| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
(Received for publication, October 11,
1995) From the
Tyrosine phosphorylation of cellular proteins occurs rapidly
upon treatment of fibroblasts with acidic or basic fibroblast growth
factors (aFGF, bFGF), suggesting a role for protein phosphorylation in
the FGF signaling pathway. Stimulation of Swiss 3T3 cells and MRC-5
fibroblasts with bFGF results in the tyrosine phosphorylation of
several proteins, of which the most prominent has been designated as
p90. The phosphorylation of p90 is observed within 30 s of treating the
cells with FGF but not with other growth factors. Microsequencing of
p90 resolved on two-dimensional polyacrylamide gel electrophoresis
indicated an N-terminal amino acid sequence which corresponded to a
protein previously named as 80K-H. Polyclonal antibodies raised against
the predicted C terminus of 80K-H recognized p90 on all Western blots.
p90 was found to bind specifically to GRB-2-glutathione S-transferase fusion protein and to be immunoreactive with
80K-H antibody. In addition, anti-phosphotyrosine antibodies
immunoprecipitated 80K-H from cell lysates of FGF-stimulated but not
from control fibroblasts. The biological function of 80K-H is yet
unknown. However, from this study and a previous observation of the
obligatory dependence of p90 phosphorylation on FGF receptor
occupation, it appears that 80K-H is involved in FGF signaling. Fibroblast growth factors (FGFs) ( FGFs elicit cellular responses by binding
to and activating high affinity FGF receptor tyrosine
kinases(2, 3) . In agreement with the general scheme
for receptor tyrosine kinases, ligand binding induces dimerization of
FGF receptors, followed by activation of the intrinsic kinase activity
and autophosphorylation of the receptor molecules(2) . Two
autophosphorylation sites in FGFR-1 have been identified. One,
Tyr-653(4) , is located in the kinase domain and could have a
regulatory role. The other, Tyr-766, has been shown to mediate the
direct binding of phospholipase C- The signal
transduction pathways following activation of FGF receptors are thought
to be primarily mediated by the tyrosine phosphorylation of key
substrates, as is the case with other receptor tyrosine kinases. In
addition to phospholipase C- Recent investigations have shown that receptor
tyrosine kinases link to downstream signaling components, like ERKs,
via adaptor proteins that have no apparent enzymatic function but
trigger off signaling cascades by assembling proteins into reactive
complexes. Protein-protein interactions occur via binding of modular
domains on the adaptor protein, such as Src homology 2 (SH2), Src
homology 3 (SH3), pleckstrin homology, and phosphotyrosine-binding
domains, to specific sequence motifs on the target
proteins(17, 18) . One such adaptor protein, GRB-2,
essentially consists of two SH3 domains flanking a solitary SH2 domain.
GRB-2 has been shown to bind to autophosphorylated receptors such as
those of EGF, PDGF, CSF-1, and insulin, as well as to
tyrosine-phosphorylated, non-receptor proteins such as the insulin
receptor substrate (IRS). Recently, GRB-2-GST fusion proteins were also
used to identify novel tyrosine-phosphorylated proteins that bind to
GRB-2(19, 20, 21, 22) . We now
report the identification of p90 by a combination of several
techniques, which include partial amino acid sequencing of the protein,
Western blotting with different antibodies, and GRB-2 binding studies.
The collective evidence points to the identity of p90 as 80K-H, a
protein whose function has not been characterized. Our studies suggest
the involvement of 80K-H in FGF signaling.
We then investigated whether this p90 response can be
elicited by other growth factors and agonists. Different ligands were
added to either Swiss 3T3 or MRC-5 cells, and the phosphotyrosine
profiles of total lysates were compared to that obtained with bFGF (Fig. 1, lane 3). Neither EGF, when added to MRC-5
cells (lane 2), nor PDGF, when added to Swiss 3T3 cells (lane 8), was able to induce the phosphorylation of p90
although these ligands induced tyrosine phosphorylation of more
proteins than bFGF in the respective cells. A similar lack of p90
phosphorylation was observed when hydrogen peroxide (a putative
activator of tyrosine kinases and/or inhibitor of tyrosine
phosphatases) was added to MRC-5 cells (lane 4). IL-6, which
causes tyrosine phosphorylation through the recruitment of Janus
kinases to its receptor components, was unable to induce
phosphorylation of p90 in Swiss 3T3 cells (lane 6). Other
ligands which are known to stimulate tyrosine phosphorylation and/or
gene induction in these fibroblasts were also unable to induce the
tyrosine phosphorylation of p90. These included both human and murine
homologues of
Figure 1:
Protein tyrosine phosphorylation
induced by different agonists in mammalian fibroblasts. Confluent MRC-5 (lanes 1-4) or Swiss 3T3 (lanes 5-8)
cultures were exposed to: diluent (lane 1), EGF (50 ng/ml, lane 2), basic FGF (10 ng/ml, lane 3), hydrogen
peroxide (10 mM, lane 4), basic FGF (10 ng/ml, lane 5), IL-6 (20 ng/ml, lane 6), diluent (lane
7), or PDGF (50 ng/ml, lane 8) for 10 min at 37 °C
and then processed for antiphosphotyrosine immunoblotting as described
under ``Experimental Procedures.'' The migration of molecular
size standards is indicated at the left-hand margin. The arrow at the right indicates the position of
p90.
Figure 2:
Subcellular localization of p90. Swiss 3T3
cells were stimulated with bFGF (10 ng/ml) for 10 min and the
fractionated extracts prepared for immunoblotting as described under
``Experimental Procedures.'' Lane 1, total cell
extracts from untreated cells; lane 2, total cell extracts
from cells treated with bFGF; lane 3, cytosolic fraction from
cells treated with bFGF; lane 4, membrane fraction from cells
treated with bFGF. The arrow indicates the location of
p90.
The identification and characterization of p90, which is so far the
only protein to undergo tyrosine phosphorylation uniquely in response
to FGF, may be relevant to understanding the specificity of FGF
signaling.
Figure 3:
Location of p90 on two-dimensional PAGE
and its identification. A, MRC-5 cells were stimulated with
bFGF (10 ng/ml) and the whole cell lysate was prepared for separation
by two-dimensional PAGE, following an enrichment by band excision
around 90 kDa, as detailed under ``Experimental Procedures.''
The two-dimensional Western blot was incubated with PY20H antibody and
visualized by ECL. The arrow indicates the location of p90. B, microsequencing of the p90 protein indicated an 18-amino
acid peptide that corresponded to the truncated N terminus of the
previously sequenced 80K-H protein(25) . The first 14 residues (bold type) are not present in mature cellular protein. The
experimentally obtained sequence is boxed within the full
human 80K-H protein. A probable calcium-binding EF-hand domain is underlined, and the C-terminal HDEL sequence, a potential
endoplasmic reticulum retention signal, is indicated in italics. C, the two-dimensional PAGE blot shown in A was stripped and reprobed with the 80K-H-1030 antibody and
visualized by ECL. The arrow indicates the location of
p90.
Rabbits were immunized with synthetic
peptides derived from the experimental N terminus and the deduced C
terminus of the protein. Two antibodies (80K-H-1029 and 80K-H-1030)
raised against the 14-residue C-terminal peptide and one (80K-H-1031)
raised against the 14-residue N-terminal peptide recognized a protein
that co-migrates on two-dimensional PAGE gel with the heavily
tyrosine-phosphorylated p90 protein in FGF-stimulated cells (Fig. 3C). Control Western blots using the respective
preimmune sera did not detect any spots at 90 kDa (data not shown). Immunoprecipitation studies carried out with crude or purified 80K-H
antibodies were not successful. However, anti-phosphotyrosine
immunoprecipitation clearly indicated that 80K-H is
tyrosine-phosphorylated only in the bFGF-stimulated but not control
cells (Fig. 4). MRC-5 cells were treated with bFGF for 10 min
and separated into cytosol and membrane fractions. The membrane pellet
was solubilized in SDS-containing buffer and incubated with 4G10
antibody conjugated to agarose. In Fig. 4, 80K-H is detected at
90 kDa in lane 4, which corresponds to tyrosine
phosphoproteins derived from bFGF-stimulated cells. No 80K-H is
detectable in immunoprecipitates obtained from unstimulated cells (lane 3). Although the amount of lysates used for
immunoprecipitation in lanes 3 and 4 is about 20-fold
higher than the loading in lanes 1 and 2, the signal
for 80K-H is stronger in the latter lanes. This implies that the
tyrosine-phosphorylated 80K-H represents only a small fraction of the
total cellular 80K-H.
Figure 4:
Detection of 80K-H in anti-phosphotyrosine
immunoprecipitates. MRC-5 cells were stimulated with bFGF(10 ng/ml) or
with diluent alone, and the membrane fraction was immunoprecipitated
with anti-phosphotyrosine antibody (4G10 conjugated to agarose). The
immunoprecipitated proteins were separated by one-dimensional PAGE,
blotted onto PVDF membrane, incubated with 80K-H antibody, and
visualized by ECL. Lanes 1 and 2 correspond to
membrane lysates from control and stimulated cells, respectively. Lanes 3 and 4 contain the phosphotyrosine proteins
from the respective lysates. The arrow indicates the position
of p90/80K-H.
Figure 5:
p90 binds to GRB-2-GST fusion protein in vitro.A, quiescent Swiss 3T3 cells were treated
with diluent, bFGF (10 ng/ml), or PDGF (50 ng/ml) for 10 min prior to
the lysates being incubated with GRB-2-GST fusion protein conjugated to
agarose beads (lanes 1-3) or to GST-agarose beads alone (lanes 4-6). The bound proteins were prepared for
immunoblotting as described under ``Experimental Procedures''
and visualized by ECL. Lanes 1 (control), 2 (bFGF),
and 3 (PDGF) are eluted from GRB-2-GST fusion protein and lanes 4 (control), 5 (bFGF), and 6 (PDGF)
are eluted from GST-agarose beads. B, Swiss 3T3 cells were
stimulated with bFGF (10 ng/ml for 10 min) and the lysate allowed to
interact with GRB-2-GST fusion protein conjugated to agarose. The
complexed proteins were eluted from the beads and separated by
two-dimensional PAGE. The membrane was incubated with PY20H antibody
and visualized by ECL. The arrow indicates the position of the
90-kDa protein that binds to GRB-2.
The 60- and 72-kDa proteins were identified as SHC and PTP1D,
respectively, by stripping the membranes and reprobing with the
appropriate antibodies (data not shown). These proteins have already
been reported to bind directly to GRB-2(29, 30) . The
90-kDa protein was investigated in experiments described below, while
the 115-kDa protein remains unidentified. We decided to compare the
two-dimensional electrophoretic behavior of the GRB-2-associating p90
with that of p90 in the total lysate to verify that they were identical
proteins. Native cell lysates from bFGF-stimulated Swiss 3T3 cells were
incubated with GRB-2-GST fusion protein conjugated to agarose beads.
The complexed proteins were eluted from the beads with a
two-dimensional PAGE loading buffer (32) and then separated by
two-dimensional PAGE. The resulting autoradiograph (Fig. 5B) shows four major protein groups, consistent
with the four main tyrosine-phosphorylated proteins observed in
one-dimensional SDS-PAGE (Fig. 5A, lane 2). In
particular, the 90-kDa spot (Fig. 5B) co-migrates with
the p90 spot in total cell lysates (Fig. 3A),
possessing a similar pI and the same oblique inclination. The membrane
was stripped and reprobed with 80K-H antibody which, upon visualization
by ECL, gives a result similar to that shown in Fig. 3C.
Figure 6:
p90 binds mainly to the SH2 domain of
GRB-2 and is identified as 80K-H. A, quiescent Swiss 3T3 cells
were treated with diluent or bFGF (10 ng/ml) for 10 min prior to the
lysates being incubated with various GRB-2/GST fusion proteins
conjugated to agarose beads or to GST-agarose beads alone. Eluates from
untreated cells are in lanes 1, 3, 5, 7, and 9, and those treated with bFGF are in lanes 2, 4, 6, 8, and 10.
The resultant immunoblots were treated as described under
``Experimental Procedures'' to reveal the
tyrosine-phosphorylated proteins. Lanes 1 and 2 are
proteins from whole cell lysates. Lanes 3 and 4 are
proteins eluted from a GRB-2 (SH2)/GST fusion protein, lanes 5 and 6 are proteins eluted from a GRB-2 (N-terminal
SH3)/GST fusion protein, lanes 7 and 8 are proteins
eluted from a GRB-2 (C-terminal SH3)/GST fusion protein, and lanes
9 and 10 are proteins eluted from GST-agarose beads
alone. The arrow indicates the location of p90. B,
MRC-5 cells were stimulated with bFGF (10 ng/ml) and subsequently
fractionated into cytosol (lanes 1 and 3) and
membrane (lanes 2 and 4) extracts and separated on
one-dimensional PAGE gels. The protein loading in the cytosolic
fraction is 5 times higher than the membrane fraction. Aliquots of the
two extracts were also incubated with GRB-2(SH2)/GST protein and
subsequently eluted and separated on the same gel (lanes 3 and 4). The separated proteins were blotted onto a PVDF membrane,
which was probed with PY20H antibody and visualized by ECL (upper
panel). The same membrane was subsequently stripped and reprobed
with 80K-H antibody (lower panel). The arrows indicate the position of p90/80K-H.
Further
proof of the identity of p90 as 80K-H and an estimation of the extent
of its tyrosine phosphorylation was obtained by examining the p90 that
bound to GRB-2(SH2)-GST fusion protein. In Fig. 6B, lanes 1 and 2 contain the proteins in cytosol and
membrane fractions of FGF-treated MRC-5 cells, respectively. Aliquots
of these lysates were incubated with GRB-2(SH2)/GST proteins, eluted
and separated in lanes 3 (cytosol) and 4 (membrane).
It is apparent that the SH2 domain of GRB-2 concentrates
tyrosine-phosphorylated p90 from membranes derived from bFGF-stimulated
cells (upper panel). The blot was subsequently stripped and
reprobed with 80K-H antibody and visualized by ECL. From the immunoblot
shown in Fig. 6B (lower panel), substantial
amounts of 80K-H protein are found in the cytosol and membrane
fractions but only a small amount of 80K-H in the membrane fraction
binds to the SH2 domain of GRB-2. This is consistent with our above
conclusion, based on immunoprecipitation with anti-phosphotyrosine
antibody, that the tyrosine-phosphorylated 80K-H represents only a
small fraction of the total cellular 80K-H. In summary, the
identification of p90 as 80K-H was initially obtained from
microsequencing of a protein resolved on two-dimensional PAGE.
Antibodies raised against 80K-H consistently recognize p90 on all
one-dimensional- and two-dimensional Western blots. The use of
anti-phosphotyrosine immunoprecipitation and GRB-2 association studies
provided proof that 80K-H is tyrosine-phosphorylated and binds to the
SH2 domain of GRB-2 in vitro. The involvement of receptor tyrosine kinases in cytokine and
growth factor signaling has been reported extensively. Many of these
studies have focused on the receptors for EGF and PDGF, which were the
first to be sequenced from this family. Relatively little is known
about the activation and downstream signaling of the FGF receptor
subfamily. Previous work had identified a few proteins that were
tyrosine-phosphorylated in the early stages of FGF-induced signal
transduction. These proteins included Src, cortactin, SHC, ERK-1, and
ERK-2(7, 8, 9, 11, 31, 32, 33) .
One prominent protein that remained unidentified was designated p90.
The interest in p90 was heightened by its apparent specificity to the
FGF signaling system, in contrast to the common activation of Src, SHC,
and ERKs by numerous different growth factors. In EGF- and
PDGF-stimulated cells, both autophosphorylated EGFR and PDGFR are,
respectively, the dominant tyrosine phosphoproteins seen in Western
blots from whole cell lysates. The phosphorylated tyrosine residues on
these receptors serve to recruit specific proteins containing
complementary SH2 domains. The protein complexes thus formed are
thought to be responsible for initiating various signaling pathways
into the interior of the cell. We and others have observed that FGF
receptors were not noticeably tyrosine-phosphorylated in response to
FGF stimulation of various cells. Similarly, the insulin receptor shows
a low level of tyrosine phosphorylation upon binding with insulin (34) . In the latter case, however, a protein known as IRS
becomes heavily tyrosine-phosphorylated after associating with the
activated insulin receptor, thereby presenting multiple docking sites
for SH2-containing signaling proteins(34) . Thus, in terms of
the low level of receptor autophosphorylation, the insulin and FGF
receptors show similar features following ligand activation. It is
therefore possible that the FGF-specific tyrosine phosphorylation of
p90 may serve a role similar to that seen with IRS in the insulin
system where it could trigger signal propagation involving the
formation of specific protein complexes. Several proteins that are
devoid of intrinsic catalytic activity can facilitate the assembly of
these signaling protein complexes. These ``adaptor'' proteins
include GRB-2, Crk, and Nck, which consist almost entirely of SH2 and
SH3 domains. A number of tyrosine-phosphorylated proteins have been
shown to be capable of binding to GRB-2 in
vivo(17, 18) . They include, among a growing
list, both the insulin receptor and IRS. In our in vitro assays, the FGF-specific p90 protein was found to associate with
GRB-2, in addition to PTP1D, SHC, and an unidentified 115-kDa protein.
Both PTP1D and SHC have well characterized SH2 domains and have been
shown previously to bind directly to GRB-2(29, 30) .
Beyond our verification that PTP1D and SHC bind only to the SH2 domain
of GRB-2, we have demonstrated that p90 also binds mainly to the SH2
domain. Since such associations were specific, they would provide a
means for enriching tyrosine-phosphorylated p90 protein from cell
lysates and aid in its identification. The 115-kDa protein, and to a
lesser extent p90, were further shown to bind to the C-terminal SH3
domain of GRB-2, possibly via their respective proline-rich motifs. The p90 protein was resolved by two-dimensional PAGE and subjected
to microsequencing. An 18-residue N-terminal sequence was obtained,
which matched exactly with the first 18 residues from the truncated N
terminus of a human protein previously designated as
80K-H(25) . Polyclonal antibodies were raised in rabbits
against the predicted C terminus (14 residues) and observed N terminus
(14 residues) of 80K-H. Several of these antibodies recognized a
protein that co-migrated with p90 on Western blots derived from one-
and two-dimensional PAGE. Additional evidence for the identity of p90
comes from anti-phosphotyrosine immunoprecipitation, in vitro GRB-2-GST association experiments, and reversed-phase high
performance liquid chromatography (data not shown) where p90 is being
recognized by the 80K-H antibody. The 80K-H protein was isolated
several years ago during an effort to identify a ubiquitous 80-87
kDa protein that is a strong substrate of protein kinase
C(35) . The main target protein of this work turned out to be
the MARCKS protein(36) , also designated 80K-L (L for light),
which is now a well characterized PKC substrate. The 80K-H protein (H
for heavy) is only a weak PKC substrate in vitro and appears
not to be a PKC substrate in vivo(35) . Aside from its
subsequent cloning(25) , the 80K-H protein was not
characterized in greater detail. Its amino acid sequence deduced from
cDNA contains several noteworthy features. Analysis performed on the
Swiss-Prot data base revealed four putative protein kinase C
phosphorylation sites, a possible calcium binding EF-hand domain, and a
prominent glutamic acid repeat around the middle of the protein, which
is seen in some proteins including tropomyosin, prothymosin,
neurofilament triple L protein, myc-transforming protein, and
adenovirus 5` terminal protein. There is a C-terminal HDEL sequence,
which is a possible endoplasmic reticulum retention signal, although
functional HDEL sequences are usually not found in mammalian
cells(37) . There are also at least three proline-rich motifs,
PXXP, which may account for the low level binding of 80K-H to
the SH3 domain of GRB-2. A consensus motif pYXNX has
been reported in various proteins such as PTP1D, SHC, EGFR, and focal
adhesion kinase, where the SH2 domain of GRB-2 binds(17) .
While the tyrosine-phosphorylated 80K-H binds the SH2 domain of GRB-2,
no such sequence was found in the former protein. Further
characterization of the 80K-H/GRB-2 interaction may help to resolve
this discrepancy. The deduced amino acid sequence of 80K-H predicts
a size of 59 kDa, which is much smaller than the 90-kDa migration in
SDS-polyacrylamide gels. The predicted isoelectric point is around 4.4,
which corresponds well with the experimental value. The size
discrepancy is not without precedence since highly acidic proteins have
been known to migrate anomalously upon SDS-PAGE. For example, the
bovine MARCKS protein, with a pI similar to that of 80K-H, migrates at
80-87 kDa although its predicted molecular mass is only 32
kDa(38) . The 80K-H sequence contains 15 tyrosine residues,
which are evenly distributed in the protein. There is, however, a
rather polar distribution of cysteine residues toward both ends of the
protein, suggesting that the native structure for 80K-H can contain
multiple intra- and/or intermolecular disulfide bridges. Molecular
modeling of 80K-H was not successful, as the protein does not contain
enough sequence homology to known structural domains in existing data
bases. We have further shown that while this protein is present in both
the cytosol and membrane, the tyrosine-phosphorylated species is found
in the membrane and can bind the SH2 domain of GRB-2 in vitro.
Based on densitometric scanning, we estimated the proportion of
tyrosine-phosphorylated 80K-H to be less than 1% of total cellular
80K-H. The physiological function of 80K-H is yet unknown. We now
report a possible role for 80K-H in FGF signaling. The biochemical
characteristics of this novel p90/80K-H protein; its expression in
different cells, tissues, and organisms; and its potential function in
FGF-induced cell signaling and development will be the subject of
further study.
Volume 271,
Number 10,
Issue of March 8, 1996 pp. 5832-5838
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
)comprise a family
of structurally related heparin-binding polypeptides of which the best
characterized members are acidic FGF (aFGF) and basic FGF (bFGF). These
polypeptides are important regulators of differentiation and
embryogenesis; they support the survival of neuronal cells and are
extremely potent inducers of DNA synthesis in ectoderm- or
mesoderm-derived cell types, including endothelial cells and smooth
muscle cells(1) .(5) .
, several other substrates have been
identified, including SHC(6) , ERK-2,
ERK-1(7, 8) , cortactin(9, 10) , and
Src(9, 11) . A number of groups have demonstrated the
prominent phosphorylation of a 90-kDa protein by both aFGF and
bFGF(7, 12, 13, 14, 15, 16) .
It was recently demonstrated that p90 phosphorylation was induced in
cells transfected with both the FGFR-1 and the keratinocyte growth
factor receptor, but not the FGFR-4(11) . The identification of
p90 would thus be relevant to our understanding of the complexity of
FGF signaling.
Reagents, Antibodies, and GST Fusion
Proteins
Monoclonal antibodies to phosphotyrosine (PY20H), SHC,
and PTP1D were obtained from Transduction Laboratories (Lexington, KY).
Anti-phosphotyrosine antibody (4G10) conjugated to agarose was obtained
from Upstate Biotechnology, Inc. (Lake Placid, NY). Horseradish
peroxidase-conjugated anti-rabbit or mouse IgGs and hydrogen peroxide
were from Sigma. CSF-1, tumor necrosis factor-
, IL-1, transforming
growth factor-
, EGF, IL-6, and PDGF were from Genzyme (Cambridge,
MA). Basic FGF was from Boehringer Mannheim. Fusion proteins consisting
of GRB-2 (whole protein), GRB-2 (SH3 domain, amino acids 1-68 or
156-199), GRB-2 (SH2 domain, amino acids 54-164), all fused
with GST and conjugated to agarose were obtained from Santa Cruz
Biotechnology (Santa Cruz, CA). ECL reagents were obtained from
Amersham (Bucks, United Kingdom) and Boehringer Mannheim.Cells and Cell Stimulation
Human MRC-5 fibroblasts
(American Tissue Culture Collection, Rockville, MD) were grown as
described previously(23) . One hour before stimulation with the
various agonists, the cells were washed twice with serum-free
Eagle's minimal essential medium and then incubated with the same
medium until the cells were lysed. Swiss 3T3 cells (American Tissue
Culture Collection) were maintained in Dulbecco's modified
Eagle's medium supplemented with 10% fetal bovine serum (HyClone
Laboratories, Logan, UT), 2 mM glutamine, 10 mM HEPES
(pH 7.3), and 100 units/ml penicillin/streptomycin. When the cells were
80-90% confluent, the medium was aspirated, the cells washed and
maintained for another 24 h in serum-free Dulbecco's modified
Eagle's medium. Various agonists were added to the quiescent
cells prior to the medium being aspirated, the cells rapidly washed in
cold PBS, and lysed for subsequent analysis.Subcellular Fractionation
After stimulation, cells
in 15-cm dishes were washed with Buffer A (25 mM Tris-HCl, pH
7.5, 250 mM sucrose, 2.5 mM magnesium acetate, 2
mM dithiothreitol, 10 mM benzamidine, 10 mM sodium fluoride). The plates were well drained of Buffer A and
replaced with 1 ml of homogenization buffer (Buffer A containing 5
mM EGTA, 5 mM EDTA plus protease inhibitors 250
µg/ml leupeptin, 150 µg/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride). The cells were scraped and
homogenized with 30 strokes of a Dounce homogenizer with a
tight-fitting pestle. The homogenate was centrifuged for 10 min at
90,000 rpm in a Beckman TLA-100 rotor at 4 °C. The supernatant
(cytosolic extract) was added with 500 µl of 5 
Laemmli
buffer and boiled for 10 min. The pellet (membrane) was extracted for
20 min on ice with 1 ml of Buffer B (25 mM Tris-HCl, pH 7.5, 5
mM dithiothreitol, 10 mM benzamidine, 10 mM sodium orthovanadate, 5 mM EDTA, and 1% Nonidet P-40 plus
other protease inhibitors used in Buffer A). The detergent-soluble
proteins were collected by centrifugation at room temperature at 12,000
rpm. The supernatant containing the detergent-soluble fraction
(membrane extract) was added with 500 µl of 5 Laemmli
buffer and boiled as above.Resolution of the p90 Protein on Two-dimensional
PAGE
Two 15-cm plates of MRC-5 cells were prepared as above for
stimulation with bFGF for 10 min. After washing with cold PBS, each
plate of cells was lysed in 500 µl of the lysis buffer used by
Coughlin et al.(13) . 200 µl of 5 Laemmli
buffer was added to each aliquot, and after boiling for 10 min the
entire lysate was loaded onto each side of a one-dimensional PAGE
minigel (Bio-Rad). Prestained molecular size markers (Bio-Rad) were
used to track the running of the gel in order to excise a section of
separated proteins between 85 and 100 kDa. The excised gel sections
were stained in 0.1% bromphenol blue and cut into small cubes using a
clean scalpel blade. Proteins were eluted from the gel pieces in a
Little Blue Tank electrophoretic elution tank (ISCO, Lincoln, NE) with
elution buffer (12.5 mM Tris base, 95 mM glycine,
0.05%, w/v, SDS). The electro-eluate was dialyzed against 1% Nonidet
P-40 in PBS before the proteins were precipitated by adding two volumes
of acetone and incubating on ice for 30 min. The precipitated protein
was collected by maximal centrifugation in a bench-top microcentrifuge
(Eppendorf) for 15 min at 4 °C. The protein pellet was solubilized
in two-dimensional PAGE sample loading buffer as described previously (24) and loaded onto the first dimension of the Millipore
Investigator two-dimensional electrophoresis system (Millipore,
Bedford, MA). The running conditions were essentially as described
previously(23) , except that the first dimensional isoelectric
focusing tubes were reduced to 7.5 cm in length and the second
dimension was run on a minigel apparatus (Bio-Rad), followed by
electroblotting in a miniblot apparatus (Bio-Rad) onto PVDF membranes.
Isoelectric point markers (Bio-Rad) were used for calibrating the
first-dimensional isoelectric focusing. Prestained molecular weight
markers (Sigma) for the second dimension were prepared in 1% agarose
and cast into capillary tubes. The membrane was stained with Amido
Black, washed in deionized water and wrapped in plastic sheet for
photocopying. The membrane was then probed with PY20H antibody and the
location of tyrosine-phosphorylated proteins visualized by ECL
(Amersham). The location of any Amido Black-stained spots coinciding
with the p90 phosphotyrosine signal was noted.Microsequencing of p90 from PVDF Membrane
A
duplicate experiment was performed with the intention of
microsequencing the p90 protein obtained from the membrane stained with
Amido Black. The putative p90 spot on the stained membrane was excised
and subjected to direct N-terminal microsequencing. The excised
membrane was loaded into a blot cartridge of a protein sequencer
(Procise, Applied Biosystems, Foster City, CA) and run using cycles
recommended by the manufacturer. The amino acid sequence obtained was
searched against protein data bases using the BLAST facility (NCBI).
The remaining membrane was probed with PY20H antibody to confirm the
removal of the p90 phosphotyrosine signal.Preparation and Purification of Polyclonal 80K-H
Antibodies
Peptides corresponding to the 14 N-terminal amino
acids (VEVKRPRGVSLTNH) and the 14 C-terminal amino acids
(PPPEAPTEDDHDEL) of the predicted human 80K-H protein sequence were
synthesized, conjugated to keyhole limpet hemocyanin, and injected into
rabbits by Neosystems Laboratoire (Strasbourg, France). Two C-terminal
antibodies designated 80K-H-1029 and 80K-H-1030, and one N-terminal
antibody designated 80K-H-1031, recognized a protein on one- and
two-dimensional immunoblots that corresponded to the
tyrosine-phosphorylated protein p90. These polyclonal antibodies were
purified on peptide affinity columns prepared by coupling the synthetic
peptide to CNBr-activated Sepharose 4B (Pharmacia, Uppsala, Sweden)
according to the manufacturer's recommendations. The column,
containing 1 ml of coupled gel, was equilibrated with five gel volumes
of PBS. 0.5 ml of the antibody-containing serum was diluted with 0.5 ml
of PBS and clarified through a 0.22-µm filter. The diluted serum
was mixed with the equilibrated gel and incubated for 1 h at 4 °C.
Unbound proteins were collected in the flow-through. The column was
then washed with 5 ml of PBST (PBS containing 0.1% Tween-20 and 0.5 M NaCl). Bound proteins were eluted in five fractions of 2 ml
of IgG elution buffer (Pierce). The fractions were neutralized
immediately with 1 M Tris-HCl to pH 7.5 and assayed by
immunoblotting cell lysates known to contain the 80K-H protein.Immunoprecipitation and Western
Blotting
Immunoprecipitations using the 80K-H antibody were
performed as follows; 80 µl of lysate (containing membrane proteins
from one 15-cm plate of MRC-5 cells) was added to 400 µl of
affinity-purified 80K-H antibody and 480 µl of 2
immunoprecipitation buffer (2% Triton X-100, 300 mM NaCl, 20
mM Tris, pH 7.4, 2 mM EDTA, 2 mM EGTA, 0.4
mM sodium orthovanadate, 0.4 mM PMSF, 1% Nonidet
P-40). The mixture was incubated for 1 h at 4 °C. 50 µl of
anti-rabbit IgG, conjugated to agarose beads were then added and the
incubation continued for another 1 h. The beads were then washed three
times in 1 immunoprecipitation buffer before boiling with 30
µl of Laemmli buffer. The supernatant from the boiled beads was
then analyzed by SDS-PAGE. Immunoblot analyses have been described
previously(27) . The commercial antibodies were used according
to manufacturers' recommendations.Binding Experiments with GRB-2 and Its SH2 and SH3
Domains
Cells were serum-starved as described above before
stimulation and lysis. The cells were treated with bFGF (10 ng/ml) or
with PDGF (50 ng/ml) or with diluent only. After washing with cold PBS,
the cells were lysed with 500 µl/plate of an Nonidet P-40 buffer
(0.5% (v/v) Nonidet P-40, 10 mM HEPES, pH 7.9, 30 mM Na
P
O
, 5 mM EDTA, 5
mM EGTA, 1 mM dithiothreitol, 1 mM PMSF, 50
mM sodium fluoride, 100 µM sodium orthovanadate,
and 10 mM genistein). The lysates were spun in a bench-top
centrifuge at full speed for 10 min, and the resultant pellet was
discarded. The protein content of the lysate was assayed with a BCA
protein assay kit (Pierce). Lysate protein (2 mg/ml) was used for each
experiment. Lysate (500 µl) was added to 500 µl of GRB-2
association buffer (2% (v/v) Triton X-100, 30 mM NaCl, 20
mM Tris-HCl, pH 7.4, 2 mM EDTA, 2 mM EGTA,
0.4 mM sodium orthovanadate, 1 mM PMSF, 1% (v/v)
Nonidet P-40) and 20 µl of GRB-2-GST protein fusion beads were
added and incubated for 2 h at 4 °C. The beads were washed three
times with 1 ml of 1 GRB-2 association buffer before the bound
proteins were released by boiling with 30 µl of 2 Laemmli
buffer for 5 min. The supernatant was subjected to one- or
two-dimensional PAGE and the separated proteins visualized by
incubating with the appropriate antibodies and ECL. Controls consisted
of GST linked to agarose beads or agarose beads alone.
A Protein Designated as p90 Is Rapidly and Uniquely
Tyrosine-phosphorylated following FGF Stimulation
Swiss mouse
3T3 or human MRC-5 fibroblasts were used to study the time course of
protein tyrosine phosphorylation induced by bFGF. Cells were lysed
after stimulation, followed by resolution of the cellular proteins on
SDS-polyacrylamide gels, electroblotting onto PVDF membranes, and
probing with anti-phosphotyrosine antibodies (PY20H). Our observations
were similar to previous reports(13) , where p90 tyrosine
phosphorylation is induced as early as 30 s after addition of bFGF and
the response is detectable at doses as low as 0.5 ng/ml (data not
shown). The level of phosphorylation peaked between 5 and 10 min and
dropped slightly by 30 min. However, others have previously shown that
p90 phosphorylation is sustained for at least 4 h and appears to be
directly dependent on the continuous occupancy of the FGF receptor (13) ./ and interferons, tumor necrosis
factor-
, IL-1, CSF-1, and transforming growth factor- (data
not shown).
Tyrosine-phosphorylated p90 Is Located in the
Membrane
The subcellular location of the tyrosine-phosphorylated
p90 protein was ascertained by fractionating the cell into cytosolic
and membrane extracts after treatment with bFGF. These fractions, along
with total cell lysates from stimulated and unstimulated cells, were
resolved by SDS-PAGE and transferred onto a PVDF membrane. The
tyrosine-phosphorylated proteins were visualized by ECL after probing
with anti-phosphotyrosine antibody (PY20H). Tyrosine phosphorylation of
p90 occurs only in the membrane fraction of bFGF-stimulated cells (Fig. 2, lane 4) as opposed to the cytosolic fraction
from the same cells (lane 3). This membrane p90 protein
co-migrated with p90 from the total cell lysate of bFGF-stimulated
cells (lane 2). It is noteworthy that the summation of lanes 3 and 4 corresponded well with lane 2.
Two-dimensional Electrophoretic Resolution and
Identification of p90
For the partial purification of p90, MRC-5
cells were stimulated with bFGF for 10 min and the cell lysates
resolved by two-dimensional PAGE (see ``Experimental
Procedures''). A prominent phosphotyrosine spot appeared on the
blot (Fig. 3A) at a molecular mass of 90 kDa and an
acidic pI of 4.5, which coincided with a group of three closely spaced
spots stained by Amido Black. The corresponding protein spots from a
parallel experiment, that does not involve PY20H probing, were excised
and used for direct N-terminal sequencing. One unambiguous amino acid
sequence was obtained (VEVKRPRGVSLTNHHFYD) which matched exactly with
the first eighteen residues from the truncated N terminus of a human
protein designated as 80K-H(25) , as shown in Fig. 3B.
p90 Binds to GRB-2-GST Fusion Protein and Co-migrates
with 80K-H
Recently, a number of novel tyrosine-phosphorylated
proteins have been identified by virtue of their specific association
with
GRB-2(21, 22, 26, 27, 28) .
Such specific interactions can significantly enrich some
tyrosine-phosphorylated proteins, thus acting as an affinity
purification method for these proteins. Since our efforts with direct
80K-H immunoprecipitation were unsuccessful, we set out to explore
other affinity methods of isolating p90 to facilitate further proof of
its identity as 80K-H. We speculated that p90, being phosphorylated on
tyrosine residues, might bind GRB-2 and therefore screened a GRB-2-GST
fusion protein for this purpose. A GRB-2-GST fusion protein, conjugated
to agarose beads, was incubated with native lysates of Swiss 3T3 cells
that were treated with optimal doses of bFGF or PDGF. After incubation,
the associated proteins were separated, transferred to a PVDF membrane,
incubated with anti-phosphotyrosine antibodies, and revealed by ECL.
Treatment with bFGF led to four prominent tyrosine-phosphorylated
proteins being associated with GRB-2 (Fig. 5A, lane
2). These proteins migrated at masses of 60, 72, 90, and 115 kDa,
three of which were in common with the binding profile obtained from
cells stimulated with PDGF (Fig. 5A, lane 3).
The 90-kDa protein appeared to be unique to lysates derived from
bFGF-stimulated cells. No proteins were found to associate
nonspecifically with the GST-agarose matrix (lanes 4-6).
80K-H Binds Mainly to the SH2 Domain of GRB-2
To
evaluate the relative contribution of the three domains of GRB-2 in
binding the four FGF-stimulated tyrosine-phosphorylated proteins,
particularly p90/80K-H, three separate GST fusion proteins that
incorporated either the N-terminal SH3, the middle SH2, or the
C-terminal SH3 domain were employed in binding experiments similar to
those described above. The resulting autoradiograph from one such
experiment is shown in Fig. 6A. Lanes 1 and 2 correspond to cell lysates from unstimulated and
FGF-stimulated cells, respectively. Lanes 3 and 4 show the tyrosine-phosphorylated proteins that bind to the SH2
domain of GRB-2 in unstimulated (lane 3) and FGF-stimulated
cells (lane 4). It is apparent that three of the four
tyrosine-phosphorylated proteins that associate with the whole GRB-2
protein are present in lane 4. The apparent amounts of
tyrosine-phosphorylated SHC, PTP1D, and p90 are enhanced when compared
to the equivalent amounts of each protein in the whole cell lysate.
Both SHC and PTP1D have been shown to bind to GRB-2 protein via
specific interactions between phosphotyrosine-containing motifs and the
SH2 domain of GRB-2(29, 30) . It is likely that p90
interacts with the GRB-2 SH2 domain in a similar manner. Lanes 5 and 7 show that neither the N- nor C-terminal SH3 domains
associate with any phosphotyrosine proteins in the control cell
lysates. Lanes 6 and 8 are the equivalent lanes
obtained from binding experiments with lysates from FGF-stimulated
cells. Only lane 8 contains a significant amount of
tyrosine-phosphorylated proteins. Notably the 115-kDa protein is
present in this lane, which implies that it binds to the C-terminal SH3
domain of GRB-2 in a specific manner. As SH3 domains interact with
proline-rich motifs, the small but detectable amount of p90 protein
present in lane 8 suggests a proline-rich sequence in p90. The
absence of tyrosine-phosphorylated proteins in lanes 9 and 10 demonstrates the absence of any nonspecific interactions
between phosphotyrosine proteins and the GST-agarose matrix.
)
We thank the following people who helped during the
course of this work: Desmond Ng and Joyce Low for technical assistance,
Dr. Thomas Klein for his enthusiasm, Robin Philp for excellent
microsequencing work, Dr. Catherine Pallen for helpful discussions, Dr.
Neeraj Jain for advice and encouragement, and the laboratory of
Professor N. Shimizu, Keio University, School of Medicine, Tokyo,
Japan, for assistance.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  A. Goldin, J. A. Beckman, A. M. Schmidt, and M. A. Creager Advanced Glycation End Products: Sparking the Development of Diabetic Vascular Injury Circulation, August 8, 2006; 114(6): 597 - 605. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. G. Levine, D. Mitra, A. Sharma, C. L. Smith, and R. S. Hegde The Efficiency of Protein Compartmentalization into the Secretory Pathway Mol. Biol. Cell, January 1, 2005; 16(1): 279 - 291. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Gkika, F. Mahieu, B. Nilius, J. G. J. Hoenderop, and R. J. M. Bindels 80K-H as a New Ca2+ Sensor Regulating the Activity of the Epithelial Ca2+ Channel Transient Receptor Potential Cation Channel V5 (TRPV5) J. Biol. Chem., June 18, 2004; 279(25): 26351 - 26357. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. S. Ridyard and S. M. Robbins Fibroblast Growth Factor-2-induced Signaling through Lipid Raft-associated Fibroblast Growth Factor Receptor Substrate 2 (FRS2) J. Biol. Chem., April 11, 2003; 278(16): 13803 - 13809. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 O. Poirier, V. Nicaud, N. Vionnet, S. Raoux, L. Tarnow, H. Vlassara, H.-H. Parving, and F. Cambien Polymorphism Screening of Four Genes Encoding Advanced Glycation End-Product Putative Receptors: Association Study With Nephropathy in Type 1 Diabetic Patients Diabetes, May 1, 2001; 50(5): 1214 - 1218. [Abstract] [Full Text]
|
|
|
|
 |
|
 S. Brûlé, F. Rabahi, R. Faure, J.-F. Beckers, D. W. Silversides, and J. G. Lussier Vacuolar System-Associated Protein-60: A Protein Characterized from Bovine Granulosa and Luteal Cells That Is Associated with Intracellular Vesicles and Related to Human 80K-H and Murine {beta}-Glucosidase II Biol Reprod, March 1, 2000; 62(3): 642 - 654. [Abstract] [Full Text]
|
|
|
|
|
|
B. C. Low, Y. P. Lim, J. Lim, E. S. M. Wong, and G. R. Guy Tyrosine Phosphorylation of the Bcl-2-associated Protein BNIP-2 by Fibroblast Growth Factor Receptor-1 Prevents Its Binding to Cdc42GAP and Cdc42 J. Biol. Chem., November 12, 1999; 274(46): 33123 - 33130. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Dell'Era, M. Mohammadi, and M. Presta Different Tyrosine Autophosphorylation Requirements in Fibroblast Growth Factor Receptor-1 Mediate Urokinase-Type Plasminogen Activator Induction and Mitogenesis Mol. Biol. Cell, January 1, 1999; 10(1): 23 - 33. [Abstract] [Full Text]
|
|
|
|
|
|
M. M. Dikov, M. B. Reich, L. Dworkin, J. W. Thomas, and G. G. Miller A Functional Fibroblast Growth Factor-1 Immunoglobulin Fusion Protein J. Biol. Chem., June 19, 1998; 273(25): 15811 - 15817. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Michelson, S Gisselbrecht, E Buff, and J. Skeath Heartbroken is a specific downstream mediator of FGF receptor signalling in Drosophila Development, January 11, 1998; 125(22): 4379 - 4389. [Abstract] [PDF]
|
|
|
|
|
|
Y. P. Lim, B. C. Low, S. H. Ong, and G. R. Guy Growth Factors Stimulate Tyrosine Dephosphorylation of p75 and Its Dissociation from the SH2 Domain of Grb2 J. Biol. Chem., November 21, 1997; 272(47): 29892 - 29898. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. J. Williams, D. J. Dunican, P. J. Green, F. V. Howell, D. Derossi, F. S. Walsh, and P. Doherty Selective Inhibition of Growth Factor-stimulated Mitogenesis by a Cell-permeable Grb2-binding Peptide J. Biol. Chem., August 29, 1997; 272(35): 22349 - 22354. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. W. Arendt and H. L. Ostergaard Identification of the CD45-associated 116-kDa and 80-kDa Proteins as the alpha - and beta -Subunits of alpha -Glucosidase II J. Biol. Chem., May 16, 1997; 272(20): 13117 - 13125. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Kanai, M. Goke, S. Tsunekawa, and D. K. Podolsky Signal Transduction Pathway of Human Fibroblast Growth Factor Receptor 3. IDENTIFICATION OF A NOVEL 66-kDa PHOSPHOPROTEIN J. Biol. Chem., March 7, 1997; 272(10): 6621 - 6628. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. K.W. Hsu, Y. Guo, G. F. Alberts, N. G. Copeland, D. J. Gilbert, N. A. Jenkins, K. A. Peifley, and J. A. Winkles Identification of a Murine TEF-1-related Gene Expressed after Mitogenic Stimulation of Quiescent Fibroblasts and during Myogenic Differentiation J. Biol. Chem., June 7, 1996; 271(23): 13786 - 13795. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||
