From the Department of Physiology, University of Michigan Medical School, Ann Arbor, Michigan 48109-0622
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ABSTRACT |
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We recently identified SH2-B as a JAK2-binding
protein and substrate involved in the signaling of receptors for growth
hormone and interferon-
. In this work, we report that SH2-B
also
functions as a signaling molecule for platelet-derived growth factor
(PDGF). SH2-B
fused to glutathione S-transferase (GST)
bound PDGF receptor (PDGFR) from PDGF-treated but not control cells.
GST fusion protein containing only the SH2 domain of SH2-B
also
bound PDGFR from PDGF-treated cells. An Arg to Glu mutation within the
FLVRQS motif in the SH2 domain of SH2-B
inhibited GST-SH2-B
binding to tyrosyl-phosphorylated PDGFR. The N-terminal truncated
SH2-B
containing the entire SH2 domain interacted directly with
tyrosyl-phosphorylated PDGFR from PDGF-treated cells but not
unphosphorylated PDGFR from control cells in a Far Western assay. These
results suggest that the SH2 domain of SH2-B
is necessary and
sufficient to mediate the interaction between SH2-B
and PDGFR. PDGF
stimulated coimmunoprecipitation of endogenous SH2-B
with endogenous
PDGFR in both 3T3-F442A and NIH3T3 cells. PDGF stimulated the rapid and
transient phosphorylation of SH2-B
on tyrosines and most likely on
serines and/or threonines. Similarly, epidermal growth factor
stimulated the phosphorylation of SH2-B
; however, phosphorylation
appears to be predominantly on serines and/or threonines. In response
to PDGF, SH2-B
associated with multiple tyrosyl-phosphorylated
proteins, at least one of which (designated p84) does not bind to
PDGFR. Taken together, these data strongly argue that, in response to
PDGF, SH2-B
directly interacts with PDGFR and is phosphorylated on
tyrosine and most likely on serines and/or threonines, and acts as a
signaling protein for PDGFR.
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INTRODUCTION |
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We recently identified the
SH21 domain-containing
molecule, SH2-B, as a substrate of JAK2 involved in signaling by
growth hormone (GH) and interferon-
(1). Receptors for GH and
interferon-
are members of the cytokine receptor family and are
known to bind JAK tyrosine kinases (JAK2 for GH and both JAK1 and JAK2
for interferon-
). After GH binding, JAK2 is activated and
tyrosyl-phosphorylates its associated GH receptor as well as JAK2
itself (2, 3). As a consequence of JAK2 autophosphorylation, SH2-B
is recruited into receptor/JAK2 complexes at least in part via the
direct interaction of the SH2 domain of SH2-B
with phosphotyrosine
containing motif(s) in JAK2 (1). GH promotes not only the association
of SH2-B
with tyrosyl-phosphorylated JAK2, but also the tyrosyl
phosphorylation of SH2-B
(1). SH2-B
also appears to be
phosphorylated on serine(s) and/or threonine(s), even in the absence of
ligand stimulation (1). These findings suggested that SH2-B
, which
contains multiple potential sites for protein-protein interaction in
addition to its SH2 domain (9 tyrosines, a pleckstrin homology (PH)
domain, and multiple proline-rich motifs) (Fig. 1A), serves
as an adapter protein and recruits additional signaling molecules into
cytokine receptor-JAK2 complexes (1).
Many signaling molecules are shared by cytokine receptors and receptor
tyrosine kinases, particularly those signaling molecules containing SH2
domains. For example, Shc, Grb2, and phosphatidylinositol 3'-kinase are
reported to play an important role in the biological actions of GH (2,
4) and platelet-derived growth factor (PDGF) (5, 6). In support of
SH2-B serving as a signaling molecule for receptor tyrosine
kinase(s), SH2-B
was found to interact with receptors for insulin
and insulin-like growth factor-1 (1, 7, 8). In this work, we
demonstrate that PDGF stimulates association of SH2-B
with PDGF
receptor (PDGFR), and phosphorylation of SH2-B
. We also show that
SH2-B
associates, not via the PDGFR, with a tyrosyl-phosphorylated,
84-kDa protein. These results provide strong evidence that SH2-B
is
a previously unknown signaling molecule for PDGF.
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EXPERIMENTAL PROCEDURES |
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Cells and Reagents--
The stock of 3T3-F442A fibroblasts was
kindly provided by H. Green (Harvard University, Cambridge, MA).
Recombinant human GH was a gift of Eli Lilly and Co. Recombinant human
PDGF-BB was from Life Technologies, Inc. Recombinant human PDGF-AA was
from Collaborative Biomedical Products. Glutathione-agarose beads were from Sigma. Recombinant protein A-agarose was from Repligen. Alkaline phosphatase, aprotinin, leupeptin, and Triton X-100 were from Boehringer Mannheim. Protein phosphatase 2A (PP2A) was from Upstate Biotechnology, Inc. Enhanced chemiluminescence (ECL) detection system
was from Amersham Corp. Antibodies to rat SH2-B (
SH2-B) were
raised against GST-SH2-B
c as described previously (1), and used at a
dilution of 1:100 for immunoprecipitation and 1:15,000 for
immunoblotting. Anti-JAK2 antiserum (
JAK2) was raised in rabbits
against a synthetic peptide corresponding to amino acids 758-776 of
murine JAK2 (9) and was used at a dilution of 1:500 for
immunoprecipitation and 1:15,000 for immunoblotting. Monoclonal anti-phosphotyrosine antibody 4G10 (
PY) and polyclonal antibody against human PDGF receptor (
PDGFR, recognizing both
and
subunits) were from Upstate Biotechnology, Inc. and used at a dilution
of 1:7500 and 1:1000 for immunoblotting, respectively.
PDGFR was
used at a dilution of 1:100 for immunoprecipitation.
Methods--
3T3-F442A fibroblasts were treated for 10 min with
25 ng/ml PDGF-BB, vehicle, or other ligands as indicated. For GST
fusion protein pull-down assays, whole cell lysates were precipitated with GST fusion proteins immobilized on glutathione-agarose beads and
subsequently immunoblotted with PDGFR or
PY as described previously (1). GST fusion proteins containing SH2-B
or mutant SH2-B
were prepared as described previously (1). Arg within the
FLVRQS motif in the SH2 domain of SH2-B
was mutated to Glu using a
site-directed mutagenesis kit (Stratagene), and the mutation was
confirmed by DNA sequencing. The mutant SH2-B
(SH2-B
(R-E)) was
subcloned into pGEX-KG to generate a GST fusion protein. For immunoprecipitations, cell lysates were incubated with the indicated antibody on ice for 2 h. The immune complexes were collected on protein A-agarose (50 µl) during a 1-h incubation at 4 °C. In some
experiments,
SH2-B immunoprecipitates were dephosphorylated by
alkaline phosphatase or PP2A as described previously (1). The
immunoprecipitates were immunoblotted with the indicated antibody. Some
membranes were stripped by incubation at 55 °C for 30-60 min in
stripping buffer (100 mM
-mercaptoethanol, 2% SDS, 62.5 mM Tris-HCl, pH 6.7) and reprobed with a different
antibody. For Far Western blotting, PDGFR was immunoprecipitated with
PDGFR from solubilized 3T3-F442A fibroblasts, subjected to SDS-PAGE, and transferred onto nitrocellulose. The nitrocellulose was incubated with GST-SH2-B
c (1.5 µg/ml) at 4 °C overnight. After extensive washing, the membrane was immunoblotted with
SH2-B. The blot was
stripped and reprobed with
PDGFR and
PY sequentially.
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RESULTS |
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SH2-B Binds to PDGFR Only from PDGF-treated Cells--
To
determine whether SH2-B
serves as a signaling molecule for PDGF, we
first tested whether SH2-B
binds to PDGFR. 3T3-F442A fibroblasts,
which express endogenous PDGFR (10), were deprived of serum overnight
and treated with 25 ng/ml PDGF-BB or vehicle for 10 min. Cell lysates
were incubated with immobilized GST or GST fusion protein containing
full-length SH2-B
, N-terminally truncated SH2-B
(SH2-B
c), or
the SH2 domain of SH2-B
(Fig. 1A), and immunoblotted with
PDGFR. GST-SH2-B
bound to PDGFR in a ligand-dependent
manner (Fig. 1B, lanes 1 and 2).
GST-SH2-B
c (Fig. 1B, lanes 3 and
4) and GST-SH2 domain of SH2-B
(Fig. 1B, lanes 5 and 6) also bound PDGFR from
PDGF- but not vehicle-treated cells. Reprobing with
PY showed that
PDGFR that is associated with SH2-B
or truncated SH2-B
is
tyrosyl-phosphorylated (data not shown). In contrast, GST alone did not
bind PDGFR (Fig. 1B, lane 7). These
results suggest that SH2-B
interacts with activated, tyrosyl-phosphorylated PDGFR, and that the SH2 domain of SH2-B
may
mediate the interaction between SH2-B
and PDGFR.
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SH2-B Binds Directly to Tyrosyl-phosphorylated PDGFR through Its
SH2 Domain--
To investigate whether SH2-B
binds PDGFR directly
or indirectly through some intermediate molecule, the ability of
GST-SH2-B
c to bind PDGFR was determined by Far Western blotting.
3T3-F442A cells were treated with 25 ng/ml PDGF-BB or vehicle. PDGFR
was immunoprecipitated with
PDGFR, resolved by SDS-PAGE, and
transferred to nitrocellulose. The nitrocellulose was incubated first
with GST-SH2-B
c, and then with
SH2-B. GST-SH2-B
c bound
directly to PDGFR from PDGF-treated (Fig.
2A, lane
2) but not vehicle-treated cells (Fig. 2A,
lane 1), although an equal amount of PDGFR was present (Fig. 2A, lanes 3 and 4).
Reprobing the same blot with
PY confirmed the
PDGF-dependent tyrosyl phosphorylation of PDGFR (Fig.
2A, lanes 5 and 6). These results
indicate that SH2-B
c binds directly to tyrosyl-phosphorylated,
activated PDGFR.
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PDGF-BB Stimulates the Association of Endogenous SH2-B with
PDGFR--
To examine whether endogenous SH2-B
associates with
endogenous PDGFR in mammalian cells, 3T3-F442A fibroblasts, shown
previously to express endogenous SH2-B
(1), were treated with
PDGF-BB or vehicle. Solubilized proteins were immunoprecipitated with
SH2-B and immunoblotted with
PDGFR. SH2-B
was observed to
coimmunoprecipitate with PDGFR in PDGF-stimulated (Fig.
3, lane 2) but not control cells (Fig. 3, lane 1), consistent with the findings (shown
in Figs. 1B and 2) that SH2-B
binds only to
tyrosyl-phosphorylated, activated PDGFR. Pre-immune serum was unable to
precipitate PDGFR from PDGF-treated cells (Fig. 3, lanes 3 and 6). Reprobing the same blot with
PY confirmed that
PDGFR associated with SH2-B
is tyrosyl-phosphorylated (Fig. 3,
lane 5). Similarly, SH2-B
was detected in
PDGFR
immunoprecipitates only when cells were stimulated with PDGF (data not
shown). SH2-B
also coimmunoprecipitated with PDGFR in NIH3T3 cells
in response to PDGF-BB (Fig. 3, lanes 7 and 8),
further demonstrating that the association of SH2-B
with PDGFR is
not cell-type-specific. Taken together, the results of Figs. 1-3
suggest that PDGF stimulates the recruitment of SH2-B
to PDGFR
presumably via a direct interaction of the SH2 domain of SH2-B
with
phosphotyrosine-containing motif(s) in the activated PDGFR.
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PDGF-BB Promotes Tyrosyl Phosphorylation of SH2-B--
SH2-B
was previously shown to be tyrosyl-phosphorylated in response to GH and
interferon-
(1). To test whether PDGF is able to stimulate tyrosyl
phosphorylation of SH2-B
, 3T3-F442A cells were treated with PDGF-BB,
and cell lysates were immunoprecipitated with
SH2-B and
immunoblotted with
PY. PDGF-BB stimulated tyrosyl phosphorylation of
SH2-B
(Fig. 4, upper
panel; Fig. 5B,
lane 4; Fig. 6,
lane 4; Fig. 7,
lane 3). Based upon the migration of proteins
immunoblotted with
SH2-B (Fig. 4, lower panel;
Fig. 5B, lane 9; Fig. 7,
lane 8), SH2-B
migrates as a diffuse band indicated by the bracket. For reasons discussed below, the
tight band, designated p84 in Figs. 4, 5B, 6, and
7, is believed to be a tyrosyl-phosphorylated protein that
coimmunoprecipitates with SH2-B
, and not a form of SH2-B
. As
predicted from Figs. 1-3, tyrosyl-phosphorylated PDGFR
coimmunoprecipitated with SH2-B
from PDGF-BB-treated cells (Fig. 4,
lanes 2-7, upper panel; Fig. 5B, lanes
4 and 5; Fig. 6, lane 4; Fig. 7, lanes
2 and 3) but not from control (Fig. 4, lane
1; Fig. 5B, lanes 1-3; Fig. 6, lane
3; Fig. 7, lane 1) or GH- or EGF-treated cells (Fig. 7,
lanes 4 and 5). When the blots were reprobed with
SH2-B, PDGF-BB was observed to cause a significant upward shift in
the mobility of SH2-B
(Fig. 4, lower panel; Fig.
5A, lane 2; Fig. 5B, lane
9; Fig. 7, lane 8), consistent with SH2-B
being
phosphorylated in response to PDGF. The PDGF-BB-induced tyrosyl
phosphorylation and shift in mobility of SH2-B
were rapid (within 1 min), transient (Fig. 4), and dose-dependent (data not
shown), indicating that phosphorylation of SH2-B
is a tightly
regulated process. Interestingly, the greatest shift in SH2-B
mobility was observed after 5 min of 25 ng/ml PDGF (Fig. 4, lower
panel), while the tyrosyl phosphorylation of SH2-B
was not
maximal until 15 min (Fig. 4, upper panel). Similarly, the
mobility shift of SH2-B
was the greatest at a dose of 5 ng/ml for 15 min, but the tyrosyl phosphorylation was not maximal until 25 ng/ml
(data not shown). Because the multiple SH2-B
bands in control and
GH-treated 3T3-F442A cells have been shown to be differentially
phosphorylated forms of SH2-B
, this discrepancy between mobility
shift and
PY signal suggests that in addition to stimulating tyrosyl
phosphorylation, PDGF-BB also promotes the phosphorylation of SH2-B
on serine(s) and/or threonine(s). Curiously, the degree of tyrosyl
phosphorylation of SH2-B
measured using
PY Western blotting is
less than that of the PDGFR, which coimmunoprecipitates with SH2-B
(Figs. 4 and 5B). Although the reason for the lower signal
is not known, it may reflect: 1) fewer phosphorylated tyrosines in
SH2-B
compared with PDGFR (reported to be phosphorylated on at least
10 tyrosines (5, 16); 2) tyrosyl phosphorylation of only a subset of
that SH2-B
that binds to PDGFR; or 3) poorer recognition by the 4G10
antibody of phosphorylated tyrosines in SH2-B
compared with those in
PDGFR.
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PDGF-BB Promotes Phosphorylation of SH2-B at Multiple
Sites--
To provide evidence that the multiple proteins recognized
by
SH2-B in PDGF-treated cells reflect different phosphorylation states of SH2-B
,
SH2-B immunoprecipitates were treated with alkaline phosphatase in the presence or absence of sodium vanadate, an
inhibitor of alkaline phosphatase. As discussed above, PDGF-BB treatment decreased the migration of SH2-B
(Fig. 5A,
lane 2). Alkaline phosphatase treatment reduced the multiple
forms of SH2-B
observed in PDGF-BB-treated cells (Fig.
5A, lane 2) to a faster migrating form (Fig.
5A, lane 3). Simultaneously, the intensity of the
faster migrating form of SH2-B
increased significantly (Fig.
5A, lane 3), indicating a shift of SH2-B
from
slower to faster migrating forms. Sodium vanadate significantly reduced the effect of alkaline phosphatase on the PDGF-BB-induced mobility shift of SH2-B
(Fig. 5A, lane 4), indicating
that the change of migration of SH2-B
by alkaline phosphatase is due
to dephosphorylation.
Multiple Tyrosyl-phosphorylated Proteins Associate with SH2-B in
Response to PDGF--
A tight, tyrosyl-phosphorylated protein band
designated p84 coimmunoprecipitated with SH2-B
(Figs. 4,
5B, 6, and 7). Interestingly, p84 aligns with neither of the
two forms of SH2-B
after dephosphorylation by PP2A (Fig.
5B, lanes 5, 10, and 11),
suggesting that p84 is an SH2-B
-interacting protein and not a form
of SH2-B
itself. PDGF stimulation increased the amount of
tyrosyl-phosphorylated p84 associated with SH2-B
(Fig. 4,
upper panel; Fig. 6, lane 4). p84 does not appear
to associate with SH2-B
via PDGFR because
PDGFR did not
immunoprecipitate p84 (Fig. 6, lane 2). Furthermore, when
SH2-B immunoprecipitates were first dissociated by boiling in
SDS-containing buffer, and then re-immunoprecipitated with
SH2-B,
p84, like PDGFR, did not coimmunoprecipitate with SH2-B
(Fig. 6,
lane 5). These data indicate that
SH2-B does not
cross-react with either PDGFR or p84, and that both PDGFR and p84
associate with SH2-B
in cells stimulated with PDGF. In addition to
PDGFR, SH2-B
, and p84, multiple other tyrosyl-phosphorylated
proteins were present in
SH2-B immunoprecipitates when cells were
treated with PDGF-BB (Fig. 4, lanes 2-6, upper
panel; Fig. 6, lane 4). Because tyrosyl-phosphorylated
proteins of similar size are also precipitated by
PDGFR and PDGFR
coimmunoprecipitates with SH2-B
, it is not clear whether these other
phosphoproteins associate with SH2-B
directly or indirectly through
their interaction with SH2-B
-bound PDGFR. It is also unclear whether
PDGF stimulates the association of p84 and/or these other
phosphoproteins with SH2-B
or SH2-B
constitutively associates
with these proteins and PDGF stimulates their tyrosyl
phosphorylation.
PDGF-AA and Epidermal Growth Factor (EGF) Stimulate Phosphorylation
and a Shift in Mobility of SH2-B--
As PDGF-BB is able to
activate both
and
subunits of PDGFR, it is not clear which
subunits in 3T3-F442A fibroblasts recruit and phosphorylate SH2-B
in
response to PDGF-BB. To begin to dissect which subunit of PDGFR
utilizes SH2-B
, 3T3-F442A cells were treated with PDGF-AA (which
activates only the
subunit of PDGFR; Ref. 17), and solubilized
proteins were immunoprecipitated with
SH2-B and immunoblotted with
PY. The extent of ligand-induced tyrosyl phosphorylation of SH2-B
and the multiple other proteins including PDGFR that
coimmunoprecipitate with SH2-B
was similar between cells stimulated
with PDGF-AA and PDGF-BB (Fig. 7, lanes 2 and 3),
with PDGF-AA being a little less effective than PDGF-BB at the same
dosage. PDGF-AA, like PDGF-BB, caused a decrease in SH2-B
mobility
(Fig. 7, lanes 7 and 8). These data suggest that
the
subunit of PDGFR recruits SH2-B
as a signaling protein in
3T3-F442A cells. Whether SH2-B
also associates with PDGFR
remains
to be determined.
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DISCUSSION |
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In the current study, we show that SH2-B binds directly to
tyrosyl-phosphorylated, but not unphosphorylated, PDGFR in both GST
fusion protein pull-down and Far Western blotting assays. The SH2
domain of SH2-B
is sufficient for binding to PDGFR in the GST fusion
protein pull-down assay. Furthermore, when the conserved Arg was
mutated to Glu within the FLVRQS motif in the SH2 domain of SH2-B
,
the ability of mutant SH2-B
to bind PDGFR was dramatically
inhibited. These results suggest that the SH2 domain of SH2-B
is
both necessary and sufficient for binding to tyrosyl-phosphorylated,
activated PDGFR. The ligand-dependent interaction of
SH2-B
with PDGFR was further confirmed by the coimmunoprecipitation
of endogenous SH2-B
with endogenous PDGFR in both 3T3-F442A and
NIH3T3 cells.
The finding that there is an upward shift in mobility of SH2-B upon
PDGF-BB treatment that is abolished by alkaline phosphatase provides
clear evidence that PDGF promotes phosphorylation of SH2-B
.
Similarly, blotting with
PY provides strong evidence that PDGF
promotes tyrosyl phosphorylation of SH2-B
. The fact that PP2A
condenses the broad SH2-B
band to two faster migrating bands
suggests that at least two tyrosines are phosphorylated. Because
SH2-B
binds directly to activated PDGFR, it is logical to
hypothesize that SH2-B
is phosphorylated directly by PDGFR. In
support of this, when coexpressed in COS cells, SH2-B
is
tyrosyl-phosphorylated by PDGFR
subunit.2
The fact that PP2A increases the migration of SH2-B in control and
PDGF-treated cells suggests that SH2-B
is phosphorylated on serines
and/or threonines. In support of PDGF and/or EGF stimulating the
serine/threonine phosphorylation of SH2-B
, there is a discrepancy between changes in SH2-B
mobility and amount of
PY binding to SH2-B
. A maximal decrease in mobility of SH2-B
occurs at shorter times and at lower PDGF-BB concentrations than the maximal increase in
tyrosyl phosphorylation as detected by
PY. In the extreme case of
EGF, no signal is detectable by
PY but a significant decrease in
SH2-B
mobility is observed. In addition, PDGF-BB stimulates a
greater decrease in SH2-B
mobility than GH, but is much less
effective than GH at stimulating tyrosyl phosphorylation of SH2-B
.
Although we favor the hypothesis that PDGF and EGF stimulate the
serine/threonine phosphorylation of SH2-B
, our data do not exclude
the possibility that SH2-B
is constitutively phosphorylated on
serines/threonines and that EGF, PDGF, and GH stimulate the
phosphorylation of different tyrosines on SH2-B
. However, one would
have to hypothesize that those tyrosines phosphorylated by EGF receptor
are not recognized by the
PY used in this study, that those
phosphorylated by PDGFR include some that are recognized by
PY and
some that are not, and that those phosphorylated in response to GH bind
PY with high affinity. It would not be surprising for SH2-B
to be
phosphorylated on multiple serines/threonines because sequence analysis
reveals that SH2-B
has 82 serines and 26 threonines, including
multiple potential phosphorylation sites for protein kinase C and a
potential site (PLSP) for mitogen-activated protein kinases
(e.g. ERK1/2). Protein kinase Cs and/or ERKs are potential
candidates for PDGF-induced serine/threonine phosphorylation of
SH2-B
, because PDGF is reported to activate multiple isoforms of
protein kinase C (18-22) and ERKs 1 and 2 (23).3
We observed a tyrosyl-phosphorylated protein with
Mr ~ 84,000 (p84) coimmunoprecipitating with
SH2-B in PDGF-stimulated cells. When the
SH2-B immunocomplex was
dissociated by boiling in SDS-containing buffer, p84 was no longer
immunoprecipitated by
SH2-B, suggesting that
SH2-B interacts with
p84 indirectly through SH2-B
rather than directly binding to p84.
p84 does not coimmunoprecipitate with PDGFR, suggesting that the
interaction of SH2-B
with p84 is not mediated by PDGFR. The identity
of p84 is not known. It is unlikely that p84 is the p85 subunit of
phosphatidylinositol 3'-kinase because p84 is not recognized by
anti-p85 in immunoblots (data not shown). Interestingly, when
SH2-B
raised from a different rabbit (rabbit 2) was used to immunoprecipitate
SH2-B
, another tyrosyl-phosphorylated protein with
Mr ~ 145,000 (p145) was observed in
SH2-B
immunoprecipitates only from PDGF-stimulated cells (data not shown). We
therefore believe that SH2-B
interacts with multiple proteins
besides PDGFR, as expected for an adapter protein involved in PDGFR
signaling. SH2-B
thereby may actively regulate PDGFR signaling by
initiating some as yet unidentified pathways.
PDGF-induced phosphorylation of SH2-B may play a significant
role in PDGFR signaling. The phosphorylated tyrosines in SH2-B
may
form docking sites for other signaling molecules which contain SH2 or
phosphotyrosine binding domains, which may include p84 and p145 as
discussed above. The significance of serine and/or threonine
phosphorylation of SH2-B
is unclear. Phosphoserine(s)/threonines in
SH2-B
could serve as a binding site for other signaling molecules such as 14-3-3 (24-29). Serine/threonine phosphorylation of SH2-B
could also inhibit tyrosine phosphorylation of SH2-B
, as reported for insulin receptor substrate-1 (30, 31), or affect the association of
SH2-B
with other signaling molecules, as reported for Sos association with Grb2 (11, 12, 32, 33).
Two isoforms of SH2-B, designated SH2-B and SH2-B
, have been
described to date (1, 34). SH2-B, along with Lnk and APS, are proposed
to form a new adapter family (35). Lnk, with an SH2 domain 68%
identical to SH2-B, is expressed preferentially in lymphoid tissues and
has been shown to bind to phosphatidylinositol 3'-kinase, Grb2, and
phospholipase C
(36). APS, with a PH domain 58% identical to that
of SH2-B and an SH2 domain 80% identical to that of SH2-B, was cloned
as a binding protein for the kinase domain of c-Kit receptor and is
predicted to play a role in B cell antigen receptor activation (35). As
the SH2 domain of SH2-B
, which is highly conserved among SH2-B
,
Lnk and APS, mediates the interaction between SH2-B
and PDGFR, we
predict that Lnk, APS, SH2-B
, or their homologues also bind to
activated PDGFR and serve as signaling molecules for PDGFR in those
cells that express both PDGFR and the SH2-B-related proteins.
In summary, we have shown that in response to PDGF, SH2-B is
recruited onto PDGFR complexes via direct interaction with PDGFR, and
is tyrosyl-phosphorylated. SH2-B
is also phosphorylated on serines/threonines. Serine/threonine phosphorylation of SH2-B
appears to be increased by PDGF and EGF stimulation. The SH2 domain of
SH2-B
is required and sufficient for the interaction of SH2-B
with tyrosyl-phosphorylated PDGFR. As a consequence of association of
SH2-B
with PDGFR, signaling molecules bound to SH2-B
such as p84
are also recruited by PDGFR. We conclude that SH2-B
is a previously
unknown signaling molecule for PDGF signaling. It will be interesting
to determine whether SH2-B
mediates some of the actions of PDGF that
cannot be accounted for by previously identified PDGF signaling
molecules.
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ACKNOWLEDGEMENTS |
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We thank M. R. Stofega and Drs. J. B. Herrington, L. S. Argetsinger, and J. A. VanderKuur for their helpful suggestions. We thank P. Du for technical assistance and B. Hawkins for assistance with the manuscript. Oligonucleotide synthesis was performed by the Biomedical Research Core Facilities, University of Michigan, supported in part by grants to the Cancer Center, Michigan Diabetes Research and Training Center (P60-DK-20572), and UM-MAC (P60-AR20557).
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant DK 34171 (to C. C.-S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Recipient of a predoctoral fellowship and a Distinguished Research
Partnership Fellowship from the Rackham School of Graduate Studies,
University of Michigan.
§ To whom correspondence should be addressed: Dept. of Physiology, The University of Michigan Medical School, Ann Arbor, MI 48109-0622. Fax: 734-647-9523; E-mail: cartersu{at}umich.edu.
The abbreviations used are: SH, Src homology; PDGF, platelet-derived growth factor; GH, growth hormone; EGF, epidermal growth factor; PDGFR, platelet-derived growth factor receptor; PP2A, protein phosphatase 2A; GST, glutathione S-transferasePAGE, polyacrylamide gel electrophoresisERK, extracellular signal regulated kinase.
2 L. Rui, A. Kazlauskas, and C. Carter-Su, unpublished data.
3 L. Rui and C. Carter-Su, unpublished data.
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REFERENCES |
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