(Received for publication, August 29, 1996, and in revised form, November 1, 1996)
From the Departments of Internal Medicine and
Physiology and Biophysics and Graduate Program in Immunology,
University of Iowa College of Medicine, Iowa City, Iowa 52246 and the
§ Laboratory of Immunology, National Institute of Dental
Research, National Institutes of Health, Bethesda, Maryland 20892
Stimulation of the IgE high affinity receptor on rat basophilic leukemia RBL-2H3 cells results in activation of protein tyrosine kinases and rapid tyrosine phosphorylation of several substrates, many of which remain unidentified. In this report, we demonstrate that the Grb2 adapter protein, when expressed as a glutathione S-transferase fusion protein, associates with four tyrosine-phosphorylated molecules (116, 76, 36, and 31 kDa) from lysates of stimulated RBL-2H3 cells. We show further that the 76-kDa protein is SLP-76, a hematopoietic cell-specific protein first identified as a Grb2-binding protein in T cells. Upon stimulation of the high affinity receptor for IgE, SLP-76 undergoes rapid tyrosine phosphorylation and associates with two additional tyrosine phosphoproteins of 62 and 130 kDa via the SH2 domain of SLP-76. Additional studies demonstrate that the SLP-76 SH2 domain also binds a protein kinase from stimulated RBL-2H3 cell lysates. Furthermore, the phosphorylation of SLP-76 requires Syk activity but is not dependent on Ca+2 mobilization. These data, together with our previous work documenting its role in T-cell activation, suggest that SLP-76 and the proteins with which it associates may play a fundamental role in coupling signaling events in multiple cell types in the immune system.
The high affinity receptor for IgE
(FcRI),1 found on basophils and mast
cells, mediates cell activation in type I allergic reactions (1).
Fc
RI is a member of a family of receptors expressed on immune
effector cells that lack intrinsic enzymatic activity but possess
cytoplasmic immune receptor tyrosine-based activation motifs that bind
cytoplasmic protein tyrosine kinases (PTKs) (2, 3). Similar to other
immune cell receptors (such as the T-cell antigen receptor (TCR) or
B-cell antigen receptor), Fc
RI is composed of multiple subunits
including a ligand binding component (
chain) noncovalently
associated with transmembrane proteins that bear the immune receptor
tyrosine-based activation motifs (
chain and a homodimer of
disulfide-linked
chains) (4, 5). The
and
cytoplasmic
domains are responsible for signal transduction (6, 7). Chimeric
receptors containing the cytoplasmic domains and an unrelated
extracellular domain mimic most of the cellular signaling events
triggered by the intact Fc
RI in the rat basophilic leukemia cell
line RBL-2H3 (7). Furthermore, stimulation of a chimeric receptor
containing the cytoplasmic domain of the TCR
chain also results in
rat basophilic leukemia cell activation, (8) suggesting a conservation
of signaling properties between T cells and mast cells.
The interaction of IgE-bound antigens with the FcRI initiates
intracellular signaling events leading to the generation of inflammatory mediators and cytokines. Proximal biochemical events include the rapid phosphorylation and activation of several PTKs including Lyn (a src family kinase) (9), p72-Syk (10, 11), and focal
adhesion kinase (12, 13, 14). Lyn associates constitutively with the
Fc
RI, whereas the interaction between Syk and the
and
subunits requires tyrosine phosphorylation of the immune receptor
tyrosine-based activation motifs after receptor ligation (15, 16). In
addition, the phosphorylated
and
subunits precipitate Shc,
Grb2, and phospholipase C-
1 (PLC
1) from RBL-2H3 cell lysates
(17).
Downstream signaling events after FcRI engagement include PLC
1
activation resulting in intracellular calcium release and protein
kinase C activation (18, 19), activation of the Ras/mitogen-activated protein kinase pathway (20, 21), and activation of nuclear factor of
activated T-cells (22), c-fos, and c-jun (23).
Proteins phosphorylated upon Fc
RI aggregation include the Fc
RI
itself, PLC
1, Nck, vav, HePTP, paxillin, and several unidentified
molecules of 72-78 kDa (9, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35). Recently we began the
characterization of SLP-76, a 76-kDa substrate of TCR-stimulated PTKs
(36, 37). We speculated that SLP-76 was one of the unidentified
substrates of Fc
RI-activated PTKs.
SLP-76, a novel hematopoietic cell-specific protein, was identified as
a protein that associates with the Grb2 adapter protein and becomes
phosphorylated after TCR ligation (36, 37). SLP-76 has several
potential tyrosine phosphorylation sites within its amino terminus
(38), a central region rich in proline residues that mediates the Grb2
association (39), and a carboxyl-terminal SH2 domain that binds to at
least two tyrosine-phosphorylated proteins and a serine/threonine
kinase after TCR ligation (39). SLP-76 seems to play a key role in
T-cell activation because its overexpression results in dramatically
enhanced TCR-mediated induction of nuclear factor of activated T-cells
and interleukin 2 promoter activity (39). It seemed likely that SLP-76
also plays a role in signaling through other immune receptor
tyrosine-based activation motif-containing receptors including the
FcRI. Here we report that SLP-76, as well as
phosphotyrosine-containing proteins of 31, 36, and 116 kDa, associate
with Grb2 in the mast cell analog RBL-2H3 after Fc
RI ligation.
SLP-76 becomes rapidly tyrosine-phosphorylated after Fc
RI engagement
and remains phosphorylated for over 1 h. Furthermore, SLP-76
phosphorylation requires Syk activity but is not dependent on
extracellular Ca+2. Stimulation of RBL-2H3 cells also
results in the phosphorylation of several other proteins and their
association with the SLP-76 SH2 domain. In addition, the SLP-76 SH2
domain binds a kinase in stimulated RBL-2H3 cells. Together these data
suggest that SLP-76 functions in several cell types to couple signaling
pathways leading to immune cell effector function.
RBL-2H3 cells were maintained
in Eagle's minimal essential medium supplemented with 15% fetal calf
serum. Jurkat (E6-1) T cells were grown in RPMI 1640 medium with 10%
fetal calf serum. All media contained penicillin (1000 units/ml),
streptomycin (1000 units/ml), and glutamine (20 mM). The
following monoclonal antibodies (mAbs) were used: anti-FcRI mAb, BC4
(30); anti-TCR mAb, C305 (40) (gift of A. Weiss, San Francisco, CA);
anti-phosphotyrosine mAb, 4G10 (gift of B. Drucker, Portland, OR); and
anti-flag mAb M2 (International Biotechnology Inc., Rochester, NY).
Anti-SLP-76 sheep antiserum 0083 was generated against amino acids
136-235 of murine SLP-76 expressed as a GST fusion protein. For
immunoprecipitation, the antiserum was bound to GammaBind Plus
Sepharose (Pharmacia Biotech Inc.) and used at 0.5 µl/20 × 106 cell equivalents. The antiserum was diluted 1:500 for
Western blotting.
GST fusion proteins were produced in bacteria as described (41). Fusion proteins containing the SH2 domain of human SLP-76 (39), the R448K mutant variant of the SLP-76 SH2 (39), and a full-length human Grb2, SH2 domain of Grb2, and full-length Grb2 with a SH2 domain loss of function mutation (R86K) have been described previously (37).
Protein Precipitations and ImmunoprecipitationsRBL-2H3
cells were stimulated with anti-FcRI mAb (BC4; 0.03 µg/ml) in
Eagle's minimal essential medium with 0.1% bovine serum albumin for
10 min unless otherwise noted. Jurkat cells were stimulated with
anti-TCR mAb (C305; 1:1000) for 1 min. For Ca2+ depletion,
cells were washed with 5 mM EGTA in PBS and stimulated in
PIPES AC (0.025 M PIPES, 0.119 M NaCl, 0.005 M KCl, 0.04 M NaOH, and 0.1% bovine serum
albumin, pH 7.4) with 0.001 M CaCl2 plus 50 µM EGTA. Cell lysates prepared in Nonidet P-40 lysis
buffer (1% Nonidet P-40, 150 mM NaCl, and 10 mM Tris, pH 7.4) including protease (50 µg/ml aprotinin,
10 µg/ml leupeptin, 50 µg/ml pepstatin A, and 1 mM
phenylmethylsulfonyl fluoride) and phosphatase (400 µM
sodium vanadate, 10 mM sodium fluoride, and 10 mM sodium pyrophosphate) inhibitors were subjected to
precipitation with GST fusion proteins or antibodies for 2 h at
4 °C. Protein complexes were washed in high-salt lysis buffer (500 mM NaCl), resolved by SDS-PAGE, transferred to
nitrocellulose, and immunoblotted with mAb or antisera followed by a
horseradish peroxidase-conjugated secondary antibody (Bio-Rad). Immunoreactive proteins were detected by ECL (Amersham Life Science, Inc.).
Immunoprecipitation complexes
were washed in 500 mM LiCl, 5 mM Tris (pH 7.4),
and water. The samples were resuspended in kinase reaction buffer (10 mM MnCl2 and 20 mM Tris, pH 7.4)
containing [32P]ATP (10 µCi/sample) for 10 min at
room temperature, washed, subjected to SDS-8% PAGE, and visualized by
autoradiography.
Ligation of the FcRI on
RBL-2H3 cells leads to rapid tyrosine phosphorylation of numerous
proteins and subsequent activation of the Ras/mitogen-activated protein
kinase pathway. Because the Grb2 adapter protein (42) links proximal
PTK activation with other signaling pathways in several systems, we
examined the tyrosine phosphoproteins that inducibly associate with
Grb2 in RBL-2H3 cells. Cells were left unstimulated or stimulated with
anti-Fc
RI mAb for 10 min. Lysates were incubated with GST/Grb2
fusion protein, and protein complexes were subjected to
anti-phosphotyrosine Western blotting. As shown in Fig.
1, a full-length GST/Grb2 fusion protein associates with
tyrosine phosphoproteins of 116, 76, 36, and 31 kDa from
Fc
RI-stimulated cells.
Our results are similar to those of Turner et al. (43), who
reported the association of 110-120-, 76-, and 31-33-kDa
phosphoproteins with a Grb2 fusion protein. These investigators also
demonstrated an association between Grb2 and tyrosine-phosphorylated
Shc in resting and FcRI-stimulated RBL-2H3 cells. Although we
detected tyrosine-phosphorylated Shc in both unstimulated and
stimulated RBL-2H3 cells, we did not find evidence for an association
between Shc and our GST-Grb2 fusion proteins in numerous in
vitro assays.
Three of the Grb2-associated proteins we detected from RBL-2H3 lysates
(pp116, pp76, and pp36) seem similar to phosphotyrosine-containing proteins from stimulated Jurkat T-cell lysates (37, 44). In T cells,
pp116 has been identified as c-cbl (45), an adapter protein with an
unknown function; pp76 is SLP-76, a hematopoietic cell-specific protein
(36) important in T-cell activation (39); and pp36 is a substrate of
the TCR-stimulated PTKs whose binding to PLC1 (46) may be critical
for its activity (47). pp36 may be lnk, a phosphotyrosine-containing
protein recently cloned from rat lymph node (48).
A GST fusion protein containing the SH2 domain of Grb2 was used to further characterize the Grb2-binding proteins from RBL-2H3 lysates. The SH2 fusion protein bound only pp31 and pp36 (Fig. 1), suggesting that pp76 and pp116 bind to the Grb2 SH3 domains. It is likely that the interaction between pp31 and pp36 with Grb2 is phosphotyrosine-dependent because mutation of the predicted phosphate binding arginine residue of the Grb2 SH2 domain to lysine (GST/Grb2R86K) completely abrogates binding of pp36 and pp31 (Fig. 1).
SLP-76 Is Expressed in RBL-2H3 Cells and Is Phosphorylated after Ligation of the FcThe Grb2/phosphoprotein associations
suggested that SLP-76 was expressed and phosphorylated in RBL-2H3
cells. To address this possibility, lysates from unstimulated and
stimulated cells were subjected to immunoprecipitation and immunoblot
analysis with anti-SLP-76 antiserum. As shown in Fig. 2,
SLP-76 is expressed in RBL-2H3 cells and undergoes an increase in
tyrosine phosphorylation within 30 s of FcRI ligation.
Phosphorylation peaks by 1 min and remains high after 30 min of
stimulation. We did not observe a decrease in SLP-76 phosphorylation up
to 60 min after stimulation (data not shown). The electrophoretic
mobility of SLP-76 from RBL-2H3 cells is faster than that of SLP-76
isolated from Jurkat T cells. This may be due to differences in the
posttranslational modification of SLP-76 or to species variation. We
are currently investigating the sites of SLP-76 phosphorylation in
RBL-2H3 and Jurkat cells to address this possibility.
SLP-76 co-immunoprecipitates with two additional tyrosine-phosphorylated proteins migrating at ~62 and ~130 kDa after RBL-2H3 cell stimulation (Fig. 2). Similar SLP-76 protein associations are seen in activated Jurkat cells (Fig. 2) (38, 39); however, pp130 migrates as a doublet in RBL-2H3 cells but as a single band in Jurkat cells. The reason for this difference is not yet clear.
The Interaction between SLP-76 and pp130 and pp62 Is Mediated by the SH2 Domain of SLP-76We speculated that the association
between SLP-76 and pp62 and pp130 may be mediated by an interaction of
the SH2 domain of SLP-76 with tyrosine-phosphorylated residues of the
other molecules. To address this possibility, GST fusion proteins
containing either the wild type SLP-76 SH2 domain or the SLP-76 SH2
domain with a point mutation of a key arginine required for
phosphotyrosine binding (R448K) were incubated with lysates from
stimulated RBL-2H3 cells. Associated proteins were resolved by SDS-PAGE
and subjected to immunoblot analysis with anti-phosphotyrosine mAb
(Fig. 3). Wild type GST/SLP-76/SH2 precipitated both
pp130 and pp62 from activated lysates, whereas GST/SLP-76/SH2R448K
failed to associate with either molecule. Thus, it seems that both
pp130 and pp62 interact with SLP-76 through its SH2 domain. We are
currently pursuing the identity of pp62 and pp130.
The possibility of other proteins binding to the SH2 domain of SLP-76
was addressed by performing an in vitro kinase assay on
protein complexes associating with the GST SLP-76 fusion proteins. As
shown in Fig. 4, the appearance of a 100-kDa
phosphoprotein suggests that the GST/SLP-76/SH2 fusion protein
associates with a protein kinase in FcRI-stimulated, but not
unstimulated, RBL-2H3 cell lysates. No kinase activity precipitates
with the GST/SLP-76/SH2R448K fusion protein. The 100-kDa phosphoprotein
seems to be similar to a SLP-76 SH2-associated molecule we found to be
phosphorylated on phosphoserine and phosphothreonine, but not
phosphotyrosine residues in stimulated Jurkat T cells (39). This
analysis is supported by the lack of a 100-kDa molecule in
anti-phosphotyrosine Western blot analysis of SLP-76
immunoprecipitations of Jurkat and RBL-2H3 cell lysates (Figs. 2 and
3). These results suggest that a serine/threonine kinase associates
either directly or indirectly with the SLP-76 SH2 domain after receptor
stimulation. Attempts are currently underway to identify the relevant
kinase in both T cells and rat basophilic leukemia cells.
Phosphorylation of SLP-76 Requires Syk Activity but Is Not Dependent on Extracellular Ca2+
We next performed
experiments to determine the requirements for SLP-76 tyrosine
phosphorylation in RBL-2H3 cells after FcRI engagement. Tyrosine
phosphorylation of many substrates in RBL-2H3 cells requires expression
and activation of the proximal signaling kinase Syk (16, 47). To test
whether SLP-76 phosphorylation depends on Syk, we examined SLP-76 after
receptor ligation in a Syk-deficient RBL-2H3 variant (TB1A2) (49). Wild
type RBL-2H3 cells, the Syk-deficient mutant, and a Syk-reconstituted
cell line (3A1) derived from TB1A2 cells were left unstimulated or were
stimulated for 10 min with BC4. Cell lysates were subjected to
immunoprecipitation with anti-SLP-76 antiserum and immunoblotted with
anti-phosphotyrosine and anti-SLP-76 antibodies. Stimulation of the
Fc
RI on wild type and Syk-reconstituted cells resulted in SLP-76
phosphorylation, whereas no phosphorylation was detected in the
Syk-deficient mutant (Fig. 5A). However, each
cell expressed similar amounts of SLP-76 protein (Fig. 5B).
These data suggest that SLP-76 phosphorylation is dependent on prior
activation of the Syk PTK.
Mast cell activation after FcRI engagement depends upon several
downstream signaling pathways including the activation of PLC
1 and
the release of intracellular calcium. A sustained increase in
intracellular Ca2+ is required for the tyrosine
phosphorylation of some substrates (such as focal adhesion kinase) but
not others (such as the Fc
RI) (14). We tested the dependence of
SLP-76 phosphorylation on sustained increases in Ca2+
concentrations by depleting Ca2+ with EGTA treatment
followed by receptor stimulation in Ca2+-free medium. Cell
lysates were subjected to SLP-76 immunoprecipitation and immunoblot
analysis with anti-phosphotyrosine antibody (Fig. 6). As
shown, Fc
RI stimulation leads to tyrosine phosphorylation of SLP-76
in the absence of extracellular Ca2+, demonstrating that
SLP-76 phosphorylation is proximal to kinase activity dependent upon
sustained increases in cellular calcium from extracellular sources.
The Grb2 adapter protein links PTKs with the Ras/mitogen-activated
protein kinase cascade and the generation of
phosphatidylinositol-derived second messengers in a variety of systems.
Thus, characterizing molecules that associate with Grb2 should provide
insight into the regulation of signal transduction. In this report, we
describe the association of four tyrosine-phosphorylated molecules with Grb2 in activated RBL-2H3 cells: pp116, pp76, pp36, and pp31. Phosphoproteins migrating at 116 kDa (c-cbl), 76 kDa (SLP-76), and 36 kDa have been shown to participate in signal transduction events in T
cells. The identity of pp116 and pp36 has not yet been confirmed in
RBL-2H3 cells. Ongoing experiments are addressing the possibility that
the 31-kDa phosphoprotein is a component of the FcRI (17).
In this report, we identify the 76-kDa band in RBL-2H3 cells as SLP-76,
an adapter protein described originally as a substrate of the
TCR-stimulated PTKs. We show further that SLP-76 is a substrate of the
PTKs activated after FcRI engagement on RBL-2H3 cells. Our data
demonstrate that SLP-76, via its SH2 domain, associates with at least
two unidentified tyrosine phosphoproteins, pp130 and pp62, in
stimulated RBL-2H3 cells. These associations are similar to those seen
after antigen receptor stimulation in T cells. However, pp130 appears
as a doublet in RBL-2H3 cells but as a single molecule in T cells,
suggesting RBL-specific modifications of pp130 or the existence of a
second molecule. In addition, the SH2 domain of SLP-76 associates
directly or indirectly with a kinase in stimulated RBL-2H3 cells,
resulting in the appearance of a 100-kDa phosphoprotein. Together,
these findings suggest that SLP-76 may play an important role in
linking the Fc
RI with distal events in mast cell activation.
RBL-2H3 cell activation after IgE binding requires the coordination of
several signaling pathways including those mediated by PLC1 and Ras.
It is not yet clear in which biochemical pathway SLP-76 functions. Our
data suggest that SLP-76 phosphorylation is dependent on Syk protein
tyrosine kinase activation; thus, SLP-76 likely acts downstream of this
proximal signaling event. In addition, SLP-76 becomes phosphorylated in
RBL-2H3 cells in the absence of sustained Ca2+
mobilization, suggesting that SLP-76 phosphorylation does not require
the activity of Ca2+-dependent kinases. Recent
studies from our laboratory suggest that in T cells, SLP-76 potentiates
signaling in the extracellular signal-regulated kinase
pathway,2 and it is likely that it has a
similar function in RBL-2H3 cells.
These data along with our studies in T cells suggest that SLP-76 plays a key role in receptor-mediated signal transduction leading to immune cell activation. We are performing structure/function analyses of SLP-76 to determine the functional significance of its associations with other potential signaling molecules in both T cells and rat basophilic leukemia cells. These experiments should help to further elucidate the role of SLP-76 in the process of immune cell activation.
We thank B. Drucker and A. Weiss for their gifts of reagents.