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Blood, Vol. 92 No. 5 (September 1), 1998:
pp. 1697-1706
SLP-76-Cbl-Grb2-Shc Interactions in Fc RI Signaling
By
Julie Chu,
Yenbou Liu,
Gary A. Koretzky, and
Donald L. Durden
From the Neil Bogart Memorial Laboratories, Division of
Hematology-Oncology, Childrens Hospital Los Angeles, Los Angeles, CA;
University of Southern California School of Medicine, Los Angeles, CA;
and the Departments of Internal Medicine, Physiology, and Biochemistry
and the Graduate Program in Immunology, University of Iowa College of
Medicine, Iowa City, IA.
 |
ABSTRACT |
SLP-76 and Cbl are complex adapter proteins that have the capacity
to bind to smaller adapter proteins, such as Grb2, which subsequently
binds the nucleotide exchange protein Sos in the transmission of
intracellular signals. SLP-76, Cbl, Shc, and Grb2 have been implicated
in immunoreceptor tyrosine-based activation motif (ITAM) signaling,
leading to activation of Ras. However, their mechanism of action has
not been determined. To date, there have been no reports of SLP-76
involvement in Fc RI-receptor signaling and no data exist for an
interaction between Cbl, Shc, and SLP-76 in vivo. We provide evidence
that SLP-76, Cbl, and Shc are tyrosine phosphorylated on
FcRI-receptor stimulation and are associated with the adapter
protein Grb2 in -interferon-differentiated U937 cells (U937IF). The
interactions between SLP-76 and Cbl and SLP-76 and Grb2 are present in
resting U937IF cells. However, the interaction between SLP-76 and Grb2
becomes augmented twofold on FcRI-receptor aggregation. Our results
provide the first evidence for a phosphorylation-dependent interaction
between SLP-76 and Shc, induced at least 10-fold on FcRI receptor
stimulation. Our data indicate that a significant portion of a
multimolecular complex containing Cbl, SLP-76, Shc, and Grb2 is
distinct from a trimolecular complex containing the Ras guanine
nucleotide exchanger Sos, Shc, and Grb2. FcRI-induced tyrosine
phosphorylation of SLP-76, Cbl, Shc, and the highly induced SLP-76-Shc
interaction provide the first evidence that SLP-76 and Cbl are involved
in FcRI signaling and suggest a functional significance for these
interactions in FcRI signal relay in the control of Ras in myeloid
cells.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
ELUCIDATING THE SIGNAL transduction
pathway through Fc receptors in monocytes and macrophages has
significant clinical implications in understanding and manipulating the
inflammatory process, autoimmune disorders, and myeloid immunity. The
FcRI receptor is a member of the immunoglobulin gene superfamily
that also includes the T-cell receptor (TCR), B-cell receptor (BCR),
and other Fc receptors.1,2 In contrast to growth factor
receptors such as insulin, epidermal growth factor (EGF), and
platelet-derived growth factor (PDGF), these receptors have no
intrinsic kinase activity. Signaling through these receptors is
mediated through a conserved stretch of amino acids consisting of
paired tyrosines and leucines in the consensus sequence
(D/E)XXYXXL(Y)6-8 YXXL, termed the immunoreceptor
tyrosine-based activation motif (ITAM).3 FcRI receptor
crosslinking occurs with stimulation by IgG molecules whose Fc
fragments interact with the FcRI subunit. This then leads to
conformational activation of the FcRI subunit that further
activates src-family kinase (SRTK) such as Hck, Lyn, and
Fgr.1,2,4,5 The activated SRTK then phosphorylates tyrosine
residues within the ITAM, which recruits binding and subsequent
activation of the Syk kinase.6-8 Activated nonreceptor
protein kinases phosphorylate complex adapter proteins that induce
protein-protein interactions leading to translocation, activation, and
regulation of Ras at the cell membrane. The mechanism by which Ras is
controlled in myeloid FcRI signaling is unknown, but is likely to
involve complex adapter proteins.
One adapter protein Shc, is thought to be involved in the activation of
Ras.9-17 It is ubiquitously expressed and occurs in two
isoforms of 46 kD and 52 kD in hematopoetic cells. The Shc protein
contains an amino-terminal phosphotyrosine-binding (PTB) domain, a
central collagen homology (CH) region, and a carboxyl-terminal SH2
domain, but no apparent catalytic domain.18-20 It is a
substrate of multiple tyrosine kinases and can transform cells when
overexpressed.18 Tyrosine-phosphorylated Shc associates
with the SH2 domain of Grb2, an adapter protein composed solely of an
SH2 domain flanked on either side by an SH3 domain and the guanine
nucleotide exchange factor Sos, localizing the molecular complex to the
plasma membrane in which it is believed that activation of Ras
occurs.9,10
Data from a number of investigators suggest a role for Cbl in the
regulation of Ras.21,22 The v-cbl oncogene was
originally described as the transforming gene of the Cas NS-1 murine
retrovirus that induces pre-B lymphomas and myeloid leukemias in
mice.23,24 The c-cbl proto-oncogene product is a
cytoplasmic protein that contains a nuclear localization domain along
with a PTB domain in its amino terminus, a ring finger domain, a
c-terminal proline-rich region, and leucine zipper
domain.25,26
Cbl is ubiquitously expressed in mammalian cells. It contains the above
mentioned domains of interest and exhibits no intrinsic enzymatic
activity.27 It has been shown to be a substrate for tyrosine kinase activity activated by the EGF and colony-stimulating factor (CSF)-1 receptors21,28,29 as well as Fc-, T-, and
B-cell receptors.30-35 Cbl is known to constitutively
interact with SH3 domains of Grb2. Recently, we described a novel
Grb2-associated protein SLP-76 (SH2 domain-containing leukocyte protein
of 76 kD) that is found only in hematopoetic cells.36 This
533 amino acid protein contains several tyrosines in its amino-terminal end, a central proline-rich region that binds the SH3 domain of Grb2,
and a single SH2 domain in the carboxy-terminal region.36 It has been shown to be a tyrosine phosphorylation substrate of ZAP-70
and plays a role in potentiating TCR-mediated induction of the nuclear
factor of activated T cells (NFAT) and interleukin-2 (IL-2) promoter
activity.37-39 Mizuno et al40 have shown an
association between SLP-76 and the phosphatase SHP-1, which may
modulate signaling through the B-cell antigen receptor. SLP-76 has been
shown to be associated with Grb2 and a 120 kD phosphoprotein in
FcRIIA signaling in platelets.41 Hendricks-Taylor et
al42 have reported SLP-76 phosphorylation on Fc RI
stimulation in rat basophilic leukemia cells.
To date there have been no reports showing a role for SLP-76 or Cbl in
FcRI signaling in myeloid cells. Herein, we report the constitutive
association of SLP-76 with Cbl and Grb2 in the myeloid cell line U937.
We also show that the interaction between SLP-76 and Grb2 is further
induced on FcRI stimulation. SLP-76 tyrosine phosphorylation is
associated with binding to the Shc adapter protein and is induced by
FcRI receptor activation. The phosphorylation dependence of some of
these associations is shown by treatment with potato acid phosphatase
(PAP), which results in disruption of SLP-76-Shc and decreases
SLP-76-Grb2, but has no effect on SLP-76-Cbl or Cbl-Grb2 interactions.
From our experiments, we conclude that the Cbl-SLP-76-Grb2 complex is
at least partly distinct from a Grb2 complex containing Sos. The
association of SLP-76, Cbl, and Grb2 along with the inducible binding
of SLP-76 to Shc provides the first evidence that a Cbl-SLP-76-Grb2-Shc complex may function in FcRI signaling in myeloid cells.
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MATERIALS AND METHODS |
Reagents and chemicals.
The FcR-specific antibodies (monoclonal antibody [MoAb] 32.2 is an
F(ab )2 fragment and 197 is whole-mouse IgG) were
generously provided by Medarex, Inc (West Lebanon, NH). The presence of
F(ab)2 fragment only without the contaminating Fc portion
of IgG is certified by Mederex based on immunoelectrophoresis and
high-pressure liquid chromotography (HPLC) analysis. The cross-linking
antibody was a rabbit antimouse F(ab)2 fragment (RM)
purchased from Organon Teknika (Durham, NC). Polyclonal rabbit anti-Cbl
and anti-Sos antibodies were obtained from Santa Cruz Biotechnology,
Inc (Santa Cruz, CA). Anti-SLP-76 antibody produced in sheep has been
described previously.37 Monoclonal antiphosphotyrosine and
anti-Shc antibodies were purchased from UBI (Lake Placid, NY).
Monoclonal anti-Grb2 antibody was obtained from Transduction Lab
(Lexington, KY). Goat antirabbit and antimouse antibodies conjugated to
alkaline phosphatase (AP) were purchased from Southern Biotechnology
Associates, Inc (Birmingham, AL). Rabbit antigoat conjugated to AP was
purchased from Sigma (St Louis, MO). PAP was purchased from Boehringer
Mannheim (Mannheim, Germany).
Cells.
The U937 and THP-1 cell lines were obtained from ATCC (Rockville, MD)
and cultured in RPMI 1640 with 10% fetal bovine serum (FBS). U937IF
cells were prepared by culturing U937 cells in RPMI 1640 with 10% FBS
and 250 U/mL human recombinant -interferon (IFN) for 4 to 5 days
(Genentech Corp, San Francisco, CA). The U937IF cells were maintained
at a concentration of 5 × 105 cells/mL and the medium was
replenished with fresh medium containing IFN every 2 to 3 days.
Stimulation of U937IF cells.
U937IF or THP-1 cells were collected and washed once in cold Hank's
Balanced Salt Solution (HBSS). Monoclonal antibodies against the
FcRI receptor (MoAb 32.2, which is an F(ab)2 fragment,
were used in all immune-precipitation experiments, Fig 1, 2, 3, and 5;
whereas MoAb 197, which is whole mouse IgG, was used in glutathione S-transferase (GST)-fusion protein-binding experiments,
Fig 4, was added to 2 × 107 cells in 500 µL of HBSS and
incubated on ice for 30 minutes. Cells were prewarmed to 37°C for 2 minutes. Secondary RM antibody F(ab)2 fragment was then
added at a concentration of 10 ug/mL and the cells were incubated at
37°C for varying periods of time. The addition of the secondary
antibody at 37°C was considered the start of stimulation. At the end
of stimulation, cells were cooled rapidly by the addition of 800 uL of
cold HBSS. The cells were then centrifuged at 1500g at 4°C
for 5 minutes and the supernatant discarded. Then either
immunoprecipitations or GST fusion protein-binding experiments were
performed as described below.
Immunoprecipitation, electrophoresis, and immunoblotting.
Immunoprecipitations were designed to preserve noncovalent
protein-protein interactions. Cells were lysed with an extraction buffer (EB buffer) and incubated at 0°C for 30 minutes. This EB buffer contained 1% Triton X-100, 10 mmol/L Tris pH 7.6, 50 mmol/L NaCl, 0.1% bovine serum albumin (BSA), 1% aprotinin, 5 mmol/L EDTA,
50 mmol/L NaF, 5 umol/L phenylarsine oxide (PAO) and 100 umol/L sodium ortho-vanadate. For immune precipitates done in the
presence of PAP, 1.8 units of PAP were added to the lysates that were
then warmed to 30°C for 10 minutes before proceeding to the next
step. Cell lysates were then centrifuged at 10,000g for 30 minutes at 4°C to bring down the cellular debris. To precipitate the
Cbl, Sos, or SLP-76 proteins along with their associated proteins, we
added polyclonal rabbit anti-Cbl, rabbit anti-Sos, or goat anti-SLP-76
antisera to these lysates and then incubated them at 0°C for 1 hour
with occassional gentle mixing. Formalin-fixed Staphylococcus
aureus, (30 µL to 50 µL of a 10% solution first washed with EB
buffer) was then added and further incubated for an additional hour at
0°C with occasional gentle mixing. The resultant immune complexes
were washed three times with EB buffer, resolved on 12.5% sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and then
transferred to nitrocellulose for immunoblot analysis. Membranes were
incubated with the specified primary antibody at a dilution of 1:1000
overnight, followed by horseradish peroxidase (1:10,000 dilution), or
alkaline phosphatase (1:5000 dilution) conjugated secondary antibody.
Proteins were visualized using an enhanced chemiluminescence detection
system (ECL, Amersham Corp, Arlington Heights, IL) or alkaline
phosphatase colorimetric development. To reprobe membranes, they were
blocked with 5% nonfat milk for 1 hour and then reblotted
with a different primary antibody.
GST-fusion Protein Experiments.
GST-fusion protein constructs representing amino acids 225-265 and
amino acids 268-416 of SLP-76 were subcloned into Escherichia coli expression plasmids pGEX. The E. coli was grown to an
optical density of 0.5 to 0.6 at 37°C. Synthesis of
GST-fusion proteins was then induced with 0.2 mmol/L isopropyl
 - -thiogalactopyranoside. After a 2-hour induction, bacteria were
harvested and lysed. GST-fusion proteins were affinity purified with
glutathione sepharose beads and then eluted with 20 mmol/L Glutathione
and dialyzed against 50 mmol/L Tris, pH 8.0. Purified proteins were
quantitated by Bradford assay and confirmed by Coomassie
blue staining on SDS/polyacrylamide gels. U937IF lysates with and
without FcRI stimulation by MoAb 197 whole-mouse IgG and RM
F(ab)2 fragment were prepared as described above. The cell
lysates were then mixed with the different GST-fusion proteins (10 µg/mL) and incubated for 1 hour at 0°C with occasional gentle
mixing. Glutathione sepharose beads were then added to the lysates and
incubated at 0°C for an additional hour with occasional gentle
mixing. The mixture was gently washed three times with pulldown-wash
buffer (50 mmol/L Tris; HCL, pH 7.5, 150 mmol/L NaCl; 1 mmol/L EDTA,
0.1% Tween-20, 10 mmol/L NaF, 1% NP-40), resolved on 12.5% SDS-PAGE
and electrotransferred to nitrocellulose paper. Immunoblots were then
performed as described above. The gel was stained with Coomassie blue
to confirm that equivalent amounts of GST and GST-protein were used in
the binding experiments.
| |
RESULTS |
SLP-76 is tyrosine phosphorylated on FcRI stimulation.
Signal transduction events through the ITAM are mediated via
associations between adapter proteins, many of which are
phosphoproteins. Ravichandran et al43 reported that these
interactions result in formation of molecular complexes containing Shc
and Grb2, which serve to localize the guanine nucleotide exchange
factor Sos to the cell membrane in which the activation of Ras likely
occurs. Recent identification of the SLP-76 phosphoprotein associated with Grb2 in TCR signaling prompted our interest in this molecule and
its potential role in FcRI signaling. We examined whether SLP-76 was
tyrosine phosphorylated after FcRI stimulation in U937 cells. U937
(2 × 107) cells differentiated in IFN for 4 to 5 days
were first incubated with MoAb 32.2 (F(ab)2 fragment of
IgG) followed by stimulation with RM F(ab)2 fragment
for 5 minutes or no stimulation. The cells were then lysed and
immunoprecipitated with polyclonal goat anti-SLP-76 antibody and
analyzed by Western Blot. Antiphosphotyrosine immunoblot was performed
(Fig 1A). Marked tyrosine phosphorylation of a 76 kD protein was detected in the U937IF cells stimulated with
anti-FcRI (Fig 1A, lane 5). The 76 kD protein was not phosphorylated in resting cells (Fig 1A, lane 4). To confirm the identity of the 76 kD
phosphoprotein, the membrane was reprobed with anti-SLP-76 antibody.
Both stimulated cells and cells at rest brought down an equivalent
amount of SLP-76 (Fig 1B, middle panel). Two bands of SLP-76 are
consistently observed in our SLP-76 immunoprecipitates. The slower
migrating isoform is the predicted tyrosine phosphorylated species. The
76 kD immunoreactive bands of the SLP-76 immunoblot superimposed on the
76 kD tyrosine phosphorylated bands on the SLP-76 immunoprecipitate.
The lower portion of the antiphosphotyrosine blot in Fig 1A was probed
with anti-Grb2, confirming that SLP-76 is associated with Grb2 in
myeloid cells (Fig 1B, lower panel). The band that appears just above
the Grb2 bands in the Fig 1B lower panel as previously described, is
identified as light chain of IgG.32 The figure also
suggests that there is an inducible component to the Grb2-SLP-76
interaction as Grb2 binding to SLP-76 is slightly increased on FcRI
activation (lanes 4 and 5). This increased binding of SLP-76 and Grb2
after receptor activation becomes more evident in Fig
2B and 3B. This
is in contrast to the Grb2-Cbl interaction in which Grb2 binding to Cbl
remains the same regardless of FcRI receptor stimulation (lanes 2 and 3). The heavy band at 120 kD in Fig 1A, lanes 2 and 3, represent tyrosine phosphorylated Cbl from Cbl immune precipitates, which superimposes on the anti-Cbl immunoblot represented in Fig 1B, upper
panel. The preimmune cells shown in Fig 1A and 1B
represent unstimulated U937IF cells immune precipitated with rabbit
IgG. Preimmune samples were made with stimulated U937IF cells immune precipitated with goat antiserum exhibit identical results (data not
shown). From these data we conclude that SLP-76 and Cbl are involved in
FcRI signaling and we hypothesize that SLP-76-Grb2 interaction may
functionally link FcRI to activation of Ras.
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| Fig 1.
[SLP-76 and Cbl are tyrosine phosphorylated upon FcRI
stimulation.] Immunoprecipitation was performed with anti-Cbl and
anti-SLP-76 antibody from lysates of resting U937IF cells or cells
stimulated by anti-FcRI cross-linking with MoAb 32.2 F(ab)2 fragment. Proteins were resolved by SDS/PAGE,
transferred to nitrocellulose membrane, and immunoblotted. (A)
Antiphosphotyrosine immunoblot. Lane 1 represents precipitation with
preimmune antisera. Lanes 2 and 3 represent anti-Cbl IP of U937IF cells
at rest and after 5-minute stimulation, respectively. Lanes 4 and 5 represent anti-SLP-76 IP of U937IF cells at rest and after 5-minute
stimulation, respectively. Lane 6 represents whole-cell lysate
(1 × 106 cell equivalents) of stimulated U937IF cells.
(B) Same membrane as in Fig 1A was blocked and reprobed. Upper panel
represents anti-Cbl immunoblot. Middle panel represents anti-SLP-76
immunoblot. Lower panel represents anti-Grb2 immunoblot.
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| Fig 2.
[Kinetics of SLP-76-Cbl interaction following
FcRI stimulation.] Immunoprecipitation was performed with
anti-SLP-76 antibody from lysates of resting U937IF cells or cells
stimulated by anti-FcRI cross-linking with MoAb 32.2 F(ab)2 fragment for varying periods of time ranging from
30 seconds to 30 minutes. Proteins were resolved by SDS/PAGE,
transferred to nitrocellulose membrane, and immunoblotted. (A)
Antiphosphotyrosine immunoblot. Lane 1 represents precipitation with
preimmune antisera. Lane 2 represents U937IF cells at rest. Lanes 3-7 represent U937IF cells stimulated for 30 seconds, 1 minutes, 5 minutes,
10 minutes, and 30 minutes, respectively. Lane 8 represents whole-cell
lysate (1 × 106 cell equivalents) of stimulated U937IF
cells. (B) Same membrane as in Fig 2A was blocked and reprobed. Upper
panel represents anti-Cbl immunoblot. Middle panel represents
anti-SLP-76 immunoblot. Lower panel represents anti-Grb2 immunoblot.
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| Fig 3.
[Biochemical characterization of SLP-76-Cbl and
SLP-76-Shc interactions.] Immunoprecipitation was performed with
anti-SLP-76 antibody from lysates of resting U937IF cells or cells
stimulated by anti-FcRI cross-linking with MoAb 32.2 F(ab)2 fragment for varying periods of time ranging from
30 seconds to 30 minutes in the presence or absence of PAP. Proteins
were resolved by SDS/PAGE, transferred to nitrocellulose membrane, and
immunoblotted. (A) Antiphosphotyrosine immunoblot. Lane 1 represents
precipitation with preimmune antisera. Lanes 2-6 represent anti-SLP-76
IP of U937IF cells at rest and after 30 seconds, 2 min, 10 min and 30 min stimulation respectively. Lanes 7-11 represent anti-SLP-76 IP of
U937IF cells at rest and after 30 seconds, 2 minutes, 10 minutes, and
30 minutes stimulation, respectively, to which 1.8 units PAP was added
to the cell lysates. Lane 12 represents whole-cell lysate
(1 × 106 cell equivalents) of stimulated U937IF cells.
(B) Same membrane as in Fig 3A was blocked and reprobed. Upper panel
represents anti-Cbl immunoblot. Second panel represents anti-SLP-76
immunoblot. Third panel represents anti-Shc immunoblot. Lower panel
represents anti-Grb2 immunoblot.
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SLP-76 associates with Cbl in a constitutive manner in myeloid cells.
A 120 kD phosphoprotein was noted to coprecipitate with SLP-76 in
FcRI-stimulated cells (Fig 1A, lane 5). This portion of the membrane
in Fig 1A was reprobed with anti-Cbl antibody. The 120 kD
immunoreactive bands on the Cbl immunoblot superimpose on the 120 kD
bands noted on the antiphosphotyrosine blot, confirming the
identification of these bands as Cbl (Fig 1B, upper panel). Cbl is
present not only in FcRI-stimulated cells but also in U937IF cells
at rest. However, Cbl is a single band at rest and a double band on
receptor stimulation (Fig 1B, upper panel, lanes 4 and 5). Extensive
studies in our lab have established that this pattern of mobility shift
of the Cbl band is due to tyrosine phosphorylation of Cbl. The sum of
the two bands in Fig 1B (upper panel, lane 5) appears to be equivalent
to the single band in Fig 1B (upper panel, lane 4).
SLP-76 immunoprecipitates performed in another myeloid cell line, THP-1
cells (data not shown), showed similar results. Taken together, these
data show that SLP-76 immunoprecipitated from FcRI-activated myeloid
cells is tyrosine phosphorylated and coprecipitates Cbl. SLP-76 is not
detected in Cbl immunoprecipitates (Fig 1A and 1B, lanes 2 and 3). The
anti-Cbl antibody used in our Cbl immunoprecipitates is directed
against the extreme carboxy-terminal region of the molecule and does
not immunoprecipitate SLP-76. The reason for our inability to
coimmunoprecipitate SLP-76 with Cbl is unclear but it is plausible that
this C-terminal region of Cbl is the region of SLP-76-Cbl binding in
vivo. These data provide the first evidence that Cbl-SLP-76 and Grb2
can form a trimolecular signaling complex in vivo.
Kinetics of SLP-76 and Cbl phosphorylation and binding in myeloid
cells.
Kinetic experiments were performed to confirm SLP-76-Cbl interaction in
myeloid cells. In Fig 2, SLP-76 was immunoprecipitated from U937IF cell
lysates as described above except that the cells were stimulated with
anti-FcRI (32.2 MoAb F(ab)2 fragment of IgG) followed
by stimulation with RM F(ab)2 fragment for varying periods of time ranging from 30 seconds to 30 minutes. Western Blot
analysis with antiphosphotyrosine was then performed. Within 30 seconds
of FcRI stimulation, SLP-76 becomes tyrosine phosphorylated. This
phosphorylation reaches a peak at 5 minutes to 10 minutes and then
gradually diminishes, but is still present at 30 minutes (Fig 2A, lanes
3-7, center bands). Similarly, Cbl also becomes rapidly tyrosine
phosphorylated on FcRI stimulation and peaks at 5 minutes to 10 minutes, but then becomes dephosphorylated on tyrosine by 30 minutes
(Fig 2A, lanes 3-7, upper bands). Importantly, the pattern of pp120
tyrosine phosphorylation observed in SLP-76 immunoprecipitates was
identical to the kinetic pattern of Cbl tyrosine phosphorylation
observed in Cbl immunoprecipitates.35 A phosphoprotein of
35 kD is also noted to coimmunoprecipitate with SLP-76 on FcRI
stimulation and follows a similar kinetic pattern of tyrosine
phosphorylation. The identity of this phosphoprotein remains unknown.
The membrane in Fig 2A probed with anti-SLP-76 antibody, confirmed
equivalent amounts of SLP-76 in all lanes except the preimmune (Fig 2B,
middle panel). The preimmune (lane 1) represents unstimulated U937IF
lysate immunoprecipitated with goat antiserum. The upper portion of the
membrane was probed with anti-Cbl antibody. Unstimulated U937IF cells
show a single-banded Cbl (Fig 2B, upper panel, lane 2). The more
rapidly migrating band of Cbl appears stronger in the early stimulation
lanes (Fig 2B, upper panel, lanes 3-4). The slower migrating band
becomes more evident as stimulation progresses (Fig 2B, upper panel,
lanes 5-7). Of note is that at 30 minutes, when the antiphosphotyrosine
band of Cbl is no longer evident, a double band persists on the
anti-Cbl blot (Fig 2A, lane 7 and Fig 2B, upper panel, lane 7). This
may represent a serine/threonine phosphorylated form of Cbl as reported
by Liu et al.44 The relative amount of Cbl in each lane
appears very similar, suggesting a constitutive interaction between
SLP-76 and Cbl but with inducible tyrosine phosphorylation. The lower portion of the membrane in Fig 2A was probed with anti-Grb2 antibody. The no stimulation lane does not contain as much Grb2 as the FcRI stimulated lanes. The quantity of Grb2 associated with SLP-76 reaches a
peak at around 5 to 10 minutes and persists at 30 minutes after
FcRI-receptor crosslinking (Fig 2B, lower panel). This pattern
suggests an inducible component to the interaction between SLP-76 and
Grb2. The kinetics of SLP-76-Grb2 interaction parallels the kinetics of
SLP-76 phosphorylation, which also parallels the kinetics of Cbl
phosphorylation. These kinetic data, along with the results shown in
Fig 1B and 3B, support the formation of a constitutive SLP-76-Grb2
complex in vivo that becomes augmented on FcRI aggregation.
Biochemical analysis of SLP-76-Cbl-Grb2-Shc interactions.
To further characterize the Cbl-SLP-76 and SLP-76-Grb2 interactions, we
determined whether these associations after FcRI-receptor stimulation were phosphorylation dependent. Lysates of U937IF cells at
rest and after varying periods of FcRI-receptor stimulation with
anti-FcRI (32.2 MoAb F(ab)2 fragment of IgG) followed
by stimulation with RM F(ab)2 fragment (from 30 seconds
to 30 minutes) were immunoprecipitated with anti-SLP-76 in the
presence and absence of PAP. PAP is known to dephosphorylate
phosphotyrosine and phosphoserine/phosphothreonine residues. Proteins
were isolated by Western Blot and antiphosphotyrosine immunoblot was
performed as described above. As expected, no tyrosine phosphorylation
was evident in the immunoprecipitates that had been previously PAP
treated (Figure 3A, lanes 7-11). Of note is that the phosphoprotein
bands migrating at 46 kD and 52 kD were also not evident in the
PAP-treated immunoprecipitates, suggesting that these bands associate
with SLP-76 via phosphorylation-dependent interactions. The membrane in
Fig 3A was then reprobed with anti-Cbl, anti-SLP-76, anti-Shc, and
anti-Grb2. The PAP-treated immunoprecipitates continue to show an
association between Cbl and SLP-76, however the slower migrating Cbl
band appears attenuated during the earlier timepoints in the presence
of PAP compared with the non-PAP-containing immunoprecipitates (Fig
3B, upper panel, lanes 2 to 6 v lanes 7-11). Equivalent amounts
of SLP-76 are confirmed by anti-SLP-76 Western Blot (Fig 3B, second
panel, lanes 2-11). The portion of the membrane in Fig 3A corresponding
to pp46 and pp52 was probed with anti-Shc antibody. The immunoreactive
bands at 46 kD and 52 kD superimpose on the 46 kD and 52 kD tyrosine
phosphorylated bands noted in Fig 3A, confirming the presence of Shc in
the SLP-76 immunoprecipitates on FcRI stimulation (Fig 3B, third
panel, lanes 3-6). In parallel experiments in which the membrane in Fig 3B (third panel) was probed with secondary antimouse antibody alone, no
Shc immunoreactive bands were observed, confirming that pp46 and pp52
in Fig 3A represent Shc (data not shown). The interaction between
SLP-76 and Shc is not detected in the PAP-treated immunoprecipitates, suggesting that the interaction is phosphorylation dependent (Fig 3B,
third panel, lanes 8-11). PAP treatment diminishes the interaction between SLP-76 and Grb2 in both stimulated and unstimulated cells (Fig
3B, lower panel, lanes 2 to 6 v lanes 7-11). These data suggest that there is a component of the SLP-76-Grb2 interaction that is
phosphorylation dependent and potentially mediated through the SH2
domain of Grb2. Alternatively, this phosphorylation-dependent component
of the SLP-76-Grb2 interaction could occur via another phosphoprotein.
Motto et al37 have mapped the Grb2-binding site on SLP-76
to amino acids 224-244 in the proline-rich region, which shows that the
association between these two molecules is mediated through the SH3
domain of Grb2. Our PAP data suggest that SLP-76 and Grb2 associate in
a constitutive manner because the interaction is observed in U937IF
cells at rest. However, this interaction becomes augmented on FcRI
receptor stimulation, suggesting an inducible association. The
anti-Grb2 blots of Fig 1B, 2B, and 3B all consistently show this
increased association between SLP-76 and Grb2 on FcRI-receptor
stimulation. This interaction possibly occurs through the Grb2 SH2
domain, likely via the interaction of another phosphoprotein that
recruits more Grb2. Based on the results in Fig 3A and 3B, a possible
candidate would be tyrosine-phosphorylated Shc that is bound to both
Grb2 and Cbl. We conclude from these data that Shc protein is tyrosine
phosphorylated after FcRI activation, which likely induces Shc to
bind to a SLP-76-Cbl-Grb2 complex.
The Grb2-binding domain of SLP-76 binds Shc but not Cbl.
To further elucidate the regions of SLP-76 involved in Cbl and Grb2
binding, pull-down binding experiments were performed. U937IF cell
lysates prepared at rest and after 1-minute stimulation of the FcRI
receptor with MoAb 197 followed by RM F(ab)2 fragment were incubated with SLP-76 GST-fusion proteins representing the Grb2
binding-domain (amino acid residues 225-265) and the proline-rich region that does not associate with Grb2 (amino acid residues 268-416)
as mapped by Motto et al.37 In FcRI-stimulated cells, a
double-banded phosphoprotein of molecular weights 46 kD and 52 kD is
pulled down by the SLP-76 GST-fusion protein representing the
Grb2-binding domain (data not shown). To identify these 46 kD and 52 kD
proteins, this region of the membrane was reprobed with anti-Shc
primary antibody and developed via an AP-colorimetric system as
described above. Both bands immunoreacted with anti-Shc antibody (Fig
4, center panel, lane 5). The data confirm
that Grb2 is pulled down by the SLP-76 GST-fusion protein corresponding to the Grb2-binding domain. This domain is also responsible for binding
Shc in FcRI- as well as FcRII-stimulated cells (Fig 4, lower
panel, lanes 4-7). Cbl does not appear to be part of this potential
trimolecular complex, nor is it associated with the proline-rich region
of SLP-76 that does not bind Grb2 (Fig 4, upper panel, lanes 4-7). The
exact Cbl-binding site on SLP-76 remains to be determined. Preliminary
GST-fusion protein data from our lab suggests that the carboxy terminal
SH2 domain of SLP-76 is not involved in Cbl binding (data not shown).
These data provide evidence that the Grb2-binding region of SLP-76 is responsible for FcRI- and perhaps FcRII-augmented association of
Shc and SLP-76 observed in Fig 3B.
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| Fig 4.
[The Grb2-binding domain of SLP-76 does not bind Cbl.]
U937IF cells at rest and stimulated by anti-FcRI cross-linking with
MoAb 197 were incubated with SLP-76 GST-fusion proteins representing
the Grb2-binding domain (amino acid residues 225-265) and the
proline-rich region that does not associate with Grb2 (amino acid
residues 268-416). Lane 1 represents precipitation with preimmune
antisera. Lanes 2 and 3 represent incubation of GST with U937IF cells
at rest and after 5 minute stimulation, respectively. Lanes 4 and 5 represent incubation of SLP-76 GST-fusion protein aa 225-268 with
U937IF cells at rest and after 5 minute stimulation, respectively.
Lanes 6 and 7 represent incubation of SLP-76 GST-fusion protein aa
268-416 with U937IF cells at rest and after 5 minutes stimulation,
respectively. Lane 8 represents whole-cell lysate
(1 × 106 cell equivalents) of stimulated U937IF cells.
Upper panel represents anti-Cbl immunoblot. Middle panel represents
anti-Shc immunoblot. Lower panel represents anti-Grb2 immunoblot.
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Characterization of SLP-76-Cbl-Grb2 and Grb2-Sos complexes in vivo.
It has been shown that Grb2 binds to the guanine nucleotide exchange
factor Sos, leading to activation of Ras.10,45,46 The
tyrosine phosphorylation of Shc, followed by its interaction with the
Grb2-SH2 domain results in recruitment of Sos to the receptor-signaling
complex.9,47 Previous data from our lab implicated Shc,
Grb2, Raf-1, and MAP Kinase in FcRI signaling in U937IF
cells.32 Herein, we observe a novel association between Cbl, SLP-76, Grb2, and Shc. Because Grb2 and Shc are known to interact
with the Ras guanine nucleotide exchange factor Sos, we set out to
determine whether Sos was also bound to this likely multimolecular
complex. U937IF cell lysates prepared at rest and after a 5 minute
stimulation of the FcRI receptor were subjected to
immunoprecipitations with anti-Sos, anti-Cbl and anti-SLP-76 antibody.
Proteins were isolated by Western Blot and then immunoblotted with
antiphosphotyrosine, anti-Sos, anti-Cbl, anti-SLP-76, anti-Shc and
anti-Grb2. The antiphosphotyrosine immunoblot is virtually identical to
Fig 1A with the exception of the very faint phosphoprotein band
migrating at 52 kD in the FcRI-stimulated lane of the anti-Sos immunoprecipitate (Fig 5A, lane 3).
Anti-Shc immunoblot of this region identifies the 52 kD band as Shc
(data not shown). SLP-76 immunoprecipitates do not contain Sos despite
the preservation of a protein complex containing SLP-76-Cbl-Grb2 and
Grb2-Shc. The reciprocal immunoprecipitates with Sos do not contain
SLP-76 (Fig 5B, first and third panel, lanes 2 and 3 and lanes 6 and 7). Grb2 is present in all immunoprecipitates, both in resting and
stimulated cells (Figure 5B, lower panel, lanes 2-7). However, in the
Sos and SLP-76 immunoprecipitates, more Grb2 is recruited in the
FcRI-receptor stimulated cells (Fig 5B, lower panel, lanes 2 and 3, 6 and 7). This pattern of increased SLP-76-Grb2 binding on
FcRI-receptor stimulation is consistent with that noted in Fig 1, 2, and 3 as described above. These results suggest that a multimolecular
complex consisting of Cbl-SLP-76-Grb2-Shc is distinct from a
trimolecular complex containing Sos-Grb2-Shc. The data provide the
first evidence that the aforementioned quatramolecular complex exists
in nature and stimulates inquiry into the potential roles of these
distinct molecular complexes in FcRI signaling.
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| Fig 5.
Characterization of SLP-76-Grb2 and Grb2-Sos signaling
complexes. Immunoprecipitation was performed with anti-Sos, anti-Cbl,
and anti-SLP-76 antibody from lysates of resting U937IF cells or cells
stimulated by anti-FcRI cross-linking with MoAb 32.2 F(ab)2 fragment. Proteins were resolved by SDS/PAGE,
transferred to nitrocellulose membrane, and immunoblotted. (A)
Antiphosphotyrosine immunoblot. Lane 1 represents precipitation with
preimmune antisera. Lanes 2 and 3 represent anti-Sos IP of U937IF cells
at rest and after 5 minutes stimulation, respectively. Lanes 4 and 5 represent anti-Cbl IP of U937IF cells at rest and after 5 minutes
stimulation, respectively. Lanes 6 and 7 represent anti-SLP-76 IP of
U937IF cells at rest and after 5 minutes stimulation, respectively.
Lane 8 represents whole-cell lysate (1 × 106 cell
equivalents) of stimulated U937IF cells. (B) Same membrane as in Fig 5A
was blocked and reprobed. Upper panel represents anti-Sos immunoblot.
Second panel represents anti-Cbl immunoblot. Third panel represents
anti-SLP-76 immunoblot. Lower panel represents anti-Grb2 immunoblot.
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| |
DISCUSSION |
Complex adapter proteins such as Cbl, Shc, and SLP-76 are substrates
for receptor-coupled tyrosine kinases implicated in ITAM-based signaling events. These proteins contain multiple domains that are
capable of forming complexes with other signaling molecules, especially
phosphoproteins and SH2 containing adapter proteins. This propensity
for phosphorylation and multimolecular complex formation serves to
regulate the proteins to which they bind and ultimately to control
signaling output from aggregated receptors. We set out to examine
specific protein-protein interactions identified as substrates for
protein tyrosine kinases induced by FcRI-receptor stimulation in
myeloid cells.
In this report, we show tyrosine phosphorylation of SLP-76, Cbl, and
Shc on FcRI-receptor stimulation and a novel constitutive interaction between Cbl and SLP-76. Phosphorylation of SLP-76 has been
reported in lymphocytes on TCR and BCR stimulation as well as myeloid
leukemia cells and platelets on FcRI- and FcRIIA-receptor stimulation, respectively.37,40-42 The kinase responsible
for SLP-76 phosphorylation after TCR stimulation is believed to be ZAP-70, a member of the Syk family kinases.48
Interestingly, Cbl also is a substrate for ZAP-70 tyrosine
phosphorylation after TCR stimulation and evidence suggests that the
association of the Cbl PTB domain with ZAP-70/Syk facilitates direct or
indirect regulation of tyrosine kinase function.25,49
Previous data from our lab and others show that the FcRI receptor
also signals through the protein tyrosine kinase Syk.6,50
The kinetics of SLP-76 phosphorylation parallels the kinetics of Cbl
phosphorylation, suggesting a link between these two adapter proteins
in the FcRI-signaling cascade.
Tyrosine phosphorylation serves to generate docking sites for
SH2-containing proteins, thereby linking upstream receptor activation to downstream effector molecules and ultimately to
transcriptional events. One such SH2-containing molecule is Shc, which
we have shown to associate with SLP-76 in a
tyrosine-phosphorylation-dependent manner. Shc is known to associate
with a molecular complex containing Grb2 and Sos, the guanine
nucleotide exchange factor responsible for converting GDP Ras to its
active GTP form.9 Data from our laboratory established that
the FcRI receptor signals through a Shc-Grb2 complex leading to
activation of Raf-1 and MAP Kinase.32,35 In the present
study, we report a novel phosphorylation-dependent association between
SLP-76 and Shc that is induced on FcRI activation. Previous
experiments in other signaling systems (EGF, B- and T-cell receptor)
have documented SLP-76-Grb2 binding but have not observed the
association between SLP-76 and Shc. It is likely that the SLP-76-Shc
interaction is mediated via the binding of the Grb2-SH2 domain to
tyrosine-phosphorylated Shc, because it is eliminated in the presence
of PAP when Shc is dephosphorylated. Further support for this
interaction is evidenced by our GST-fusion protein-binding data (Fig 4)
indicating that the Grb2-binding domain of SLP-76 (amino acid residues
225-265) also associates with Shc under conditions of stimulation.
The interaction between SLP-76, Shc, and Grb2 would suggest that SLP-76
may also be found in a multimolecular complex containing Sos, which
also associates with Shc and Grb2. However, our data indicate that
SLP-76 is not found in such a multimolecular Ras-activating complex.
The association of Grb2 and Shc with SLP-76 and Cbl may serve as a
repository for Grb2 and Shc, regulating their association with Sos and
subsequent activation of Ras. There are several lines of evidence in
support of the involvement of Cbl in the regulation of Ras. First, in
Caenorhabditis elegans, sli-1, a Cbl homolog acts as a negative
regulator of the Ras homolog, Let60, possibly by regulating the
activity of Sem5, a Grb2 homolog.51 Second, Liu et
al22 reported that transient overexpression of a
transforming Cbl mutant was able to increase NFAT activity, showing
that Cbl is involved in Ras-dependent T-cell-signaling pathways
leading to transcriptional activation of IL-2. Third, Cbl
overexpression in conjunction with a Ras-sensitive AP1 reporter
resulted in inhibition of TCR-induced ERK2 activation and a T-cell
activation-induced exchange of Cbl for Sos on Grb2.52 The
above mentioned data from Rellahan et al,52 along with data
from our lab, can be interpreted as evidence that Cbl functions as an
adapter shield or exchanger for Grb2 in regulation of
Grb2-Sos.35 Evidence also exists in TCR signaling
supporting involvement of SLP-76 in the regulation of Ras. A functional
link between SLP-76 and activation of Ras and calcium pathways has been
suggested by Wardenburg et al48 who have shown that
overexpression of wild-type SLP-76 leads to a hyperactive TCR, whereas
expression of a SLP-76 molecule that is unable to be tyrosine
phosphorylated results in attenuated TCR function. Musci et
al53 have shown that SLP-76 functions upstream of ERK,
which is downstream of Ras and does not involve calcium-dependent
pathways. All three of the major SLP-76 domains (amino terminal
phosphotyrosines, central proline-rich Grb2-binding domain, and carboxy
terminal SH2 domain) are required for optimal activation of T cells.
Others have linked SLP-76 to transcriptional activation of the IL-2
gene through association with the SH2 domain of
Vav.37,38,54 Cbl has also been shown to associate with the
SH2 domain of Vav, which also contains a guanine nucleotide exchange
factor (GNEF) domain.55
Our data provides the first evidence for an inducible interaction
between SLP-76 and Shc, occurring only on FcRI-receptor activation
and a constitutive interaction between SLP-76 and Cbl. We postulate
that these complex adapter proteins through their protein-protein
interactions, function as a repository for key molecules involved in
the activation of Ras, thereby serving to regulate Ras. Work is
underway to elucidate the functional significance of these
protein-protein interactions and determine the structural and
functional motifs required for SLP-76-Cbl and SLP-76-Grb2-Shc interactions that contribute to the regulation of Ras in myeloid cells.
| |
FOOTNOTES |
Submitted February 11, 1998;
accepted April 27, 1998.
Supported in part by grants from the National Institutes of Health
(R01CA75637-01 and R01GM53256) and performed in the Neil Bogart
Memorial Laboratories, which are supported by the T.J. Martell
Foundation for Leukemia, Cancer, and AIDS Research. D.L.D. is supported
by the Childrens Hospital Career Development Award, a grant from the
Robert E. and May R. Wright Stop Cancer Foundation, and a
grant from the American Cancer Society (RPG-98-244-01-LBC). G.A.K. is an Established Investigator for the American Heart
Association.
Address correspondence to Donald L. Durden, MD, PhD, Department of
Pediatrics, Division of Hematology-Oncology, Childrens Hospital Los
Angeles, 4650 Sunset Blvd MS#57, Los Angeles, CA 90027; e-mail:
ddurden%smtpgate{at}chlais.usc.edu.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors thank Drs Wade Kyono, Rae Kil Park, and Anat Epstein for
their suggestions and careful review of the manuscript.
| |
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Abstract
Introduction
Abstract