(Received for publication, June 7, 1995; and in revised form, September 21, 1995)
From the
Engagement of many cell surface receptors results in tyrosine
phosphorylation of an overlapping set of protein substrates. Some
proteins, such as the adaptor protein Shc, and a frequently observed
Shc-associated protein, p145, are common substrates in a variety of
receptor signaling pathways and are thus of special interest.
Tyrosine-phosphorylated Shc and p145 coprecipitated with anti-Shc
antibodies following B cell antigen receptor (BCR) cross-linking or
interleukin-4 (IL-4) receptor activation in B cells, and after
lipopolysaccharide (LPS) treatment or IgG Fc receptor (FcR)
cross-linking in macrophages. In the case of BCR stimulation, we have
shown that this represented the formation of an inducible complex.
Furthermore, in response to LPS activation or Fc
R cross-linking of
macrophages and BCR cross-linking (but not IL-4 treatment) of B cells,
we observed a similar tyrosine-phosphorylated p145 protein associated
with the tyrosine kinase Syk. We did not detect any Shc associated with
Syk, indicating that a trimolecular complex of Shc, Syk, and p145 was
not formed in significant amounts. By several criteria, the
Syk-associated p145 was very likely the same protein as the previously
identified Shc-associated p145. The Syk-associated p145 and the
Shc-associated p145 exhibited identical mobility by SDS-polyacrylamide
gel electrophoresis and identical patterns of induced tyrosine
phosphorylation. The p145 protein that coprecipitated with either Shc
or Syk bound to a GST-Shc fusion protein. In addition, a monoclonal
antibody developed against Shc-associated p145 also immunoblotted the
Syk-associated p145. The observations that p145 associated with both
Shc and Syk proteins, in response to stimulation of a variety of
receptors, suggest that it plays an important role in coordinating
early signaling events.
Receptors for antigens, cytokines, and growth factors utilize tyrosine phosphorylation of proteins to initiate and propagate intracellular events that result in cellular responses. As many of these receptors lack intrinsic tyrosine kinase activity, this increase in cellular phosphorylation can result from the recruitment and activation of cytoplasmic tyrosine kinases including Syk, ZAP-70, Src family tyrosine kinases(1) , and JAK family tyrosine kinases(2) . Targets of these kinases include enzymes that generate second messengers, regulators of Ras and other Ras-like G proteins, transcription factors, and a variety of other proteins that are believed to play a role in receptor signaling(3) .
Recently, attention has focused on the Shc protein, which is a
ubiquitously expressed adaptor protein that is tyrosine-phosphorylated
following stimulation of B or T lymphocyte antigen receptors (BCR ()and TCR)(4, 5, 6) , growth
factor receptors(7) , and cytokine
receptors(8, 9, 10, 11) . The SHC1 gene encodes the two major Shc isoforms,
p52
and p46
. These two
proteins are produced by utilization of two in-frame translation
initiation sites. Each Shc isoform contains a C-terminal SH2 domain, a
proline-rich central domain with multiple collagen-
1-type repeats,
and at the N terminus, a recently identified phosphotyrosine
interaction domain(12, 13, 14, 15) .
All of these domains are likely to be important for mediating
protein-protein interactions(12) . Phosphorylation at Tyr-317
of the central domain of Shc directs binding of the Grb-2
SOS-1
complex to Shc via the SH2 domain of Grb-2(16) . By virtue of
this association, Shc has been implicated in Ras activation. The
localization of SOS-1 to the plasma membrane is necessary for the
activation of Ras(17) . Although Shc is not itself a membrane
protein, it can bind to tyrosine-phosphorylated activated IL-2
receptor(18) , erythropoeitin receptor(10) , or
TCR(5) . Presumably this binding, coupled with Grb-2 binding to
phosphorylated Shc, would direct Grb-2
SOS complexes to the
membrane following receptor activation. Other reports have implicated
Shc in Ras activation in non-hematopoietic cells as
well(19, 20) .
In different cell types activated by
a variety of stimuli, Shc also associates with a highly
tyrosine-phosphorylated protein of approximately 145
kDa(4, 11, 21, 22) . Depending upon
the cell type analyzed, this protein has been referred to as p140,
p145, or p150. It can appear as a single band or as several closely
spaced bands. These Shc-associated proteins may well be the same
protein or very similar isoforms of the same protein. In this report,
we used BCR-stimulated B lymphocytes as a model system to demonstrate
that the p145Shc association is an inducible event and that
complex formation and maintenance correlates with levels of tyrosine
phosphorylation of these proteins. In examining responses via other
signaling receptors, we found association of tyrosine-phosphorylated
p145 with Shc in response to IL-4 treatment of B lymphocytes and
Fc
R cross-linking or LPS treatment of macrophages. To address
which tyrosine kinases may phosphorylate Shc and p145, we looked for
direct association of these substrates with specific kinases. We found
that Syk kinase coprecipitated with the p145 protein following most but
not all of the same stimuli that induced p145 to associate with Shc.
These observations suggest that p145 may have an important role in
coordinating signaling pathways from a number of receptors.
Immunoprecipitations, SDS-PAGE, and immunoblotting were carried out as described previously(26) . Briefly, lysates were precleared by incubation with 25 µl of protein A-Sepharose for 1 h at 4 °C. Precleared lysates were incubated with anti-Shc polyclonal antibody, used at a concentration of 1 µg of antibody/mg of cell lysate protein. Anti-Syk polyclonal antiserum was used at a concentration of 5 µl of antiserum/mg of cell lysate protein. For anti-phosphotyrosine immunoblotting, 4G10 hybridoma supernatant was used at a 1:5 dilution. Alternatively, anti-Shc monoclonal antibody was used at 1 µg/ml or anti-Syk antiserum was used at a 1:1000 dilution. Blots were incubated with HRP-conjugated secondary antibody (1 µg/ml in TBST) for 30-60 min. Bands were visualized using the Renaissance chemiluminecence detection system (DuPont).
In order to better understand the nature
of this complex and, hence, its role in B cell responses, it was
important to determine first whether Shcp145 was present as a
complex in unstimulated B cells or whether the association of p145 and
Shc required stimulation of the cells. Therefore, we immunoprecipitated
Shc from either resting or BCR-stimulated
S-labeled cells (Fig. 1A). Biosynthetically labeled p145 only
coprecipitated with Shc after cell stimulation, demonstrating that the
association between these proteins was inducible. Other Shc-associated
proteins were also observed in this way. The prominent 70-kDa species
visible only in stimulated lysates was detected by anti-mouse Ig
immunoblotting and therefore is likely to be endogenous immunoglobulin
µ heavy chain that was immunoprecipitated by the stimulating
antibody. In addition to p145, proteins of 40, 45, 100, and 110 kDa
coprecipitated with Shc at 15 and 30 min following stimulation. The
identity of these bands was unknown, and none were recognized by
anti-phosphotyrosine antibodies. However, anti-phosphotyrosine
immunoblotting of the same samples showed the prominent doublet of
Shc-associated tyrosine-phosphorylated proteins at 140 and 145 kDa (Fig. 1B). In addition, a weaker
tyrosine-phosphorylated band of 150 kDa was sometimes seen. To confirm
that the inducibly associated 140- and 145-kDa proteins were indeed the
tyrosine-phosphorylated species observed, the
S-labeled
Shc immunoprecipitates were dissociated by treatment with 2% SDS and
then reimmunoprecipitated with anti-phosphotyrosine antibody. The
metabolically labeled protein doublet at 140-145 kDa was
reprecipitated in this way (data not shown), indicating that these
bands corresponded to the p145 tyrosine-phosphorylated proteins. We
believe that these bands were all isoforms of the same protein as each
of these proteins exhibited the same properties in all of the
experiments reported here. The nature of the changes that caused
differential migration are not known at this time. These bands retained
their different mobilities after de-phosphorylation with calf
intestinal phosphatase (data not shown), suggesting that some property
other than phosphorylation was responsible for the differential
mobility of these bands.
Figure 1:
Activation-induced
association of a 145-kDa, tyrosine-phosphorylated protein with Shc in B
cells. WEHI-231 cells (5 10
cells/point) were
labeled with [
S]methionine and
[
S]cysteine for 4 h. Cells were stimulated for
the indicated times during cell labeling such that all time points were
harvested after 4 h of metabolic labeling. Shc immunoprecipitates were
resolved by SDS-PAGE, transferred to nitrocellulose, exposed to film (A), and subsequently immunoblotted with anti-phosphotyrosine
antibody (B). The position of the
S-labeled p145
doublet (p140, p145) is indicated in A, and the positions of
the tyrosine-phosphorylated Shc and p145 doublet proteins are indicated
in B. The Shc proteins were not readily apparent in these
labeling experiments (A), although they can be detected by
anti-Shc immunoblotting. The anti-phosphotyrosine immunoblotting
signals were comparable to those from unlabeled cells. In panel
B, the band below p52
was also seen with
protein A-Sepharose and the sheep anti-mouse Ig secondary reagent and
hence is not specifically immunoprecipitated or
tyrosine-phosphorylated. The p46 form of Shc is concealed by this
background band. The migration positions of molecular weight markers
are indicated.
Figure 2: A complex of Syk with p145 but not Shc. Lysates from WEHI-231 cells stimulated through the antigen receptor (as in Fig. 1) were subjected to immunoprecipitations using anti-Shc or anti-Syk polyclonal antiserum (A). Shc complexes were isolated from 2 mg of lysate proteins, whereas 4 mg of lysate proteins were used for each of the anti-Syk immunoprecipitations. Isolated proteins were separated by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with anti-phosphotyrosine antibody. The positions of Syk (72 kDa), Shc, and associated p145 are indicated. In addition, Shc and Syk immunoprecipitates from stimulated and unstimulated WEHI-231 cells were resolved by SDS-PAGE (B) and the possible presence of a coprecipitating component was assessed by immunoblotting in parallel with polyclonal anti-Syk antibodies (upper section) or monoclonal anti-Shc antibodies (lower section).
The
p145 protein associated with Syk appeared to be the same as the p145
associated with Shc both by electrophoretic mobility and pattern of
anti-phosphotyrosine blotting. To determine in a more direct manner
whether these tyrosine-phosphorylated p145 proteins were indeed the
same protein, we examined whether Shc could associate with p145 protein
that was isolated by virtue of its association with Syk. For this
purpose, we made use of previous observations that recombinant proteins
containing full-length Shc fused to GST (GST-Shc) can interact with
Shc-associated p145 resolved by SDS-PAGE and transferred to
nitrocellulose(13) . Shc or Syk immunoprecipitates containing
either Shc-associated or Syk-associated p145 respectively were resolved
on SDS-PAGE, transferred to nitrocellulose, and probed with P-labeled GST-Shc proteins. As previously reported,
GST-Shc proteins associated with p145 that had been coprecipitated with
anti-Shc antibodies. Importantly, GST-Shc also associated with p145
that had been coprecipitated with anti-Syk antibodies (Fig. 3A). The signal associated with Syk-associated
p145 was comparable to that seen with Shc-associated p145. This
observation provides evidence that the p145 proteins that associated
with Syk and with Shc were the same.
Figure 3:
Syk-associated p145 can bind to Shc.
Lysates from anti-IgM-stimulated or unstimulated BalI7 cells
were subjected to immunoprecipitation with Shc- or Syk-specific
antibodies (2 10
or 13
10
cells/point, respectively), resolved by SDS-PAGE, and transferred
to a nitrocellulose membrane. Membranes were incubated with
P-labeled GST-Shc fusion proteins, washed, and exposed to
film for 11 h (A). After exposure, the same membranes were
stripped, blocked, and immunoblotted with anti-phosphotyrosine antibody (B). Note that lysate from more cells was used for the
anti-Syk immunoprecipitation to achieve similar amounts of
tyrosine-phoshorylated p145 on the filters.
Finally, the Shc-associated and Syk-associated p145 proteins were immunologically related. Hybridomas were generated from spleen cells of mice immunized with purified, Shc-associated p145. Monoclonal antibodies were selected on the basis of reactivity with Shc-associated p145 by immunoblotting. One monoclonal antibody, 4U2, reacted in an immunoblot with both the Shc-associated, and Syk-associated p140, p145, and p150 phosphotyrosine containing proteins (Fig. 4). This monoclonal also reacted with a protein of 165 kDa, which did not appear to be tyrosine-phosphorylated. The identity of this protein is not known. The anti-p145 reactivity did not represent anti-phosphotyrosine immunoreactivity since this monoclonal antibody still immunoblotted p145 that was dephosphorylated by calf intestinal phosphatase (data not shown).
Figure 4:
Monoclonal anti-Shc-associated p145
antibody immunoblots both Shc- and Syk-associated p145. Shc or Syk
immunoprecipitates from anti-IgM-stimulated WEHI-231 cell lysates were
separated by SDS-PAGE and transferred to nitrocellulose. Membranes were
immunoblotted with anti-phosphotyrosine (P-Y) antibody (3
10
cells/lane) or 4U2, an anti-p145 antibody (13
10
cells/lane) and reactions were detected by use
of an HRP-conjugated anti-mouse secondary antibody, which did not
produce any signal by itself (not shown).
As p145 had not been previously reported to coprecipitate
with Syk, it was important to demonstrate that the observed
coimmunoprecipitation of Syk and p145 in activated B cells was not due
to anti-p145 reactivity in the anti-Syk antiserum. To address this
issue, we made use of a chicken B cell line (S1.10) in which Syk is not
expressed because the syk genes had been disrupted by
homologous recombination (31) . The knockout cells responded
poorly to BCR stimulation, as previously reported, but they exhibited
appreciable tyrosine phosphorylation of cellular proteins following
treatment with the tyrosine phosphatase inhibitor pervanadate. In the
parental Syk-expressing chicken B cell line, DT40, pervanadate
treatment resulted in tyrosine phosphorylation of p145 and its
coimmunoprecipitation with Shc and to a lesser extent with Syk (Fig. 5). The p145 protein appeared as a single band in these
chicken cells, a pattern that has been reported previously in some
mammalian cell types(13, 21) . In the
pervanadate-treated syk cells (S1.10),
phosphorylated p145 was coprecipitated by the anti-Shc antiserum but
not by the anti-Syk antiserum (Fig. 5). Thus, anti-Syk antiserum
only immunoprecipitated p145 from cells expressing Syk protein,
demonstrating that the coprecipitation of the two proteins was not due
to a cross-reactivity of the anti-Syk antiserum with p145 or some other
p145-associated protein.
Figure 5:
Immunoprecipitation of p145 with anti-Syk
requires Syk expression. Chicken B cells DT40 (parental) and S1.10 (syk) were stimulated with or without added
pervanadate solution (400 µM) for 4 min. Since total
cellular phosphorylation was consistently lower in the syk
cells as compared to the parental B
cells, a greater amount of protein of the former was used for the
immunoprecipitations to give similar phosphotyrosine signals. Cells
were lysed and subjected to immunoprecipitation with anti-Shc antibody
(0.6
10
cells or 1.8
10
cells/point) or anti-Syk antibody (1.5
10
or
4.5
10
cells/point). Immunoprecipitates were
resolved by SDS-PAGE and then analyzed by anti-phosphotyrosine
immunoblotting. The migration positions of the tyrosine-phosphorylated
p145, Syk, and Shc proteins are indicated.
Figure 6: IL-4 receptor-induced tyrosine phosphorylation of Shc and p145. WEHI-231 cells were stimulated with medium(-), anti-IgM at 25 µg/ml for 3 min (BCR), or IL-4 at 100 units/ml for 5 or 10 min. Lysis, immunoprecipitation, and immunoblotting were performed as in Fig. 1. The position of the Shc and p145 proteins are indicated. Longer exposures revealed a p145 doublet induced by IL-4, although only one band of the doublet is visible in the data shown. IL-4 responses were maximum at 100 units/ml IL-4. Tyrosine phosphorylation induced by IL-4 was roughly equivalent to that induced through the antigen receptor using 1-3 µg/ml anti-IgM.
Figure 7:
A
145-kDa tyrosine-phosphorylated protein coimmunoprecipitates with Shc
and Syk in macrophages. RAW 264.7 cells (5 10
cells/ml) were exposed to LPS (5 µg/ml) for 5 min at 37
°C. Alternatively, cells stimulated through the Fc
R were
incubated on ice for 30 min (10
10
cells/ml) with
purified monoclonal antibody (2.4G2) against murine Fc
R (40
µg/ml). The latter cells were warmed to 37 °C and exposed to a
cross-linking mouse anti-rat Ig antibody (10 µg/ml). Anti-Shc and
anti-Syk immunoprecipitated complexes were analyzed by SDS-PAGE and
anti-phosphotyrosine immunoblotting. In panel A, Shc and Syk
complexes were isolated from 3 mg of untreated, LPS, or
cross-linked-Fc
R-stimulated cells. The positions of Syk, Shc, and
the p145 protein are indicated. In a separate experiment (B),
Shc and Syk proteins were immunoprecipitated from lysates of
FcR-stimulated cells. As in A, cells were incubated with
anti-FcR antibody on ice. After warming to 37 °C, half the cells
were exposed to cross-linking antibody (``+'') and half
were not (``-'').
Another potent stimulator of macrophages is
bacterial LPS. LPS treatment of macrophages induces them to produce
numerous cytokines and proinflammatory
mediators(39, 40) . LPS binding to the
glycosylphosphatidylinositol-linked protein CD14 leads to increased
tyrosine phosphorylation in macrophages, and this event is important
for downstream responses(41, 42, 43) .
Although CD14 lacks any cytoplasmic tail sequences for direct
association and activation of cytoplasmic protein kinases or signaling
proteins, LPS rapidly activates the Src family tyrosine kinases Lyn,
Hck, and Fgr in human monocytes suggesting an important role for these
tyrosine kinases in LPS responses(44, 45) .
Interestingly, RAW 264.7 cells stimulated with LPS exhibited induced
tyrosine phosphorylation of Shc and Shc-associated p145 (Fig. 7A). The induced tyrosine phosphorylation and
association of Shc and p145 was greater than that which occurred in
response to FcR cross-linking. Moreover, these were rapid events
in macrophages, being evident as early as 1 min and peaking at about 10
min after stimulation (data not shown). Unexpectedly, LPS signaling
also induced tyrosine phosphorylation of Syk and its association with
p145 (Fig. 7A). As was true for p145
Shc
association, the p145
Syk association (as detected by
anti-phosphotyrosine immunoblotting) was greater in response to LPS
than in response to Fc
R cross-linking. This is most likely due to
the relatively low level of Fc
R expression on these cells.
Cross-linking of higher levels of Fc
R expressed on other
macrophage lines elicited a signal equivalent to or greater than that
induced by LPS. Syk and Shc did not appear to associate in a
precipitable complex from Fc
R- or LPS- stimulated macrophages,
similar to the situation with BCR-stimulated B cells (data not shown).
Although it has been reported that Syk becomes tyrosine-phosphorylated
in response to Fc
R cross-linking and is important in
phagocytosis(46, 47) , a role for Syk in LPS receptor
signaling in macrophages had not been previously suggested. These
observations were particularly interesting, as few downstream targets
of tyrosine kinases in LPS-activated macrophages have been identified.
Aside from the observations regarding Syk and Shc reported here, the
only other reported early substrate for tyrosine kinases activated by
LPS is the proto-oncogene product Vav(48) . Further
characterization of the role of Shc and Syk in LPS-induced signaling in
macrophages is currently under way and will be the subject of another
report.
In agreement with previous reports(4, 49) ,
we have found that BCR stimulation of B lymphocytes leads to tyrosine
phosphorylation of Shc and appearance of a tyrosine-phosphorylated
protein doublet of apparent molecular weight 145,000 in Shc
immunoprecipitates. We have additionally found that this association
between Shc and p145 was induced upon BCR signaling. The quantity of
Shcp145 complexes in BCR-stimulated B cells decreased
coincidently with the amount of tyrosine phosphorylation of the
immunoprecipitated Shc and p145. These observations suggest that
tyrosine phosphorylation of p145 and/or Shc was important for complex
formation and maintenance. Kavanaugh et al.(13) found
that recombinant Shc protein expressed in insect cells could associate
with phosphorylated but not with dephosphorylated p145 immobilized on
nitrocellulose membranes. Taken together with these results, our
findings suggest that BCR engagement induced the tyrosine
phosphorylation of p145 and this in turn led to Shc binding.
The
mechanisms by which activation of the BCR-induced tyrosine
phosphorylation of Shc and p145 are not known. One possibility is that
intracellular tyrosine kinases activated by the BCR interacted directly
with these signaling components. For example, Lyn and Fyn have been
shown to associate with phosphatidylinositol 3-kinase in some
situations where the latter component becomes
activated(50, 51) . In addition to Src family tyrosine
kinases, Syk and the highly related ZAP-70 tyrosine kinases play
critical roles in signaling by antigen receptors(52) , and by
FcRI and Fc
R(46, 53) . Upon
immunoprecipitation of Lyn and Syk from BCR-stimulated B cells, we
found tyrosine-phosphorylated p145 associated with Syk but not Lyn. In
contrast, we did not detect any association of Shc with Syk. Recently,
an interaction between Syk and Shc was detected following
overexpression of Syk in B cells(54) . It could be that a very
small amount of Shc
Syk complex did form in vivo in our
BCR-stimulated B cells but was below our limit of detection. Such a
small amount of Shc would not be sufficient to account for the
prominent association of p145 with Syk, however, so p145
Syk
association must either be direct or mediated via an association of
both Syk and p145 with a protein or proteins other than Shc. In any
case, the association of p145 with Syk is provocative and may reflect a
role for Syk in phosphorylating p145 and possibly Shc as well.
The Syk-associated 145-kDa doublet appeared to be the same as the Shc-associated p145 doublet by several criteria. First, the distinctive pattern of tyrosine phosphorylation of the Shc-associated p145 was identical to that produced by the Syk-associated p145. In murine cells, the upper band was a more highly phosphorylated and more abundant protein than the lower band. In chicken B cells, the banding pattern was different from that found in mammalian B cells, yet the Syk-associated p145 again resembled the Shc-associated p145. Second, the association of p145 with Syk or Shc occurred with similar kinetics (data not shown). Third, murine GST-Shc protein was able to bind Shc-associated p145 or Syk-associated p145 immunoprecipitated from both murine and chicken cells and immobilized on nitrocellulose. And finally, a monoclonal antibody specific for Shc-associated p145 was reactive with Syk-associated p145 protein as well. Thus, the Syk-associated p145 and the Shc-associated p145 behaved identically in many respects, strongly suggesting their identity.
The tyrosine
phosphorylation of Shc and the association of Shc with
tyrosine-phosphorylated p145 were also seen in B cells stimulated
through the IL-4 receptor, a member of the cytokine/hematopoietic
receptor superfamily, and in macrophages stimulated either through
FcRs or by LPS. We observed that Fc
R cross-linking or LPS
stimulation of macrophages also induced tyrosine phosphorylation of Syk
and its association with p145. IL-4 stimulation of B cells did not lead
to Syk phosphorylation or its association with tyrosine-phosphorylated
p145. Thus, association of p145 with Syk correlated with the tyrosine
phosphorylation of Syk and not with the tyrosine phosphorylation of
p145. While the mechanism by which p145 and Syk associate with each
other remains to be determined, these results suggest that Syk may not
associate via its SH2 domains with p145 or that the SH2 domains of Syk
are inaccessible when the kinase is inactive. In support of the first
suggestion, we have not detected an interaction between a
GST-Syk(SH2)
fusion protein and p145 immobilized on
membranes. (
)
The Sykp145 association observed in
response to BCR-, Fc
R- and LPS-mediated activation suggests a role
for Syk in phosphorylating either p145 or Shc. If that is the case then
the IL-4 receptor presumably utilizes a different mechanism for
phosphorylating p145 and Shc. Additionally, our results comparing BCR
cross-linking and IL-4 receptor signaling in B cells indicated that
different p145 associations were induced by kinases activated through
different receptors within the same cell. This result raises the
possibility that phosphorylated p145, although it is an early substrate
of tyrosine kinases, may function in separate and distinct signaling
pathways depending on the nature of the signaling receptor.
The
functional significance of the p145Shc and p145
Syk
complexes are not yet known, although several possibilities can be
considered. These complexes appeared to be primarily membrane-bound in
BCR-stimulated B cells. (
)Association with p145 might
concentrate Shc in a particular location in the cell, such as the
plasma membrane. Thus, p145 could function to bring Shc to the membrane
where its subsequent tyrosine phosphorylation and association with
Grb-2
SOS-1 complex could promote Ras activation. In agreement
with this idea, Saxton et al.(4) have shown that
Shc
p145 complexes are present in cytosolic and membrane fractions
of cells and that BCR stimulation resulted in elevated levels of Shc
complexes in the membrane-bound fraction. It is unclear whether p145 or
some other component is responsible for this membrane localization.
Moreover, studies in other cell types have given different results
regarding the molecular nature of the interactions between Shc, p145,
and Grb-2, the intracellular localization of these complexes, and
whether these proteins are within a single
complex(4, 11, 21, 22) . It is
unclear at this time whether these differences are due to the different
experimental approaches utilized or, as reflected in our own results
regarding Syk
p145 and Shc
p145 complexes, whether the exact
nature of the complex may vary with the cell type and route of
stimulation.
It is possible that p145 membrane localization is due
to its association with tyrosine-phosphorylated receptor cytoplasmic
domains, as has been reported for Syk. In this model, the
coprecipitation of Syk and p145 would reflect a trimolecular complex
consisting of phosphorylated receptor tails interacting with both p145
and Syk. Interestingly, it has recently been reported that stimulation
of the FcRI on mast cells resulted in coimmunoprecipitation of a
tyrosine-phosphorylated protein of 145 kDa with the Fc
RI
chain (53) . In this system, a GST-Syk(SH2)
fusion
protein was used to precipitate not only the
and
chains of
the Fc
RI, but also a tyrosine-phosphorylated doublet resembling
p145 (53, 55) . These observations may reflect
associations of GST-Syk(SH2)
and p145 with the Fc
RI
chain and
chain cytoplasmic domains. Interestingly, we have
observed a small amount of p145 protein to coprecipitate with Ig-
from lysates of BCR-stimulated B cells.
Thus, one possible
scenario is that BCR and Fc
R stimulation leads to p145 association
with tyrosine-phosphorylated receptor tails, followed by tyrosine
phosphorylation of p145, dissociation of Syk, and subsequent
association of Shc with p145. This could be a means whereby Shc would
achieve recruitment to the membrane and subsequent phosphorylation on
Tyr-317. Phosphorylated Shc could then be bound by the Grb-2(SH2)
domain of a Grb-2
SOS-1 complex resulting in activation of Ras at
the membrane. The exact nature of the interactions between Syk,
ITAM-containing receptor chains and p145 and whether this association
is used as a mechanism for Ras activation remain to be determined.
The observations reported here suggest that p145 may be an important
signaling component. The membrane localization of p145 and its
association with Shc in B cells stimulated through the BCR are
consistent with p145 playing an important role in Ras activation. It is
equally possible that p145 is a signaling effector that is regulated by
Shc and therefore represents a non-Ras Shc-signaling pathway. Two of
the receptors that induced Shc tyrosine phosphorylation and association
with p145, IL-4R in B cells and FcR in macrophages, have not been
reported to result in Ras activation. LPS may activate Ras in monocytes
and macrophages, although this has not been uniformly observed to date (56, 57) . Thus the downstream consequences of Shc and
p145 tyrosine phosphorylation in B cells or in macrophages remains
unknown. The cloning of the gene for p145 and subsequent
characterization of its primary sequence may shed light on these
issues. In any case, the ability of p145 to interact with the
cytoplasmic signaling components Shc and Syk suggests that it plays an
important role in the initiation of signaling events in activated B
cells and macrophages.