From the
Activation of growth factor receptor protein tyrosine kinases
frequently results in the binding of numerous proteins to their
tyrosine-phosphorylated cytoplasmic domains. These interactions involve
the SH2 domains of the binding proteins and phosphorylated tyrosines on
the receptor molecules, with the specificity of interaction dictated by
the amino acid composition surrounding the phosphorylated tyrosine. In
the case of the platelet-derived growth factor (PDGF) receptor, the
major binding site for Src family tyrosine kinases is in the
juxtamembrane domain and includes tyrosine 579 (Mori, S.,
Rönnstrand, L., Yokote, K., Engström, Å., Courtneidge,
S. A., Claesson-Welsh, L., and Heldin, C-H. (1993) EMBO J. 12,
2257-2264). To analyze in more detail which amino acids
surrounding the phosphorylated tyrosine at position 579 were important
for high affinity interaction with Src family kinases, we synthesized a
series of phosphopeptides corresponding to this binding site in which
single amino acids were individually changed and tested their ability
to compete with the PDGF receptor for binding of Fyn. We found that not
only the three residues carboxyl-terminal to the phosphorylated
tyrosine were important but that also residues at positions
Growth factor receptors have the function of binding
extracellular signaling molecules and elicit responses within the cell.
In the case of tyrosine kinase type receptors, they are transmembrane
proteins composed of an extracellular ligand binding domain, a
transmembrane domain, and a cytoplasmic domain harboring the tyrosine
kinase activity (for review, see Ref. 1). Ligand binding leads to
dimerization of the receptor and autophosphorylation in its cytoplasmic
domain. The phosphotyrosine residues serve as binding sites for
numerous proteins with SH2 domains. The SH2 domain, composed of
approximately 100 amino acids, binds to tyrosine-phosphorylated
proteins, where the affinity of the binding is dictated by the amino
acids in the vicinity of the phosphorylated tyrosine. SH2 domains are
found in a wide variety of polypeptides, many of which are known to
function in the transduction of growth factor signals to cellular
responses (for review, see Ref. 2). The preferences of the various SH2
domains for phosphorylated polypeptides differ
(3, 4, 5) . In particular, the three amino acids
carboxyl-terminal to the phosphotyrosine have been shown frequently to
be important for the affinity of the interaction
(4, 5) .
The Src family tyrosine kinases cSrc, Fyn,
and cYes are among the proteins that bind, via their SH2 domains, and
become phosphorylated by the receptor for platelet-derived growth
factor (PDGF)
The binding site for Src family tyrosine kinases
on the PDGF receptor has been mapped to a tyrosine in the juxtamembrane
region
(12) . To characterize sequence requirements for the
interaction of this group of cytoplasmic tyrosine kinases with receptor
kinases in greater detail, we have used an in vitro competition assay.
For immunoblotting experiments, transfer of proteins to
nitrocellulose (BA85, Schleicher and Shuell) was performed using a
semi-dry apparatus according to the manufacturer's instructions
(Millipore). Following two rinses in phosphate-buffered saline, the
membrane was incubated in blocking solution (3% bovine serum albumin,
fraction V (ICN) in phosphate-buffered saline) for 1 h. The incubation
with the antibodies was carried out for 1 h in blocking solution,
followed by horseradish peroxidase-coupled antibodies, and detected
using the ECL detection reagent (Amersham Corp.).
To
measure the activation of repressed cSrc by tyrosine-phosphorylated
peptides, Src was generated in the repressed state by incubating 6 ng
of purified cSrc with or without 90 ng of purified Csk in 20 µl
modified kinase buffer (kinase buffer containing 0.1% Nonidet P-40 and
5 µM ATP) at 30 °C for 10 min. Proteins were purified
using cell lysate of Sf9 overexpressing cSrc and Csk, respectively, as
previously described
(16) . The mixture was then diluted to give
120 µl of 4
Previous studies have demonstrated that tyrosines 579
and 581 of the PDGF receptor are involved in its association with Fyn,
based on the observations that mutation of these residues reduces the
binding of Src family kinases to the receptor and that phosphopeptides
modeled on these sequences bind Src family kinases
(12) . To
assay whether other phosphorylation sites on the PDGF receptor could
also be involved, we used an in vitro competition assay. We
synthesized a series of phosphotyrosine-containing peptides based on
sequences in the PDGF receptor (Fig. 1 A). The
phosphorylated and non-phosphorylated versions of the peptides were
tested, initially at a concentration of 100 µM, for their
ability to compete the association of Fyn with the PDGF receptor in
vitro. For that purpose, insect cell extracts containing high
levels of Fyn were incubated first with the different peptides and
subsequently with the immobilized PDGF receptor (which had been
isolated from the baculovirus-infected insect cells by
immunoprecipitation). After extensive washing, bound proteins were
labeled by an in vitro kinase reaction using
[
The question of the specificity in the sequence to bind
any particular SH2 domain has been previously addressed using a
phosphopeptide library in which the three amino acids following the
pTyr were varied. With this method, it was found that the optimal
sequence for binding the SH2 domain of the Src family was pYEEI
(4) . A peptide containing this sequence was shown to be able to
inhibit the binding of the Src SH2 domain to the PDGF receptor. The
crystal structure of Src SH2 complexed with a peptide containing the
pYEEI sequence has been solved. In this structure, the peptide binds in
an extended conformation, making primary interactions with the SH2
domain at six amino acids (from
The interaction of Src, Fyn, and Yes with the PDGF receptor
is mediated by the SH2 domains of the Src family kinases and the
sequence surrounding the phosphorylated tyrosines at positions 579 and
581 in the juxtamembrane region of the receptor
(12, 15) . Even though a peptide phosphorylated only on
tyrosine 579 displayed a reasonable affinity for Fyn in our competition
assay, we found that both tyrosines had to be phosphorylated to create
a high affinity binding site. The optimal sequence carboxyl-terminal to
the pTyr to bind to the Src SH2 domain has been determined from a
library of phosphopeptides and was found to be pTyr-Glu-Glu-Ile
(pYEEI). The selectivity of the Src SH2 domain was highest for a large
aliphatic amino acid at position +3, with slightly lower
selectivities at positions +1 and +2
(4) . The
residues carboxyl-terminal to tyrosine 579 of the PDGF receptor,
Ile-Tyr-Val, only match this sequence at the +3 position, whereas
the amino acids at position +1 and +2 do not confirm with the
selected sequence. Our observation that tyrosines 579 and 581 have to
be phosphorylated simultaneously provides an explanation for this
discrepancy: phosphorylation of the tyrosine at +2 introduces a
negative charge, making the peptide resemble pYEEI more closely.
In
the crystal structure of the Src SH2 domain with the pYEEI peptide, an
interaction of the glutamic acid at position +1 with lysine 200 of
cSrc can be observed
(23) . Thus, a likely explanation for the
increased affinity of the doubly phosphorylated PDGF receptor peptide
is that the phosphate on tyrosine 581 makes contacts with lysine 200,
thereby adding to the stability of the complex. The SH2 domains of the
Src family members Src, Fyn, Lck, and Fgr all prefer negatively charged
amino acids at position +1, lending support to the idea that this
interaction is of significance
(4) . Co-crystallization of the
PDGF receptor peptide with the Fyn SH2 domain will be needed to test
this prediction. The SH2 domains of other proteins, such as Abl, or the
amino-terminal SH2 domain of PLC
Phosphopeptides containing the major
autophosphorylation site of Src, tyrosine 416, or the regulatory
tyrosine 527 were not capable of competing the binding of Fyn to the
PDGF receptor (data not shown). Furthermore, we found that Fyn could
bind to the pTyr-527 peptide (poorly when compared to the pTyr-579
peptide) but not to the pTyr-416 peptide (data not shown).
Phosphopeptides containing either Tyr-416 or Tyr-527 have been
described to compete, with the same affinity, the binding of the SH2
domain of Src to a phosphopeptide containing Tyr-527
(28) . In
our system, the Tyr-527 phosphopeptide competed at high concentration
(IC
Using a binding competition assay, we identified a
tyrosine residue in the CSF-1 receptor homologous to Tyr-579 in the
PDGF receptor as the binding site of Src family tyrosine kinases. A
peptide containing this pTyr residue, Tyr-561, efficiently inhibited
the association of Fyn with the CSF-1 receptor (Fig. 2 B)
and also with the PDGF receptor (data not shown). Moreover, CSF-1
receptor mutants in which tyrosine 561 was changed to a phenylalanine
had a reduced ability to associate with Src family members in vivo (Fig. 3), strongly supporting the idea that this tyrosine
residue is primarily responsible for the association of Src kinases
with the CSF-1 receptor. This peptide differs from the PDGF receptor
peptide in that it has a phenylalanine instead of tyrosine at position
+2. When assayed in the competition assay, its affinity is lower
than that of the doubly phosphorylated PDGF receptor peptide yet higher
than the affinity of all other peptides tested. In the activation
assay, the peptide efficiently activated the repressed form of cSrc at
30 µM. Higher concentrations of the peptide did not result
in further activation. However, higher concentrations of the
non-phosphorylated CSF-1 receptor peptide had inhibitory effects on
cSrc activity, so the apparent maximal activation may be
underestimated. Thus, even though the binding site for Src family
kinases on the CSF-1 receptor differs from that on the PDGF receptor in
that it involves only a single phosphorylation event, it still is of
sufficiently high affinity to bind the SH2 domain of repressed forms of
Src.
Different SH2 domains bind best to different pTyr-containing
sequences
(3, 4, 25, 29, 30) .
The question of which sequence binds to any particular SH2 domain has
been addressed before using a phosphopeptide library in which the three
residues following the pTyr were degenerated
(4) (see also
above). The importance of amino acids at positions other than these
three was not considered in this analysis. We focused our attention on
the specificity of the sequence from the PDGF receptor known to bind
the SH2 domain of Src family kinases. We used a series of
phosphopeptides, all of which contain Tyr-579, in which single amino
acids have been changed. To design these mutants, we made an alignment
of the sequence of PDGF receptor, the CSF-1 receptor, and the
carboxyl-terminal tail of Src, known to bind the SH2 domain of Src.
Most of the homologies among these sequences are found in the region
amino-terminal to the pTyr, where acidic residues are found in
positions
A previous
study has reported activation of Src by the pYEEI peptide in vitro (11) . The increase in activity could only be seen at
peptide concentrations 2 orders of magnitude above the ones required
for activation in our experiments. Most likely, the fact that Liu
et al. (11) used immunoprecipitated cSrc, whereas we
employed soluble purified cSrc, accounts for this difference. Indeed,
we have observed inhibition of enzymatic activity brought about by
antibody binding
(37) . Also, the lack of activation by the
doubly phosphorylated peptide from the PDGF receptor reported by Mori
et al. (12) may be because the authors had to rely on
cSrc immunoprecipitated from mammalian cell extracts.
Src family
kinases become transiently activated when the receptor for PDGF is
activated by ligand binding
(7, 8) . At the same time,
they bind the receptor and become phosphorylated on tyrosines in the
amino-terminal half of the protein
(6, 7, 8, 15) . Microinjection
experiments have shown that the engagement of Src family kinases is
crucial for the mitogenic effect of PDGF
(9) , yet little is
known about the actual mechanism of activation. With the demonstration
that PDGF receptor-based peptides can activate Src-like tyrosine
kinases, the most parsimonious explanation at the moment is that
binding to the receptor directly leads to the observed activation. It
remains possible, however, that the scenario is more complex and that
amino-terminal phosphorylations or dephosphorylation at tyrosine 527
serve to amplify the activation. Another known binding site for the SH2
domain of Src family kinases is the autophosphorylation site of focal
adhesion kinase
(38) . The sequence surrounding this tyrosine
meets all of the criteria identified to be necessary for high affinity
binding by Songyang and collaborators and by us
(4) . Thus, the
suggestion that focal adhesion kinase can activate Src family kinases
by binding to their SH2 domains appears plausible in the light of our
data.
Fyn was preincubated with
increasing concentrations of the different pTyr-containing peptides
before the immobilized PDGF receptor was added. Upper panel, mutant
peptides containing pTyr 579; lower panel, phosphopeptides based on the
PDGF receptor binding site and high affinity peptides of different
length. The tyrosine-phosphorylated residue is highlighted. Associated
Fyn was detected as described in Fig. 1 B. The quantitation was
carried out using a PhosphorImager. For every concentration, the amount
of Fyn was normalized to the amount of PDGF receptor present. aa, amino
acids.
We thank Martine F. Roussel for the cell lines
expressing the 723F and 561F mutant receptors, Domique Nalis and
Murielle LeBreton for generation of the phosphopeptides, and Serge
Roche and Peter Lock for critical reading of the manuscript.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
1
and +4 relative to the tyrosine were required. Phosphorylation of
both tyrosines 579 and 581 significantly increased competition
efficiency. The activated colony stimulating factor-1 (CSF-1) receptor,
which is known to associate with Src family kinases, has a sequence in
its juxtamembrane region similar to that surrounding Tyr-579 of the
PDGF receptor, and a phosphopeptide modeled on this sequence competed
the association of Fyn with the receptor in vitro.
Furthermore, mutational analysis demonstrated that these sequences were
required for the efficient association of Src family kinases with the
activated CSF-1 receptor in vivo. Phosphopeptides
corresponding to the Src family binding sites of both PDGF and CSF-1
receptors activated Src kinase activity in vitro. These
observations support a model in which the enzymatic activity of Src
family tyrosine kinases is controlled by intra- and intermolecular
interactions of tyrosine-phosphorylated peptides with the SH2 domain of
the kinases.
(
)(6, 7, 8) . Concomitant with
receptor activation, a transient increase in the specific activity of
the Src family kinases is observed
(7, 8) . The
recruitment of these kinases seems to be indispensable for the
mitogenic effects of the PDGF receptor
(9) . In addition to
binding to activated growth factor receptors, the SH2 domain of Src
family tyrosine kinases has been proposed to regulate their activity;
down-regulation of kinase activity by phosphorylation at a
carboxyl-terminal tyrosine residue involves binding of this
phosphotyrosine to the SH2 domain of the molecule, thereby resulting in
a conformation unfavorable for kinase activity (reviewed in Ref. 10).
Binding of the SH2 domain to the autophosphorylated PDGF receptor may
directly cause activation of Src family tyrosine kinases by repelling
the phosphorylated tail from the SH2 domain, thereby unfolding the
repressive conformation of the kinase. In accordance with this, an
artificial tyrosine-phosphorylated peptide with a high affinity for the
Src SH2 domain has been shown to activate Src in vitro (11) .
Antibodies
Antibodies that recognize
the cSrc, Fyn, and cYes (anti-cst.1), the PDGF receptor (anti-PR4), and
the CSF-1 receptor have been previously described
(8, 13) . Antibodies to pTyr (4G10) were purchased from
Upstate Biotechnology Inc. and used according to manufacturer's
recommendations.
Growth Factors
Recombinant human CSF-1
was the kind gift of Dr. Steven Clark (Genetics Institute, Cambridge,
MA) and was used at 8000 units/ml. Stimulation of quiescent cells with
CSF-1 was conducted at 37 °C for 5 min prior to cell lysis.
Cell Lines
NIH-3T3 cells expressing
mutant CSF-1 receptors, the kind gift of Dr. M. Roussel, were
maintained in Dulbecco's modified Eagle's medium containing
10% fetal calf serum. They were growth arrested at confluence for 48 h
and then incubated overnight in serum-free Dulbecco's modified
Eagle's medium supplemented with 5 µg/ml insulin and 5
µg/ml transferrin prior to stimulation with CSF-1. The expression
levels of the different mutant receptors were checked by immunoblot
analysis.
Baculovirus and Insect Cell
Infections
The construction of a recombinant baculovirus
expressing Fyn and the methods for infection and lysis of insect cells
have been described
(14, 15) . Briefly, the cells were
seeded at 3 10
cells ml
and
infected with the virus for 1 h. The inoculum was removed, and fresh
medium was added. Cells were harvested after 2-4 days and then
washed and lysed as described below.
Biochemical Analyses
Methods for
immunoprecipitation of proteins, kinase assay, SDS-PAGE, and
immunoblotting have all been described before
(8, 15) .
Briefly, cells were rinsed twice with cold TBS (20 mM Tris, pH
7.5, 150 mM NaCl, 1 mM DTT, 100 µM
sodium orthovanadate) and then lysed by scraping in LB (20 mM
Tris, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 1% aprotinin, 20
µM leupeptin, 1 mM DTT, 100 µM
sodium orthovanadate, 10 mM NaF). Lysed cells were transferred
to microfuge tubes, vortexed, incubated 10 min, and centrifuged for 10
min at 10,000 g to remove insoluble material. Lysates
were incubated with antisera for 60 min, centrifuged before transfer to
tubes containing 10 µl of protein A-Sepharose, and incubated for 30
min. All incubations and centrifugations were carried out at 4 °C.
Immunoprecipitates were washed four times with LB and once with TBS.
Competition Assay
The whole process was
carried out at 4 °C. Lysates from Sf9 cells overexpressing Fyn were
incubated with 100 µM peptides (unless otherwise stated)
for 90 min. The PDGF receptor was immunoprecipitated with anti-PR4 from
overexpressing Sf9 cells and added to the mixture of lysate and
peptide. To allow binding, the mixture was incubated for 1 h. After 3
washes in buffer containing 1% Nonidet P-40, 10 mM Tris, pH
7.5, 150 mM NaCl, 100 µM sodium orthovanadate, 1
mM DTT and one wash in TBS, an in vitro kinase assay
was carried out by incubating the immunocomplexed material in kinase
buffer (20 mM Hepes, pH 7.5, 10 mM MnCl,
1 mM DTT) containing 2-10 µCi
[
-
P]ATP for 4 min at 30 °C. The
proteins were resolved in a 7.5% SDS-PAGE and detected by
autoradiography. Quantitation was carried out using a PhosphorImager
(Molecular Dynamics). To determine the IC
of the different
pTyr-containing peptides, increasing concentrations were used in a
competition assay. Alternatively, the bound material was detected by
immunoblot. After the binding reaction and washes, the proteins were
resolved by SDS-PAGE and transferred to nitrocellulose. The upper part
of the blot was then probed with anti-PR4 antibody while the lower part
was probed with anti-cst.1 antibody, followed by incubation with
horseradish peroxidase-coupled protein A and detection by ECL.
concentrated modified kinase buffer. 5 µl of
this were incubated with 10 µl of peptides dissolved at different
concentrations in 100 mM Hepes, pH 7.5, or Hepes only for 60
min on ice. The reaction was started by addition of 5 µl of heat
and acid-denatured enolase containing 2-10 µCi
[
-
P]ATP per reaction. After 4 min, reaction
products were analyzed as described above. In our hands, enolase is not
a substrate for Csk; therefore, the inclusion of Csk in the reaction
had no influence on the phosphorylation of enolase measured.
Tyrosines 579 and 581 of the PDGF Receptor, When
Phosphorylated, Create a High Affinity Binding Site for Src Family
Kinases
After activation of the PDGF receptor by ligand
binding, a complex is formed between Src family tyrosine kinases and
the receptor
(8) . To study this interaction in more detail, we
used PDGF receptors and Fyn protein generated by expression in insect
cells using baculovirus vectors. High levels of PDGF receptor
expression is achieved in insect cells, such that the receptor
dimerizes and becomes activated independent of PDGF
(17) .
Furthermore, we have previously shown that Fyn produced in insect cells
will associate with the PDGF receptor in vitro in a manner
indistinguishable from their association in mammalian cells
(15) .
-
P]ATP, and the reaction products were
analyzed by SDS-PAGE and autoradiography. The Fyn protein obtained from
insect cells is not phosphorylated on its regulatory tyrosine so that
it is not further activated by binding to the PDGF receptor. Under
these conditions, the autophosphorylation of Fyn is a direct measure of
the amount of protein bound to the receptor. The peptide containing
phosphorylated tyrosine 579 inhibited Fyn binding to the PDGF receptor
in a dose-dependent manner (Fig. 1, B and D).
This finding is in agreement with the suggestion that this residue, in
the juxtamembrane region of the receptor, is the target site for Src
family kinases. Interestingly, the most efficient inhibitor of the
association was a phosphopeptide that had both tyrosine 579 and 581
phosphorylated at the same time (see Fig. 5 B). At 100
µM, this peptide inhibited the association of Fyn with the
PDGF receptor more than 90% while the peptide singly phosphorylated on
tyrosine 579 inhibited only 70% of the binding (Fig. 1 B,
see also Fig. 5). No significant inhibition was seen with a
phosphopeptide phosphorylated only on tyrosine 581 (see
Fig. 5B). Thus, the sequence around tyrosine 579 is the
most efficient target site for Src family kinases, and, in contrast to
the known target sites of all other SH2 domains, two phosphorylations
are necessary to create a high affinity binding site for Fyn on the
PDGF receptor. We also observed that phosphorylated peptides modeled on
Tyr-740 and, to a lesser extent, Tyr-751 inhibited the binding of Fyn
to the PDGF receptor (Fig. 1 B). Tyrosines 740 and 751
are located in the kinase insert region and correspond to the binding
sites for phosphatidylinositol 3-kinase
(18) . Tyrosine 740 may
be a second, minor binding site for Src family kinases on the receptor.
Immunoblot analyses were also performed to confirm that the extent of
autophosphorylation of Fyn indeed reflected the amount of protein
present in the assay. The same result was obtained using this approach
(Fig. 1 C).
Figure 1:
Inhibition of the association of Fyn
with the PDGF receptor by tyrosine-phosphorylated peptides. A,
list of peptides modeled on the different tyrosine residues present in
the PDGF receptor ( PR peptides). Numbers indicate the
position of the pTyr in the receptor. The tyrosine-phosphorylated
residue is in bold. B, inhibition of the association
of Fyn with the PDGF receptor by different peptides. Fyn was incubated
with the different peptides at a concentration of 100 µM
before the immobilized PDGF receptor was added. After extensive
washing, both the receptor and the associated Fyn were labeled by an
in vitro kinase assay using
[-
P]ATP and analyzed by SDS-PAGE and
autoradiography. The positions of the PDGF receptor ( PR) and
Fyn are indicated on the left.
, non-phosphorylated
peptide; +, phosphorylated peptide. Molecular weight markers are
shown on the right. C, inhibition of the association of Fyn
with the PDGF receptor by different peptides detected by immunoblot.
After the binding reaction and washes, the proteins were resolved in a
7.5% SDS-PAGE and transferred to nitrocellulose. The upper panel of the blot was probed with anti-PR4 antibody,
whereas the lower panel was probed with anti-cst.1
antibody and detected as described in under ``Materials and
Methods.'' The positions of the PDGF receptor ( PR) and
Fyn are indicated on the left.
, non-phosphorylated
peptide; +, phosphorylated peptide. D, inhibition of the
association of Fyn with the PDGF receptor by tyrosine 579 peptide. Fyn
was preincubated with buffer, increasing concentrations of
phosphorylated tyrosine 579 peptide (from 1 to 500 µM), or
500 µM non-phosphorylated tyrosine 579 peptide. Analysis
of associated Fyn was carried out as described in
B.
Figure 5:
Inhibition of the association of Fyn with
the PDGF receptor by pTyr-containing peptides based on the PDGF
receptor binding site and high affinity peptides. A, list of
peptides based on the PDGF Tyr-579 sequence and high affinity peptides.
The tyrosine-phosphorylated residue is in bold. Numbers in parentheses indicate the length of the peptides.
B, inhibition of the association of Fyn with the PDGF receptor
by peptides. Fyn was incubated with the different peptides at a
concentration of 100 µM before the immobilized PDGF
receptor was added. The associated Fyn was detected as described in
Fig. 1 B. The positions of the PDGF receptor ( PR) and
Fyn are marked on the left. Molecular weight markers are shown
on the right.
Binding Site for Fyn on the CSF-1
Receptor
The CSF-1 receptor is in the same subfamily of
receptor tyrosine kinases as the PDGF receptor
(19) . The
proteins that can bind the activated CSF-1 receptor are less well
characterized than in the case of the receptor for PDGF. So far, only
phosphatidylinositol 3-kinase
(20) , members of the Src family
(13) , and Grb2
(21) have been found in a complex with
the activated receptor. To study the binding site of Src family kinases
to the CSF-1 receptor, we used the same experimental approach as that
for the PDGF receptor by using peptides modeled in CSF-1 receptor
sequence (Fig. 2 A). Only the phosphorylated peptide
corresponding to tyrosine 561 was able to compete the association of
Fyn with the CSF-1 receptor, with 100 µM peptide
inhibiting 80% of the binding (Fig. 2 B). Interestingly,
tyrosine 561 lies in the juxtamembrane region of the CSF-1 receptor, as
does tyrosine 579 in the PDGF receptor, and there is some similarity
between the two sequences. We noticed an increased amount of Fyn when
the assay was conducted in the presence of non-phosphorylated peptides
based on the sequences around tyrosine 556 and tyrosine 809. This may
be due to the low solubility of these particular peptides leading to
nonspecific aggregation problems. Indeed, when Fyn was preincubated
with these peptides, it also associated with immune complexes made with
preimmune serum (data not shown).
Figure 2:
Inhibition of the association of Fyn with
the CSF-1 receptor by pTyr-containing peptides. A, list of
peptides modeled on the different tyrosine residues present in the
CSF-1 receptor ( CR peptides). Numbers indicate the
position of the pTyr on the receptor. The tyrosine-phosphorylated
residue is in bold. B, inhibition of the association
of Fyn with the CSF-1 receptor by different peptides. Fyn was incubated
with the different peptides at a concentration of 100 µM
before the immobilized CSF-1 receptor was added. The associated Fyn was
detected as described in Fig. 1 B. The positions of the CSF-1
receptor ( CR) and Fyn are indicated on the left.
, non-phosphorylated peptide; +, phosphorylated peptide.
Molecular weight markers are shown on the
right.
From the peptide competition
assays, tyrosine 561 seemed to be responsible for the binding of the
Src family kinases to the CSF-1 receptor. To test in vivo whether this was the case, we utilized a mutant receptor (a kind
gift of M. Roussel) in which tyrosine 561 was replaced with
phenylalanine (561F) and expressed in NIH-3T3 cells. As a control, we
used a cell line that contained equivalent levels of a CSF-1 receptor
in which tyrosine 723 (the binding site for phosphatidylinositol
3-kinase
(22) ) was replaced with phenylalanine (723F).
Following CSF-1 stimulation, lysates were immunoprecipitated using
anti-cst.1, an antibody that recognizes Src, Fyn, and Yes
(Fig. 3, lanes 1, 2, 5, and
6) and proteins revealed by anti-phosphotyrosine
immunoblotting. To test the stoichiometry of the association with the
CSF-1 receptor, 5% of the lysate was immunoprecipitated with the anti
CSF-1 receptor antibody (Fig. 3, lanes 3,
4, 7, and 8). After CSF-1 stimulation, in
the cells expressing the 723F mutant, there was an association of a few
percent of this mutant receptor with Src family tyrosine kinases,
whereas in the cells expressing the 561F mutant this association was
severely reduced (Fig. 3, compare lanes 2 and
6). This was not due to the presence of a non-functional
receptor, since cells bearing either type of mutant receptor could
undergo autophosphorylation when stimulated with CSF-1 (Fig. 3,
compare lanes 4 and 8). This lack of
association of Src kinases with the 561F mutant receptor was
consistently seen in this kind of assay (data not shown). The results
of these two experimental approaches strongly suggest that tyrosine 561
is the primary binding site for the Src family of kinases on the CSF-1
receptor.
Figure 3:
Association of Src family kinases with
mutant CSF-1 receptors. Immunoprecipitates were prepared from NIH-3T3
cells expressing mutant CSF-1 receptors (723F, 561F) that were either
left quiescent () or stimulated with CSF-1 (+) using an
antibody against Src family kinases (anti-cst.1, lanes 1, 2, 5, and 6) and anti-CSF-1
receptor antibody ( lanes 3, 4, 7,
and 8). The immunocomplexes were subjected to SDS-PAGE,
transferred to nitrocellulose, and probed with an antibody specific for
pTyr. The position of the receptor ( CR), Src kinases, and the
imunoglobulin heavy chain ( H chain) are marked. 20
times more lysate was used in lanes 1, 2,
5, and 6 compared to 3, 4,
7, and 8. Molecular weight markers are shown on the
right.
Contribution of the Amino Acids around the pTyr to
SH2 Domain Binding
To learn more about the contribution of
the amino acids around the pTyr in the binding of peptides to the SH2
domain of Src family kinases, we compared the sequences surrounding
tyrosine 527 in Src, tyrosine 579 in the PDGF receptor, and tyrosine
561 in the CSF-1 receptor. The residues at positions 9,
6,
4, and +4, relative to the pTyr, are highly
conserved. To test the possible contribution of these residues in the
binding to the SH2 domain, we synthesized a series of mutant peptides,
all of them containing Tyr-579 from the PDGF receptor sequence but with
single changes in the conserved residues (Fig. 4 A). We
also changed the amino acid at position +3 because it has been
described to be important for binding in several SH2 domains
(3, 4) , as well as the amino acid in the
1
position, a negatively charged amino acid in the sequence of the PDGF
receptor. To compare the contribution of any position to the binding,
the concentration of peptide needed to inhibit 50% of the binding
(IC
) was calculated for each peptide (,
upper panel). Whenever a peptide loses its ability to compete,
the mutated residue has to be considered important.
Figure 4:
Inhibition of the association of Fyn with
the PDGF receptor by mutant versions of tyrosine 579-containing
peptides. A, list of the mutant peptides based on tyrosine
579-containing peptide; the tyrosine-phosphorylated residue is
underlined, and the mutated residues are in bold.
Numbers in parentheses indicate the position of the
mutated residue compared to the pTyr. B, inhibition of the
association of Fyn with the PDGF receptor by tyrosine 579 mutant
peptides. Fyn was incubated with the different peptides at a
concentration of 100 µM before the immobilized PDGF
receptor was added. The associated Fyn was detected as described in
Fig. 1 B. The positions of the PDGF receptor ( PR) and
Fyn are marked on the left. , non-phosphorylated
peptide; +, phosphorylated peptide. Molecular weight markers are
shown on the right.
The
phosphorylated non-mutated pTyr-579 peptide inhibited association of
Fyn with the receptor with an ICof 46 µM.
Peptides with changes in positions
9,
6, or
4
still competed for binding to the same extent as the wild type peptide
when tested at a concentration of 100 µM
(Fig. 4 B, lanes 4, 6, and
8). By contrast, peptides with single changes in positions
1, +3, or +4 were strongly reduced in their ability to
inhibit the binding (Fig. 4 B, lanes 12, 14, and 16). Each had an IC
of approximately 200 µM, suggesting an equivalent
contribution of all of these positions in the binding. A peptide in
which all of the negatively charged amino acids, at positions
9,
4,
1, and +4, were changed to positively charged
amino acids could not inhibit the binding efficiently
(Fig. 4 B, lane 10). This peptide has
an IC
of 400 µM, almost 10 times more than
the wild type peptide. These experiments suggest that the amino acids
in positions
1, +3, and +4 relative to the pTyr
contribute equally to the specificity of the interaction with the Fyn
SH2 domain.
2 to +3)
(23) .
However, from the data we obtained with the mutant peptides, the amino
acid in the +4 position also seems to play an important role in
the binding. We decided to test peptides containing the sequence pYEEI
in our system and compare them with Tyr-579-containing peptides. We
synthesized a 9-amino acid pYEEI peptide (shown to interfere with the
binding of Src SH2 domain with the PDGF receptor) and a 6-amino acid
peptide (shown to make primary interactions with the SH2 domain in the
crystal structure). Additional Tyr-579-containing peptides of 9 and 6
amino acids were also synthesized, phosphorylated at tyrosine 579 and
581 either singly or in combination (Fig. 5 A). We tested
the ability of these peptides to inhibit competitively the binding of
Fyn to the PDGF receptor (Fig. 5 B), and the IC
values of all of them were determined (, lower panel). Deletion of 7 amino acids in the amino-terminal
region of the Tyr-579-containing peptide (from
10 to
4)
did not affect its ability to inhibit the association of Fyn with the
PDGF receptor (, lower panel). The
9-amino acid pYEEI peptide inhibited the association of Fyn with the
receptor better (IC
= 3 µM) than the
two peptides containing singly phosphorylated Tyr-579 (IC
= 46 µM for the original 16-amino acid
peptide and 60 µM for the 9-amino acid peptide). However,
the peptide doubly phosphorylated at Tyr-579 and Tyr-581 competed the
binding with the same efficiency as the pYEEI peptide of the same
length, whereas the phosphorylated 581 peptide did not inhibit the
binding (IC
= 350 µM). The ability of
the 6-amino acid peptides to inhibit the binding was clearly decreased
compared with the corresponding 9-amino acid peptides
(Fig. 5 B); the IC
for the 6-amino acid-long
pYEEI peptide was 28 µM, and for the Tyr-579 peptide, it
was 192 µM (, lower panel).
This latter value is similar to the one for the Tyr-579 peptide mutated
in the +4 position (), underscoring the importance of
the +4 position.
Activation of Src by
Phosphopeptides
Negative regulation of Src family tyrosine
kinases by carboxyl-terminal tyrosine phosphorylation is thought to
depend on binding of the pTyr to the SH2 domain
(24, 25, 26, 27) . PDGF stimulation of
cells leads to an increase in the activity of Src family tyrosine
kinases concomitant with binding of these kinases to the activated
receptor
(8) . Although the details of the activation mechanism
are not known, it is possible that binding of the cytoplasmic kinases
to the receptor directly leads to their activation by repelling the
interaction of the phosphorylated tail with the SH2 domain. Indeed, it
has been shown that the high affinity pYEEI peptide can cause an
increase in activity of cSrc
(11) . We purified cSrc and Csk
from overexpressing insect cells
(16) and generated active or
repressed forms of cSrc in vitro. To test if the binding sites
for Src on the growth factor receptors had the potential to increase
Src activity, we incubated the repressed form of Src with high affinity
phosphopeptides and determined the effect on its kinase activity. As
shown in Fig. 6, incubation with the pYEEI peptide resulted in
significant activation of Src activity, whereas non-phosphorylated
peptides had no effect. The PDGF receptor peptide phosphorylated only
on tyrosine 579, as well as the doubly phosphorylated peptide, and the
phosphopeptide modeled on the CSF-1 receptor binding site also
activated Src activity, albeit to lower levels (Fig. 6). A
peptide phosphorylated only on tyrosine 581 or phosphopeptides
containing tyrosine 527 of cSrc did not activate (data not shown).
Thus, binding of Src family kinases to the phosphotyrosines of
activated growth factor receptors may directly lead to activation of
the cytoplasmic kinases.
Figure 6:
Activation of cSrc by high affinity
phosphopeptides. cSrc purified from insect cells was phosphorylated
with pure Csk to generate the repressed form or incubated with ATP only
to generate the non-repressed form as described under ``Materials
and Methods.'' After incubation of the enzyme with
non-phosphorylated peptides (300 µM, lanes 2, 6, and 13) or phosphopeptides (3
µM, lanes 3, 7, 10,
and 14; 30 µM, lanes 4,
8, 11, and 15; 300 µM,
lanes 5, 9, 12, and 16) on
ice for 60 min, Src activity was determined in a kinase assay using
enolase as a substrate followed by SDS-PAGE and analysis on a
PhosphorImager. The peptide concentrations refer to the final
concentration after the addition of enolase. Error bars represent standard deviations derived from three separate
experiments. For the sequence of the peptides, see Fig. 1. Units
represent -fold activation of repressed Src activity. Error bars on the activity of repressed Src are derived from
the variation of activity compared to non-repressed
Src.
also showed preferences for
negatively charged amino acids in position +1 or +2
(4) . The possibility exists that for some of these proteins,
binding sites exist that involve more than one phosphorylation.
However, so far Src family kinases are the only example for this kind
of interaction.
350 µM), but the one containing
Tyr-416 did not compete, even at 1 mM (data not shown).
Possibly, differences in the setup of the competition assay account for
this discrepancy.
9 and
4, and a serine in position
6.
However, these amino acids do not appear to be of any significance to
the affinity of SH2 domain binding, since substitution of these
residues has no effect on the ability of the peptides to compete with
the PDGF receptor in SH2 domain binding. Negatively charged amino acids
are frequently found in the amino-terminal region of phosphorylated
tyrosines
(31) . Indeed, in the case of the binding site of Src
family kinases to the PDGF receptor, this
1 position was of
importance for high affinity binding. Furthermore, the aspartic acid
residue at position +4 appeared to be equally important. That
specificity of SH2 domain binding involves sequences other than the
three amino acids carboxyl-terminal to the pTyr has also been suggested
for other proteins, like Shc
(5, 32, 33) , Nck
(34) , or PLC
1
(35, 36) .
Table:
Inhibition of the binding of Fyn to the PDGF
receptor by pTyr-containing peptides
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.