(Received for publication, September 12, 1996, and in revised form, November 8, 1996)
From the The c-fps/fes proto-oncogene encodes
a 92-kDa protein-tyrosine kinase that is expressed at high levels in
macrophages. We have previously shown that overexpression of
c-fps/fes in a CSF-1-dependent macrophage cell
line (BAC1.2F5) partially released these cells from their factor
dependence and that this correlated with the tyrosine phosphorylation
of a subset of proteins in a tissue-specific manner. We have now
identified one of the macrophage substrates of Fes as the
crk-associated substrate (Cas) and a second substrate as a
130-kDa protein that has been previously described as a T cell
activation-dependent substrate and is unrelated to Cas.
Both of these proteins, which have optimal consensus sequences for phosphorylation by Fes, were tightly associated with this kinase through its SH2 domain, suggesting that they were direct substrates of
Fes. Remarkably, when the Fes SH2 domain was used as an affinity reagent to identify potential substrates of endogenous Fes in control
BAC1.2F5 cells, the phosphotyrosyl proteins that were recognized were
the same as those that were specifically phosphorylated when Fes was
overexpressed in the same cells. We conclude that the substrates we
identified may be structurally related or identical to the
physiological targets of this kinase in macrophages. The known
functions of Cas and p130 suggest that Fes kinase may play a role in
signaling triggered by cell adhesion and cell-cell interactions during
immune responses of macrophages.
The c-fps/fes proto-oncogene encodes a non-receptor
protein-tyrosine kinase (p92c-fes) (1, 2) that has
been repeatedly transduced by RNA tumor viruses (3). In the adult,
c-fps/fes is preferentially expressed in hematopoietic cells
of the myeloid lineage and in endothelial cells (1, 4-6), whereas in
the embryo, a wider pattern of expression has been observed (7).
The tissue specificity of Fes expression has suggested that this kinase
may play a role in myelopoiesis or in specialized functions of myeloid
cells (1, 5, 6). This idea is supported by several reports describing
the involvement of Fes in cytokine receptor signaling (8, 9), in
myeloid differentiation (6, 10), and in inhibition of apoptosis during
granulocytic differentiation (11). However, the mechanism of action and
biological role of this kinase in its target tissues is not well
understood.
Since identification of the substrates of Fes kinase is one of the keys
to elucidate its biological role, efforts have been made to uncover
these substrates. One approach has been to express this kinase in
different cell types and to correlate biological activity with
phosphorylation of specific cellular targets. In established murine
fibroblasts, where Fes is normally not present, expression of this
kinase at high levels causes tumorigenic transformation (12, 13). This
is mediated by tyrosine phosphorylation or activation of several well
known mitogenic targets such as GTPase-activating protein and its
associated proteins p62 and p190 (12-14), Bcr/Grb2 (15), Shc/Grb2 (16,
17), and phosphatidylinositol (PI)1
3-kinase (18), suggesting that activation of the ras and PI 3-kinase pathways is involved in the mechanism of transformation by
Fes.
By contrast, the biological and biochemical activity of ectopically
expressed p92c-fes in macrophages, a cell type
where this kinase is normally expressed at the highest levels, was more
restricted. Fes expression in the CSF-1-dependent BAC1.2F5
cell line resulted in only partial relief of factor dependence, and Fes
phosphorylated a limited subset of proteins on tyrosine, including a
130-kDa (p130) and a 75-kDa (p75) protein, which have so far not been
implicated in major mitogenic pathways (19). The lack of a strong
proliferative effect of Fes in macrophages and the failure to
phosphorylate or activate targets frequently involved in mitogenic and
oncogenic signaling suggest that, in contrast to its effect on cell
proliferation in fibroblasts, in macrophages Fes may participate in a
specialized function of these cells.
In this paper we have identified Cas and p130 as two distinct
tissue-specific substrates of Fes in macrophages. Their known functions
suggest a possible role of Fes during signaling in response to cell
adhesion and interactions with other cells of the immune system.
The CSF-1-dependent murine macrophage
cell line BAC1.2F5 has been described (20). BAC1.2F5 cells and derived
subclones were maintained in The retroviral expression
vector pFF, which encodes human p92c-fes, has been
described (2, 12). The bacterial pGEX-3X vector (21), which allows the
expression of bacterial proteins as glutathione transferase (GST)
fusion proteins, was obtained from Pharmacia Biotech Inc. The GST-SH2
fusion construct encoding the SH2 domain of Fes has been described
(19).
pGEX-3X control
and pGEX-SH2 bacteria were induced with 0.1 mM
isopropyl-1-thio- Polyclonal and monoclonal antibodies to Fes
proteins have been described (2, 19). The rabbit polyclonal antiserum
directed against p130 has been described (22). Monoclonal antibodies directed against Cas were obtained from Transduction Laboratories (Lexington, KY), and rabbit polyclonal antibodies directed against Cas
were obtained from H. Hirai (Tokyo) (23), and from Amy Bouton (University of Virginnia, Charlottesville). Monoclonal antibodies directed against phosphotyrosine (4G10) were obtained from UBI (Lake
Placid, NY).
Preparation of cell lysates for protein analysis was
carried out as described (19) in a buffer containing 50 mM
Hepes-KOH, pH 7.4, 1% (v/v) Triton X-100, 150 mM NaCl, 2.5 mM EDTA, 10% (v/v) glycerol, 10 mM sodium
pyrophosphate, 100 mM NaF, 1 mM sodium orthovanadate, and 2% (v/v) Trasylol.
The analysis of proteins by immunoprecipitation, the in
vitro protein kinase assay, electrophoresis in 8.5% sodium
dodecyl sulfate-polyacrylamide gels (SDS-PAGE), and Western blot
analysis by enhanced chemiluminescence (Amersham Corp.) have been
described (1, 13, 19). For immunoprecipitation cell lysates containing 200 µg of protein were immunoprecipitated with the indicated
antisera, and the immunoprecipitates were collected on Protein
A-Sepharose (Pharmacia) as described (19). For adsorption to bacterial
fusion proteins, cell lysates containing 200 µg of protein were
incubated with 1-2 µg of bacterial protein immobilized on
glutathione-agarose for 2 h at 4 °C. Adsorbed pellets were
washed five times in cell lysis buffer and analyzed by SDS-PAGE,
followed by Western blot analysis using the indicated antibodies.
To identify potential physiological substrates of Fes in
macrophages, we have overexpressed this kinase in BAC1.2F5, a
CSF-1-dependent macrophage cell line (20) capable of
carrying out specialized immune functions such as activation in
response to bacterial lipopolysaccharide and interferon-
In BAC1.2F5 cells where Fes was ectopically expressed (BAC1-Fes), this
kinase was tightly associated with several tyrosine-phosphorylated proteins. The most prominent bands had apparent molecular masses of
130, 115, 95, and 65-70 kDa (Fig. 2, lane
1). To determine if the 130-kDa species was related to Cas, we
used specific anti-Cas antibodies. As shown in Fig. 2, lane
2, the anti-Cas antibody precipitated tyrosine-phosphorylated
Cas from lysates of BAC1-Fes cells, and direct immunoblotting of Fes
immunoprecipitates with anti-Cas antibody confirmed that Cas
coprecipitated with Fes (Fig. 2, lane 3). However, we were
surprised to find that anti-Cas antibodies did not recognize the major
130-kDa species (Fig. 2, lanes 2 and 4) and that
Cas and p130 had different electrophoretic mobilities, suggesting that
they were different proteins. p130 and Cas were not differentially
phosphorylated forms of the same protein: anti-Cas antibody
precipitated the different phosphorylated forms of Cas but not p130
(Fig. 2). Similar results were obtained using two other different
antibodies raised to bacterially expressed Cas (obtained from H. Hirai
and from A. Bouton) (data not shown). The phosphotyrosyl Cas protein in
BAC1-Fes cells had a higher electrophoretic mobility than the
phosphorylated Cas protein in v-src-transformed cells (data
not shown), which may reflect differences in the state of
phosphorylation of Cas in Fes-expressing macrophages and
v-src-transformed fibroblasts. Although the Cas product is often referred to as p130cas (23), in the present study we
refer to it as Cas to avoid confusion with the protein we call
p130.
We have previously shown that the SH2 domain of Fes had high
specificity for the substrates of this kinase and that it mediated binding of Fes to some of its substrates (19). To determine if the Fes
SH2 domain mediated the association of Fes with tyrosine-phosphorylated Cas, we incubated lysates of BAC1-Fes cells with a bacterial fusion protein encoding the Fes SH2 domain (GST-SH2), followed by immunoblot analysis of the adsorbed proteins with anti-Cas antibody. As shown in
Fig. 3, lanes 2 and 3, GST-SH2 but
not control GST vector protein was able to precipitate Cas, suggesting
that the tight association of Fes with phosphorylated Cas was mediated
through the Fes SH2 domain. This association and the presence in Cas of
two putative tyrosine phosphorylation sites (YDXV) (23) that
are close to the optimal consensus sequence for Fes phosphorylation and
binding to its SH2 domain (YEXV) suggest that Cas may be a
direct substrate of Fes in BAC1-Fes cells.
From these results we conclude that Cas or a closely related protein is
one of the substrates phosphorylated by Fes in BAC1-Fes cells and that
this substrate remains tightly associated with Fes kinase through the
Fes SH2 domain.
The results presented above
indicated that Cas was distinct from the phosphotyrosyl p130 protein we
originally described (19). Therefore we sought to identify p130 using
antisera to known signaling proteins in the same molecular weight
range. One of the candidates we tested was a 120-130-kDa protein that
is selectively expressed in lymphoid and myeloid cells and becomes
tyrosine-phosphorylated during T cell activation (22). Like our p130
substrate, this T cell protein was also recognized by 4F4 antibody
(28), but the use of specific polyclonal antibodies raised against
purified p120/130 has recently revealed that p120/130 was unrelated to Cas (22). In addition, DNA sequence analysis of cloned p120/130 cDNA confirmed that this protein had no homology to Cas or any other protein in the data bank.2 Therefore,
we used this specific anti-p120/130 antiserum to determine if the
120-130-kDa T cell protein and the p130 substrate of Fes were related
proteins. As shown in Fig. 4, anti-p120/130 precipitated a prominent tyrosine-phosphorylated 130-kDa protein from lysates of
BAC1-Fes cells, which co-migrated with the phosphotyrosyl p130 present
in anti-Fes immunoprecipitates (Fig. 4). This suggested that the
120-130-kDa protein identified in T cells was the same or related to
the p130 protein phosphorylated in BAC1-Fes cells. Using specific
antibodies to other proteins in the same molecular weight range of p130
and Cas, we also determined that the
We then examined whether phosphotyrosyl p130 was recognized by the Fes
SH2 domain. Cell lysates from BAC1-Fes cells were adsorbed with
bacterially expressed GST-SH2 fusion protein, and the adsorbed proteins
were analyzed by anti-phosphotyrosine immunoblotting. GST-SH2 but not
control GST vector protein was able to precipitate several
tyrosine-phosphorylated proteins including p130 and p75 from cell
lysates of Fes overexpressing cells (Fig. 5, lanes
1 and 2). Immunoblotting of GST-SH2 precipitates with
anti-p120/130 antibody confirmed the identity of the phosphotyrosyl
p130 protein recognized by the Fes SH2 domain as p120/130 (Fig. 5,
lane 5). Fig. 5, lane 3, also shows that in
BAC1.2F5 cells, p130 consists of two closely migrating species.
We conclude that the 120-130-kDa substrate identified in T cells and
the p130 substrate of Fes are the same or related proteins and that
p130 and Cas are two different substrates of Fes in BAC1-Fes cells. DNA
sequence analysis of full-length p120/130 cDNA clones revealed the
presence of two phosphotyrosine motifs (YDDV) that are close to the
optimal consensus sequence for Fes phosphorylation,2 which
is consistent with the idea that p130 may be a direct substrate of Fes.
Further analysis will be required to identify the Fes tyrosine
phosphorylation sites in p130.
Since the Fes SH2 domain appears to have high
specificity for the substrates of this kinase (19), we reasoned that
this SH2 domain might have affinity for potential physiological targets of endogenous p92c-fes in control macrophages. To
determine if the Fes SH2 domain recognized any phosphotyrosyl proteins
in BAC1.2F5, cell lysates were adsorbed with GST-SH2 fusion protein,
and the adsorbed proteins were analyzed by anti-phosphotyrosine
immunoblotting. As shown in Fig. 6A, lanes 3 and 4, GST-SH2 but not GST recognized a number of
phosphotyrosyl proteins from control BAC1.2F5. Remarkably, these
phosphotyrosyl proteins had the same electrophoretic mobilities as
those that were identified as the major substrates of Fes in BAC1-Fes
cells (Fig. 6A, lanes 1, 2, 4, and 5).
Immunoblotting of the Fes-SH2-adsorbed proteins from control BAC1.2F5
cells with anti-p120/130 antibody confirmed that p120/130 was one of
the proteins recognized by the Fes SH2 domain (Fig. 6B, lanes
3 and 5).
Taken together, these results are highly suggestive that the Fes
substrates we identified are structurally related or identical to the
physiological substrates of Fes kinase in BAC1.2F5 macrophages.
In this study we have identified Cas and p130 as two different
tissue-specific substrates of Fes in macrophages.
Overexpression of Fes kinase in BAC1.2F5 cells resulted in tyrosine
phosphorylation of a subset of proteins, which were different from
known adapter proteins associated with mitogenic signaling. These
proteins were tightly bound to Fes through its SH2 domain and contained
optimal consensus sequences for phosphorylation by Fes kinase and
binding to its SH2 domain, suggesting that they were direct substrates
of this kinase.
Using peptide library technology it was previously shown that the
optimal consensus sequences for phosphorylation by non-receptor tyrosine kinases were very similar to the optimal consensus sequences for binding to the SH2 domains of the corresponding kinases (34, 35).
Thus, the catalytic and SH2 domains of tyrosine kinases have co-evolved
so that some of the phosphorylated substrates can be retained by their
SH2 domains. This mechanism may allow for phosphorylation of additional
sites in the retained substrates or in other proteins present in the
phosphotyrosyl complex. Our results are consistent with some of the
predictions of this model. We showed that some of the Fes substrates in
BAC1-Fes cells remain associated with Fes after phosphorylation and
that this association was mediated through the Fes SH2 domain. Although
the Fes tyrosine phosphorylation sites in Cas and p130 have not yet
been mapped, these two proteins contain optimal Fes tyrosine
phosphorylation sites, which may be phosphorylated first and mediate
subsequent binding to the Fes SH2 domain. Cas contains several
additional tandem YDPY motifs that do not conform to the optimal
sequences for Fes phosphorylation. Yet, the detection of multiple Cas
forms in the Fes·Cas complex, which may represent different
phosphorylated forms of Cas, suggests that some of these non-canonical
sites may also be phosphorylated by Fes, perhaps facilitated by the formation of the initial complex between Fes and its substrate. Further
analysis using mutants of Fes and its substrates should clarify the
mechanism used by Fes to select and phosphorylate its targets.
Our previous work (12, 13, 19) and the results presented here suggest
that the biological and biochemical activity of Fes is tissue-specific.
In established murine fibroblasts, Fes expression led to cell
transformation mediated through phosphorylation or activation of
proteins involved in mitogenic and oncogenic signaling such as the
GTPase-activating protein·p62· p190 complex (12-14, 36), Shc
(16), and PI 3-kinase (18). On the other hand, overexpression of
p92c-fes had only a modest effect on the
proliferative capacity of BAC1 cells, and Fes kinase phosphorylated a
subset of proteins which so far have not been implicated in mitogenic
pathways (19). This is in contrast to the action of other tyrosine
kinases present in the same cells. In BAC1.2F5, the CSF-1 receptor is
involved in the control of cell proliferation, and this is mediated
through activation of the Shc/Grb2/Ras and PI 3-kinase pathways (19, 37). Thus, in macrophages Fes may not be involved in mitogenic signaling but participate in specialized signaling pathways that operate in these cells.
One of the most interesting findings of this study was the observation
that the phosphotyrosyl proteins recognized by the SH2 domain of Fes in
control BAC1.2F5 cells were very similar to the substrates identified
by overexpression of Fes in the same cells. Thus the Fes SH2 domain,
which does not recognize the substrates of other tyrosine kinases
present in BAC1.2F5 (19), appears to be a very specific reagent for Fes
substrates. Taken together, our results are consistent with the idea
that some of the substrates phosphorylated by ectopically expressed Fes
in macrophages may be structurally related or identical to its
physiological substrates.
The identity of the two substrates described in this study provides
insight into the possible site of action of Fes kinase in macrophages.
Cas has an SH3 domain that is a binding site for focal adhesion kinase
(38),3 a tyrosine kinase that is activated
by integrin engagement (39). Cas also binds tensin (40) and is believed
to play a role in reorganization of the actin cytoskeleton and other
signaling events induced by cell adhesion and cell-cell interactions.
Moreover, Cas is phosphorylated on tyrosine following integrin
engagement (41-43). This suggests that in macrophages and leukocytes,
which carry out functions that involve close interactions with other cells of the immune system and with the cell matrix (e.g.
cell adhesion during inflammatory responses), Fes may relay some of the
signals generated during these processes. Similarly, the identity of
p130 as a Fes substrate also suggests a role for Fes during cell-cell
interactions. The expression of p130 in T cells and in myeloid cells
and its tyrosine phosphorylation during engagement of receptors during
T cell activation (22) suggest that this protein may play a role in the
generation of bi-polar signals in interacting immunocompetent cells. In
T cells p130 is believed to be phosphorylated by Fyn (22), whereas in
macrophages Fes may be one of the kinases involved in the
phosphorylation of this protein. The identity of the macrophage
substrates uncovered in this study suggests that Fes kinase may play a
role in signaling triggered by cell adhesion and cell-cell interactions
during immune responses of macrophages.
Excellent technical assistance by Margaret
Tate is greatly appreciated. We thank Amy Bouton, J. T. Parsons, and H. Hirai for generously providing us with antibodies against Cas, and E. Richard Stanley for a gift of CSF-1.
Department of Microbiology and Immunology,
University of Maryland School of Medicine, Baltimore, the
Medical Biotechnology Center, University of Maryland
Biotechnology Institute, Baltimore, Maryland 21201, ¶ the Division of
Tumor Immunology,
Cells
-minimum essential medium supplemented
with 10% (v/v) fetal calf serum (Life Technologies, Inc.) (
-minimum
essential medium-fetal calf serum) and 36 ng/ml human recombinant CSF-1 (Chiron Corp., Emeryville, CA). BAC1.2F5 cells that overexpress c-fps/fes (BAC1-Fes) were obtained by introduction of the
retroviral expression vector pFF as described previously (19).
-D-galactopyranoside as described (21). Bacteria were lysed by sonication in a buffer containing 50 mM Hepes-KOH, pH 7.4, 1% (v/v) Triton X-100, 150 mM NaCl, 10% (v/v) glycerol, and 2% (v/v) Trasylol (FBA
Pharmaceuticals, NY), and the crude bacterial extracts were clarified
by centrifugation at 15,000 × g for 10 min at 4 °C.
Control GST and GST-SH2 fusion proteins were purified by adsorption to
glutathione-agarose (Sigma) as described (19, 21).
Fes Kinase Phosphorylates the crk-associated Substrate
(Cas)
. Human
p92c-fes was introduced into these cells by
retroviral-mediated gene transfer using the retroviral expression
vector pFF (12, 19). As previously shown, this vector induced the
expression of p92c-fes at levels 30-50 times
higher than background levels found in control cells (19). Ectopically
expressed Fes protein was enzymatically active as shown by its in
vitro kinase activity (Fig. 1A) and by
the induction of tyrosine phosphorylation of several proteins that were
largely undetectable in control BACI.2F5 cells (Fig. 1B).
The most prominent were two phosphotyrosyl proteins of 130-kDa (p130)
and a 75-kDa (p75) that we have previously described (19). p75 did not
cross-react with known signaling proteins in the same molecular weight
range (19) and may be a novel protein, whereas p130 was recognized by
an antibody (4F4) directed to a known substrate of v-src
(19, 24-26). This v-src substrate has been cloned and named
crk-associated substrate (Cas) because of its association with the v-crk oncoprotein in v-crk-transformed
cells (23, 27). Since the original antibody used to characterize p130
also recognized other tyrosine-phosphorylated proteins (19), we sought
additional evidence that p130 and Cas were the same protein. To this
end we used specific antibodies raised to the bacterially expressed Cas
protein.
Fig. 1.
Tyrosine phosphorylation of cellular proteins
in BAC1.2F5 cells that overexpress Fes kinase. A, control
BAC1.2F5 cells (lane 1), and BAC1.2F5 cells that overexpress
Fes (BAC1-FES) (lanes 2 and 3) were lysed in
Triton X-100 buffer. Cell lysates were immunoprecipitated with anti-Fes
(lanes 1 and 2) or with preimmune serum
(NIS) (lane 3), and immunoprecipitates were
assayed for in vitro kinase activity followed by SDS-PAGE
and autoradiography, as described under "Materials and Methods."
B, control BAC1.2F5 (lane 4) and BAC1-Fes
(lane 5) cells were starved of CSF-1 by incubation for
16 h at 37 °C in CSF-1-free -minimum essential medium-fetal
calf serum. Cells were lysed in Triton X-100 buffer, and total cell
lysates containing 50 µg of protein were analyzed by SDS-PAGE,
followed by Western blot analysis using anti-phosphotyrosine (P.Tyr) antibodies as described under
"Materials and Methods." The position of molecular weight markers
is indicated on the right.
[View Larger Version of this Image (30K GIF file)]
Fig. 2.
The crk-associated substrate
(Cas) is a substrate of Fes and is tightly associated with this
kinase. Cell lysates of CSF-1-starved BAC1-Fes cells were
immunoprecipitated (IP) with anti-Fes (Fes)
(lanes 1 and 3) or anti-Cas (Cas)
(lanes 2 and 4) antibodies, and the
immunoprecipitates were analyzed by SDS-PAGE followed by Western blot
(WB) analysis using anti-phosphotyrosine (lanes 1 and
2) or anti-Cas (lanes 3 and 4)
antibodies.
[View Larger Version of this Image (44K GIF file)]
Fig. 3.
The SH2 domain of Fes recognizes
tyrosine-phosphorylated Cas. Cell lysates of CSF-1-starved
BAC1-Fes cells were immunoprecipitated (IP) with anti-Cas
(Cas) antibody (lane 1) or adsorbed
(Ads) with either bacterial GST protein (GST)
(lane 2) or with a GST fusion protein encoding the Fes SH2
domain (GST-Fes-SH2) (lane 3). The precipitates
were analyzed by SDS-PAGE followed by immunoblotting with
anti-phosphotyrosine (lane 1) or anti-Cas (lanes
2 and 3) antibodies. The position of Cas proteins is
indicated at the right.
[View Larger Version of this Image (19K GIF file)]
subunit of
granulocyte/macrophage-CSF receptor (29), JAK kinases (30), focal
adhesion kinase (31), phospholipase C-
(32), and the pp120
v-src substrate (33) were not detectably
tyrosine-phosphorylated in BAC1-Fes cells and that therefore these were
not primary substrates of Fes kinase in these cells (19, data not
shown). However, we cannot rule out that there are other substrates of
Fes in this molecular weight range in BAC1-Fes cells, which were not
identified in this study.
Fig. 4.
p130 cross-reacts with a known 120-130-kDa
protein that is tyrosine-phosphorylated during T cell activation.
Cell lysates of CSF-1-starved BAC1-Fes cells were analyzed either
directly () (lane 1) or after immunoprecipitation with
anti-p130 (130K) (lane 2) or anti-Fes
(Fes) (lane 3) antibodies. Samples were analyzed by SDS-PAGE followed by immunoblotting with anti-phosphotyrosine antibodies.
[View Larger Version of this Image (20K GIF file)]
Fig. 5.
The SH2 domain of Fes recognizes
tyrosine-phosphorylated p130. Cell lysates of CSF-1-starved
BAC1-Fes cells were either adsorbed with bacterial GST (lanes
1 and 4), with GST fusion protein encoding the Fes SH2
domain (GST-SH2) (lanes 2 and 5), or were immunoprecipitated with anti-p130 (-p130) antibodies (lane
3). The precipitates were analyzed by immunoblotting with
anti-phosphotyrosine (lanes 1 and 2) or with
anti-p130 antibodies (lanes 3-5).
[View Larger Version of this Image (27K GIF file)]
Fig. 6.
The Fes SH2 domain recognizes the same set of
tyrosine-phosphorylated proteins in BAC1-Fes and control BAC1.2F5
cells. A, BAC1-Fes (lanes 1, 2, and 5)
and control BAC1.2F5 (lanes 3 and 4) cells were
incubated for 16 h in the absence of CSF-1, and cells were lysed
in Triton X-100 buffer. Total cell lysates (TCL) of BAC1-Fes
cells were analyzed either directly (lanes 1 and
5) or after adsorption to GST-Fes SH2 (GST-SH2)
fusion protein (lane 2). Cells lysates from control BAC1.2F5
cells were adsorbed with either bacterial GST (lane 3) or
GST-SH2 fusion protein (lane 4). Total cell lysates and
precipitated samples were analyzed by anti-phosphotyrosine
(P.Tyr) immunoblotting using the enhanced chemiluminescence system (lanes 1-5). Exposure time for
lanes 1, 2, and 5 was 10 s. Exposure time
for lanes 3 and 4 was 2 min. B, cell
lysates of CSF-1-starved BAC1.2F5 cells were adsorbed with bacterial
GST (lanes 1 and 4), with GST-Fes-SH2 domain
(lanes 2 and 5), or were immunoprecipitated with
anti-p130 antibody (lane 3). The precipitates were analyzed
by immunoblotting with either anti-phosphotyrosine (P.Tyr)
(lanes 1 and 2) or anti-p130 (-p130) (lanes 3-5) antibodies.
[View Larger Version of this Image (23K GIF file)]
*
This work was supported by National Institutes of Health
Grant R29 CA 55293. 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 Deutsche Forschungsgemeinschaft Fellowship from the
FRG. Present address: Heinrich-Pette-Institut für Experimentelle Virologie und Immunologie an der Universität, Hamburg,
Martinistrasse 52, 20251 Hamburg, Federal Republic of Germany.
**
To whom correspondence should be addressed.
1
The abbreviations used are: PI,
phosphatidylinositol; Cas, crk-associated substrate; PAGE,
polyacrylamide gel electrophoresis; GST, glutathione
S-transferase; CSF, colony stimulating factor; WB, Western
blot.
2
A. J. da Silva, submitted for publication.
3
E. Golemis, personal communication.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.