(Received for publication, March 16, 1995; and in revised form, June 13, 1995)
From the
A major pathway for mitogenicity is gated via the small
GTP-binding protein Ras. Receptor tyrosine kinases couple to Ras
through the Src homology 2 (SH2) domain protein Grb2. The activated
fibroblast growth factor receptor-1 (FGFR-1) expressed in L6 myoblasts
did not bind Grb2 directly, but indirectly, through the small adaptor
protein Shc, which was tyrosine-phosphorylated in response to
fibroblast growth factor-2 (FGF-2) stimulation. A FGFR-1 mutant in
which Tyr, a known autophosphorylation site, was changed
to Phe, mediated less efficient tyrosine phosphorylation of Shc. FGF-2
stimulation of mutant FGFR-1-expressing cells still allowed formation
of complexes containing Shc, Grb2, and the nucleotide exchange factor
Sos and mediation of a mitogenic signal. Another pool of Grb2 was found
in complex with a tyrosine-phosphorylated 89-kDa component after FGF-2
stimulation. Stimulation with other growth factors did not lead to
tyrosine phosphorylation of p89. As shown by ``far-Western''
analysis, p89 bound directly to the Grb2 SH2 domain, and this
interaction was inhibited by a peptide containing the Y(P)-X-N
motif. Tyrosine-phosphorylated p89 was found exclusively in the
membrane fraction, indicating its role in bringing Grb2, as well as
Sos, to the plasma membrane. These data support the concept of growth
factor-specific coupling of Grb2 to the Ras pathway.
Fibroblast growth factors (FGFs) ()comprise a family
of heparin-binding polypeptides that, to date, include nine distinct
gene products, FGF-1 to FGF-9(1, 2, 3) . FGFs
are mitogenic for a wide variety of cells and are also implicated in
regulation of differentiation, cell motility, and transformation. In vivo studies describe a regulatory role of FGFs in
physiological processes such as neovascularization, wound healing, and
development(4, 5, 6) .
FGFs bind extracellularly to high affinity transmembrane tyrosine kinase receptors and low affinity heparan sulfate proteoglycan co-receptors. The high affinity FGF receptors form a family of four structurally related members, FGFR-1 to FGFR-4(7, 8) . Each of these contain two or three extracellular immunoglobulin-like loops, a characteristic stretch of acidic amino acids between the first and second loop, a single transmembrane region, and an intracellular kinase domain split by a 14-17-amino acid kinase insert. Alternative splicing generates a multitude of structural variants that differ in ectodomain regions known to be involved in ligand binding(8) .
Autophosphorylated tyrosine residues and adjacent amino acids
provide specific binding sites for signal transducing proteins
containing Src homology 2 (SH2) domains(9) . Recruitment of
signal-transducing molecules to activated tyrosine kinase receptors
triggers signaling pathways, which eventually result in specific
cellular responses. This far, two intracellular autophosphorylation
sites have been identified in the FGFR-1, at Tyr and
Tyr
, located in the kinase domain and the C-terminal
tail, respectively(10) . Whereas it is possible that
Tyr
is involved in regulation of kinase activity,
phosphorylated Tyr
has been shown to provide a binding
site for phospholipase C-
(PLC-
)(11) . The Y766F
mutant FGFR-1 has been shown to be internalized at a decreased rate in
transfected rat myoblasts(12) , but still mediates a mitogenic
signal (13, 14) and allows neurite outgrowth when
expressed in PC-12 cells(15) . Thus the role of PLC-
in
FGFR-1 signaling is not well understood; however, PLC-
-mediated
activation of PKC in FGF-stimulated cells could play a negative
regulatory role, i.e. in Src-dependent downstream
signaling(16) . A number of other signal transduction molecules
have been shown not to interact with or be affected by the activated
FGFR-1. These include phosphatidylinositol 3`-kinase (17) and
RasGAP(18) .
Activation of FGFR-1 leads to a potent mitogenic signal. Conversion of the Ras protein from its inactive GDP-bound state to its active GTP-bound state appears to be a key event in intracellular signaling from several activated tyrosine kinase receptors to the nucleus. The small adaptor proteins Grb2 and Shc provide a link between growth factor receptors and Ras, via the nucleotide exchange factor Sos. Grb2 has been shown to interact directly with growth factor receptors, but also indirectly, via Shc or the tyrosine phosphatase PTP1D/Syp(19) . A number of other proteins are described to interact with the Grb2 SH2 domain, such as BCR-ABL(20) , focal adhesion kinase (FAK)(21) , and a novel 36-38-kDa protein in T-lymphocytes(22) .
In this work we have characterized coupling of FGFR-1 to Ras via Grb2 and Shc. Whereas Grb2 did not interact directly with the FGFR-1, an indirect interaction via the adaptor protein Shc was evident. In addition, a separate pool of Grb2 was in complex with a tyrosine-phosphorylated component of 89 kDa in FGF-2 stimulated cells. We propose a role for this novel component in FGFR-1-mediated Ras activation.
Figure 1: Grb2 SH2 fusion protein does not interact with intact FGFR-1. Lysates from stimulated (+) or unstimulated (-) L6 cells transfected with wild type FGFR-1 were incubated with Grb2 SH2 fusion protein coupled to glutathione-Sepharose. Immunoprecipitation of the receptor was included as a control. The subsequent filter was immunoblotted with FGFR-1 antiserum. Prec., precipitation; Ib, immunoblotting.
Figure 8: FGF-2, but not EGF or PDGF-BB, stimulates association of tyrosine-phosphorylated p89 to Grb2 SH2. Murine brain endothelial cells expressing endogenous receptors for FGF-2, EGF, and PDGF-BB were incubated with 100 ng/ml growth factor for 10 min, lysed, and precipitated with Grb2 SH2 fusion protein bound to glutathione-Sepharose. Samples were separated by SDS-PAGE and immunoblotted with Tyr(P) antibody. Ip, immunoprecipitation; Ib, immunoblotting.
In contrast, Shc,
which is known to anchor Grb2 to receptor tyrosine kinases, was
tyrosine-phosphorylated in response to FGF-2 treatment of FGFR-1
expressing L6 myoblasts (Fig. 2A). In L6 cells
expressing a mutant FGFR-1, Y766F, Shc was still phosphorylated,
although to a lesser extent. The reduction in extent of phosphorylation
of Shc was not due to impaired kinase activity of the mutant, as
compared with the wild type receptor (Fig. 2B).
Analysis of Shc SH2 domain fusion protein interactions in vitro with immobilized FGFR-1 synthetic peptides, phosphorylated or not,
showed that the peptide containing phosphorylated Tyr provided the most efficient binding site (Fig. 2C). The mitogenic signaling capacities of the
wild type FGFR-1, the mutant Y766F, and in addition, the mutant Y463F,
were compared. The incorporation of
I-labeled
deoxyuridine in serum-starved, FGF-2-stimulated L6 cells expressing
wild type or mutant (Y463F or Y766F) receptors showed that both mutants
were less efficient in stimulating DNA synthesis (Fig. 2D). Thus, although Tyr
appears to
be involved in coupling FGFR-1 to mitogenic signal transduction, other
pathways for mitogenicity must exist, depending e.g. on
Tyr
for their initiation. We were, furthermore, not able
to demonstrate direct interaction between the receptor and Shc in
vivo (Fig. 3A). Immunoprecipitation using Shc
antiserum followed by immunoblotting using phosphotyrosine antibody
showed instead that Shc associated with a tyrosine-phosphorylated
145-kDa component after FGF-2 stimulation (Fig. 3B).
This component was not recognized by antisera against the receptor,
Sos, or PLC-
(data not shown) and may correspond to the 145-kDa
component described by Kavanaugh and Williams(28) .
Figure 2:
A, tyrosine phosphorylation of Shc after
FGF-2 stimulation. L6 cells expressing wild type and mutated FGFR-1
were incubated in the presence (+) or absence(-) of 100
ng/ml FGF-2 for 10 min, lysed, and immunoprecipitated with Shc
antiserum. Samples were separated by SDS-PAGE and immunoblotted with
Tyr(P) antibodies. As determined by densitometric scanning, tyrosine
phosphorylation of Shc proteins was reduced in cells expressing the
Y766F mutant FGFR-1, to approximately 30% of the level detected in
cells expressing wild type and Y463F mutant FGFR-1. B,
phosphorylation of wild type and mutated FGFR-1 in FGF-2-stimulated
cells. Immunoprecipitation using FGFR-1 antiserum was performed on cell
lysates from FGF-2 stimulated (+) or unstimulated(-) L6
transfectants. The samples were separated by SDS-PAGE and immunoblotted
with Tyr(P) antiserum, as indicated. C, binding of Shc SH2
domain fusion protein to FGFR-1 synthetic peptides. 5 µg of GST-Shc
SH2 was incubated with a series of nonphosphorylated or phosphorylated
FGFR-1 synthetic peptides coupled to AH Sepharose 4B. Material bound to
the beads was separated by SDS-PAGE and immunoblotted with GST
antiserum. Ip, immunoprecipitation; Ib,
immunoblotting. D, mitogenic capacity of wild type and mutated
FGFR-1. Serum-starved L6 cells expressing wild type (triangles), mutated Y463F (circles), or Y766F (squares) were stimulated with different concentrations of
FGF-2 in the presence of I-labeled deoxyuridine. The
extent of incorporation of radiolabeled iododeoxyuridine was assessed
using a
-counter and plotted as fold increase as compared with
unstimulated cells of each type.
Figure 3: Analysis of Shc complex formation in vivo. L6 cells expressing wild type FGFR-1 were incubated in the presence (+) or absence(-) of 100 ng/ml FGF-2 for 10 min, lysed, and immunoprecipitated with Shc or FGFR-1 antiserum. Samples were separated by SDS-PAGE and immunoblotted with FGFR-1 antiserum (A) and Tyr(P) antibody (B). Ip, immunoprecipitation; Ib, immunoblotting.
Figure 4:
Shc-Grb2 interactions and downstream
events in FGF-2-stimulated cells. L6 cells expressing wild type or,
where indicated, mutated FGFR-1 were incubated in the presence (+)
or absence (-) of 100 ng/ml FGF-2 for 10 min, lysed, and
immunoprecipitated with Shc antiserum (A and C) or
Sos antiserum (B, C, E). Material bound to beads was separated
by SDS-PAGE and imunoblotted with antiserum as indicated (A and B, Grb2 antiserum; C and E, Sos
antiserum). D, Raf1 immunoprecipitates were subjected to
kinase assay in the presence of 5 µCi of
[-
P]ATP and 0.3 mg of recombinant
catalytically inactive MEK B. Kinase reactions were performed for 30
min at 30 °C and samples were separated by SDS-PAGE and analyzed by
autoradiography. Ip, immunoprecipitation; Ib,
immunoblotting.
Figure 5: Association of an 89-kDa component to Grb2. A, L6 cells expressing wild type FGFR-1 were incubated in the presence (+) or absence(-) of 100 ng/ml FGF-2 for the time periods indicated, lysed, and immunoprecipitated with Grb2 antiserum. Material bound to beads was separated by SDS-PAGE and immunoblotted with Tyr(P) antibody to reveal Grb2 in vivo interactions. B, cell lysates from stimulated (+) or unstimulated(-) L6 cells expressing wild type FGFR-1 were incubated with 6 µg of Grb2 SH2 fusion protein or GST control protein coupled to glutathione-Sepharose. Material bound to the beads was separated by SDS-PAGE and immunoblotted with Tyr(P) antibody. Ip, immunoprecipitation; Ib, immunoblotting.
Figure 6: Direct interaction between Grb2 SH2 domain and p89. A, L6 cells expressing wild type FGFR-1 were incubated in the presence (+) or absence(-) of 100 ng/ml FGF-2 for 10 min and lysed. The lysates were preincubated with or without synthetic FGFR-1 peptides and an EGFR peptide comprising the known Grb2 binding site, Y(P)1068, for 45 min before addition of glutathione-Sepharose bound Grb2 SH2 fusion protein and a continued 2-h incubation. After washing, samples were separated by SDS-PAGE and immunoblotted with Tyr(P) antibody to show that only the Y(P)1068 peptide was able to compete for complex formation between Grb2 and p89. B, to show direct binding of Grb2 SH2 to p89, a far-Western analysis was performed. Cells were treated as above and lysates incubated with Grb2 SH2 fusion protein bound to glutathione-Sepharose. Samples were separated by SDS-PAGE and transferred to nitrocellulose filter. The filter was incubated with 0.1 µg/ml Grb2 SH2 fusion protein for 1 h before immunoblotting with GST antiserum. C, analysis of tyrosine-phosphorylated proteins associated with FGFR-1 after FGF-2 stimulation was performed by treating cells with FGF-2 as described above and immunoprecipitation with FGFR-1 antiserum. Samples were subjected to SDS-PAGE and immunoblotting with Tyr(P) antibody. Ip, immunoprecipitation; Ib, immunoblotting.
Figure 7: A, localization of Grb2 SH2-bound p89 in the membrane fraction. L6 cells expressing wild type FGFR-1 were incubated in the presence (+) or absence(-) of 100 ng/ml FGF-2 for 10 min. Cells were scraped into PBS and homogenized. Nuclei were pelleted and discarded, while the supernatants were subjected to Airfuge centrifugation to pellet the particulate (P) fraction. The particulate and soluble (S) fractions were adjusted to equal volumes in Nonidet P-40 lysis buffer and incubated with Grb2 SH2 fusion protein-bound to glutathione-Sepharose. Material bound to beads was separated by SDS-PAGE and immunoblotted with Tyr(P) antibody. B, complex formation of p89 with Sos after FGF-2 stimulation. L6 cells expressing wild type FGFR-1 were incubated in the presence (+) or absence(-) of 100 ng/ml FGF-2 for 10 min, lysed, and incubated with either Sos antiserum or Grb2 SH2 fusion protein. Samples were separated by SDS-PAGE and immunoblotted with Tyr(P) antibody. Prec., precipitation; Ib, immunoblotting.
In this work, we have characterized proteins interacting with
Grb2 in FGF-2-stimulated cells. Growth factor receptors are thought to
couple to Ras via Grb2 in several ways; directly, by binding of the
Grb2 SH2 domain to a specific motif in the receptor, or indirectly, via
binding to different adaptor proteins. Thus far, two such adaptor
molecules have been thoroughly characterized, Shc (33) and PTP
1D/Syp (27) . We could not detect direct interaction between
FGFR-1 and Grb2, which is consistent with the lack of consensus binding
site for Grb2, Y(P)-X-N, in the FGFR-1 sequence(34) .
We further showed that PTP 1D/Syp is not tyrosine-phosporylated in
FGF-2-stimulated cells. PTP 1D/Syp is in complex with and tyrosine
phosphorylated by the PDGF -receptor in response to PDGF-BB, but
this seems to be of consequence for Ras activation only in certain cell
types(35) .
In agreement with previous reports, we detected
tyrosine-phosphorylated Shc proteins in FGF-2-stimulated
FGFR-1-expressing cells(15, 36, 37) . We
failed to identify in vivo complex formation between the
FGFR-1 and Shc, but tyrosine phosphorylation of Shc proteins was to
some extent dependent on Tyr in FGFR-1. This is the only
known binding site in the receptor for SH2 domain-containing proteins,
and it is required for binding of phospholipase
C
(13, 14) . The sequence surrounding Tyr
is compatible with the known consensus sequence for Shc binding
(Y-hydrophobic-X-hydrophobic) in the PDGF
-receptor(38) .
A mutant FGFR-1 in which Tyr
was changed to Phe still
mediated tyrosine phosphorylation of Shc, indicating that also other
sites in the receptor are important or that association of Shc with the
receptor is not required for this adaptor protein to become
phosphorylated. Binding of Shc to the PDGF
-receptor has been
shown to involve several autophosphorylated tyrosine residues, which
bind Shc with low affinity(38) . Moreover, tyrosine
phosphorylation of Shc in response to EGF is largely independent of
individual autophosphorylation sites and occurs even when several or
all autophosphorylation sites have been removed from the
receptor(39, 40) . Recent data indicate that Shc is
critically involved in Ras activation(41) , although a function
for Shc in other pathways cannot be excluded. The Y766F mutant FGFR-1
expressed in L6 myoblasts was indeed less efficient in mediating a
mitogenic signal. This could be due to impaired tyrosine
phosphorylation of Shc, but also to the lack of binding and activation
of PLC-
in cells expressing the Y766F receptor. The role of
PLC-
in mitogenic signaling is unclear, and previous reports have
shown intact mitogenic capacity of the Y766F mutant
receptor(13, 14) . That several parallel pathways for
mitogenicity exists is indicated by the fact that an Y463F mutant
FGFR-1, expressed in L6 cells, also was impaired in mitogenic signaling
(see Fig. 2D).
In two different cell types, L6 myoblasts expressing FGFR-1 after transfection, and murine brain endothelial cells, we found in vivo complexes containing Grb2 and an 89-kDa protein. This protein was tyrosine-phosphorylated after FGF-2 stimulation, but not after EGF or PDGF-BB stimulation. Tyrosine phosphorylation apparently creates a consensus binding site for Grb2 SH2, which, as shown by far-Western analysis, allowed direct complex formation between the 89-kDa component and Grb2. Interestingly, cell fractionation showed that the 89-kDa component was localized exclusively in the membrane fraction in FGF-2-stimulated cells, whereas Shc proteins remained in the cytoplasm. Ras activation has been shown to depend on translocation of the Grb2-Sos complex to the plasma membrane, to allow interaction with Ras (32) . A role for the 89-kDa component in Ras activation was indicated by the presence of 89-kDa protein-Sos complexes in vivo in FGF-stimulated cells. This pool appeared to be separate from the Shc-Grb2-Sos complexes, since we failed to detect the 89-kDa component in Shc immunoprecipitations. Fig. 9shows schematically the composition of the different Grb2-containing complexes in FGF-stimulated cells. We also detected another tyrosine-phosphorylated protein of 40 kDa interacting with Grb2 SH2 in FGF-2-stimulated cells. This far, we have only been able to visualize the 40-kDa component in vitro.
Figure 9: Schematic representation of possible Grb2 interactions after FGF-2 stimulation. Dimerized and activated FGFR-1, with FGF-2 bound to the extracellular domain, mediates tyrosine phosphorylation of Shc through several sites in the FGFR-1. Tyrosine phosphorylation of p89 is mediated directly or indirectly by FGFR-1. Tyrosine-phosphorylated, membrane-bound p89 is found in complex with Grb2-Sos, and is therefore, together with Shc, implicated in Ras activation.
Tyrosine phosphorylation of the 89-kDa component was seen immediately in response to FGF-2 and remained in FGF-2-stimulated cells for several hours. The fact that the 89-kDa molecule is tyrosine-phosphorylated rapidly after FGF-2 stimulation argues that it is phosphorylated directly by the receptor. Whether the phosphorylation involves complex formation with the receptor is presently not clear. Tyrosine phosphorylation of 80-90-kDa proteins in FGF-2-stimulated cells has been described previously(42, 43) . One of these proteins has been identified as cortactin, a cytoskeleton-associated protein(44) . We do not believe that the 89-kDa component described here corresponds to cortactin or to other known signal transduction molecules. Thus, cortactin lacks a potential Grb2 binding site(45) . Furthermore, as shown by immunoblotting analyses, p89 does not correspond to the SH2 domain containing transcription factors denoted ``signal transducer and activator of transcription'' (STAT; data not shown)(46) . Other possible candidate proteins, such as the regulatory subunit p85 of phosphatidylinositol 3`-kinase, do not fulfil the criterion of being tyrosine-phosphorylated only after FGF-2 and not after PDGF-BB or EGF stimulation(47) . Yet other signal transduction molecules with an appropriate mass, such as Eps8(48) , also lack a potential Grb2 SH2 consensus binding site. In order to further characterize the 89-kDa component, we have undertaken a purification procedure. In agreement with a role for p89 in signal transduction, we have detected only very low levels of p89 binding to Grb2 SH2, in the cell types examined this far (data not shown). We aim to extensively increase the starting material, and using microsequencing techniques, we hope to deduce the structure of p89.