©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Sequence Requirements for Binding of Src Family Tyrosine Kinases to Activated Growth Factor Receptors (*)

Gema Alonso (§) , Manfred Koegl , Natalia Mazurenko , Sara A. Courtneidge (¶)

From the (1) Differentiation Programme, European Molecular Biology Laboratory, Postfach 10.2209, Meyerhofstrasse 1, 69012 Heidelberg, Germany

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 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.


INTRODUCTION

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)()(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) .

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.


MATERIALS AND METHODS

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 10cells mland 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.

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.).

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 ICof 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.

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 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.


RESULTS

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) .

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 [-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 ICof 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 ICof 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.

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 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 ICvalues 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 ICfor 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.




DISCUSSION

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 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.

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 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.

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 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 PLC1 (35, 36) .

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.

  
Table: Inhibition of the binding of Fyn to the PDGF receptor by pTyr-containing peptides

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.



FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
A recipient of the Spanish Ministry of Education and Science fellowship.

Present address: SUGEN Inc., 515 Galveston Dr., Redwood City, CA 94063. To whom correspondence should be addressed. Tel.: 415-306-7600; Fax: 415-306-7613; E-mail: sarac@sugen.sf.ca.us.

The abbreviations used are: PDGF, platelet-derived growth factor; CSF-1, colony stimulating factor-1; PAGE, polyacrylamide gel electrophoresis; pTyr, phosphorylated tyrosine; TBS, Tris-buffered saline; DTT, dithiothreitol.


ACKNOWLEDGEMENTS

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.


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