(Received for publication, October 6, 1995; and in revised form, November 22, 1995)
From the
Fibroblast growth factors (FGFs) transduce a variety of biological signals via four distinct tyrosine kinase receptors. We have characterized the phosphorylation of FGF receptor 4 (FGFR-4) and its association with a putative substrate, p85, using transfected L6 myoblast and NIH3T3 fibroblast cell lines. FGFR-4 was phosphorylated in vivo and in vitro mainly on serine and threonine residues in several peptides and to a lower degree on tyrosine residues. When analyzed further by in-gel kinase assay, immunoprecipitates of ligand-activated FGFR-4 contained a serine autophosphorylated polypeptide doublet of 85 kDa. Analysis of the major autophosphorylation site Y754F mutant of FGFR-4 showed that binding of p85 and its serine phosphorylation were independent of receptor autophosphorylation at this site. Okadaic acid treatment increased the basal autophosphorylation activity of p85 but decreased FGFR-4 tyrosine phosphorylation. In contrast, orthovanadate treatment increased the tyrosine phosphorylation of FGFR-4. These data show that a serine kinase is associated with activated FGFR-4 and suggest a role for serine phosphorylation in FGFR-4 function.
Distinct but structurally related receptor tyrosine kinases
FGFR-1 ()to FGFR-4 (1, 2) are responsible
for the specificity and redundancy of FGF signaling for e.g. mitogenesis, neural differentiation, and inhibition of myoblast
differentiation(2, 3) . Alternative splicing of
transcripts generates further FGFR variants, which differ in their
ligand binding capacities. Stimulation of tyrosine kinase receptors by
their ligands leads to activation of the receptors and consequent
phosphorylation of specific tyrosyl residues of the cytoplasmic domain.
However, FGFR-4 tyrosine autophosphorylation is substantially weaker
than that of the other FGF receptors in stimulated
cells(4, 5) . In general, tyrosyl phosphorylation of
the receptors generates binding sites for cellular substrates
containing Src homology 2 (SH2) domains(6) , and these
interactions further trigger signal transduction pathways resulting in
biological responses. This far only one conserved FGFR tyrosyl residue
has been identified as a binding site for cellular substrates. This
residue, corresponding to Tyr-766 in FGFR-1, has been shown to be a
binding site for phospholipase C-
(PLC-
)(7) .
A
number of substrates are known that bind several types of activated
receptor tyrosine kinases. These include PLC- and docking or
adaptor proteins Grb2/Sem-5 and SHC as well as Ras GTPase-activating
protein, phosphatidylinositol 3-kinase, and tyrosine phosphatase SYP
(for a review, see (8) ). It has been shown that after aFGF
stimulation of FGFR-1-transfected cells, PLC-
and SHC are
prominently phosphorylated, whereas FGFR-4 stimulation leads to a weak
phosphorylation of these signal transducers(4, 5) .
Further downstream, Raf-1 and mitogen-activated protein kinases are
activated after FGFR-1 stimulation. In contrast, only a weak
phosphorylation of Raf-1 and mitogen-activated protein kinases is
detected after FGFR-4 activation. While FGFR-1 induced a mitogenic
signal in BaF3 cells, no proliferation was obtained after FGFR-4
activation. However, in L6 and U2OSDr1 cells also FGFR-4 activation
sufficed for DNA synthesis and
proliferation(4, 5, 9, 10) .
In
contrast to PLC-, several signaling molecules have been shown not
to interact with FGF receptors. These include SHC, Grb2, Ras
GTPase-activating protein, phosphatidylinositol 3-kinase, and
SYP(4, 5, 11) . Recently, it was reported
that a novel 89-kDa protein is tyrosine-phosphorylated in
FGF-2-stimulated FGFR-1-expressing cells and binds to
Grb2(11) . It was suggested that p89 couples activated FGFR-1
to the Ras pathway.
In this report we have characterized the in vitro and in vivo phosphorylation of FGFR-4 and show that this receptor associates with a putative novel 85-kDa polypeptide having serine kinase activity.
For two-dimensional
phosphopeptide mapping, the washed filters were incubated overnight in
50 mM NHHCO
containing 5 µg/ml
sequencing grade trypsin (Promega). The supernatants were dried,
oxidized in 50 µl of 50 mM performic acid for 1 h, diluted
with 500 µl of water, frozen, dried, dissolved in 50 µl of 50
mM NH
HCO
, sonicated in a water bath
for 10 min, and subjected to overnight digestion with 5 µg/ml
trypsin. After the second trypsin digestion, 140 µl of
electrophoresis buffer was added, and samples were dried and dissolved
again in 7 µl of electrophoresis buffer. Two-dimensional cellulose
thin layer analysis was performed according to Boyle et al.(13) using the pH 1.9 buffer for electrophoresis and
isobutyric acid buffer for chromatography. The chromatography plates
were analyzed in a Fuji BAS 1000 Bio-Imager.
Figure 1:
Tryptic phosphopeptide maps of FGFR-4.
Shown are the maps of in vivo labeled, unstimulated
(R4-) and aFGF-stimulated (R4+) as well as in vitro labeled WT and Y754F mutant FGFR-4. Confluent 10-cm dishes of
FGFR-4 expressing L6 cells were labeled with
[P]PO
for 4 h and stimulated for 10
min, after which FGFR-4 was immunoprecipitated and analyzed by
two-dimensional phosphopeptide mapping (A and B).
Alternatively, receptor immunoprecipitates from unlabeled cells were
subjected to in vitro kinase reaction followed by the mapping (D and E). White spots in C represent ligand-induced phosphopeptides (a, b, e, and f), and black spots represent
constitutively phosphorylated ones (c and d); the
origin is marked with an asterisk.
When cells expressing FGFR-4 were treated with the phosphotyrosyl phosphatase inhibitor, 200 µM orthovanadate, for 30 min prior to ligand stimulation, FGFR-4 tyrosyl phosphorylation assessed by anti-Tyr(P) Western blotting was increased 2-3-fold (Fig. 2A, upper panel). This suggested that the weak tyrosyl phosphorylation of FGFR-4 in comparison with serine phosphorylation was due to tyrosyl phosphatase activity. However, when the cells were first treated for 2 h with 1 µM okadaic acid, an inhibitor of protein phosphatase types 1 and 2A, and then stimulated with ligand, tyrosyl phosphorylation of FGFR-4 was decreased (Fig. 2A). Neither orthovanadate nor okadaic acid inhibited FGFR-4 kinase activity substantially in vitro (Fig. 2B).
Figure 2:
Effect of sodium orthovanadate and
okadaic acid on tyrosine phosphorylation of FGFR-4. Unstimulated and
aFGF-stimulated FGFR-4 were immunoprecipitated and analyzed by Western
blotting using antiphosphotyrosine (P-Tyr) antibodies (A). The same filter was reprobed with anti-FGFR-4 antiserum (lower panel). In B, the in vitro kinase (IVK) activity of FGFR-4 was analyzed after sodium
orthovanadate and okadaic acid treatment (upper panel). The
immunoprecipitates were also analyzed by Western blotting using FGFR-4
antiserum (B, lower
panel).
Figure 3: In vitro kinase activity of WT and kinase defective (K503A) FGFR-4. FGFR-4 immunoprecipitates from L6 cells were analyzed by in vitro kinase (IVK) assay (A, upper panel) and by anti-FGFR-4 Western blotting (lower panel). The in vitro labeled WT FGFR-4 band was subjected to phosphoamino acid analysis (PAA). In B, the kinase activity of equivalent amounts of WT and K503A mutant FGFR-4 expressed in L6 and NIH3T3 cells was compared using poly(Glu-Tyr) as substrate. LR4, L6 FGFR-4; NR4, NIH3T3 FGFR-4.
Figure 4: A 85-kDa serine kinase associates with FGFR-4. Immunocomplexes of FGFR-4 WT and K503A mutant from L6 cells (A) and WT and Y754F mutant from NIH3T3 cells (C) were analyzed by in-gel kinase assay. Phosphoamino acid analysis (PAA) of p85 from immunoprecipitates (IP) of aFGF-stimulated FGFR-4 WT is shown in B.
As can be seen from Fig. 4C, also FGFR-4 immunoprecipitated from transfected NIH3T3 cells associated with ligand-activated p85 kinase. Similar results were obtained when the previously described phosphorylation mutant Y754F (5) was analyzed. These results show that the association of FGFR-4 and p85 kinase occurs at least in two different cell types and this association is not mediated by the previously described autophosphorylation site, Tyr-754.
Figure 5: Okadaic acid increases the basal activity of p85. FGFR-4 was immunoprecipitated from untreated or okadaic acid-treated NIH3T3 cells. The activity of p85 in the immunocomplexes was then measured by in-gel kinase assay. IP, immunoprecipitate.
In the present study we have obtained evidence that a serine kinase is associated with FGFR-4. The receptor itself is prominently serine-phosphorylated in vivo and in vitro; a 85-kDa polypeptide having serine kinase activity associates with the receptor, and in vitro autophosphorylation of this p85 protein is induced by FGFR-4 activation. These conclusions are based on in vivo labeling results as well as on in vitro kinase reactions done either directly with FGFR-4 immunocomplexes or after SDS-PAGE.
The low tyrosine/serine phosphorylation ratio of FGFR-4 was altered in favor of phosphotyrosine, when cells were treated with the tyrosine phosphatase inhibitor sodium orthophosphate. Thus, tyrosine phosphatases are at least partly responsible for the weak tyrosine phosphorylation detected. After treatment with the serine phosphatase inhibitor okadaic acid, the tyrosine phosphorylation or FGFR-4 was decreased. However, the receptor was still prominently autophosphorylated in vitro. This suggests that the activity of the tyrosine kinase was not substantially altered and that the decrease of FGFR-4 tyrosine phosphorylation is caused by an inhibition of the turnover of serine phosphorylation.
The in-gel kinase assay
showed that FGFR-4 associates with a 85-kDa serine kinase. The
autophosphorylation activity of p85 was clearly increased when FGFR-4
was stimulated with aFGF, but no such stimulation was seen associated
with the kinase-deficient FGFR-4 mutant. Thus, stimulation of FGFR-4
causes the activation of p85 in the system used. However, when cells
were treated with okadaic acid, the activity of p85 was increased to
the level seen after aFGF stimulation. This shows that serine
phosphorylation also apparently regulates the activity of the p85
kinase. It is thus possible that after aFGF stimulation, serine
autophosphorylation or tyrosine phosphorylation by FGFR-4 increases the
activity of p85. We have previously shown that a
tyrosine-phosphorylated band of 85 kDa is detected in FGFR-4
immunoprecipitates from L6 cells(5) . A very weakly labeled
polypeptide of similar mobility is also evident in FGFR-4
immunoprecipitates from cells metabolically labeled with
[P]PO
. However, without specific
antisera or molecular cloning it is not possible to establish whether
the tyrosine-phosphorylated 85-kDa band is the p85 serine kinase.
The C-terminal tyrosine residue Tyr-766 has been previously reported
as the major in vivo and in vitro phosphorylation
site of FGFR-1 and when phosphorylated, it forms the binding site for
PLC-(7) . This tyrosine residue is conserved within the
FGF receptor family, and mutation of this site abolishes most of FGFR-4
autophosphorylation and all detectable PLC-
binding(5) .
Furthermore, the tyrosine residue Tyr-754 of FGFR-4 is the only
reported in vivo phosphorylation site thus far. Therefore, we
analyzed if phosphorylation of this site is required for the
association of FGFR-4 and p85. The in-gel kinase assay showed that the
ligand-stimulated Y754F mutant and WT FGFR-4 induced equally well the
autophosphorylation of p85, indicating that phosphorylation of Tyr-754
is not critical for the association. This suggests that the association
involves some other tyrosine residue or is mediated by mechanisms where
increased receptor tyrosine phosphorylation is not critical. The
hypothesis that tyrosine phosphorylation of FGFR-4 is not critical for
its association with p85 is further supported by the result that p85
activity was detected in immunoprecipitates of unstimulated FGFR-4
after okadaic acid treatment.
It has been reported that FGFR-1
activation leads to tyrosine phosphorylation of a 89-kDa protein and to
complex formation between this p89 and the Grb2 docking
protein(11) . p89 and p85 reported here are distinct proteins,
since no autophosphorylation activity was associated with p89. It also has been recently shown that a serine/threonine kinase,
protein kinase B/Rac, is activated upon basic FGF stimulation in Rat-1
cells. However, it was concluded that phosphatidylinositol 3-kinase was
essential for protein kinase B activation by platelet-derived growth
factor and insulin(17, 18) . The present results
suggest that the differential signal transduction mechanisms of FGFR-4
involve a serine kinase differing from protein kinase B in molecular
weight and properties. The characterization of this kinase will be an
important task for further studies.