of
inflammatory mediators. This pathway involves the activation of several
protein-tyrosine kinases including Lyn, Syk, Btk, and Fak that induce
the tyrosine phosphorylation of various proteins (1-3). This results
in the stimulation of phospholipase A2, C, and D, mobilization of
Ca2+ from intracellular and extracellular sources, and
activation of serine and threonine kinases (4, 5). There is also
activation of the Ras pathway that may be important for the release of
arachidonic acid and its metabolites (6). In the Ras pathway, best
elucidated in non-mast cells, stimulation of growth factor receptors or
antigen receptors on T and B cells results in the tyrosine
phosphorylation of Shc and the formation of a complex containing Shc,
the Grb2 adapter protein, and the nucleotide exchange factor Sos (7, 8). This complex activates Ras which then, by a pathway that involves
Raf1, induces phosphorylation and stimulation of mitogen-activated protein kinase (9, 10). A 145-kDa tyrosine-phosphorylated protein is
present in association with Shc and Grb2 (11, 12). Recently this
145-kDa tyrosine-phosphorylated protein was found to be an SH2
domain-containing inositol-polyphosphate 5-phosphatase, which has been
called SHIP (13). In addition to the SH2 domain, SHIP has several
tyrosine phosphorylation sites that may interact with other
SH2-containing proteins such as Shc and the Syk protein-tyrosine kinase
(13, 14).
RI) on mast cells and basophils is
a tetrameric structure composed of the IgE binding
chain, a
subunit, and disulfide-linked homodimeric
chains (15). The
COOH-terminal cytoplasmic domains of the
and the
contain a
motif with the amino acid sequence
(D/E)X2YX2LX6-7YX2(L/I) that is critical for cell activation (16-18). This
immunoreceptor tyrosine-based
activation motif (ITAM) is also present in the
subunit of the T-cell receptor complex and in Ig
and Ig
of the B-cell receptor and is important for cell activation (19-23). In B
cells and mast cells, cross-linking of ITAM-containing receptors to
Fc
RIIB results in down-regulatory signals (24). The cytoplasmic domain of Fc
RIIB contains the immunoreceptor tyrosine-based
inhibitory motif that recruits negative regulatory molecules such as
SHIP and protein-tyrosine phosphatase SHP-1 (25-27).
but not the
subunit of the high affinity IgE
receptor. Tyrosine phosphorylation of SHIP and its association with the
receptor may play an important role in down-regulating receptor-mediated signal transduction in mast cells.
Materials
and
subunits of the rat Fc
RI (15) have been described
previously (28, 29). The sequence of the Fc
RI
peptide was
KVPDDRLYEELHVYSPIYSALEDTR, and that of the Fc
RI
peptide was
REKSDAVYTGLNTRNQETYETLKHEK. Some of the peptides were also biotinylated
as described previously (29).
RI-mediated activation cells were stimulated with
either anti-Fc
RI
monoclonal antibody (CA5) or with antigen after
overnight culture with antigen-specific IgE. In some experiments cells
were stimulated with either 0.5 µM calcium ionophore or
40 nM PMA as described previously (34, 35). After
stimulation, the monolayers were rinsed twice with ice-cold
phosphate-buffered saline and solubilized by adding lysis buffer (10 mM Tris, pH 7.5, containing 1.0% Triton X-100, 1 mM Na3VO4, 150 mM NaCl,
50 mg/ml leupeptin, 0.5 unit/ml aprotinin, 2 mM pepstatin
A, 1 mM phenylmethylsulfonyl fluoride). The plates were
left on ice for 10 min. The cells were then scraped, and the
supernatants were collected and centrifuged for 30 min at 16,000 × g at 4 °C as described previously (28). In experiments designed to deplete extracellular Ca2+, the monolayers were
washed with calcium-free Eagle's minimal essential medium containing
10 mM EDTA and stimulated in this medium (34).
Tyrosine Phosphorylation of Inositol Phosphatase SHIP after FcRI
Aggregation
RI was stimulated by
preincubating the cells with antigen-specific IgE and then adding
antigen (see below). As has been reported previously, immunoblot
analysis demonstrated the association of SHIP with Shc and Grb2
(data not shown).
Fig. 1.
SHIP was tyrosine-phosphorylated by FcRI
aggregation. RBL-2H3 cells were either non-stimulated or
stimulated with anti-Fc
RI
monoclonal antibody for 30 min. Lysates
were then immunoprecipitated with either 2 µg of anti-SHIP antibody
or normal rabbit IgG prebound to 20 µl of protein A-agarose beads.
The precipitates were washed, eluted by boiling, separated by SDS-PAGE
on 12% gels, and analyzed by immunoblotting with anti-phosphotyrosine
antibody (Anti-PY) followed by anti-SHIP antibody
(Anti-SHIP).
[View Larger Version of this Image (31K GIF file)]
Fig. 2.
SHIP was coprecipitated with anti-Shc
antibody and tyrosine-phosphorylated after FcRI aggregation.
RBL-2H3 cells were stimulated with anti-Fc
RI
monoclonal antibody
for the indicated times, and the cell lysates were immunoprecipitated
with anti-Shc antibody (2 µg of anti-Shc monoclonal antibody prebound
to 20 µl of rabbit anti-mouse IgG coupled to protein A-agarose
beads). The precipitates were separated by SDS-PAGE on 12% gels. After transfer the membrane was blotted with anti-phosphotyrosine antibody (Anti-PY), stripped, cut into three parts, and immunoblotted
with anti-SHIP polyclonal antibody (Anti-SHIP), anti-Shc
polyclonal antibody (Anti-Shc), and anti-Grb2 monoclonal
antibody (Anti-Grb2).
[View Larger Version of this Image (27K GIF file)]
Fig. 3.
Tyrosine phosphorylation is required for the
association of SHIP with Shc. RBL-2H3 cells were stimulated with
anti-FcRI
monoclonal antibody for 30 min. The cell lysates were
prepared in the presence or absence of vanadate and immunoprecipitated with anti-Shc antibody (2 µg of anti-Shc polyclonal antibody prebound to protein A beads). The precipitates were analyzed by immunoblotting with anti-phosphotyrosine antibody (Anti-PY), stripped, cut
into three parts, and immunoblotted with anti-SHIP polyclonal antibody (Anti-SHIP), anti-Shc polyclonal antibody
(Anti-Shc), and anti-Grb2 monoclonal antibody
(Anti-Grb2).
[View Larger Version of this Image (26K GIF file)]
RI aggregation whereas
others are phosphorylated at later stages after a rise in intracellular
calcium and/or after the activation of protein kinase C (2).
Stimulation of cells with either IgE and antigen or with
anti-Fc
RI
antibodies resulted in an increase in SHIP tyrosine
phosphorylation (Fig. 4A). However, there was no increase in SHIP tyrosine phosphorylation after direct activation of
protein kinase C by the addition of PMA or by the addition of the
calcium ionophore A23187. To further define the role of Ca2+ in Fc
RI-mediated tyrosine phosphorylation of SHIP,
cells were stimulated in a Ca2+-free medium containing EDTA
(Fig. 5). The absence of extracellular Ca2+
did not affect Fc
RI-mediated tyrosine phosphorylation of SHIP. Therefore, the tyrosine phosphorylation of SHIP is an early
receptor-mediated event that is upstream of the rise in the
intracellular calcium and/or the activation of protein kinase C.
Fig. 4.
Tyrosine phosphorylation of SHIP was a
receptor-mediated event not induced by calcium ionophore or PMA and did
not require Syk. Cells used were either RBL-2H3 (A) or
the Syk negative TB1A2 (B, C) and were either non-stimulated
or stimulated for 30 min with 0.5 µM calcium ionophore
A23187 (A23187), 40 nM PMA (PMA),
anti-FcRI
monoclonal antibody (@Fc
RI), or IgE
antigen (Ag). Lysates were then immunoprecipitated with
either anti-phosphotyrosine polyclonal antibody (A, B) or
with anti-Shc polyclonal antibody (C). The
immunoprecipitates were separated by SDS-PAGE (8%) and analyzed by
immunoblotting with anti-SHIP antibody (Anti-SHIP) and
anti-phosphotyrosine antibody (Anti-PY). Note that the lower percent PAGE of these gels results in better separation of the anti-SHIP immunoblotted 145- and 140-kDa proteins.
[View Larger Version of this Image (21K GIF file)]
Fig. 5.
Tyrosine phosphorylation of SHIP did not
require calcium in the medium. RBL-2H3 cells were stimulated with
anti-FcRI
monoclonal antibody for 30 min in the presence or
absence of calcium in the medium. Lysates were then immunoprecipitated
with anti-Shc antibodies and analyzed by immunoblotting with
anti-phosphotyrosine antibody (Anti-PY), anti-SHIP
polyclonal antibody (Anti-SHIP), anti-Shc polyclonal
antibody (Anti-Shc), and anti-Grb2 monoclonal antibody
(Anti-Grb2).
[View Larger Version of this Image (22K GIF file)]
RI-mediated
degranulation. Although the
and
subunits of Fc
RI are tyrosine-phosphorylated in a Syk-deficient variant of the RBL-2H3 cells, histamine release and phosphorylation of downstream molecules such as phospholipase C are not observed (33). Histamine release and
phosphorylation of other substrates were reconstituted by the
transfection of Syk into these negative cells (33). These Syk negative
cells were used to evaluate the tyrosine phosphorylation of SHIP (Fig.
4B). Cells were stimulated by receptor aggregation, the
calcium ionophore A23187, or PMA, and the lysates were then immunoprecipitated with anti-phosphotyrosine antibody. There was some
constitutive tyrosine phosphorylation of SHIP in the non-stimulated Syk
negative cells. After either IgE-antigen or anti-Fc
RI
antibody stimulation there was increased tyrosine phosphorylation of SHIP (Fig.
4B). In some experiments this was stronger after IgE-antigen than with anti-Fc
RI
antibodies. Similar results were obtained when SHIP was coimmunoprecipitated with anti-Shc antibody (Fig. 4C). The addition of PMA resulted in dephosphorylation of
SHIP and a decrease in its coprecipitation with Shc. Therefore, both the constitutive and the Fc
RI-induced tyrosine phosphorylation of
SHIP are independent of the presence of Syk in cells. Furthermore, the
receptor-mediated phosphorylation of SHIP was an early event upstream
of Syk.
RI
but Not the
ITAM of Fc
RI
Precipitated SHIP from Lysates of RBL-2H3
Cells
subunit of Fc
RI (29). As SHIP coprecipitates with Shc, we investigated whether there was an association of SHIP with Fc
RI. Synthetic phosphorylated and non-phosphorylated peptides based on the ITAM of the
and
subunits of Fc
RI were used for precipitation studies (Fig. 6). SHIP was precipitated only by the
phosphorylated peptide based on the ITAM of Fc
RI
. The immunoblots
also suggested that the
-phosphorylated ITAM peptide was
precipitating two isoforms of SHIP with slightly different
characteristics in migration in SDS-PAGE. To further define the
interaction of SHIP with the ITAM synthetic peptide, we compared the
proteins precipitated with anti-Shc or anti-SHIP antibodies with those
precipitated by the tyrosine-phosphorylated
ITAM peptide (Fig.
7). Anti-SHIP antibodies precipitated 145-, 140-, and
105-kDa proteins; the 145-kDa form was the most strongly tyrosine
phosphorylated in these non-stimulated cells (Fig. 7A). This
145-kDa form of SHIP was the major form associated with Shc. In
contrast, the
-phosphorylated ITAM precipitated all the isoforms of
SHIP (Fig. 7B). Therefore, the SHIP associated with Shc was
predominantly tyrosine phosphorylated, whereas the
ITAM
precipitated both non-phosphorylated and phosphorylated SHIP.
Altogether these data strongly suggest that the precipitation of SHIP
by the
-phosphorylated ITAM is due to direct interaction.
Fig. 6.
SHIP was precipitated by diphosphorylated
synthetic peptide based on the ITAM of the subunit of Fc
RI.
RBL-2H3 cells were either non-stimulated or stimulated with
anti-Fc
RI
monoclonal antibody, and lysates from 1.5 × 107 were then incubated for 90 min at 4 °C with 1 nmol
of the different biotinylated ITAM peptides that had been prebound to
20 µl of streptavidin beads. For controls streptavidin beads were
used without any peptide. The precipitates were washed, eluted by
boiling, and separated by SDS-PAGE on 10% gels. After transfer the
membrane was blotted with anti-SHIP polyclonal antibody
(Anti-SHIP). In the nomenclature used here
refers to
Fc
RI
and
to Fc
RI
. The
YY is
unphosphorylated, whereas
PP is the diphosphorylated synthetic peptide based on the ITAM of Fc
RI
. Similarly, for the
ITAM based on the
subunit
YY is the unphosphorylated
peptide,
PP is the same peptide with both tyrosines
phosphorylated, and
PY and
YP are
monophosphorylated peptides with phosphorylation of either the first or
second tyrosine.
[View Larger Version of this Image (20K GIF file)]
Fig. 7.
Comparison of SHIP immunoprecipitated by
anti-SHIP or anti-Shc antibodies to that precipitated by phosphorylated
synthetic ITAM peptide. Lysates from 1.5 × 107 non-stimulated RBL-2H3 cells were precipitated with
anti-Shc or anti-SHIP polyclonal antibodies (A), with 3 nmol
of biotinylated phosphorylated ITAM of Fc
RI
peptide
(
PP), or with anti-Shc polyclonal antibody
(B). The precipitates were analyzed by immunoblotting with
anti-phosphotyrosine antibody (Anti-PY) followed by
anti-SHIP polyclonal antibody (Anti-SHIP).
[View Larger Version of this Image (23K GIF file)]
RI aggregation. This tyrosine phosphorylation occurred after receptor stimulation but not when cells
were activated with either calcium ionophore or with PMA. Therefore,
tyrosine phosphorylation of SHIP was not secondary to the increase of
intracellular calcium or the activation of protein kinase C. Interestingly, the phosphorylation of SHIP was present even in Syk
negative cells indicating that this is an early event upstream of the
activation of Syk.
1 and phospholipase C
2, which results in the
formation of inositol phosphates that release Ca2+ from
intracellular sources. This is followed by an influx of Ca2+ from extracellular sources mediated by the
Icrac channel. Therefore, by decreasing the concentration
of inositol 1,3,4,5-tetraphosphate SHIP would limit calcium influx.
subunit of Fc
RI would localize it to
membrane sites where there is activation of signaling pathways.
RI aggregation results in the
activation of mitogen-activated protein kinase, which is important for
the release of arachidonic acid and in regulating nuclear events that
result in the synthesis of cytokines (6, 8, 40, 41). In the case of
antigen receptors on T and B cells, activation of this pathway is
initiated by the tyrosine phosphorylation of Shc which then forms a
complex with Grb2-Sos and activates p21ras (7, 8). However, Shc
is constitutively tyrosine-phosphorylated in non-stimulated RBL-2H3
cells, and this phosphorylation may be increased after Fc
RI
aggregation (37). Shc also associates with the tyrosine-phosphorylated
subunit of Fc
RI (29). Therefore, the tyrosine-phosphorylated
subunit of Fc
RI could bind SHIP either directly or indirectly by
binding mediated by Shc. The extent of the tyrosine phosphorylation of
the
subunit would determine how much SHIP was recruited to the
receptor and thereby regulate and/or limit the extent of the signals
generated by the receptor.
RI is the activation of protein-tyrosine kinase, probably Lyn, which results in tyrosine phosphorylation of the
receptor subunits (42). The COOH-terminal cytoplasmic domain of
Fc
RI
and the cytoplasmic domain of Fc
RI
contain ITAMs that
once they are tyrosine-phosphorylated preferentially bind different
downstream signaling molecules (29). Thus, we recently observed that a
synthetic tyrosine-diphosphorylated peptide based on the ITAM sequence
of the
subunit of Fc
RI precipitated Shc, phospholipase C
1,
and Lyn, whereas the similar peptide based on the ITAM of
did not
bind these molecules but was very effective in binding Syk (29). The
binding of Syk to the tyrosine-phosphorylated ITAM results in a
conformational change in Syk with an increase in its enzymatic activity
and the downstream propagation of signals such as the tyrosine
phosphorylation of phospholipase C
1, phospholipase C
2, and the
influx of calcium (28). Therefore, tyrosine phosphorylation of the ITAM
of the
subunit recruits Syk, which is critical in downstream
activating signals. In contrast the ITAM based on the
subunit, once
it is phosphorylated, recruits other molecules such as Shc and SHIP,
which are important for activating and regulating signaling events.
RIIB (43). Such coclustering results in decreased
signaling from the B-cell receptor. In mast cells, SHIP associated with
the Fc
RIIB that was coclustered with Fc
RI (44). These are
conditions that resulted in a decrease in Fc
RI-mediated signaling
(45). The coclustering of the immune receptor with Fc
RIIB probably
results in the tyrosine phosphorylation of the cytoplasmic domain of
Fc
RIIB and the recruitment of SHIP. In the present experiments we
found that in fact the
subunit of Fc
RI has a domain that can
recruit SHIP to the receptor. SHIP most probably binds to the amino
acid sequence SPIYSAL that is similar to the
(T/S)XXYXX(L/I) immunoreceptor tyrosine-based
inhibitory motif present in Fc
RIIB (24, 26). However, unlike this
inhibiting motif in Fc
RIIB, we could not detect any binding of the
protein-tyrosine phosphatase SHP-1 to the
-phosphorylated ITAM
peptide.2 Therefore, unlike B cells where
the antigen receptor and coreceptors such as Fc
RIIB or CD22 each
recruit molecules with opposite effects, the Fc
RI contains subunits
that recruit molecules that activate and inhibit signal
transduction.
*
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
To whom correspondence should be addressed: Laboratory of
Immunology, Bldg. 10, Rm. 1N106, NIDR, NIH, Bethesda, MD 20892-1188. Tel.: 301-496-5105; Fax: 301-480-8328; E-mail: tk51w{at}nih.gov.
1
The abbreviations used are: FcRI, high
affinity IgE receptor; ITAM, immunoreceptor tyrosine-based activation
motif; SHIP, SH2 domain-containing inositol-polyphosphate
5-phosphatase; PMA, phorbol 12-myristate 13-acetate; PAGE,
polyacrylamide gel electrophoresis.
2
T. Kimura, H. Sakamoto, E. Apella, and R. P. Siraganian, unpublished observations.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.