From the
Antigenic cross-linking of the high affinity IgE receptor
(Fc
Antigenic cross-linking of the high affinity immunoglobulin E
(IgE) receptor (Fc
Members of the TAM-based receptor family are typically coupled to
two subfamilies of PTKs. The Fc
A second, essential,
PTK-controlled signaling pathway to originate from the TCR involves the
guanine nucleotide-binding proteins p21ras
(23) . The mechanism
of TCR coupling to p21ras was proposed to involve the guanine
nucleotide exchange protein Sos, the homologue of the Drosophila ``son of sevenless'' gene product
(24, 25) . Sos is known to complex with the adapter
protein Grb2/Sem 5 which is composed of one SH2 domain and two SH3
domains
(26, 27) . In many cell systems, the SH3 domains
of Grb2 bind to Sos whereas the interaction between the Grb2 SH2 domain
and tyrosine-phosphorylated molecules such as the epidermal growth
factor (EGF) receptor or Shc may regulate the function and cell
localization of Sos
(27, 28, 29, 30) .
Studies in T lymphocytes have identifed a number of
tyrosine-phosphorylated proteins which can potentially bind to Grb2.
These include Shc, a membrane-localized tyrosine phosphoprotein of
36-kDa and 75- and 116-kDa molecules
(25, 31, 32, 33, 34) . The 75-
and 116-kDa molecules are constitutively associated with the SH3
domains of Grb2 analogous to the Grb2/Sos association and are
substrates for TCR-activated PTKs
(32, 33) . The p75 is
apparently a hematopoietic cell-specific protein. Its structure and
protein sequence are not known, but on the basis of its association
with the Grb2 SH3 domains, p75 is a candidate for a second Grb2
downstream effector molecule. Shc and p36 when tyrosine phosphorylated
bind to the SH2 domain of Grb2. In TCR-activated T cells, both Shc and
p36 are tyrosine phosphorylated and could potentially form a complex
with Grb2 SH2 domains. However, the predominant complexes that can be
detected are composed of p36-Grb2-Sos
(25, 35) . Studies
in B cells have generated evidence for regulation of Grb2 complexes by
the BCR. In this system both Shc tyrosine phosphorylation and the
formation of Shc-Grb2-Sos complexes are observed in response to BCR
ligation
(36, 37) The pattern of PTK activation in
response to BCR or Fc
The data presented here show that the
SH3 domain of Grb2 binds Sos and 75-, 120-, and 140-kDa tyrosine
phosphoproteins whereas the major Grb2 SH2 domain-binding proteins are
Shc and an unknown 33-kDa protein. Two of the Grb2-binding proteins
described herein are tyrosine phosphorylated in response to Fc
The resolved proteins were transferred to a polyvinylidene
difluoride (PVDF) membrane (Millipore) which was blocked in 5% non-fat
milk for 1 h. The membrane was probed with a specific antibody followed
by an appropriate second stage horseradish peroxidase-conjugated
anti-rabbit or anti-mouse IgG (Amersham). Reactive bands were detected
using the enhanced chemiluminescence system (Amersham).
The experiment in Fig. 2explores also the SH
domain specificity of Fc
In many cells the adapter molecule Shc is tyrosine phosphorylated in
response to receptor stimulation and forms a complex with Grb2 SH2
domains. Western blot analysis with Shc antisera has shown that RBL2H3
cells express two isoforms of Shc. The 55-kDa isoform is predominant
and the 46-kDa Shc protein is a minor component of the Shc population
in these cells. The data in Fig. 4 B show that a tyrosine
phosphoprotein of 55 kDa is coprecipitated with endogenous Grb2 by the
GST-Sos fusion protein from both quiescent and Fc
To further
analyze the tyrosine phosphorylation of Shc in RBL2H3 cells, we used a
tyrosine phosphopeptide Trk-Y490, which corresponds to a high affinity
binding site for Shc SH2 domains in the Trk subunit of the nerve growth
factor receptor. This was used as a reagent to affinity purify Shc from
RBL2H3 cells. The data in Fig. 5 A show an anti-Shc Western
blot of proteins affinity purified with the Trk-Y490 peptide from
RBL2H3 cells. The 55- and 46-kDa Shc isoforms bind to the Trk-Y490
peptide but not to the EGFR-Y1068 peptide used here as a specificity
control. The data in Fig. 5 B show an anti-phosphotyrosine
Western blot of Trk-Y490 peptide precipitates isolated from quiescent
and Fc
The data presented here show that in RBL2H3 mast cells, as in
many cell systems, the guanine nucleotide exchange factor for p21ras,
Sos, is complexed to the SH3 domains of Grb2. The data show also that
there are Fc
It has been
described in many cell systems that tyrosine-phosphorylated Shc forms a
complex with Grb2
(28, 39) . In the present study, Shc
was observed to be tyrosine phosphorylated in quiescent RBL2H3 cells,
and no increases in Shc tyrosine phosphorylation were detected in
response to Fc
In
the absence of inducible tyrosine phosphorylation of Shc, a possible
candidate for linking signals transduced by the Fc
One
well-documented role of Grb2 is to couple tyrosine kinases to the
guanine nucleotide exchange protein Sos and hence to p21ras signaling
pathways
(45) . We have observed that triggering of the
Fc
The role of the Fc
We thank Dr. Narin Osman and Dr. Larry Samelson for
valuable advice and discussions and Nicola O'Reilly and Elisabeth
Li for peptide syntheses.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
R1) on mast cells results in protein tyrosine kinase
activation. The object of the present study was to explore the
regulation of the SH2 and SH3 domain containing adapter molecule Grb2
by Fc
R1-stimulated PTK signal transduction pathways. Affinity
purification of in vivo Grb2 complexes together with in
vitro experiments with Grb2 glutathione S-transferase
fusion proteins were used to analyze Grb2 complexes in the mast cell
line RBL2H3. The data show that in RBL2H3 cells several different
proteins are complexed to the SH3 domains of Grb2. These include the
p21ras guanine nucleotide exchange factor Sos, two basally
tyrosine-phosphorylated 110- and 120-kDa molecules, and a 75-kDa
protein that is a substrate for Fc
R1-activated PTKs. By analogy
with Sos, p75, p110 and p120 are candidates for Grb2 effector proteins
which suggests that Grb2 may be a pleiotropic adapter. Two Grb2
SH2-binding proteins were also characterized in RBL2H3 cells; the
adapter Shc and a 33-kDa molecule. Shc is constitutively tyrosine
phosphorylated in unstimulated cells and Fc
R1 ligation induces no
changes in its phosphorylation or binding to Grb2. In contrast, p33 is
a substrate for Fc
R1-activated PTKs and binds to Grb2 SH2 domains
in Fc
R1 activated but not quiescent cells. The
subunit of
the Fc
R1 is a 33-kDa tyrosine phosphoprotein, but the p33
Grb2-binding protein described in the present report is not the
Fc
R1
chain and its identity is unknown. The present report
thus demonstrates that there are multiple Grb2 containing protein
complexes in mast cells of which a subset are Fc
R1-regulated. Two
other of the Grb2-binding proteins described herein are tyrosine
phosphorylated in response to Fc
R1 ligation: the 75-kDa protein
which binds to Grb2 SH3 domains and the 33-kDa protein that associates
with the Grb2 SH2 domain. We propose that protein complex formation by
Grb2 is an important consequence of Fc
R1 cross-linking and that
this may be a signal transduction pathway which acts synergistically
with calcium/PKC signals to bring about optimal mast cell end function.
R1) on mast cells and basophils results in
expression of the production of cytokines and exocytotic secre-tion of
allergic mediators
(1) . The stimulation of PTKs
(
)
by the activated Fc
R1 is an immediate membrane
proximal event crucial for Fc
R1 signal transduction and hence mast
cell end function
(2, 3, 4) . The Fc
R1
complex is composed of three subunits, the 45-kDa
and 30-kDa
chains and a homodimer of two disulfide-linked 10-kDa
chains
(5) . The intracellular tails of the
and
subunits contain a common motif, termed the tyrosine-based activation
motif (TAM) of the general sequence
EX
YX
L/IX
YX
L/I,
(6) which is thought to couple the Fc
R1 to intracellular
PTKs. TAMs are found also in the invariant subunits of the B cell and T
cell antigen receptors (BCR and TCR) and thus appear to be an
evolutionarily conserved sequence motif essential for the activation of
lymphocytes
(7, 8, 9, 10, 11) .
R1 associates with the src-family
tyrosine kinase p56lyn and a 72-kDa PTK, Syk
(3, 12) .
The BCR is similarily associated with p56lyn
(13) and Syk
whereas the TCR is predominantly associated with the src-family p59fyn
and a kinase homologous to Syk, ZAP70
(14, 15) . It has
also been reported that Fc
R1 cross-linking induces tyrosine
phosphorylation and activation of the atypical src-like Bruton tyrosine
kinase
(16) suggesting that a third category of PTKs may
contribute to Fc
R1 signaling. One common response to triggering of
the Fc
R1, TCR, and BCR is tyrosine phosphorylation and activation
of phospholipase C-
1
(2, 17, 18) . This
permits these receptors to control inositol polyphosphate and
diacylglycerol production which in turn modulate intracellular calcium
concentration and the activation of PKC, respectively. Calcium flux and
PKC play a critical role in TCR signal transduction
(19, 20) and are important signals for mast cell activation and the
secretion of granule components such as histamine and
5-hydroxytryptamine
(21, 22) .
R1 triggering is similar, particularly in the
stimulation of the 72-kDa PTK Syk
(12) . However, there has been
no analysis of the regulation of adapter molecules such as Grb2 and Shc
in Fc
R1-stimulated cells. Accordingly, the object of the present
studies was to examine whether Grb2 and associated molecules are
regulated by the Fc
R1.
R1
ligation: the 75-kDa protein which binds to Grb2 SH3 domains and the
33-kDa protein that associates with the Grb2 SH2 domain. Shc is
apparently not a substrate for Fc
R1-activated PTKs and hence its
association with Grb2 is not controlled by cell activation. The present
report thus demonstrates that there are multiple Grb2-containing
protein complexes in mast cells of which a subset are
Fc
R1-regulated.
Antibodies
The anti-phosphotyrosine monoclonal
antibody 4G10 was purchased from Upstate Biotechnology Inc (Lake
Placid, NY). The anti-phosphotyrosine antibody FB2 (W. Fantl,
University of California) was purified from hybridoma supernatant.
Polyclonal anti-Shc antibody was purchased from TCS Biologicals.
Monoclonal anti-Dinitrophenyl (mouse IgE isotype) was purchased from
Sigma. Anti-mouse Sos1 and anti-Grb2 were purchased from Upstate
Biotechnology Inc. and Affiniti (Nottingham, United Kingdom),
respectively. The monoclonal antibody JRK1, with specificity for the
chain of Fc
R1
(38) , was a generous gift of Dr. Juan
Rivera (NIAMS, Bethesda, MD).
Peptide Reagents and Fusion Proteins
The
EGFR-Y1068 peptide sequence is PVPEpYINQS. The Trk-Y490 peptide
sequence is IENPQpYFSDA. For use in affinity purification these
peptides were coupled to Affi-Gel 10 beads (Bio-Rad). The following
glutathione S-transferase (GST) fusions were used and have
been previously described
(28, 32) . GST-alone, GST-Grb2
(full-length Grb2), GST-µSH3 49L/203R double SH3 mutant,
GST-Grb2NSH3 isolated amino-terminal SH3 domain (amino acids
1-58), GST-Grb2 CSH3 isolated carboxyl-terminal SH3 domain (amino
acids 159-217) and GST-Sos (mSos1 carboxyl-terminal residues
1135-1336). The fusion proteins were coupled to
glutathione-agarose beads (Sigma) for affinity purification
experiments.
Cell Culture
The rat basophilic leukemia cell line
RBL2H3 was a gift of Dr. Roberto Solari, Glaxo Research and
Development, U.K. They were maintained in RPMI 1640 medium supplemented
with 10% (v/v) heat-inactivated (56 °C for 30 min) fetal bovine
serum and 2 m
M glutamine. Only the adherent fraction of the
cell cultures was passaged or used in experiments.
Cell Stimulation and Lysis
RBL2H3 monolayer cells
were detached from the culture flask using a cell scraper, washed once,
and primed in suspension with 1 µg/ml IgE anti-dinitrophenol (IgE
anti-DNP) in RPMI 1640, 10% fetal bovine serum for 1 h at 37 °C.
Receptor cross-linking was effected using 10 µg/ml keyhole limpet
hemocyanin (KLH)-DNP conjugate (Calbiochem) at 37 °C. After
stimulation all cells were placed on ice and pelleted immediately in a
microfuge. The cells were lysed in a buffer containing 50 m
M HEPES (pH 7.4), 1% (w/v) Nonidet P-40, 150 m
M NaCl, 20
m
M NaF, 20 m
M iodoacetamide, 1 m
M phenylmethylsulfonyl fluoride, 1 µg/ml protease inhibitors
(leupeptin, pepstatin A, and chymotrypsin), and 1 m
M NaVO
.
Affinity Purifications and Western
Blotting
Lysates (1.5 10
RBL2H3) were
precleared with Protein A-insoluble suspension (Sigma) for 15 min at 4
°C. Lysates for fusion protein affinity purifications were then
precleared with glutathione-agarose bead suspension (Sigma). Lysates
for peptide precipitations (Trk-Y490 and EGFR-Y1068) were cleared with
Affi-Gel 10 (Pharmacia). Affinity purifications with specific reagents
were carried out for 2 h at 4 °C with constant rotation. The beads
were washed three times in 1 ml of lysis buffer and boiled in reducing
SDS sample buffer for 10 min. Samples were resolved on 11% SDS-PAGE.
Tyrosine Phosphorylation of Multiple Grb2 Binding
Proteins Is an Immediate Consequence of Fc
The data in Fig. 1 show a Western blot analysis
with anti-phosphotyrosine antibodies of total cell lysates from
quiescent and FcR1
Cross-linking
R1-stimulated RBL2H3 cells. The experiment
demonstrates that there is prominent Fc
R1-induced tyrosine
phosphorylation of proteins at 80, 70, and 55 kDa. This phosphorylation
is rapid, detected within 2 min and sustained for a period of 30 min.
In control experiments no increase in tyrosine phosphorylation of any
proteins in RBL2H3 cells which have been incubated with monomeric IgE
but not exposed to cross-linking multivalent antigen was observed.
Binding experiments with a GST-fusion protein of wild type Grb2 were
carried out in order to establish whether Fc
R1-induced tyrosine
phosphoproteins include a subset of Grb2-binding proteins. The data
from this experiment are shown in Fig. 2. In precipitates purified from
RBL2H3 cell lysates with GST-Grb2, there are multiple tyrosine
phosphoproteins in both quiescent and activated cells. Many proteins
are observed which are basally tyrosine phosphorylated in unstimulated
cells. However, there is an Fc
R1-induced increase in tyrosine
phosphorylation of proteins migrating at 120, 110, 75, 65, and
30-33 kDa.
R1-induced tyrosine-phosphorylated
Grb2-binding proteins. Affinity binding experiments were performed with
truncated or mutated Grb2 fusion proteins comprising the isolated Grb2
NH
-terminal and COOH-terminal SH3 domains, called
GST-Grb2NSH3 and GST-Grb2CSH3, respectively. In addition, GST-Grb2 with
mutations in both SH3 domains which leave the SH2 domain intact
(GST-Grb2 µSH3) was used. Fig. 2shows a Western blot
analysis with anti-phosphotyrosine antibodies on proteins affinity
purified from RBL2H3 lysates using the panel of GST-Grb2 variants in
comparison with wild-type GST-Grb2. The data show that a prominent
Fc
R1-induced tyrosine phosphoprotein of 75 kDa was detected in the
precipitates isolated with wild type GST-Grb2, GST-Grb2NSH3, or
GST-Grb2CSH3 fusion proteins. Both GST-Grb2 SH3 domain fusion proteins
also purified tyrosine-phosphorylated proteins of 38, 52-55, 120,
and 140 kDa. Cross-linking of the Fc
R1 induced tyrosine
phosphorylation of 33- and 65-kDa proteins which bound to the wild type
GST-Grb2 fusion protein. These species did not bind the isolated Grb2
SH3 domains. In addition to the isolated SH3 domains, further
characterization of domain specificities was possible using the
GST-Grb2 µSH3 molecule. This is a full-length Grb2 construct which
has point mutations introduced into each of the SH3 domains but an
intact SH2 domain. The Fc
R1-induced 75-kDa tyrosine phosphoprotein
did not bind to the GSTGrb2 µSH3 fusion protein, nor did the
110-kDa tyrosine phosphoprotein. However, Fc
R1-induced tyrosine
phosphoproteins of 33 and 65 kDa bound efficiently to the GST-Grb2
µSH3 protein. As well, a 55-kDa tyrosine phosphoprotein and a
doublet of high molecular mass tyrosine-phosphorylated proteins at
120-140 kDa were detected binding to GST-Grb2 µSH3. The
latter species were inducibly tyrosine phosphorylated in
Fc
R1-activated RBL2H3 to varying degrees between experiments.
Figure 2:
Anti-phosphotyrosine Western blot showing
SH domain specificity of tyrosyl proteins binding to Grb2 in RBL2H3
cells. 2 10
RBL2H3 cells/point were primed as
described and stimulated with 10 µg/ml KLH-DNP for the time
indicated in minutes at 37 °C. Lysates were affinity purified for 2
h with the Grb2 fusion protein indicated. The panel of GST fusion
proteins used were the wild type GST-Grb2, the single SH3 domain
truncations GST-Grb2NSH3 and GST-Grb2CSH3, and the double SH3 mutant
GST-Grb2 µSH3. GST alone coupled to glutathione-agarose beads was
included as a negative control. Samples were resolved on 11% SDS-PAGE
under reducing conditions before transfer to PVDF membrane, blocking
with non-fat milk, and Western blot analysis with
4G10.
From these binding studies it can be concluded that there are
FcR1-induced tyrosine phosphoproteins of 33 and 65 kDa which can
bind to Grb2 SH2 domains. In addition, there is an Fc
R1-induced
tyrosine phosphoprotein of 75 kDa which binds to Grb2 SH3 domains.
Other tyrosine phosphoproteins that associate with Grb2 SH3 domains and
SH2 domains have molecular masses of 110-120 and 120-140
kDa, respectively. The binding experiments using truncated and mutated
Grb2 fusion proteins show that the proteins migrating in the
110-120 kDa range may in fact be resolved into both basally and
Fc
R1-induced tyrosine-phosphorylated proteins. Similarly, there
are multiple tyrosine-phosphorylated proteins at 52-55 kDa
binding to the intact GST-Grb2 fusion protein that resolve into both
basally phosphorylated proteins and Fc
R1-induced tyrosine
phosphoproteins upon analysis with the truncated and mutant Grb2 fusion
proteins.
Tyrosine Phosphoproteins That Associate with the SH3
Domains of Endogenous Grb2 in RBL2H3 Cells
Initial experiments
showed that there is a complicated pattern of FcR1-induced
tyrosine phosphoproteins which are capable of binding to Grb2 fusion
proteins. Therefore, to establish whether there are Fc
R1-induced
complexes between tyrosine-phosphorylated proteins and endogenous Grb2,
we used affinity purification protocols to isolate Grb2 and associated
proteins from RBL2H3 cell lysates. This technique has been described in
detail previously
(32) and has been used effectively to examine
Grb2 complexes in TCR-activated T-lymphocytes. Briefly, a synthetic
tyrosine phosphopeptide corresponding to the autophosphorylation site
Tyr-1068 in the carboxyl-terminal tail of the epidermal growth factor
receptor (EGFR-Y1068) was used to precipitate Grb2 from RBL2H3 cells.
The EGFR-Y1068 peptide binds to the Grb2 SH2 domain with nanomolar
affinity and thus competitively blocks associations between cellular
proteins and the SH2 domains of Grb2. Use of this peptide allows the
copurification of Grb2 and proteins complexed with Grb2 SH3 domains.
Grb2 was also purified from RBL2H3 cells using a GST fusion protein of
a proline-rich fragment from the carboxyl terminus of murine Sos
(GST-Sos). The GST-Sos fusion protein used in these experiments binds
the Grb2 SH3 domain and therefore isolates Grb2 and proteins associated
with the Grb2 SH2 domain. GST-Sos competitively prevents the
copurification of Grb2 SH3-binding proteins. The data in Fig. 3 show an
anti-Grb2 Western blot of the cellular proteins purified with GST-Sos
or EGFR-Y1068 and indicate that these two reagents isolate equivalent
levels of endogenous Grb2. Moreover, a comparison of the levels of Grb2
purified with EGFR-Y1068 or GST-Sos with Grb2 levels in total cell
lysates (Fig. 3 A) shows that these reagents are capable
of affinity purifying the majority of endogenous Grb2 from RBL2H3
cells. The data in Fig. 3 B show that it is possible to
copurify Sos with Grb2 when the EGFR-Y1068 motif but not GST-Sos is
used as an affinity matrix.
Figure 3:
The
EGFR-Y1068 peptide and GST-Sos fusion protein affinity purify Grb2 from
RBL2H3 cell lysate. RBL2H3 cell lysates containing 2 10
cell equivalents were precleared as described and affinity
purified with either EGFR-Y1068 coupled to Affi-Gel-10 or GST-Sos
fusion protein coupled to glutathione-agarose beads. Post-nuclear
proteins were acetone precipitated from 2
10
RBL2H3
(total lysate). The samples were resolved on 11% SDS-PAGE under
reducing conditions before transfer to PVDF and blocking of the
membrane. A, the membrane was then probed with 0.5 µg/ml
anti-Grb2 for 1 h and developed with anti-mouse Ig-horseradish
peroxidase second stage antibody. B, the PVDF membrane was
blocked and probed with 1 µg/ml anti-mSos1 for 2 h before
development with anti-rabbit Ig-horseradish peroxidase second stage
antibody.
To determine whether any of the tyrosine
phosphoproteins observed in GST-Grb2 affinity purifications bind to
endogenous Grb2 via its SH3 domains, we performed Western blot analysis
with phosphotyrosine antibodies on Grb2 complexes isolated with
EGFR-Y1068 peptide. As shown in Fig. 4 A, the EGFR-Y1068
peptide precipitates a 75-kDa tyrosine phosphoprotein from
FcR1-activated but not quiescent RBL2H3 cells. The EGFR-Y1068
peptide also purified two tyrosine phosphoproteins of 110 and 120 kDa
which were apparently equally phosphorylated in quiescent and
Fc
R1-activated cells. These tyrosine phosphoproteins appear to
bind to endogenous Grb2 via its SH3 domains. They do not coprecipitate
with Grb2 which has been isolated using the GST-Sos fusion protein to
compete out Grb2 SH3-binding proteins (Fig. 4 B). They
also comigrate with the 75-, 110-, and 120-kDa tyrosine phosphoproteins
detected in the binding experiments using GST-Grb2 single SH3 domain
fusion proteins (Fig. 2). Affinity purification with the GST-Grb2
fusion protein produces the characteristic pattern of tyrosine
phosphoproteins observed in previous experiments. It should be noted
that there was a variable nonspecific binding of a 50-52-kDa
doublet of tyrosine phosphoproteins to control affinity purifications
comprising GST alone or Affi-Gel beads alone.
Figure 4:
A,
the EGFR-Y1068 phosphopeptide purifies tyrosine phosphoproteins from
stimulated RBL2H3. RBL2H3 were primed with IgE anti-DNP and stimulated
with KLH-DNP for 15 min as described. Lysates were made, and affinity
purifications were carried out on 2 10
cell
equivalents/lane with the following reagents: Affi-Gel-10 beads alone,
EGFR-Y1068 coupled to Affi-Gel-10, GST-Grb2 fusion protein, and GST
alone coupled to glutathione-agarose beads. The samples were resolved
under reducing conditions on 11% SDS-PAGE before transfer and blocking
as described above. The membranes were then probed with 1 µg/ml
4G10 for 1 h and developed with anti-mouse Ig-horseradish peroxidase.
B, binding of 55- and 33-kDa tyrosine phosphoproteins to
endogenous Grb2 in RBL2H3. RBL2H3 cells were stimulated and lysed as
described previously. Lysates from 2
10
cells/point
were precleared and affinity purified for 2 h with either GST-Grb2,
GST-Grb2 µSH3, or GST-Sos. After 11% SDS-PAGE and transfer to PVDF
membrane, Western blot analysis was carried out with 1 µg/ml 4G10.
The membrane was then stripped and reprobed with 1 µg/ml anti-Shc
overnight at 4 °C and developed with anti-rabbit Ig-horseradish
peroxidase.
Tyrosine Phosphoproteins of 33 and 55 kDa Associate with
Endogenous Grb2 SH2 Domains
The data in Fig. 4 B show anti-phosphotyrosine Western blots of the endogenous Grb2
complexes isolated from RBL2H3 cells with GST-Sos. These data indicate
that the 120-, 140-, and 65-kDa tyrosine phosphoproteins observed
binding to the normal and the double SH3 mutant GST-Grb2 fusion
proteins do not coprecipitate with endogenous Grb2. However, an
FcR1-induced tyrosine phosphoprotein of 33 kDa was detected in the
endogenous Grb2 complexes purified by GST-Sos. In addition, a tyrosine
phosphoprotein of 55 kDa was observed to bind to endogenous Grb2 in
both quiescent and Fc
R1-stimulated RBL2H3. These p33 and p55
molecules appeared to bind to Grb2 SH2 domains based on their pattern
of binding to the panel of Grb2 fusion proteins. As well, they did not
coprecipitate with the Grb2 complexes purified with the EGFR-Y1068
peptide, and their interaction with endogenous Grb2 was competed by the
EGFR-Y1068 peptide that binds to the Grb2 SH2 domain (data not shown).
R1-stimulated
RBL2H3 cells. This protein had a slightly lower mobility than the
nonspecific 50-52-kDa proteins described earlier and could thus
possibly represent tyrosine phosphorylated 55-kDa Shc isoform.
Accordingly, the blots were reprobed with a Shc antiserum. Fig. 4 B shows that the 55-kDa Shc isoform binds to the wild type and
SH3-mutated Grb2 fusion proteins and also can be copurified with
endogenous Grb2 by GST-Sos. The binding of Shc to endogenous Grb2 was
not influenced by the activation state of the RBL2H3.
R1-triggered RBL2H3 cells. The 55-kDa Shc isoform is
tyrosine phosphorylated in both quiescent and stimulated cells. These
data demonstrate also that the 55-kDa tyrosine phosphoprotein binding
to GST-Grb2 and GST-Sos comigrates with the tyrosine-phosphorylated
55-kDa Shc protein purified with Trk-Y490. The nonspecific
50-52-kDa protein doublet can once again be observed binding to
control Affi-Gel beads alone but with a different mobilty to either of
the Shc isoforms.
The 33-kDa Grb2 SH2 Domain-binding Tyrosine
Phosphoprotein Is Not the
The
accumulated evidence from the experiments in Figs. 1-5 show that
Fc Subunit of the Fc
R1
R1 triggering results in tyrosine phosphorylation of a 33-kDa
protein which binds to endogenous Grb2 in an SH2 domain-mediated
association. The
subunit of the Fc
R1 is 30-33 kDa and
is known to be tyrosine phosphorylated in response to receptor
triggering. Accordingly, we examined whether the p33 Grb2-binding
protein was the
subunit of the Fc
R1 (Fig. 6). Western blot
analysis with the Fc
R1
chain antibody JRK1 showed that the
chain can be immunoprecipitated with the anti-phosphotyrosine
antibody FB2 from Fc
R1-stimulated but not quiescent cells
(Fig. 6). Furthermore, it was possible to observe a
hyperphosphorylated population of JRK1-immunoreactive
chain
molecules in total lysates from Fc
R1-activated RBL2H3. This latter
population comigrated on SDS-PAGE with the population selected for by
the FB2 anti-phosphotyrosine immunoprecipitation. However, the 33-kDa
Grb2-binding tyrosine phosphoprotein detected by anti-phosphotyrosine
Western blot analysis of GST-Grb2 affinity purifications from
FceR1-stimulated RBL2H3 was not immunoreactive with the JRK1 antibody
and is thus not the
subunit of the Fc
R1. It is apparent,
moreover, that the SDS-PAGE mobility of p33 is different from the
tyrosine-phosphorylated
chain.
Figure 6:
The p33 tyrosine phosphoprotein binding
the Grb2 SH2 domain in RBL2H3 cells is not the FcR1
chain.
Affinity purifications of lysates from 2
10
quiescent or Fc
R1-stimulated RBL2H3 were carried out using
GST-Grb2. Post-nuclear protein from 2
10
stimulated
or unstimulated RBL2H3 cells was acetone-precipitated.
Immunoprecipitations using 2 µg/point of the anti-phosphotyrosine
antibody FB2 were carried out on lysates from 2
10
quiescent or Fc
R1-stimulated RBL2H3. The samples were
resolved and transferred as described. The membrane was probed with the
mouse monoclonal antibody JRK1, which is reactive for the Fc
R1
chain, overnight at 4 °C. The Western blot was then developed
with an anti-mouse Ig-horseradish peroxidase second stage antibody. An
arrow marks the position of antibody light chain in the
anti-phosphotyrosine immunoprecipitations.
R1-induced complexes between tyrosine phosphoproteins
and the adapter molecule Grb2. These include two basally
tyrosine-phosphorylated 110- and 120-kDa molecules and a 75-kDa protein
substrate for Fc
R1-activated PTKs which binds to the Grb2 SH3
domain. Two protein substrates for Fc
R1-activated tyrosine kinases
were observed to bind GST fusion proteins of Grb2 but more importantly
were also found in association with endogenous Grb2. A 75-kDa protein
which was tyrosine phosphorylated in activated but not quiescent RBL2H3
cells was associated with the SH3 domains of Grb2. We also detected a
33-kDa tyrosine phosphoprotein complexed to Grb2 SH2 domains in
Fc
R1 stimulated but not quiescent cells. Previous studies of
Grb2-binding proteins in T cells have illustrated how experiments that
identify Grb2-binding proteins solely on the basis of affinity
purifications with GST-Grb2 fusion proteins can be misleading. This
point is made again by the present study. Hence wild type GST-Grb2
fusion protein experiments suggested that in RBL2H3 lysates there are
tyrosine phosphoproteins of 33, 55, 65, 120, and 140 kDa which are
capable of binding to Grb2 SH2 domains. The 33- and 65-kDa proteins in
particular are substrates for Fc
R1-activated PTKs as evidenced by
their inducible tyrosine phosphorylation. However, only the
constitutively tyrosine-phosphorylated 55-kDa and the Fc
R1-induced
33-kDa tyrosine phosphoproteins were detected in association with
endogenous Grb2 SH2 domains. Similarly, only a subset of the
tyrosine-phosphorylated proteins identified as Grb2 SH3-binding
proteins by the in vitro binding experiments were present in
the in vivo Grb2 complexes isolated from RBL2H3 cells: a
75-kDa Fc
R1-induced tyrosine phosphoprotein and two proteins of
110 and 120 kDa, respectively, which were equivalently tyrosine
phosphorylated in quiescent and activated cells and appeared to
associate constitutively with Grb2 SH3 domains.
R1 triggering. This suggests that Shc is not a major
substrate for Fc
R1-activated tyrosine kinases in RBL2H3 cells. In
accordance with the lack of Fc
R1 control of Shc tyrosine
phosphorylation, no Fc
R1-induced changes in Shc-Grb2 complex
formation were observed in RBL2H3 cells although Shc was constitutively
associated with Grb2 in an SH2 domain-mediated association. It has been
previously noted that there is no significant formation of Shc-Grb2
complexes in response to TCR ligation in T cells
(25, 35) . In contrast, in B cells ligation of the BCR
induces tyrosine phosphorylation of Shc and induces a Shc-Grb2-Sos
complex that is proposed to link the BCR to p21ras
(36, 37) . Interestingly, Shc is a reasonably abundant
protein in both T cells and mast cells. Moreover, in both cell types
cytokines such as interleukin-2 and interleukin-3 can stimulate high
levels of Shc tyrosine phosphorylation and induce Shc-Grb2 complexes
indicating that there are no technical problems in detecting such
complexes in these cells
(40, 41, 42) .
R1 to the Grb2
signaling pathway is a 33-kDa tyrosine phosphoprotein. This p33 is
tyrosine phosphorylated in response to Fc
R1 stimulation and binds
Grb2 SH2 domains. The
subunit of the Fc
R1 has a similar
molecular weight to p33 and is tyrosine phosphorylated in response to
receptor ligation
(17) . We have shown, however, that the p33
tyrosine phosphoprotein observed to bind the SH2 domain of endogenous
Grb2 is not the
chain of the Fc
R1. This does not exclude an
indirect interaction between Grb2 and the Fc
R1. The
Fc
R1-induced p33 molecule appears functionally analogous to the
TCR-induced p36 phosphoprotein observed as the major
tyrosine-phosphorylated species to bind Grb2 SH2 domains in activated T
cells
(25, 35) . It remains to be determined, however,
whether p36 and p33 share any structural homology. In this context,
studies in a variety of cell systems have identified many tyrosine
kinase substrates with apparently diverse functions which can bind to
Grb2 SH2 domains. These include adapters such as Shc, tyrosine
phosphatases such as R-PTP
and Syp, and growth factor receptors
and receptor subunits such as the EGF receptor and IRS-1
(27, 43, 44) . Accordingly, it appears that
multiple mechanisms have evolved to couple cellular PTKs to Grb2, of
which the p36 and now p33 molecules may be one type.
R1 in RBL2H3 cells results in activation of p21ras and
downstream effector molecules such as Raf-1 and the MAP kinase
ERK2.
(
)
The current data demonstrate that Sos is
constitutively associated with Grb2 SH3 domains in RBL2H3 cells.
Accordingly, the Fc
R1-induced 33-kDa tyrosine phosphoprotein that
binds to Grb2 SH2 domain may be an adapter that couples the Fc
R1
to Grb2-Sos complexes thereby permitting Fc
R1 stimulation of
p21ras. However, it has been recognized increasingly that Grb2 may link
PTKs to molecules other than Sos. For example, a second p21ras guanine
nucleotide exchange protein C3G binds to Grb2 SH3 domains in PC12 cells
(46) . As well, the GTPase dynamin can bind to and be activated
by Grb2 SH3 domains in vitro and in vivo (47) . In the present report, we have shown that in RBL2H3
cells Grb2 SH3 domains do not only bind the p21ras guanine nucleotide
exchange protein Sos. There are also tyrosine-phosphorylated proteins
of 75, 110, and 120 kDa which associate with endogenous Grb2 SH3
domains. By analogy with Sos, C3G, and dynamin, the p75, p110, and p120
molecules are candidates for alternate Grb2 effector molecules. The p75
molecule is of particular interest in this context because it is a
substrate for Fc
R1-activated PTKs. One of the major substrates for
Fc
R1-activated PTKs has been recently cloned and is a 75-kDa
protein termed SPY or HS1, which has an SH3 domain and also contains
proline-rich sequences reminiscent of SH3-binding domains characterized
in molecules such as Sos
(48) . Western blot analysis of
endogenous Grb2 complexes in both T cells and RBL2H3 cells demonstrated
that HS1 is not the Grb2-associated p75 described in this
study.
(
)
Associations between Grb2 and p75 have
not been detected in fibroblasts suggesting that this molecule has a
limited tissue distribution and hence function
(32) . In
preliminary analysis the Fc
R1-induced p75 appears identical to a
75-kDa protein that is a substrate for TCR-activated PTKs and is
constitutively associated with Grb2 SH3 domains in T cells
(32) . We propose that p75 may be a common signaling element for
the TAM-based receptor family that includes the TCR and the Fc
R1.
Recently, p120
, which is a substrate for TCR-activated
PTKs, was shown to bind to the NH
-terminal SH3 domain of
Grb2 fusion proteins
(34) . The constitutively
tyrosine-phosphorylated 120 kDa protein described herein to associate
with endogenous Grb2 complexes was not p120
although,
p120
was detected in the Grb2 fusion protein complexes
isolated from RBL2H3 cells and corresponded to the 120-kDa
Fc
R1-induced tyrosine phosphoprotein seen in these complexes (data
not shown).
R1 is to regulate the exocytosis
of mast cell granules and to modulate cytokine gene expression. The
present data have shown that the Fc
R1 induces tyrosine
phosphorylation of multiple Grb2-binding proteins, strongly suggesting
that this adapter protein is involved in coupling the Fc
R1 to one
or more signal transduction pathways. It has been demonstrated that
calcium and PKC have an important role in Fc
R1-regulated cellular
responses
(21) . We would propose that Grb2-coupled signaling
pathways may also be important for Fc
R1-induced mast cell
activation.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.