| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
*
Neil Bogart Memorial Laboratories, Division of Hematology-Oncology, Children’s Hospital Los Angeles Research Institute and University of Southern California School of Medicine, Norris Cancer Center, Los Angeles, CA 90027; and
Department of Microbiology and Immunology, Wonkwang University School of Medicine, Iksan, Korea
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
RI receptor signal transduction. We
study the FcRI receptor, an immunoreceptor tyrosine activation motif
(ITAM)-linked signaling pathway, using IFN--differentiated U937
myeloid cells, termed U937IF cells. CBL is constitutively associated
with both GRB2 and the ITAM-containing receptor subunit, FcRI of
FcRI, providing direct evidence that CBL functions in myeloid ITAM
signaling. FcRI cross-linking of U937IF cells induces the tyrosine
phosphorylation of CBL that is associated with an altered CBL-GRB2
interaction. Both GRB2-SH3 and SH2 domains bind CBL in resting cell
lysates; upon FcRI stimulation, phosphorylated CBL binds exclusively
to the GRB2-SH2 domain. Glutathione-S-transferase fusion protein data
demonstrate that the constitutive interaction of CBL with GRB2 and CRKL
is mediated via two discrete regions of the CBL C terminus. The
proximal C terminus (residues 461–670) binds to GRB2 constitutively,
and under conditions of receptor activation binds to the
tyrosine-phosphorylated SHC adapter molecule. The distal C terminus of
CBL (residues 671–906) binds the CRKL adapter protein. The data
demonstrate that the CBL-GRB2 and GRB2-SOS protein complexes are
distinct and mutually exclusive in U937IF cells, supporting a model by
which the CBL-GRB2 and GRB2-SOS complexes function in separate pathways
for myeloid FcRI signaling. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
CBL is tyrosine phosphorylated following the activation of receptors
belonging to the Ig gene superfamily (TCR, B cell receptor, and Fc
receptors). These multisubunit receptors signal through an
immunoreceptor tyrosine activation motif
(ITAM)3
(YXXLX6–8YXXL, consensus) (8, 9, 10, 11). CBL is known to
interact with adapter proteins (e.g., GRB2, CRK, CRKL, NCK) that
regulate the guanine nucleotide exchange factors, "son of
sevenless" (SOS) and C3G in mammalian cells following the activation
of the TCR (12, 13, 14). Marcilla et al. reported that stimulation of
multiple FcR classes (FcRI, FcRII, and FcRIII) in HL-60
cells with IgG/anti-IgG complexes induces the tyrosine
phosphorylation of CBL (9). Stimulation with these immune complexes
results in the activation of FcRI, FcRIIA, and FcRIII
receptors, making it more difficult to interpret these results. Matsuo
et al. and Tanaka et al. subsequently implicated CBL in FcRII/III
signaling in macrophages and THP-1 cells, respectively (14, 15). The
function of CBL tyrosine phosphorylation and/or the interaction of CBL
with adapter proteins as it relates to specific signaling through the
FcRI receptor in myeloid cells and the regulation of RAS have not
been thoroughly studied.
We investigated the role of the CBL adapter protein interaction
following specific cross-linking of the FcRI receptor in myeloid
signaling. CBL is bound to the FcRI subunit of the FcRI
receptor in myeloid cells, providing direct evidence that CBL is
involved in ITAM signaling. Our data demonstrate that CBL binds in
vitro to GRB2 and CRKL molecules via different domains of the CBL C
terminus. CBL is tyrosine phosphorylated after FcRI cross-linking,
and this phosphorylation is associated with an altered CBL-GRB2
interaction. At the same time, the GRB2-SH2 domain inducibly binds to
SHC after FcRI stimulation. These events are associated with the
conversion of GDPras to
GTPras (unpublished observation). Taken
together, our data support a model by which the CBL-GRB2 interaction
may modulate the interaction between GRB2 and SOS in FcRI signaling
in myeloid cells.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The FcRI
-specific cross-linking Abs were generously
provided by Medarex (West Lebanon, NH). The mAb 197 and mAb 32.2 are
specific for the FcRI subunit; mAb 32.2 is a
F(ab')2 fragment of IgG. The cross-linking Ab was a
rabbit anti-mouse F(ab')2 fragment purchased from
Organon Teknika (West Chester, PA). Anti-CBL Ab was purchased from
Santa Cruz Biotechnology (Santa Cruz, CA). Anti-phosphotyrosine,
anti-SHC Abs, and anti-CRKL antisera were purchased from
Upstate Biotechnology (Lake Placid, NY), and the anti-GRB2 mAb
(G16720) was obtained from Transduction Laboratories (Lexington, KY).
GRB2 immunoprecipitations were performed with polyclonal anti-GRB2
(C-231) against residues 195–217 of human GRB2 molecule from Santa
Cruz Biotechnology. The anti- subunit (FcRI) antisera 5927
was prepared in our laboratory, as described (16, 17), and the 4D8
anti- mAb was generously provided by J. Kochan (Hoffman-La
Roche, Nutley, NJ) (18). Preimmune immunoprecipitations were performed
with an equal amount of purified rabbit IgG.
Differentiation and stimulation of U937 cells
U937 cells were maintained in RPMI 1640 with 10% FCS and
differentiated with 250 U/ml human rIFN- (obtained from Genentech,
San Francisco, CA) for 4 days (termed U937IF cells). U937IF cells were
cultured at a concentration of 5 x 105 cells/ml,
and the medium was replenished with fresh IFN- (250 U/ml) every 2
days, as described (16, 19). At the time of performing cross-linking
experiments, the U937IF cells are 48 h from the addition of fresh
IFN-. Flow-cytometric analysis of U937IF cells demonstrated the
expression of the FcRI and FcRII receptors on these cells (data
not shown). For stimulation of FcRI receptors on U937IF cells, cells
were washed twice in cold HBSS and adjusted to a concentration of
4 x 107 cells/ml; 0.5-ml aliquots were incubated on
ice for 30 min with anti-FcRI Abs (0.25 µg/sample).
Cross-linking Abs used in Figure 1
were the 32.2 (F(ab')2
fragment) and 197 (whole IgG) anti-FcRI mAbs. Experiments shown
in Figures 2, 3, 6, and 7 were conducted with the 32.2
(F(ab')2 fragment), and Figures 4 and 5 were performed with
197 Ab cross-linking, as described (19). In both 32.2 and 197
cross-linking experiments, we added 10 µg/ml rabbit anti-mouse
(F(ab')2 fragment) Ab at 37°C for different times.
Stimulated cells were cooled rapidly with cold HBSS and centrifuged at
500 x g for 5 min in a cold centrifuge. The cell
pellet was lysed with 800 µl of Triton X-100 extraction buffer (EB
buffer) on ice for 30 min or resuspended in 25 µl of 1x sample
buffer per 1 x 106 cells for whole cell lysates.
|
|
|
|
|
|
|
Cell lysates were prepared in a lysis buffer (EB) containing 1% Triton X-100, 10 mM Tris, pH 7.6, 50 mM NaCl, 0.1% BSA, 1 mM PMSF, 1% aprotinin, 5 mM EDTA, 50 mM NaF, 0.1% 2-ME, 5 µM phenylarsine oxide, and 100 µM vanadate. Lysates were cleared by centrifugation at 15,000 x g for 45 min at 4°C. For precipitation of specific protein, we added 3 to 10 µl of the appropriate Ab to clarified cell lysates. After incubation on ice for 2 h, 100 µl of a 10% suspension of Formalin-fixed Staphylococcus aureus was added to the immunoprecipitate (IP) and incubated on ice for 1 h. The absorbed immune complexes were washed three times in EB buffer and resuspended with 25 µl of 1x sample buffer. After boiling at 98°C for 5 min, samples were resolved by SDS-PAGE.
Electrophoresis and immunoblotting
Immunoprecipitates were resolved on 10 or 15% acrylamide, 0.193% of bisacrylamide gels by SDS-PAGE. Proteins were transferred to nitrocellulose membranes (1 mAh/cm2) using a dry transfer system (Ellard, Seattle, WA), as described (16). The blot was incubated with blocking solution (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 5% powered milk) for 1 h at room temperature and then incubated with specific anti-phosphotyrosine, anti-CBL, anti-SHC, anti-GRB2, or anti-CRKL for 2 h at room temperature with continuous agitation. After three washes in rinse solution (10 mM Tris-HCl, pH 7.5, and 150 mM NaCl), the membranes were incubated at room temperature for 1 h with secondary Ab conjugated with horseradish peroxidase for enhanced chemoluminescence (ECL; Amersham, Arlington Heights, IL) or conjugated with alkaline phosphatase for colorimetric development. To reprobe the membrane, we stripped membrane with 0.1 M glycine, pH 2.5, at room temperature for 30 min and then reblotted with primary Ab.
GST fusion protein experiments
The C terminus of CBL was defined as the domain containing 11
PXXP motifs, as previously described (1, 2). We prepared GST
fusion constructs representing the entire C terminus of the CBL
molecule (residues 461–906) or the proximal C terminus (residues
461–670) or the distal C terminus (residues 671–906). We subcloned
these cDNAs into the pGEX using a two-staged PCR reaction initially to
clone into the pBS and subsequently into pGEX2T vector for expression
in Escherichia coli. Preparation of GST-GRB2 fusion
constructs were as previously described by Lioubin et al. (20). We
performed DNA sequence analysis to confirm the identity and fidelity of
the N-terminal GST-GRB2-SH3, C-terminal GRB2-SH3 domain, GRB2-SH2
domain, and CBL C-terminal fusion constructs. GST fusion proteins were
affinity purified from cell lysates of E. coli DH5 by
adsorption to glutathione Sepharose beads. Sepharose-bound GST fusion
proteins were washed several times and stored -80°C. Sepharose-bound
fusion proteins were added to lysates (EB lysis conditions) of resting
or FcRI-stimulated U937IF cells, incubated for 1 h at 4 degrees
C. Beads were then washed with EB buffer without vortexing, and bound
proteins were eluted with 2x SDS sample buffer at 95°C, and resolved
by SDS-PAGE. The glutathione Sepharose-bound GST fusion protein (10
µg) was confirmed by Bradford protein assay and by performing
SDS-PAGE on an aliquot of the Sepharose-bound GST fusion proteins
eluted from the beads. Equivalent amounts of each GST fusion protein
were used in each experimental group confirmed by Coomassie blue
staining of the protein gels after transfer of protein.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
RI cross-linking
Our results demonstrate that CBL is tyrosine phosphorylated in
resting U937IF cells (Fig. 1
A,
lane 2; Fig. 2A,
lane 2; and Fig. 5A, lane 2)
and that FcRI cross-linking induces the augmented tyrosine
phosphorylation of CBL (Fig. 1A, lanes 3–6; Fig. 2A, lanes 4–8; and Fig. 5A,
lanes 3 and 4). CBL tyrosine
phosphorylation is rapid following FcRI cross-linking, occurring 1
min after stimulation using two different FcRI-specific mAbs (Fig. 1A, lanes 3 and 5). Other
GRB2-binding proteins (e.g., SLP-76, VAV, and SHC) are not tyrosine
phosphorylated in resting U937IF cells, and become markedly
phosphorylated upon FcRI stimulation (unpublished observation) (21).
The CBL immunoprecipitates contain a prominent 76-kDa
tyrosine-phosphorylated protein noted to associate with CBL under
conditions of FcRI stimulation (Fig. 1A, lanes
4–6). Anti-CBL immunoblots confirm that equivalent amounts
of CBL are immunoprecipitated (Fig. 1B, lanes
2–6; Fig. 2B, lanes 2–8; and Fig. 5B, lanes 2–4) and confirm the marked
mobility shift of CBL following FcRI stimulation. Preimmune Ab does
not immunoprecipitate CBL or the tyrosine-phosphorylated pp76 or pp120
molecules (Fig. 1, A and B, lane
1). Upon FcRI cross-linking, CBL is observed to undergo
a mobility shift on SDS-PAGE (Fig. 1, A and B,
lanes 3–6). The upper CBL isoform appears as a
tyrosine-phosphorylated band only in stimulated cell lysates,
suggesting that the shift is driven by the phosphorylation of the CBL
protein. The kinetics of CBL tyrosine phosphorylation differs with the
two different FcRI-specific mAbs (32.2 and 197) used to cross-link
the receptor (Fig. 1A, compare lanes 3–4 with
5–6). CBL is more extensively tyrosine
phosphorylated 1 min following 197 stimulation as compared with
stimulation with 32.2 cross-linking (Fig. 1A, compare
lane 3 with 5). Our observation that the
197 mAb is a stronger stimulus for CBL tyrosine phosphorylation is
consistent with previous reports from our lab showing that the 197 Ab
is a more potent activator of SYK, SHC, GRB2, RAF-1, and MAP kinase
(21, 22). The effect of 197 Ab could be via a dual binding of 197 to
the Fc binding region of FcRI subunit as well as binding through
F(ab')2 region of 197 to another epitope of FcRI.
This may increase the efficiency of cross-linking, but should remain
relatively FcRI specific, as FcRI is the only Fc receptor with
affinity for binding monomeric IgG (23, 24). The FcRI-induced CBL
mobility shift seen on anti-CBL immunoblots does not differ under
conditions of 32.2 or 197 cross-linking, demonstrating that the altered
mobility of CBL on SDS-PAGE does not correlate with the simultaneous
extent or kinetics of tyrosine phosphorylation (Fig. 1B,
compare lanes 3–6). From these data, we conclude
that the mobility shift in CBL is unrelated to its tyrosine
phosphorylation state in myeloid cells. Lane 7 of Figure 1B represents whole cell lysate of U937IF cells (1 x
106 cell equivalents of protein). The CBL band is not
apparent in lane 7 due to the short exposure time used to
clearly demonstrate the CBL mobility shift in lanes 3 to
6. From these data, we conclude that the mobility shift in
CBL is unrelated to its induced tyrosine phosphorylation state in
myeloid cells.
We then explored in more detail the kinetics of CBL phosphorylation
following FcRI stimulation (Fig. 2A). The tyrosine
phosphorylation of CBL is tightly controlled in U937IF following
FcRI stimulation (Fig. 2A, lanes 4–8).
PMA stimulation of U937IF cells induced a mobility shift in CBL similar
to that induced by FcRI stimulation, in the absence of tyrosine
phosphorylation (Fig. 2, A and B, lane
3). PMA stimulation induced the dephosphorylation of CBL
coincident with a pronounced mobility shift. In contrast, FcRI
stimulation of U937IF cells induces a rapid tyrosine phosphorylation of
CBL (Fig. 2A, lanes 2–8) with complete
tyrosine dephosphorylation observed 15 min after receptor activation
(Fig. 2A, lane 6). The CBL mobility shift
is maximal at 15 min and disappears 30 to 60 min following FcRI
stimulation (Fig. 2B, lanes 6–8).
CBL is bound to FcRI subunit in U937IF cells
Anti-CBL immunoprecipitations performed on U937IF cell lysates
were probed with anti- subunit antisera (Fig. 2C). We detected the presence of the FcRI
subunit, an ITAM-containing receptor subunit, in CBL immunoprecipitates
(Fig. 2C, lanes 2–8). FcRI subunit
protein is detected readily in CBL immunoprecipitates from resting,
PMA-, and FcRI-stimulated cells (Fig. 2C, lanes
2–8) (16, 18, 23, 24). We previously reported that FcRI
stimulation induces a mobility shift on SDS-PAGE in the FcRI
subunit, forming 0 and 1 bands (16).
Phosphoamino acid analysis demonstrated that this
0/1 pattern is due to the FcRI-induced
tyrosine and serine/threonine phosphorylation of the 1
protein (16, 17). In particular, the 1 isoform is
predominantly serine phosphorylated upon FcRI stimulation in U937IF
cells (16). The characteristic 0 and 1
bands of FcRI on SDS-PAGE previously reported to occur after PMA
and FcRI stimulation were clearly observed in the CBL IPs (Fig. 2C, lanes 3 and 4). The pattern
of 0 and 1 observed to
coimmunoprecipitate with CBL in both resting and FcRI-stimulated
U937IF cells (Fig. 2C, lanes 2–7) is
similar to the pattern observed in our anti- subunit IPs
performed on FcRI-stimulated cells, as previously described (16).
Importantly, the detection of the coimmunoprecipitating FcRI
protein shown in Figure 2C, lanes 2 to
8, is blocked completely by preincubation of the
immunoblotting antisera (5927) with a FcRI-specific peptide
(NQETYETLKHEKPPQ) (Fig. 3A,
lane 2), confirming the identity of the
coprecipitating subunit protein (16). The decreased quantity of
FcRI bound to CBL at 60 min after receptor stimulation shown in
Figure 2C is not a consistent finding in all experiments
performed (Fig. 2C, lane 8). We did not
detect CBL or FcRI in preimmune immunoprecipitations performed on
the same lysates (Fig. 2, A and C, lane
1). These IPs showed strong background signal when probed
with goat anti-rabbit secondary (Fig. 1B, lane
1; Fig. 2B, lanes 1 and
9); however, at multiple exposure times using ECL we
did not observe a CBL-specific band in these blots nor did we observe
the coprecipitation of FcRI (Fig. 2C, lane
1).
Similar results were obtained when we performed anti- subunit
immunoprecipitations using the 4D8 anti- mAb and probed these
blots for the CBL protein (Fig. 4A, lanes
1–3). We previously reported that the 4D8 anti-
subunit Ab coimmunoprecipitates SYK and FcRI (24). We used a
series of FcRI-specific peptides to determine the binding
specificity of the 4D8 mAb (Fig. 3, A and
B). We discovered that the 4D8 mAb binds to a defined
peptide within the FcRI protein (Fig. 3, A and
B) (SDGVYTGLSTR) and that this peptide blocks the
immunoprecipitation of by 4D8 and not IP by 5927 antisera (Fig. 3A, compare lane 7 to 3). Using
this information, we designed a separate set of experiments to
determine the specificity of the FcRI-CBL interaction in U937IF
cells (Fig. 4C). Immunoprecipitation performed with
4D8 Ab coimmunoprecipitates CBL and the subunit under
conditions of rest or FcRI stimulation. The addition of the
4D8-specific peptide (SDGVYTGLSTR) to cell lysates completely abrogates
the coimmunoprecipitation of both CBL bands (Fig. 4C,
lane 2) and the subunit (Fig. 3A,
lane 7). Other peptides, lanes 3 to
6, corresponding to regions of FcRI that do not affect
4D8 immunoprecipitation of (Fig. 3A, lane
2) do not affect the coprecipitation of CBL (Fig. 4C, lanes 1 and 4–6). The
peptide KSDGVY(PO4)TGLSTRNQETYETLKHEKPPQ, which is
synthetically phosphorylated on the first tyrosine of the ITAM, does
not block the immunoprecipitation of subunit by the 4D8 Ab and
correspondingly fails to block the coimmunoprecipitation of the CBL
protein (Fig. 4C, lane 5). The reasons for
this result are unclear and under active investigation. The
addition of peptide (NQETYETLKHEKPPQ) in lane 3 was observed
to increase the coprecipitation of CBL and FcRI (Fig. 4C, lane 3). The mechanism for this
augmented CBL- coprecipitation in vitro is unclear. The preimmune
lane in Figure 4C shows high background at the exposure used
to demonstrate both CBL-specific bands in lanes 1 to
6; at earlier exposures we observe no CBL-specific bands in
these preimmune IPs using the ECL system. The reciprocal IP of CBL and
FcRI (Figs. 2 and 4), combined with data from the peptide block
experiments (Figs. 3 and 4), provide convincing evidence that CBL and
FcRI form a complex in vivo in myeloid cells.
The CBL-GRB2 interaction is modulated during FcRI stimulation
Immunoprecipitation of CBL from resting and FcRI-stimulated
cells (Fig. 5, A and
B) confirmed the tyrosine phosphorylation of CBL and
the constitutive CBL-GRB2 association (Fig. 5C). In
this and other experiments, we observe a small decrease in the amount
of GRB2 protein bound to CBL under conditions of FcRI stimulation at
1 and 5 min following receptor cross-linking (Fig. 5C,
compare lanes 2–4; Fig. 6,
lanes 1 and 2). The negative immunoblot in
Figure 5, lane 5, occurs as result of short exposure time
used to see mobility shift in CBL; at longer exposure, the positive
control lysates show distinct CBL bands. We were interested in defining
the modules of GRB2 (SH3 and SH2 domains) that associate with CBL under
conditions of rest vs FcRI stimulation (Fig. 5, A–D). GST fusion protein constructs representing the
N- and C-terminal GRB2-SH3 domains and the GRB2-SH2 domain were used to
characterize the in vitro binding of CBL to GRB2 during FcRI
stimulation of U937IF cells (Fig. 5D). Both the
C-terminal GRB2-SH3 domain and the GRB2-SH2 domain bind CBL in resting
cell lysates (Fig. 5D). Upon FcRI stimulation, the
C-terminal GRB2-SH3 domain no longer binds CBL present in the U937IF
cell lysates. The tyrosine-phosphorylated CBL remained exclusively
bound to the GRB2-SH2 domain in the FcRI-stimulated myeloid cells
(Fig. 5D). In these experiments, the N-terminal
GRB2-SH3 domain was noted to bind a small quantity of the SOS molecule,
but did not bind detectable levels of CBL in resting or stimulated cell
lysates (Fig. 5D). Lane 5 of Figure 5, A and B, represents U937IF cell lysates from
1 x 106 cell equivalents as compared with 20 x
106 cell equivalents loaded per lane in the CBL IP
(lanes 1–4).
CBL-GRB2 and SOS-GRB2 protein complexes are distinct in U937IF cells
Based on the data shown in Figure 5D, we postulate that
CBL tyrosine phosphorylation modulates the CBL-GRB2 interaction in vivo
and that the physical interaction between CBL and GRB2 may regulate the
capacity of GRB2 to bind SOS. We then asked whether the CBL-GRB2
protein-protein complexes are distinct from GRB2-SOS complexes in
myeloid cells (Fig. 6). CBL immunoprecipitates were noted to contain
GRB2, but no detectable SOS (Fig. 6, lanes 1 and
2). In contrast, GRB2 IPs contain SOS and minimal CBL
(Fig. 6, lanes 5 and 6), and SOS IPs
contain GRB2, but no CBL (Fig. 6, lanes 3 and
4). The quantity of GRB2 bound to CBL decreases
following FcRI stimulation (10–15% change) (Fig. 5C,
lanes 3 and 4; Fig. 6, lanes 1 and
2), and CBL IPs contain several-fold greater amounts
of GRB2 as compared with amount of CBL detected in GRB2 IP, suggesting
that most of GRB2 in the cell is not bound to CBL. Although the CBL and
SOS IP brought down similar amounts of GRB2, we were unable to detect
evidence of SOS and CBL in the same protein-protein complex (Fig. 6, compare lanes 1 and 2 with 3 and
4). The lack of detection of CBL
coimmunoprecipitating with the anti-GRB2 antisera (Fig. 6, lanes 5 and 6) could be explained by the
immunoreactivity of the polyclonal anti-GRB2 antisera used to IP
GRB2 (C-231 binds to residues 197–217 in the C terminus of GRB2, the
region mediating the GRB2-CBL interaction shown in Fig. 5D).
Differential binding of CBL C terminus to GRB2 and CRKL adapter molecules
Previous reports suggested that CBL can bind to CRKL and
GRB2 in hemopoietic cells (25). To define the region of CBL-mediating
constitutive binding of these adapter proteins in myeloid cell lysates,
we divided the C terminus of CBL into two proline-rich subdomains and
prepared GST bacterial fusion protein constructs of each. Data from our
GST-CBL pull-down experiments are shown in Figure 7. Our GST fusion constructs consisted of
the entire C terminus of CBL protein (residues 461–906), the proximal
C terminus (residues 461–620, containing a PPVPPR consensus), and
the distal C terminus (residues 621–906, containing 2 YXXPXXP motifs).
These fusion proteins were purified using glutathione Sepharose beads
and used to characterize binding to the GRB2 and CRKL adapter proteins
in U937IF cell lysates prepared from resting or FcRI-stimulated
cells. Equivalent amounts of GST (10 µg) or GST fusion proteins are
incubated with cell lysates prepared from resting or FcRI-stimulated
U937IF cells, followed by adsorption of GST with glutathione Sepharose
beads. Bound proteins are resolved by SDS-PAGE, transferred to
nitrocellulose, and blotted with specific antisera in Western blot
analysis (immunoblot Ab shown on left border of Fig. 7).
Figure 7 shows the immunoblot analysis for CBL, SHC, CRKL, and GRB2 of
proteins that bind to GST vs GST-CBL fusion constructs under conditions
of rest or FcRI stimulation. We demonstrate that the proximal CBL C
terminus, CBL (PC), binds the GRB2 molecule (Fig. 7, lanes 5
and 6), whereas the more distal CBL C terminus, CBL
(DC), selectively binds to CRKL (Fig. 7, lanes 7 and
8). The GST protein alone did not bring down CBL,
SHC, GRB2, or CRKL (Fig. 7, lanes 1 and
2), and CBL (PC) did not bind CRKL; the CBL (DC) did
not bind GRB2. Interestingly, the presence of CRKL is required for the
C terminus of CBL to bring down the endogenous CBL molecule in these
pull-down experiments, suggesting that CRKL may serve as the molecular
bridge between GST-CBL (DC) and endogenous CBL by virtue of its
capacity to bind the CBL (DC) via CRKL-SH3 and endogenous CBL via the
CRKL-SH2 domain. In contrast, the interaction of CBL with SHC only
occurs following FcRI stimulation and is mediated via the GRB2
binding region of CBL (PC) region (Fig. 7, lanes 4 and
6). The SHC binding noted in Figure 7 correlates well
with the kinetics of tyrosine phosphorylation of SHC and the
interaction of tyrosine-phosphorylated SHC with the GRB2-SH2 domain
(21) (data not shown). Lane 9 is a whole cell lysate of
U937IF cells stimulated with FcRI cross-linking used to test the
integrity of the immunoblots.
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
RI signaling pathway (27).
Matsuo et al. reported that cross-linking FcRII receptor in THP-1
cells induces the tyrosine phosphorylation of CBL, and Tanaka et al.
implicated CBL in murine FcRII/FcRIII signaling in macrophages
(14, 15). There are no reports of CBL tyrosine phosphorylation
following FcRI stimulation. In this work, we provide the first
experiments implicating CBL and the CBL-GRB2 interaction in FcRI
signaling. CBL is tyrosine phosphorylated in resting
IFN--differentiated U937 cells and undergoes increased
tyrosine phosphorylation (10-fold increase) following FcRI
stimulation (Figs. 1A, 2A, and
5A). In other experiments, we observe that other
GRB2-binding proteins, SLP-76, VAV, and SHC, are only tyrosine
phosphorylated upon FcRI stimulation (21) (unpublished observation),
suggesting a specific role for the basal level of CBL tyrosine
phosphorylation observed in myeloid cells. This basal level of CBL
phosphorylation is also observed in primary cultures of human bone
marrow-derived macrophages and non-IFN-differentiated THP-1 myeloid
cells (data not shown). We have also performed similar biochemical
experiments in IFN-starved U937 and THP-1 cells and in primary bone
marrow-derived human macrophages with similar results, suggesting that
the signaling events reported in this work are not the immediate
consequence of IFN stimulation. Our data demonstrate that PMA induces a
mobility shift in CBL under conditions in which CBL is dephosphorylated
(Fig. 2, A and B, lane 3). This
mobility shift is reversed 30 to 60 min following FcRI stimulation
(Fig. 2B, lanes 7 and 8). Other
experiments show that potato acid phosphatase treatment of CBL IPs
results in a dephosphorylation of CBL and a concomitant loss of the
mobility shift, and that the omission of the tyrosine phosphatase
inhibitors, phenylarsine oxide (PAO) and vanadate, from the EB lysis
buffer results in tyrosine dephosphorylation of CBL with no alteration
in the CBL mobility shift (unpublished observation). These data are
consistent with the data of Liu et al. demonstrating that
phosphorylation of two serine residues, S619 and S629, in the CBL C
terminus serves as binding site for the 14-3-3
protein in T cells
(26). We conclude that FcRI receptor aggregation results in the
tyrosine phosphorylation of CBL in myeloid cells. Additional analysis
will be required to prove that CBL is a substrate for serine/threonine
kinases activated by FcRI cross-linking.
We sought additional lines of evidence for role for CBL in FcRI
signaling. We surmised that if CBL is directly involved in FcRI
signaling, the FcRI receptor complex would contain significant
amounts of CBL protein. Our data demonstrate that
tyrosine-phosphorylated CBL is constitutively bound to FcRI
subunit in resting and stimulated U937IF cells (Fig. 2, A
and C, lanes 2–8). The FcRI-CBL
interaction is confirmed using anti- and anti-CBL
immunoprecipitations and peptides to block the immunoprecipitation of
(4D8 Ab) (Figs. 3 and 4) and the detection of by 5927 antisera
in anti-CBL IPs (
Figs. 2–4). We used a series of
-chain-specific peptides to determine the binding site for the
anti-FcRI mAb (4D8) originally described by Schoeneich et al.
(Fig. 3) (18), and then used the 4D8-specific peptide to confirm the
specificity of the -CBL coprecipitation in U937IF cells (Figs. 3 and 4). In several systems, the ITAM-containing receptor subunit binds
constitutively to the nonreceptor protein tyrosine kinase SYK (29, 30, 31),
suggesting that the constitutive CBL- subunit interaction may be
mediated by a multimeric protein complex containing the FcRI
subunit, SYK and CBL. The constitutive association of CBL with
FcRI in IFN-primed U937 cells is consistent with the data from
our laboratory showing that SYK binds to the FcRI subunit in a
constitutive manner (22). Constitutive binding of SYK to the ITAM motif
occurs in both platelets and B cells (31, 32). The data of Iwashima et
al. demonstrate that the tyrosine phosphorylation of SYK or ZAP-70 is
not required for their association with ITAM receptor subunits (30).
The aggregation of the FcRI receptor complex activates SYK kinase
activity, which may result in the tyrosine phosphorylation of CBL (22, 33). Lupher et al. reported that CBL contains a PTB motif capable of an
inducible binding to the tyrosine-phosphorylated ZAP-70 kinase in
activated T cells (34). These data along with the report of Fournel et
al., demonstrating that ZAP-70 and SYK can bind to CBL and that CBL is
a substrate for SYK in COS cells, support a potential interaction
between SYK and CBL in FcRI signaling (35). Ota and Samelson
reported that CBL interacts with the SYK kinase, alters SYK-ITAM
signaling, and regulates ITAM function in myeloid cells (27). From
these combined data, we conclude that CBL is associated with FcRI
subunit and that the CBL-GRB2 interaction functions in FcRI
signaling in myeloid cells. Our preliminary experiment and the results
of Ota et al. (27) suggest that the CBL-FcRI interaction is not
direct and that an indirect FcRI-SYK-CBL interaction exists in
myeloid FcRI signaling.
The GRB2-SOS interaction is a critical event in the activation of RAS
in many cell types (36, 37, 38). Previous studies from our laboratory
implicated SHC, GRB2, RAF-1, and MAP kinase in FcRI signaling,
suggesting a role for RAS in this signaling pathway (21). In
hemopoietic cells, four major GRB2-binding proteins undergo rapid
tyrosine phosphorylation upon ITAM stimulation: 1) CBL, 2) SLP-76, 3)
LNK, and 4) VAV (33, 39, 40, 41, 42). The role of these complex adapter
proteins in ITAM signaling and the molecular consequences of their
tyrosine phosphorylation and binding to GRB2 or other adapter proteins
(CRK, CRKL, NCK, SHC) remain to be determined. Buday et al. reported
upon T cell activation, CBL rapidly dissociates from GRB2 and binds to
CRKL (25). This work demonstrated that the capacity of CBL to bind to
the N- and C-terminal GRB2 SH3 domains in vitro is strongly reduced in
activated T cells. We performed experiments with GST fusion proteins
representing different modular domains of the GRB2 molecule to test the
hypothesis that phosphorylation of CBL could alter its interaction with
GRB2 following FcRI stimulation in myeloid cells (Fig. 5D). We observed at a very early time point following
FcRI stimulation, a qualitative change in the interaction between
the domains of the GRB2 molecule and CBL in vitro (Fig. 5D). In U937IF cells, CBL is constitutively bound to
GRB2 (Fig. 5C). Our in vitro data demonstrate that
this interaction is mediated via the combined GRB2-SH3 and GRB2-SH2
domains (Fig. 5D). The GST-GRB2 fusion constructs
used in these experiments bind to a free pool of CBL not already
complexed to GRB2 in the U937IF lysates. Importantly, this pool of
cellular CBL that binds to GRB2 in our in vitro experiments would not
be the same species of CBL bound to the GRB2-SH3 and SH2 domains in
vivo. Our results therefore reflect an altered potential for
interaction between CBL and GRB2 under conditions in which free CBL in
the lysate becomes tyrosine phosphorylated. Tyrosine phosphorylation of
CBL is associated with the decrease in GRB2-SH3-CBL interaction,
leaving CBL bound to the GRB2-SH2 domain (Fig. 5D).
Tyrosine-phosphorylated CBL could be conformationally altered such that
it will not bind to the GRB2-SH3 domain. Alternatively, CBL tyrosine
phosphorylation may result in the binding of CBL to another molecule,
thereby preventing the CBL-GRB2-SH3 interaction. The data reported by
Buday et al. in T cells demonstrate that upon TCR activation, CBL is
tyrosine phosphorylated and the GRB2-SH3-CBL interaction is reduced
dramatically at the same time that the CBL-CRKL complex is augmented
(25). In our experiments, the CBL-GRB2 interaction is qualitatively
altered (Fig. 5D) with mild reduction in the total
quantity of CBL-GRB2 binding (Fig. 5C, lane 3;
Fig. 6, lanes 1 and 2).
Other laboratories have observed the binding of CBL to GRB2-SH2 domain,
but the mechanism and importance of this binding are not understood
(5). Experiments performed in C. elegans support a model by
which the sli-1 homologue of CBL modulates signaling events
through its direct interaction with the GRB2 molecule (3). The SLI-1
protein contains a conserved consensus GRB2-SH2 binding motif (YXNX)
and directly binds the GRB2 homologue in C. elegans. In
contrast, mammalian CBL does not contain a direct binding site for the
GRB2-SH2 domain and most likely interacts via another phosphoprotein in
our system. The CBL C terminus is composed of a proline-rich region
containing 11 PXXP motifs that bind to SH3 domain-containing proteins.
Our GST fusion protein data demonstrate that the constitutive
interaction of CBL with GRB2 and CRKL is mediated via two discrete
regions of the CBL C terminus. The proximal C terminus (residues
461–670) binds to GRB2 constitutively, and under conditions of
receptor activation binds to the tyrosine-phosphorylated SHC adapter
molecule (Fig. 7, lanes 5 and 6). The
distal C terminus of CBL (residues 671–906) binds the CRKL adapter
protein and endogenous CBL (Fig. 7, lanes 7 and
8). CBL contains 22 potential tyrosine
phosphorylation sites, 14 in the N terminus and 8 within the C terminus
(1, 43). Based on our data and the data of Buday et al. and Meisner et
al., we suggest that CBL may serve an exchange function as an adapter
shield in resting cells, modulating the onloading of SOS to GRB2 (Fig. 8) (5, 25). Cell fractionation
experiments from our laboratory support this hypothesis in that we
observe a 6- to 10-fold recruitment of GRB2 and SOS to form a complex
in membrane fractions prepared under conditions of FcRI stimulation
(unpublished results). Similar data suggest that 90 to 100% of GRB2
complexed with CBL is present within these membrane fractions. The
existence of discrete complexes containing either CBL-GRB2 or GRB2-SOS
(Fig. 6), as also observed by Meisner et al. and Donovan et al., in T
cells (5, 10) is consistent with an exchange function for the CBL-GRB2
complex in the regulation of RAS in myeloid cells. The decrease in
GRB2-SH3-CBL interaction could promote the binding of GRB2-SH3 to SOS
within the same signaling complex. To date, an exchange of SOS for CBL
in a receptor complex containing GRB2 has not been demonstrated. Our
results (Fig. 6) and other data from our lab, including CBL and SOS
immunodepletion experiments, do not support the existence of a
trimolecular protein complex containing CBL, GRB2, and SOS in myeloid
cells. It is of course possible that different lysis buffer conditions
will support the detection of trimolecular complex between CBL, GRB2,
and SOS in U937IF cells. It is also plausible that the CBL-GRB2
interaction could function in a parallel pathway for the activation of
RAS via a nucleotide exchange factor other than SOS, or that the
CBL-GRB2-SOS trimolecular complex is unstable and difficult to detect
in FcRI signaling. These possibilities are currently under
exploration in our laboratory to understand the role of CBL and
CBL-GRB2 interaction in the regulation of RAS in myeloid cells.
|
|
Acknowledgments |
|---|
RI and FcRI subunits, respectively. We thank Dr.
Anat Epstein for careful reading of the manuscript before
submission. |
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Donald L. Durden, Department of Pediatrics, Division of Hematology-Oncology (M/S #57), Children’s Hospital Los Angeles, 4650 Sunset Blvd., Los Angeles, CA 90027. E-mail address:
3 Abbreviations used in this paper: ITAM, immunoreceptor tyrosine activation motif; ECL, enhanced chemiluminescence; GST, glutathione-S-transferase; IP, immunoprecipitate; MAP, mitogen-activated protein; SOS, son of sevenless.
Received for publication September 2, 1997. Accepted for publication January 21, 1998.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
receptors. J. Biol. Chem. 270:9115. receptor signaling. FEBS Lett. 382:11.[Medline]
-subunit after activation of the high-affinity Fc receptor for immunoglobulin G. Biochem. J. 299:569.
RI receptor. Exp. Cell Res. 211:150.[Medline]
RI subunit with novel cell surface polypeptides. J. Immunol. 148:2181.[Abstract]
RI receptor signals through the activation of HCK and MAP kinase. J. Immunol. 154:4039.[Abstract]
RI signal relay. J. Biol. Chem. 271:13342.RI receptor signaling. Blood 84:2102.- dimers of Fc receptors as connectors to signal transduction. Curr. Opin. Immunol. 4:43.[Medline]
receptor on human platelets through an immunoreceptor tyrosine-based activation motif. J. Biol. Chem. 271:10775. receptor I (FcRI) and receptor II (FcRII) on monocytic cells activates a signal transduction pathway common to both Fc receptors that involves the stimulation of p72 Syk protein tyrosine kinase. J. Biol. Chem. 268:24442.This article has been cited by other articles:
|
|
 |
|
  A. M. Kant, P. De, X. Peng, T. Yi, D. J. Rawlings, J. S. Kim, and D. L. Durden SHP-1 regulates Fcgamma receptor-mediated phagocytosis and the activation of RAC Blood, August 13, 2002; 100(5): 1852 - 1859. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. S. Kim, X. Peng, P. K. De, R. L. Geahlen, and D. L. Durden PTEN controls immunoreceptor (immunoreceptor tyrosine-based activation motif) signaling and the activation of Rac Blood, January 15, 2002; 99(2): 694 - 697. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R.-K. Park, A. Erdreich-Epstein, M. Liu, K. D. Izadi, and D. L. Durden High Affinity IgG Receptor Activation of Src Family Kinases Is Required for Modulation of the Shc-Grb2-Sos Complex and the Downstream Activation of the Nicotinamide Adenine Dinucleotide Phosphate (Reduced) Oxidase J. Immunol., December 1, 1999; 163(11): 6023 - 6034. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Sato, M.-K. Kim, and A. D. Schreiber Enhancement of Fc{gamma} Receptor-Mediated Phagocytosis by Transforming Mutants of Cbl J. Immunol., December 1, 1999; 163(11): 6123 - 6131. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R.-K. Park, K. D. Izadi, Y. M. Deo, and D. L. Durden Role of Src in the Modulation of Multiple Adaptor Proteins in Fcalpha RI Oxidant Signaling Blood, September 15, 1999; 94(6): 2112 - 2120. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. T. Kyono, R. de Jong, R. Kil Park, Y. Liu, N. Heisterkamp, J. Groffen, and D. L. Durden Differential Interaction of Crkl with Cbl or C3G, Hef-1, and {gamma} Subunit Immunoreceptor Tyrosine-Based Activation Motif in Signaling of Myeloid High Affinity Fc Receptor for IgG (Fc{gamma}RI) J. Immunol., November 15, 1998; 161(10): 5555 - 5563. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. A. Bonilla, R. M. Fujita, V. I. Pivniouk, A. C. Chan, and R. S. Geha Adapter proteins SLP-76 and BLNK both are expressed by murine macrophages and are linked to signaling via Fcgamma receptors I and II/III PNAS, February 15, 2000; 97(4): 1725 - 1730. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
