(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) ()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) .
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-(5) .
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-, 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.
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 /
and
interferons, tumor necrosis
factor-
, IL-1, CSF-1, and transforming growth factor-
(data
not shown).
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.