From the
We have recently identified the protein product of the c- cbl proto-oncogene as an SH3 binding protein expressed in macrophages.
To investigate the possibility that p120
Cellular receptors for the Fc domain of immunoglobulins
(FcRs)
Previous studies have shown that biological responses mediated by
FcRs require tyrosine phosphorylation
(4) . Because FcRs lack
intrinsic kinase activity, substrate phosphorylation must result from
activation of coupled kinases. Two members of the Src family of protein
tyrosine kinases, p56 and p62, have been implicated in FcR signal
transduction by their physical association with receptors in rat and
mouse cell lines
(5) . p56 has been found in association with
the
The cbl gene was initially discovered as the transforming component of Cas
NS-1, a tumorigenic murine retrovirus
(11) . The cbl proto-oncogene is mainly expressed in hematopoietic cells
(11, 12) , and its protein product, p120
A number of substrates have been identified for
protein-tyrosine kinases involved in the pathways initiated by FcR
engagement. These include individual components of the multimeric Fc
receptors
(18, 19) as well as the kinases p56, p62
(5) , and p72
(8, 10, 20) . In the
present study, we have shown that p120
The
involvement of tyrosine phosphorylation in recruiting molecules
containing SH2 domains to signaling complexes has been well documented
(22) . For example, tyrosine phosphorylation of receptor
activation motifs of Fc
Previous studies
have shown that p120
We thank Jeanne H. Sameshima for her help in preparing
the manuscript and Dr. Silvio Gutkind for helpful discussions.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
is involved in
signaling pathways initiated by cell surface receptors for IgG
(Fc
R), lysates of HL60 cells were examined for tyrosine
phosphorylation of p120
upon Fc
R engagement. Our
findings demonstrate that p120
is tyrosine-phosphorylated
upon Fc
R engagement and that this molecule represents the major
tyrosine kinase substrate in this signaling pathway. Protein complexes
containing p120
, p72, and p56 were observed either in
resting or activated cells. In vitro studies showed that the
direct association between p120
and p56 was mediated by
the SH3 domain of p56.
(
)
are widely expressed on the surface of
immune cells. FcRs belong to the immunoglobulin superfamily and include
high affinity receptors for IgE (Fc
RI), IgG (Fc
RI), and IgA
as well as low affinity IgG receptors (Fc
RII and RIII)
(1) . In the presence of antigen-antibody complexes, these
receptors initiate a variety of immune responses such as exocytosis of
inflammatory mediators and phagocytosis
(2) . Recent studies of
a murine strain deficient in Fc
Rs have shown a markedly attenuated
inflammatory response to immune complexes, directly demonstrating a
requirement for Fc
receptors in this process
(3) .
subunit of Fc
RI and p72, also a protein-tyrosine kinase,
with its phosphorylated
subunit
(6, 7) . Recent
studies have shown the physical association of p72 with activated
Fc
RII in platelets
(8) . According to a current model for
signaling through Fc
RI, p56 associates with the
subunit of
the receptor and becomes active when the receptor is engaged. Activated
p56 kinase then phosphorylates the
subunit of the receptor, which
in turn serves as a docking motif for the SH2 domains of p72
(6) . This model has also been suggested for the Fc
R in
modulating B-cell activation triggered by the surface immunoglobulin
complex
(9) . The contribution of p72 kinase to Fc
R
signaling is not well understood, but it has been shown to associate
with tyrosine-phosphorylated Fc
RIIA acting as a catalyst in early
events of platelet activation
(8) . Furthermore, p72
coprecipitates with a 120-kDa protein which has been suggested to be a
possible substrate in myelomonocytic cells
(10) .
,
has been recently isolated from murine macrophages by virtue of its
ability to bind SH3 elements
(13) . Other recent studies have
shown that conversion of c- cbl to a transforming gene involves
tyrosine phosphorylation of its protein product
(14) . These and
other observations prompted us to examine p120
as a
potential player in the Fc receptor signaling pathway.
Cells and Antibodies
The human promyelomonocytic
cell line HL60
(15) was maintained as described previously
(10) . Goat anti-mouse and goat anti-rabbit antibodies coupled
to horseradish peroxidase, human IgG, and goat anti-human IgG
F(ab)`antibodies were obtained from Cappel Organon Teknika
Corp. Monoclonal anti-phosphotyrosine (4G10) coupled to agarose beads
and polyclonal anti-p56 antibodies were purchased from Upstate
Biotechnology Inc. Polyclonal anti-p120
(C15) and
polyclonal anti-p72 (SC 573) antibodies were purchased from Santa Cruz
Biotechnology. Monoclonal anti-phosphotyrosine (PY20) antibody coupled
to horseradish peroxidase was obtained from ICN. Anti-p72 serum was
obtained by immunizing rabbits with a peptide representing residues
622-635 of p72.
Expression of Glutathione S-Transferase Fusion
Proteins
A fragment containing the SH3 domain of lyn (amino acids 60-104) was amplified by polymerase chain
reaction from a full-length human lyn cDNA
(16) ,
kindly provided by Joseph Bolen, and ligated into the BamHI
and EcoRI sites of pGEX-4T-3 (Pharmacia Biotech Inc.). The
MscI- NcoI (Klenow end-filled) fragment of lyn containing its SH2 domain (amino acids 105-226) was
subcloned into SmaI-cut pGEX-4T-3. Purification of GST fusion
proteins has been described previously
(17) .
Fc
Receptors were engaged as
described
(10) . Briefly, cells were suspended at a
concentration of 1 R Cross-linking
10
/ml in serum-free RPMI 1640
containing 0.1% bovine serum albumin and 1 m
M sodium vanadate.
Cells were treated with 10 µg/ml human IgG on ice for 10 min and
washed once with phosphate-buffered saline. Cells were then resuspended
in serum-free RPMI medium prewarmed to 37 °C and incubated with 20
µg/ml goat anti-human IgG F(ab)`
for periods indicated.
After washing with cold phosphate-buffered saline, cells were lysed at
4 °C for 15 min in a buffer containing 50 m
M Tris-HCl, pH
7.6, 5 m
M EDTA, 150 m
M NaCl, 1% Nonident P-40, 1
m
M sodium vanadate, and a mixture of protease inhibitors which
included 2 m
M diisopropylfluorophosphate, 1 m
M
phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, 5 µg/ml
leupeptin, 0.7 µg/ml pepstatin A, 0.5 m
M
4-(2-aminoethyl)benzenesulfonyl fluoride, and 2 m
M EDTA.
Lysates were clarified at 100,000
g for 20 min at 4
°C, and their protein concentrations were determined by the method
of Bradford using commercially available reagents (Bio-Rad).
Supernatants were processed for either sodium
dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and
Western blotting or for immunoprecipitation.
Immunoprecipitation and Western Blotting
Analysis
For immunoprecipitation cell lysates were incubated 1 h
on ice with indicated antibodies and for an additional 30 min with 10
µl of GammaBind G-Sepharose beads (Pharmacia Biotech Inc.), washed
twice with cold lysis buffer and once with lysis buffer containing 1
M NaCl (high salt buffer). Total cell lysates and
immunoprecipitates were fractionated by SDS-PAGE, and proteins were
transferred to PVDF membranes (Immobilon-P, Millipore). Filters were
blocked for 1 h at 37 °C in 10 m
M Tris-HCl, pH 7.4, NaCl
150 m
M (TNB), containing 4% bovine serum albumin and blotted
for 1 h at room temperature with TNB containing the antibodies
indicated. After exposure to secondary antibodies coupled to
horseradish peroxidase, proteins were visualized with the aid of an
enhanced chemilumiscence kit (Amersham Corp.).
Adsorption of Proteins with GST Fusion Proteins
5
µg of each GST fusion protein bound to GSH-agarose beads (Sigma)
were incubated for 60 min at 4 °C with 5 mg of cell lysates. After
two washes with cold lysis buffer and one with lysis buffer containing
1
M NaCl (high salt buffer), proteins were eluted from the
beads by boiling in sample buffer and processed for SDS-PAGE as
described above.
p120 Is Tyrosine-phosphorylated upon Engagement of
Fc
Several lines of evidence have raised the
possibility that p120 Receptors
might be involved in Fc
receptor signaling. To test this hypothesis, lysates from cells treated
sequentially with human IgG and goat anti-human IgG F(ab)`
to cross-link IgG-bound Fc
receptors were analyzed by
immunoblotting with anti-phosphotyrosine antibodies (anti-PY). As
expected
(10) , proteins of 120, 72, and 55-60 kDa were
tyrosine-phosphorylated in activated but not untreated HL60 cells,
demonstrating successful stimulation of the Fc
R signaling pathway
(Fig. 1 A). When the same lysates were first
immunoprecipitated with a previously characterized antibody capable of
recognizing p120
(13) , the protein was detected as
a highly tyrosine-phosphorylated species in lysates from activated but
not control cells. Analysis of these same immunoprecipitates and cell
lysates with anti-p120
antibody demonstrated that similar
amounts of p120
were present in activated and resting
cells (Fig. 1 B). Specific and independent stimulation of
Fc
RI and II (undifferentiated HL60 cells do not express
Fc
RIII) resulted in tyrosine phosphorylation of p120
,
demonstrating that both receptors participate in the event after
stimulation of the cells with IgG (data not shown). Thus, p120
is highly phosphorylated on tyrosine in response to Fc
R
stimulation.
Figure 1:
p120 is tyrosine
phosphorylated upon engagement of Fc
Rs in HL60 cells. Protein
extracts (5 mg) from untreated (-) or stimulated (+) HL60
cells were immunoprecipitated with anti-p120
antibodies,
fractionated by 10% SDS-PAGE, and blotted onto PVDF membranes. Total
cell lysate (60 µg/lane) was similarly electrophoresed and blotted.
Filters were treated with anti-phosphotyrosine ( Panel A) or
anti-p120
( Panel B) antibodies. Proteins were
visualized by chemiluminescence. The positions of molecular mass
markers in kilodaltons and p120
are
indicated.
p120 Is the Major Protein Tyrosine-phosphorylated in
Response to Fc
To determine what portion of the
120-kDa band detected by anti-PY was represented by p120R Engagement
,
this latter molecule was first depleted from lysates of stimulated
cells by immunoprecipitation. As shown in Fig. 2,
anti-p120
antibodies did not detect the protein after
three rounds of depletion by immunoprecipitation
(Fig. 2 A, compare lanes 2 and 3). When
blots containing lysates devoid of p120
were incubated
with anti-PY, the intensity of the p120 band was notably decreased
(Fig. 2 B, compare lanes 2 and 3).
Depleted lysates were also examined by immunoprecipitation with
anti-PY. Under these conditions the intensity of p120 was decreased
whereas the intensity of p72 was not appreciably affected
(Fig. 2 B, compare lanes 4 and 5).
Thus, depletion of p120
was selective. We conclude from
these results that p120
represents a major portion of the
120-kDa proteins tyrosine-phosphorylated upon Fc
R engagement.
Figure 2:
Immunodepletion of
p120 decreases detectability of the phosphoprotein
p120. A, protein extracts (1 mg) from activated HL60 cells
were subjected to one ( lane 2) or three ( lane 3)
rounds of immunoprecipitation with anti-p120
antibody,
fractionated by 10% SDS-PAGE, and immunoblotted with anti-p120
antibody. Immunoprecipitates using a nonreactive serum served as
a control ( lane 1). B, identical samples subjected to
one ( lanes 2 and 4) or three ( lanes 3 and
5) rounds of immunoprecipitation were either blotted directly
onto filters ( lanes 2 and 3) or reimmunoprecipitated
with anti-PY ( lanes 4 and 5). Immunoprecipitates
using nonreactive serum ( lane 1) served as a control. After
fractionation and blotting, filters were probed with anti-PY, and
proteins were visualized by chemiluminescence. The positions of
molecular mass markers in kilodaltons, p120
, and p72 are
indicated.
p120 Associates with p56and p72Tyrosine Kinases
To determine whether p56 or p72, two
protein tyrosine kinases involved in the FcR signaling pathway,
might be responsible for phosphorylation of p120
, lysates
from resting or activated cells were immunoprecipitated with either
anti-p56 or anti-p72 and examined for the presence of
p120
. As shown in Fig. 3, p120
was
detected clearly associated with p56 ( lanes 5 and 6)
and in less amount with p72 ( lanes 7 and 8). The
formation of this complex was not dependent upon Fc
R
cross-linking, suggesting that the associations between p56, p72, and
p120
are not mediated by tyrosine phosphorylation. When
the same blots were stripped and reblotted with anti-p72 antibodies,
very little p72 was detected in anti-p120
immunoprecipitates, and no p72 was present in anti-p56
immunoprecipitates (data not shown). In reciprocal experiments, p56 was
detected in anti-p120
immunoprecipitates. When the
kinetics of p56-p120
association was analyzed, a slight
increase in the intensity of p56 was observed 10 min after Fc
R
activation. By 60 min, p56 was less apparent (Fig. 4). We
conclude that a portion of p56 is physically associated with
p120
in resting cells and that by 60 min after activation,
the number of p56 molecules complexed with p120
was
reduced.
Figure 3:
p120 associates with
p56
and p72
. Protein extracts (10
mg) from resting (-), or activated (+) HL60 cells were
immunoprecipitated with either a non reactive serum ( NRS),
anti-p120
( Anti-Cbl), anti-p56
( Anti-Lyn), or anti-p72 ( Anti-Syk) antibodies.
Immunoprecipitates were fractionated by 10% SDS-PAGE, blotted onto
filters and immunodetected with anti-Cbl antibody. The positions of
molecular mass markers in kDa, p120
and immunoglobulin
heavy chain ( IgG H) are indicated.
Figure 4:
Kinetics of the
p120-p56
association after
Fc
R engagement. Protein extracts (5 mg) from HL60 cells stimulated
for the times indicated, were immunoprecipitated with anti-p120
( Anti-Cbl) antibody, or with a nonreactive serum
( NRS). Immunoprecipitates as well as cell lysates
( None) were fractionated by electrophoresis, blotted onto
filters and probed with anti-p56 ( Anti-Lyn) antibody. The
positions of molecular mass markers in kilodaltons and p56 are
indicated.
To study the tyrosine phosphorylation state of p120complexed with p56 and p72, immunoprecipitates from lysates of
resting and activated HL60 cells were examined by anti-PY
immunoblotting. As shown in Fig. 5, tyrosine phosphorylated
p120
was detected in either anti-Lyn or anti-Syk
immunoprecipitates, as well as in lysates, from activated cells. These
findings demonstrated that the population of p120
who was
associated with p56 or p72 was tyrosine-phosphorylated upon Fc
R
engagement.
Figure 5:
p120 coprecipitated
with p56
and p72
is tyrosine
phosphorylated upon Fc
R engagement. Protein extracts (5 mg) from
resting (-) or activated (+) HL60 cells were
immunoprecipitated with anti-p56 ( Anti-Lyn), or anti-p72
( Anti-Syk) antibodies, and fractionated by electrophoresis.
Immunoprecipitates and total cell lysates ( None) similarly
fractionated were blotted onto filters, and probed with
anti-phosphotyrosine (anti-PY) antibody. The positions of molecular
mass markers in kilodaltons, p120
, p72, and p56 are
indicated.
To determine whether p56 or p72 kinases might
phosphorylate p120in vitro, lysates from resting
or activated cells were immunoprecipitated with anti-p56, anti-p72, or
anti-p120
antibodies and assayed in vitro for
kinase activity, but no phosphorylation in vitro was detected
(data not shown). These results provided no direct evidence that
p120
was a substrate for either p56 or p72 kinases.
p120 Associates with the SH3 Domain of p56
To further examine the nature of the interaction between p56
and p120, we tested the ability of the SH2 and SH3 domains
of p56 to bind p120
in vitro. GST fusion proteins
containing these domains were attached to glutathione agarose beads and
incubated in the presence of lysates from resting or activated HL60
cells. The associated proteins were then fractionated by SDS-PAGE and
immunoblotted with anti-p120
antibody. p120
specifically associated with the SH3 domain of p56 (Fig. 6,
lanes 5 and 6), but not with GST ( lanes 1 and 2) or the SH2 domain of p56 ( lanes 3 and
4). Moreover, the amount of p120
that bound to
Lyn-SH3 did not vary after 10 min of Fc
R cross-linking ( lane
6), consistent with the level of association observed in assays
in vivo (Figs. 3 and 4).
Figure 6:
p120 binds in
vitro to the SH3 domain of p56
. Protein extracts
(5 mg) from resting (-) or activated (+) HL60 cells were
incubated with 5 µg of the indicated GST fusion proteins coupled to
glutathione-agarose beads (Sigma). GST protein ( GST) and
anti-p120
(anti-(
) Cbl) immunoprecipitate
were included as controls. Samples were fractionated in 10% SDS-PAGE
and analyzed by Western blotting using anti-p120
as a
probe. The positions of molecular mass markers in kilodaltons and
p120
are indicated.
Phosphorylation of p120 and Its Associated Proteins
in Vivo
To gain information regarding the sequence of
association and tyrosine phosphorylation events among
p120, p72, and p56, we performed a series of time course
experiments. Each of these molecules was immunoprecipitated and
examined by Western blotting using anti-PY as a probe. As shown in
Fig. 7B, tyrosine phosphorylation of p56 varied by
2-3-fold during the time course, reaching a maximum at 5-10
min. Associated proteins of 72 and 120 kDa were also observed, their
intensities peaking at 5-10 min after activation
(Fig. 7 B). A molecule of 120 kDa (likely
p120
), also associated with p72, is seen peaking in its
detectability at 20 min (Fig. 7 C). Phosphorylation of
p120
(Fig. 7 D) was also maximal at 20 min
and closely followed the pattern observed for p120 in total cell
lysates (Fig. 7 A). These experiments demonstrate
transient tyrosine phosphorylation of p56, p72, and p120
upon Fc
R activation. The data also confirm the formation of
a complex consisting of p56, p72, and p120
.
Figure 7:
Tyrosine phosphorylation of
p120 and its associated proteins. Protein extracts
(5 mg) from HL60 cells stimulated for the times indicated were
immunoprecipitated with anti-p56 ( Anti-Lyn) ( Panel
B), anti-p72 ( Anti-Syk) ( Panel C), or
anti-p120
( Anti-Cbl) ( Panel D) and
fractionated by electrophoresis. Immunoprecipitates as well as total
cell lysates similarly fractionated ( Panel A), were blotted
onto PVDF membranes, and analyzed with anti-PY antibody.
Electrophoretic mobility of molecular mass markers in kilodaltons is
indicated.
is phosphorylated
on tyrosine in response to Fc
R engagement and is the most
prominent of all tyrosine-phosphorylated proteins in this pathway. The
lack of obvious p120 tyrosine phosphorylation upon Fc
RI activation
in rat basophilic leukemia cells
(4)
(
)
suggests that phosphorylation of p120
might
distinguish Fc
from Fc
receptor signaling pathways.
Nevertheless, recent studies have shown tyrosine phosphorylation of
p120
in response to T cell-receptor activation as well
(21) . Thus, p120
emerges as an important player in
immune recognition receptor signaling in hematopoietic cells.
(6, 7) and probably
Fc
receptors
(8) serves as the mechanism by which p72
binds activated receptor components. However, the involvement of SH3
domains in the formation of FcR signaling complexes has not been
previously described. In this study we show that p120
associates with the SH3 domain of p56, as it has been observed
for other tyrosine kinases
(13, 21) , and this
association is not affected after Fc
R cross-linking. Furthermore,
the p120
-p56 complex is constitutive in vivo and
is present in both resting and activated cells. The amount of
p120
coprecipitated with anti-p56 did not vary upon
Fc
R cross-linking suggesting that Lyn-SH2 domain does not
contribute to the stability of the complex. Consistent with this
finding, Lyn-SH2 domain was unable to bind p120
in
vitro, even after activation of the cells. This result differs
from the observed in T cells, in which the association of p59 and a
phosphorylated p120, probably p120
, requires both the SH3
and SH2 elements
(23) . The small amount of p120
associated with p72 in vivo would suggest a nondirect
interaction between these two molecules. The steady state association
of p120
with p56 and p72, in combination with its tyrosine
phosphorylation, might suggest that these kinases directly
phosphorylate p120
, but in vitro kinase assays
failed to confirm this point. It is possible that the binding of
p120
to p56, a known membrane-attached protein, might
carry this molecule to the proximity of an active tyrosine kinase
located in the plasma membrane, where p120
would became
phosphorylated. Further studies will be required to identify the kinase
responsible for p120
phosphorylation.
physically binds to p47 in vitro and in vivo (13) . Thus, p47 may also contribute
fundamentally to the ability of p120
to associate with the
activated signaling complex. The role of p120
in the
Fc
R signaling complex is not clear. Its known associations with
p47 and p56 are consistent with the idea that p120
serves
as a bridge together with p47 to position p56 for activation after FcR
engagement. Alternatively, p120
may be responsible for
initiating biologic responses when phosphorylated. The ability of
oncogenic forms of p120
to subvert growth regulatory
pathways in fibroblasts and hematopoietic cells
(14, 21) attests to its biological potential, an area that warrants
further investigation.
R, receptors for IgE; Fc
,
receptors for IgG; GST, glutathione S-transferase; SH, Src
homology regions; PAGE, polyacrylamide gel electrophoresis; PVDF,
polyvinylidene difluoride; PY, phosphotyrosine.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.