(Received for publication, November 13, 1995; and in revised form, February 1, 1996)
From the
The Src family of kinases are held in an inactive state by interaction of their SH2 domain with a C-terminal phosphotyrosine. Dephosphorylation of this site can reactivate Src; however, recent evidence suggests that activation can also occur without dephosphorylation. In this study, platelet-derived growth factor receptor phosphorylation of Src on Tyr-213 specifically blocked binding of its SH2 domain to a phosphopeptide corresponding to the C-terminal regulatory sequence, while binding to other sequences, such as the platelet-derived growth factor receptor or a peptide from the epidermal growth factor receptor, was unaffected. Consequently, Src was activated over 50-fold. This is the first demonstration of regulation of a SH2 domain specificity by post-translational modification and is likely to be a general mechanism for regulation of all Src-like kinases.
pp60 (Src) was the first
identified protein-tyrosine kinase and is the prototype of the Src
family of membrane-associated protein-tyrosine kinases(1) . All
of the members of this family have a kinase domain, a domain that binds
phosphotyrosine-containing sequences, and a domain that associates with
proline-rich motifs (for reviews see (2, 3, 4) ). These domains are known as Src
homology (SH) domains or SH1, SH2, and SH3 domains, respectively. The
kinase activity of this family is down-regulated by phosphorylation of
a C-terminal tyrosine (residue number 527 in Src) by members of the
C-terminal Src kinase (Csk) family of kinases(5) . Interaction
of the SH2 domain with the C-terminal phosphotyrosine appears to be
responsible for this inhibition(4) . The Src-like kinases can
be reactivated by dephosphorylation of this tyrosine(6) ;
however, recent reports suggest that reactivation can occur in the
absence of dephosphorylation. For example, T lymphocytes, lacking the
protein-tyrosine phosphatase CD45, have elevated levels of Lck and Fyn
activity even though their negative regulatory sites are
hyperphosphorylated(7, 8) . In addition, Src is
observed to be activated in vitro by phosphorylation with CDC2
only when Tyr-527 is fully phosphorylated(9) .
The binding
specificity of many SH2 domains have been identified
recently(10) . These domains are involved in targeting proteins
to specific locations and in regulating the catalytic domains with
which they are associated(3, 11) . However, the
possibility that the binding of SH2 domains might be regulated has not
yet been explored in depth. There is considerable data demonstrating
that Src is important for growth factor signaling. Treatment of cells
with various growth factors triggers Src to associate with the
corresponding receptor protein-tyrosine kinase (i.e. ErbB2(12) , EGF ()receptor(13) , colony
stimulating factor receptor(14) , PDGF receptor(15) ,
and others). Treatment of cells with PDGF or EGF elevates Src
activity(16, 17) , while overexpression of Src
enhances EGF-induced signaling(18) . We have recently
demonstrated that Src directly phosphorylates the EGF receptor to
create additional docking sites for specific SH2-containing proteins (13) . In contrast, Src is phosphorylated on tyrosine at a
non-autophosphorylation site in PDGF-stimulated cells(16) . We
now demonstrate that PDGF induces phosphorylation of Src within its SH2
domain (tyrosine 213) in vivo. Direct phosphorylation of Src,
by the PDGF receptor in vitro, alters the specificity of its
SH2 domain so that it no longer binds the C-terminal inhibitory
phosphotyrosine sequence, while its binding to other ligands is
unaffected. This results in reactivation of Csk-inactivated Src without
dephosphorylation of Tyr-527. Moreover, conservation of the
phosphorylated tyrosine suggests that phosphorylation of this site may
be a regulatory mechanism that is common for all of the Src-like
kinases(19, 20, 21) .
Confirmation of peptide identity and position of phosphorylation was done by tandem MS-MS. The ion at mass/charge ratio 649 (Fig. 1B) was selected in the first quadrapole. Fragment ions were generated by collision-induced dissociation in an Argon collision chamber, and analyzed by the third quadrapole.
Figure 1:
A, determination of the stoichiometry
of phosphorylation of Src by the PDGF receptor. One µg of Src was
incubated for 2 h at room temperature with
[P]ATP and, where indicated, with 10 µM selective Src inhibitor (CGP 59272) and 100 ng of PDGF receptor.
Src was analyzed by SDS-PAGE and visualized using a PhosphorImager. Src
was excised from the gel, and radioactivity was measured in
scintillation fluid. Src, from lane 2, contained 8,525 cpm
while Src from lane 3 had 56,486 cpm; therefore, the amount of
radioactivity due to PDGF receptor phosphorylation was 47,961 cpm or 12
pmol of phosphate in 17 pmol of Src. Assuming a single site of
phosphorylation this would amount to approximately 70% phosphorylation. B, identification of the PDGF receptor phosphorylation site in
Src. Fifty µg of purified Src was incubated with approximately 10
µg of affinity-purified PDGF receptor in the presence of
[
P]ATP (5 µCi/nmol ATP) and CGP 59272.
Phosphorylated Src was isolated by SDS-PAGE, and in-gel digestion with
trypsin was performed. Tryptic fragments were separated by
reverse-phase HPLC, and 10 µl of each fraction was used for
scintillation counting of radioactivity. The single major peak of
radioactivity (12,438 cpm, 10-fold greater than the next largest peak)
was analyzed by Electro-spray mass spectrometry (MS). C,
confirmation of peptide identity and position of phosphorylation tandem
MS-MS. The ion at mass/charge ratio 649 from B was selected in
the first quadrapole. Fragment ions were generated by collision-induced
dissociation in an Argon collision chamber and analyzed by the third
quadrapole. y ions 1-10 are marked and represent C-terminal
fragments of the parent peptide. Phosphotyrosine is identified by the
difference in mass of 243.5 daltons between the y4 and the y5
ions.
To simplify the identification of sites of tyrosine phosphorylation in vivo, Src was treated with both protein phosphatase 1 and protein phosphatase 2a (gifts from Dr B. Hemmings, Basel, Switzerland), before SDS-PAGE separation, to remove phosphate on serine and threonine. There was no difference in the tryptic phosphopeptide maps of Src, phosphorylated in vitro by Csk and PDGF receptor, treated or untreated with these phosphatases, indicating that they do not affect the level of tyrosine phosphorylation (data not shown). In each case, two-dimensional phosphoamino acid analysis was used to verify that no phosphoserine or phosphothreonine remained (data not shown).
In order to assess the possible result of phosphorylation of Tyr-213 on the peptide binding of the c-Src SH2 domain, these structural models were manipulated by adding phosphate to the appropriate tyrosine. The resulting phosphotyrosine was then revolved around its axis to determine if an interaction between the phosphate and the binding peptide could occur. In both structures, the phosphate could potentially interact with Arg-205; however, only the low affinity pYQPGE peptide could directly interact (via the glutamate in the +4 position) with either Arg-205 or Tyr-213 itself. In either case, the addition of phosphate to Tyr-213 would only be likely to have a negative effect on binding, either by direct interaction of its similar negative charge with Glu in the +4 position of the binding peptide or by competition for binding to Arg-205. Both models were prepared using the molecular modeling program Insight II (Biosym Corp.).
In order to determine how PDGF stimulation causes Src to be activated, we phosphorylated Src with the PDGF receptor under conditions where Src is inhibited and does not autophosphorylate. Approximately 0.7 mol of phosphate/mol of Src were incorporated (Fig. 1A). Tryptic fragments of phosphorylated Src were separated by reverse-phase HPLC, and the single major peak of radioactivity was analyzed by mass spectrometry (MS) (Fig. 1B). The mass spectrum revealed the presence of several peptides, one of which had the mass of a tryptic peptide containing Src residues 207-217 plus 80 daltons (the extra mass expected for addition of a phosphate). The molecular weights of the other peptides were consistent with them being nonphosphorylated tryptic peptides of Src. A complete series of ions representing C-terminal fragments of the peptide was generated and detected by tandem MS-MS (Fig. 1C); this confirmed the identity of the peptide and revealed that Tyr-213 was phosphorylated.
Phosphopeptide maps of Src immunoprecipitated from Balb/c cells and then treated by protein phosphatases 1 and 2a to remove Ser/Thr phosphate, revealed that only a single tyrosine, comigrating with the site confirmed to be phosphorylated by Csk in vitro, was phosphorylated in vivo (data not shown). However, after PDGF treatment, two spots are present, one comigrating again with the Csk site and the other with the PDGF receptor site identified in vitro. This indicates that there is only one major site of phosphorylation by PDGF receptor both in vitro and in vivo.
Comigration of phosphopeptides from Src phosphorylated by PDGF receptor in vitro and of Src isolated from Balb/c cells 10 min after treatment with PDGF, revealed that this same site is phosphorylated in vivo (Fig. 2). Because the site of phosphorylation of Src by the PDGF receptor was originally reported to be on an insoluble tryptic peptide that was only solubilized after thermolysin digestion(16) , we also used both trypsin and thermolysin to examine whether an additional phosphopeptide could be detected. In vitro phosphorylated Src, digested with both trypsin and thermolysin, still had only two soluble phosphopeptides, indicating that thermolysin did not render any additional major phosphorylation sites soluble (Fig. 2).
Figure 2:
Each
of the panels shown above is a two-dimensional phosphopeptide map of
Src digested with trypsin (A-D) or trypsin plus
thermolysin (E and F), as described under
``Materials and Methods.'' The first dimension
electrophoresis moved the peptides from left to right, while the second
dimension chromatography ran from bottom to top. To simplify the
identification of sites of tyrosine phosphorylation, Src was treated
with both protein phosphatase 1 and protein phosphatase 2a, before
SDS-PAGE separation, to remove phosphate on serine and threonine. A, Src (5 µg) was phosphorylated by Csk in the presence of
CGP 59272 to block Src autophosphorylation. B, Src (5 µg)
was phosphorylated by PDGF receptor (200 ng) in the presence of CGP
59272. C, Src was immunoprecipitated (50 µg of Ab327
Oncogene Science) from cell extracts, 10 min after PDGF stimulation of P-loaded Balb/c cells (10
cells). D,
phosphopeptide samples from A-C were combined such that
each contributed an equal share of the total radioactivity. E,
Src was phosphorylated in vitro with Csk and PDGF receptor as
in A and B and digested with both trypsin and
thermolysin. F, Src was immunoprecipitated from
P-loaded cells as in C and digested with both
trypsin and thermolysin.
Tyr-213 lies within the SH2 domain of Src, which has been crystallized with both a low affinity peptide (pYVPML) (19) and a high affinity peptide (EPQpYEEIPIYL)(20) . In the crystal structure of the SH2 domain of Src with the high affinity peptide, Tyr-213 lies near the peptide binding site; however, the proline in the low affinity peptide causes it to bend so that the peptide lies much closer to Tyr-213 (Fig. 3, A and B). Using the coordinates of the Src SH2 domain, crystallized in the presence of low affinity peptide, we made a model of the Src SH2 domain bound to the C-terminal phosphotyrosine sequence (pYQPGE). This peptide also has a proline in the +2 position (relative to the phosphotyrosine) that causes the peptide to bend, and, as a result, the glutamic acid in position +4 lies in close proximity to Arg-205. This arginine probably helps stabilize the binding of this peptide since it is the only positively charged moiety with which the carboxylate of the glutamic acid can interact with. When phosphorylated, the negative charge of Tyr(P)-213 may form a stable electrostatic interaction with Arg-205 and thereby disrupt the interaction between Arg-205 and the glutamic acid in the +4 position of the peptide.
Figure 3: A, model of the Src phosphopeptide, corresponding to the C-terminal regulatory sequence ((pY)QPGE), bound to the SH2 domain of Src with Tyr-213 (the PDGF receptor phosphorylation site) phosphorylated. The model is based on the crystal structure of the SH2 domain of Src complexed with the low affinity peptide (pY)VPML(19) . B, model of (pY)DGIP bound to the SH2 domain of Src with Tyr-213 phosphorylated. The model is based on the crystal structure of the SH2 domain of Src complexed with the high affinity peptide (pY)EEIP from the EGF receptor(20) . Both models were prepared using the molecular modeling program Insight II (Biosym Corp.).
We replaced the sequence of the high affinity peptide with the sequence of the binding site for Src in the EGF receptor (pYDGIP, Fig. 3B) in our model(13) . This peptide is related to the high affinity peptide by the absence of a proline in the +2 position, and, therefore, would be expected to have a similar extended conformation. However, it has the advantage of being a demonstrated physiological binding sequence. Src reportedly binds to phosphotyrosines 579 and/or 581 in the PDGF receptor (pYIpYVDPV)(25) , neither of which are followed by a proline in the +2 position. However, double phosphorylation of this peptide makes it a more complex peptide for doing kinetics with and for interpreting molecular models. Because of the linear conformation of the EGF receptor peptide, its binding would not be expected to be affected by phosphorylation of Tyr-213 (Fig. 3B).
Based on this model, we predicted that phosphorylation of Src by the PDGF receptor would specifically disrupt binding of Src to a phosphopeptide corresponding to its C terminus and to other peptides with similar sequence. However, we expected that Src would still be able to bind to relatively high affinity peptides such as those found in the PDGF receptor and EGF receptor. The first test of this hypothesis was to look for activation of Csk-inactivated Src by phosphorylation with the PDGF receptor. This would indicate that the intramolecular interaction of the N-terminal SH2 domain with the C-terminal phosphotyrosine had been blocked. Two forms of Src were prepared from recombinant baculovirus-infected Sf9 cells, as described previously(9, 13) . Purified Src was dephosphorylated with alkaline phosphatase to yield the first form (simply referred to as Src). A portion of this Src was phosphorylated with Csk and isolated by separation on a Mono Q column (referred to as Csk-inactivated Src, because its activity is 50-100-fold less than that of nonphosphorylated Src).
Src was rapidly, yet modestly, activated by PDGF receptor phosphorylation, to a maximum of approximately twice its initial activity. On the other hand, Csk-inactivated Src was relatively slowly reactivated; however, it was activated to approximately 50-fold its initial activity. The slow phosphorylation of Src by PDGF receptor was surprising, but it is consistent with the model of Src inhibition by Csk. Inhibition occurs as a result of the SH2 binding the C-terminal phosphotyrosine (phosphorylated by Csk). This interaction could be expected to inhibit phosphorylation of a site so close to the binding pocket of the SH2 domain (Tyr-213). To ensure that this result was not due to a contaminating phosphatase dephosphorylating the Tyr-527 site, the experiment was repeated in the opposite sequence. Specifically, Csk-induced inhibition was reduced by prior phosphorylation of Src by the PDGF receptor (data not shown). This demonstrates that PDGF receptor phosphorylation blocks inhibition of Src by Csk, and that this effect is independent of the sequence of phosphorylation by the two kinases. This activation was probably not due to binding of Src to the PDGF-R because PDGF-R was present at only 1/20 the molar concentration of Src. To further test this possibility, autophosphorylated PDGF-R (disabled by 1 µM specific PDGF-R inhibitor, CGP53716) was added to Csk-inactivated Src with little to no effect on activity (data not shown). While activation of Src by phosphorylation of Tyr-213 was predicted by our model, we did not predict the 2-fold activation of the nonphosphorylated form of Src by PDGF receptor. The mechanism of activation for this form of Src is unclear. However, the majority of Src is phosphorylated on Tyr-527 in vivo(4) , suggesting that this phenomena may not be of much physiological importance.
Reactivation of Csk-phosphorylated Src by the PDGF
receptor suggests that phosphorylation of Tyr-213 disrupts the
interaction of the Src-SH2 domain with Tyr(P)-527, as predicted. To
further test this hypothesis, we determined the effect of
phosphorylation on the affinity of Src for P-phosphopeptides (Fig. 4B).
Nonphosphorylated Src bound phosphopeptides corresponding to the Csk
phosphorylation site (PQpYQPGE (pYQPGE), K
= 400 µM), to a segment of the EGF receptor
(PpYDGIPASEISSILEK (pYDGIP), K
= 5
µM), and to the PDGF receptor itself. Phosphorylation of
Src by the PDGF receptor did not affect binding to the pYDGIP peptide
or the autophosphorylated PDGF receptor, as predicted. However, PDGF
receptor-phosphorylated Src bound 70% less pYQPGE peptide, which
corresponds to the fraction of Src phosphorylated (Fig. 1A), indicating that the phosphorylated form of
Src does not bind the pYQPGE phosphopeptide. This conclusion is
supported by restoration of the phosphopeptide binding capacity after
treatment of Src with a protein-tyrosine phosphatase (CD45). This
result demonstrates that phosphorylation of the SH2 domain of Src by
the PDGF receptor specifically blocks binding to the C-terminal
regulatory phosphorylation site, while allowing association with other
high affinity ligands.
Figure 4:
A, the effect of PDGF receptor
phosphorylation on Src or Csk-inactivated Src was tested. The two forms
of Src were prepared described under ``Materials and
Methods.'' Src or Csk-inactivated Src (2 µg) were incubated
with PDGF receptor (50 ng) and 20 µM ATP for the indicated
period of time. The reaction was stopped by addition of a selective
PDGF-receptor inhibitor (1 µM CGP 53716)(23) . The
Src activity was assessed after a 5-min incubation with 20 µM [P]ATP and 10 µg of poly(Glu-Tyr). Ten
µl of the reaction was spotted onto P81 paper and the radioactivity
was measured. Each time point is the mean of 3 determinations with the
standard deviation shown by error bars. B, the ability of
immobilized Src (filled) or PDGF receptor-phosphorylated Src (open) to bind to either P(pY)DGIPASEISSILEK (circles) or to PQ(pY)QPGE (triangles) was assessed.
Phosphopeptides were diluted to 7 different concentrations ranging from
0.01 µM to 10,000 µM. After a 2 min
incubation with the antibody-complexed Src, the unbound peptides were
removed by washing with 5 ml of wash buffer. The amount of peptide
bound was calculated based on a 50% Cerenkoff counting efficiency after
transferring the antibody complex to scintillation vials. The K
values for each peptide are designated
by dotted lines.
Platelet-derived growth factor (PDGF) primarily stimulates the protein-tyrosine kinase activity of the PDGF receptor(26) ; however, it also elevates the activity of a second tyrosine kinase, Src(16) . Src has previously been shown to associate with the PDGF receptor (15) and to be phosphorylated within its N-terminal segment, following treatment of cells with PDGF(27) . Hunter et al.(28, 29) have reported that mutation of Tyr-138 to Phe abolishes phosphorylation of Src by the PDGF-R in vitro. However, direct observation of Tyr(P)-138 (mass spectrometry, sequencing, or co-migration with synthetic peptides) has not been reported. Furthermore, the ability of mutated Tyr-138 to abolish the in vivo phosphorylation has not been demonstrated. In fact, mutation of either Tyr-138 or Tyr-133 to Phe interferes with mitogenicity of both PDGF and EGF, suggesting that modification of this region of the SH3 domain may block its function in a non-phosphorylation-dependent manner. This hypothesis is supported by data demonstrating that EGF-R does not phosphorylate Src in vitro or in vivo (in response to EGF). Therefore, we conclude that phosphorylation of Tyr-138 is not the relevant site of phosphorylation by PDGF-R in vivo.
In this report, we
demonstrate by direct tandem mass spectrometric peptide sequencing that
the PDGF receptor phosphorylates Src near the binding pocket of the SH2
domain at Tyr-213 in vitro and that the in vivo site
co-migrates by two-dimensional peptide mapping to a synthetic peptide
containing Tyr-213. Our identification of this site is further
strengthened by results by Couture et al.(30) ()demonstrating that Lck is phosphorylated
at the equivalent site (Tyr-192) by Syk and ZAP-70. Molecular modeling
correctly predicted that a phosphate at this site would specifically
disrupt binding of the SH2 domain to phosphopeptides with sequences
similar to the C-terminal regulatory phosphotyrosine of Src (proline in
the +2 position).
Disruption of the Src-SH2 interaction with its C-terminal regulatory phosphotyrosine may affect more than just its enzymatic activity. Treatment of Balb/c cells with PDGF causes Src to translocate from the plasma membrane to the cytosol(31) . It has recently been shown that mutation of Tyr-527 results in a redistribution of Src to focal adhesions sites(32) . Because phosphorylation of Tyr-213 affects both the availability and the specificity of Src's SH2 domain, we speculate that phosphorylation by the PDGF receptor may induce both translocation and activation of Src.
Tyrosine 213 of Src is conserved in the SH2
domain of all Src-like kinases, as is the C-terminal regulatory
phosphorylation site(21) . The activation of Lck and Fyn in T
lymphocytes lacking CD45 correlates with the increase in tyrosine
phosphorylation of these kinases(7) , suggesting that they are
also activated by tyrosylphosphorylation. Furthermore, Lck is
phosphorylated on Tyr-192, the residue corresponding to Tyr-213 of Src,
in response to treatment of cells with anti-CD3 antibodies(8) .
Phosphorylation of this site correlates with increased activity of
pp56 in COS cells co-transfected with pp56
and pp72
(30) . Additionally,
phosphorylation alters the binding properties of its SH2 domain in a
manner similar to what we observe for PDGF receptor phosphorylated Src
(Couture et al.(1995)). These findings, in conjunction with
the results of our study, suggest that this may be a general mechanism
for activating Src family kinases.
Recent investigations suggest that Src can be activated by a number of mechanisms. Src can be activated by dephosphorylation of Tyr-527, ligand occupation of the SH2 binding site, phosphorylation by CDC2, and/or phosphorylation by the PDGF receptor. All of these events act to disrupt the interaction of the SH2 domain with Tyr-527, supporting the standard model of repression. Because these different activating mechanisms also affect the availability and at least in one instance the specificity of the SH2 domain, we speculate that they may result in differential localization and/or protein-protein interactions in addition to activation.