(Received for publication, October 21, 1994; and in revised form, November 22, 1994)
From the
SH2 domains bind to specific phosphotyrosine-containing sites in
a fashion dictated by the amino acids flanking the phosphotyrosine.
Attention has focused on the role of the three COOH-terminal positions
(+1 to +3) in generating specificity. Autophosphorylation of
Tyr in the tail of the
-receptor for
platelet-derived growth factor creates a specific binding site for the
COOH-terminal SH2 domain of phospholipase C (PLC)-
1. We show that
the residues 4 and 5 amino acids COOH-terminal to Tyr
(+4 Leu and +5 Pro) are required for efficient
PLC-
1 binding, and that their replacement with the corresponding
residues from a phosphatidylinositol 3`-kinase binding site abrogates
stable association with PLC-
1. In contrast, replacement of the
+3 Pro with Met produces a Tyr
site with mixed
specificity that binds both PLC-
1 and phosphatidylinositol
3`-kinase. This motif is rendered specific for phosphatidylinositol
3`-kinase by further substitution of the +4 Leu. These results
indicate that the +4 and +5 residues are important for the
selective binding of specific SH2 domains. This study suggests that
phosphotyrosine sites can be tailored to bind one or more SH2 domains
with high affinity, depending on the combination of residues in the
+1 to +5 positions.
The Src homology 2 (SH2) domains of intracellular signaling
proteins bind phosphotyrosine-containing sites on a variety of
autophosphorylated growth factor receptors and cytoplasmic
phosphoproteins(1, 2) . Such complexes are important
for interactions of receptor tyrosine kinases with their
targets(3) , for the activation of lymphoid cells by
antigen(4) , and for the regulation of gene expression by
cytokine and interferon receptors(5) . SH2 domains possess a
common ability to bind phosphotyrosine, but distinguish different
phosphorylated sites through the recognition of residues flanking the
phosphotyrosine(6) . Hence, SH2 domains bind
phosphotyrosine-containing peptides of optimal sequence with a K of
10-100 nM,
whereas their affinities for phosphopeptides of random sequence are up
to 1000-fold lower(7) . Experiments employing specific
phosphopeptides and degenerate phosphopeptide libraries have focused
attention on the 3 residues immediately COOH-terminal to the
phosphotyrosine (the +1 to +3 residues), and have shown that
each SH2 domain has a distinct selectivity for these amino
acids(8, 9, 10, 11) . The Src and
Lck SH2 domains, for example, bind preferentially to peptides with the
sequence pTyr-Glu-Glu-Ile (pYEEI). Structural data have shown that
these SH2 domains contain two pockets, one of which is lined by basic
residues that bind phosphotyrosine, while the second is a hydrophobic
pocket that accommodates the side chain of the Ile at the +3
position(12, 13) . The +1 Glu, in contrast,
contacts the surface of the domain. Hence, although the Src and Lck SH2
domains touch the peptide backbone at the -1 and +4
positions, specific contacts are restricted to the phosphotyrosine and
the side chains of the +1 and +3
residues(12, 13) .
In contrast to the Src SH2
domain, that binds preferentially to hydrophilic residues at the
+1 position, the SH2 domains of phospholipase C (PLC)-1, (
)the SH2-containing phosphotyrosine phosphatases, and
phosphatidylinositol 3`-kinase (PI3K) select hydrophobic residues at
both the +1 and +3 positions(10) . We have previously
suggested that residues in addition to the 3 amino acids directly
following the phosphotyrosine are required to stabilize the interaction
between PLC-
1 and the Tyr
autophosphorylation site
of the
-receptor for platelet-derived growth factor
(
-PDGFR)(14) . This notion has received support from
structural analysis of the COOH-terminal PLC-
1 SH2 domain (C-SH2)
and the NH
-terminal Syp phosphotyrosine phosphatase SH2
domain both of which possess an extended hydrophobic groove that
accommodates the +1 to +3 hydrophobic peptide residues, as
well as residues in the +4 and +5
positions(15, 16) . Contacts with the +6 residue
are also detected for PLC-
1 SH2-C domain(15) .
The
-PDGFR possesses autophosphorylation sites in the juxtamembrane
region, the kinase insert, and the COOH-terminal tail that bind to
SH2-containing proteins such as Src, PI3K, Nck, GAP, Syp, and
PLC-
1 (17, 18, 19, 20, 21, 22, 23) .
In most cases these interactions are relatively specific. For example,
PLC-
1 binds the receptor upon phosphorylation of Tyr
in the C-terminal
tail(14, 24, 25, 26) . Substitution
of this Tyr with Phe abrogates PLC-
1-binding and PDGF-induced
hydrolysis of phosphatidylinositol 4,5-biphosphate, but has no effect
on binding of other SH2-containing signaling proteins(27) .
However, mounting evidence suggests that not all SH2-binding sites are
specific for a single signaling protein. For example, both Nck and PI3K
reportedly bind Tyr
in the
-PDGFR(28) . In
the Met receptor tyrosine kinase, two closely spaced tyrosine residues
bind multiple SH2-containing proteins, suggesting that a single
tyrosine phosphorylation site might under some circumstances couple to
multiple signaling pathways(29) . Using the PLC-
1 binding
site of the
-PDGFR, we have investigated the determinants that
restrict a phosphotyrosine-containing site to a single binding partner,
or can allow binding to multiple SH2 proteins.
The PLC-1-binding site at Tyr
in the tail
of the
-PDGFR is contained within the sequence
pYIIPLPD(30) . In contrast, the SH2 domains of the PI3K p85
subunit bind Tyr
and Tyr
in the kinase
insert, which conform to the consensus for PI3K-binding,
pYXXM(10, 27) . GST fusion proteins
containing the kinase insert or COOH-terminal tail of the
-PDGFR
were phosphorylated on tyrosine, and incubated with lysates of Rat-2
fibroblasts. Under these conditions, the kinase insert binds the p85
subunit of PI3K, but not PLC-
1 (Fig. 1, A and B, lanes 1). In contrast, the tail binds PLC-
1
but fails to associate with PI3K (Fig. 1, A and B,
lanes 2). To investigate which residues COOH-terminal to
Tyr
in the tail were required for specific PLC-
1
binding, the residues at the +3, +4, and +5 positions
were substituted with the amino acids found at the corresponding
positions of the Tyr
PI3K-binding site (which has the
sequence pYMDMSKD). Hence, the +3 Pro of the Tyr
PLC-
1-binding site was changed to Met, the +4 Leu to
Ser, and the +5 Pro to Lys, within the context of the GST tail
fusion protein. Since both PLC-
1 and PI3K binding sites have Asp
at +6, this residue was changed to Ala. Wild type and mutant
fusion proteins were expressed and phosphorylated on tyrosine to
similar levels as described under ``Materials and Methods,''
and assayed for binding to PLC-
1 and PI3K. Substitution of the
+3 Pro by Met, to yield a
-PDGFR tail with the sequence
pY
IIMLPD, had little discernible effect on
PLC-
1-binding (Fig. 1C). In contrast,
substitutions of the +4 Leu to Ser (to give pYIIPSPD) or
the +5 Pro to Lys (to pYIIPLKD) both greatly reduced
PLC-
1 binding activity (Fig. 1C). Substitution of
the +6 Asp by Ala had no detectable effect in these assays.
Figure 1:
In vitro binding of p85 and
PLC-1 to the kinase insert (KI) or the carboxyl-terminal
tail (Tail) of the
-PDGF receptor. Panels A and B, GST fusion proteins containing the tyrosine-phosphorylated
-PDGF receptor KI (1), COOH-terminal tail (2),
or a mutated COOH-terminal tail with Pro
(at the +3
position relative to Tyr
) changed to Met (3)
were immobilized on beads and incubated with Rat-2 cell lysate. Binding
of p85 (A) or PLC-
1 (B) to immobilized GST
fusion proteins was detected by immunoblotting with specific antibodies
(``Materials and Methods'') and
I-protein A. As
a control, p85 (panel A, lane 4) and PLC-
1 (panel B,
lane 4) were also immunoprecipitated with specific antibodies. C, wild type and mutant GST fusion proteins containing the
tyrosine-phosphorylated COOH-terminal tail of the
-PDGF receptor
were purified on beads and then incubated with Rat-2 cell lysate.
Fusion proteins contained either the wild type sequence surrounding
Tyr
(pYIIPLPD) or had substitutions at position +3
(pYIIMLPD), +4 (pYIIPSPD), +5 (pYIIPLKD), or +6
(pYIIPLPA). Binding of PLC-
1 was monitored by immunoblotting with
anti-PLC-
1 antibodies, followed by
I-protein A. The
expression and tyrosine phosphorylation of bacterial fusion proteins
was equivalent in each case.
These results indicate that the +4 and +5 residues of the
-PDGFR Tyr
site are both important for high
affinity PLC-
1 binding. In each case their replacement with amino
acids from the cognate positions of the PI3K-binding site effectively
abolished high affinity binding to PLC-
1, but did not induce PI3K
binding (data not shown). However, replacing the +3 Pro with Met
had little effect on PLC-
1 binding, but induced a novel binding
activity for the p85 subunit of PI3K (Fig. 1, A and B, lanes 3). Substitution of the +3 Pro with Met
therefore appears to broaden the binding specificity of the
Tyr
site by increasing its affinity for PI3K, without
markedly affecting binding to PLC-
1. Although the SH2 domains of
PLC-
1 and p85 select phosphopeptides with different amino acids at
the +1 to +3 positions from a degenerate peptide library
screen, they both bind preferentially to hydrophobic residues at
+1 and +3(10) . Consistent with this finding, they
both possess a Cys or Ile at SH2 residue
D5, which appears to be
diagnostic of SH2 domains that bind residues COOH-terminal to the
phosphotyrosine in a hydrophobic groove(10) . This being the
case, it seems likely that the PLC-
1 SH2 domains can accommodate a
+3 Met in the phosphopeptide ligand without substantial loss in
binding affinity, provided that the appropriate residues are present at
the +4 and +5 positions. Although it does not inhibit
PLC-
1 binding, this substitution does create a PI3K-binding site,
consistent with the observation that the binding of p85 SH2 domains is
particularly dependent on a +3 Met. The residues normally present
at the +4 and +5 positions of the PLC-
1-binding site are
apparently compatible with PI3K, once a +3 Met has been
introduced. Consistent with this observation, a
-PDGFR tail mutant
in which the +1 to +3 residues of the PLC-
1-binding site
are converted from Ile-Ile-Pro to Met-Asp-Met (i.e.pYMDMLPD) binds both PLC-
1 and p85 (Fig. 2).
Figure 2:
Modification of Tyr SH2
binding specificity. GST fusion proteins containing the
-PDGF
receptor tail with the wild type binding site for PLC-
1
(Y
IIPL), or with mutated binding sites
(Y
MDML, Y
MDMS) were phosphorylated on
tyrosine, purified on beads, and incubated with Rat-2 cell lysate. A
GST fusion protein containing the
-PDGFR kinase insert, with
binding sites for PI3K (Y
MDMS, Y
VPML) was
also phosphorylated, immobilized, and incubated with Rat-2 cell lysate.
Association of PLC-
1 and p85 with immobilized GST fusion proteins
was detected by immunoblotting with specific antibodies and
I-protein A.
These results predict that further substitution of
the pYMDMLPD sequence at the +4 position, by replacement of
the +4 Pro with a Ser, should discriminate against PLC-1 but
not against PI3K, and thereby create a p85-specific binding site. Fig. 2shows that the phosphorylated
-PDGFR tail containing
the sequence pYMDMSPD binds p85, but shows no detectable
PLC-
1 binding activity. These data indicate that PLC-
1 does
not bind to the Tyr
PI3K-binding site in the kinase
insert because the +4 and +5 residues in the natural PI3K
site antagonize association with PLC-
1.
To investigate in more
detail the notion that the residues at the +4 and +5
positions of the Tyr
-PDGFR autophosphorylation
site are important for binding to PLC-
1, we synthesized
phosphopeptides corresponding to the Tyr
site (residues
1018-1029 of the
-PDGFR). These peptides contained either
the wild type residues, or variant sequences in which the +4 Leu
or +5 Pro were replaced with Ala. The wild type and variant
phosphopeptides were analyzed for their ability to compete for the
binding of the phosphorylated
-PDGFR tail to PLC-
1 (Fig. 3A). The wild type phosphopeptide inhibited
binding of PLC-
1 to the phosphorylated tail of the
-PDGFR
with an IC
of approximately 10 nM, reflecting a
high affinity interaction. In contrast, a variant peptide with Ala at
+4 was about 16-fold less efficient in competing with the
-PDGFR tail for binding to PLC-
1 (IC
, 160
nM). Substitution of the +5 Pro by Ala resulted in a
3-fold decrease in peptide inhibition of PLC-
1 binding to the
-PDGFR tail (IC
, 30 nM). When the data were
expressed as the relative percent of PLC-
1 bound in the absence of
phosphopeptide, all three competition curves showed a Hill coefficient
of 1.0, suggesting only one site of interaction for PLC-
1 on the
-PDGFR tail (Fig. 3B). This is consistent with the
fact that in a previous study(14) , we have demonstrated that
Tyr
in the GST-PDGF receptor tail was the major
phosphorylation site by the Elk kinase domain in the bacterial cells.
Finally, these results indicate that the residues at the +4 and
+5 positions of the Tyr
site are indeed required
for optimal binding, and cannot be replaced even with another
hydrophobic residue without affecting SH2-binding efficiency.
Figure 3:
Phosphopeptide inhibition of PLC-1
binding to the
-PDGF receptor tail. A,
tyrosine-phosphorylated GST fusion protein containing the wild type
-PDGF receptor COOH-terminal tail was immobilized on beads and
incubated with a Rat-2 cell lysate. The cell lysate was preincubated
for 5 min with increasing concentrations of the following
phosphopeptides: wild type Tyr
(DNDpYIIPLPDPK) (
),
Tyr
substituted at position +4 (DNDpYIIPAPDPK)
(
) or +5 (DNDpYIIPLADPK) (
), and a phosphopeptide
Tyr
(SSNpYMAPYDNYK) (
) corresponding to the
recognition site for GAP in the
-PDGF receptor. PLC-
1 binding
to the GST fusion protein was detected by immunoblotting with
anti-PLC-
1 antibodies and
I-protein A (A).
The amount of PLC-
1 bound in the presence of increasing
concentrations of phosphopeptides was expressed as a relative
percentage of PLC-
1 bound in the absence of phosphopeptide (B).
The
experiments discussed above employed full-length PLC-1 isolated
from fibroblast cell lysates. Since the binding of PLC-
1 to the
phosphorylated
-PDGFR tail is apparently mediated by one or the
other of the two PLC-
1 SH2 domains, we have directly measured the
association of these SH2 domains with a phosphopeptide containing the
Tyr
site. For this purpose, wild type Tyr
phosphopeptide was immobilized on the sensor chip of a Pharmacia
BIAcore instrument(37, 38) . Surface plasmon resonance
was used to measure the binding of these immobilized phosphopeptides to
bacterial GST fusion proteins containing the PLC-
1
NH
-terminal SH2 domain (N-SH2), the COOH-terminal SH2
domain (C-SH2), or both SH2 domains together (N+C-SH2). The
combined N+C-SH2 domains of PLC-
1 bound the wild type
Tyr
peptide with the highest affinity (Table 1).
When the two SH2 domains were examined individually, substantial
differences in their affinities for the wild type
-PDGFR
phosphopeptide were observed. The C-SH2 domain bound Tyr
with a higher affinity than the N-SH2 domain (Table 1).
These results suggest that the COOH-terminal SH2 domain is primarily
responsible for specific PLC-
1-binding to the Tyr
autophosphorylation site in the tail of the
-PDGFR, and are
consistent with predictions based on screening of a degenerate
phosphopeptide library(10) . Furthermore, this finding
strengthens the value of models based on the NMR structure of the
PLC-
1 C-SH2 domain complexed to a phosphopeptide representing the
-PDGFR Tyr
autophosphorylation site(15) .
However, these results suggest that the two SH2 domains of PLC-
1
might be acting cooperatively to support optimal affinity binding of
PLC-
1 to the
-PDGF receptor in vivo.
To assess
the importance of residues at positions +4 and +5 in the
Tyr phosphopeptide for the binding of the combined
N+C-SH2 domains of PLC-
1 using the BIAcore system,
Tyr
phosphopeptides with alanine substitutions at the
+4 or +5 positions were used. Substitution of Leu for Ala at
position +4 decreased the affinity of this peptide for
N+C-SH2 domains by about 10-fold (Table 1). Substitution of
Pro by Ala at position +5 resulted in a 2-fold decrease in binding
affinity for GST-SH2 fusion protein containing both SH2 domains (Table 1). The principal kinetic effect of the +4 and
+5 substitutions was on the off-rate (data not shown). These
results indicate that the isolated SH2 domains of PLC-
1 bind with
relatively high affinity to a phosphopeptide spanning the Tyr
site of the
-PDGFR. In addition, substituting residues
+4 and +5 with Ala had similar effects on binding to isolated
PLC-
1 SH2 domains, measured by surface plasmon resonance, and
association with full-length PLC-
1, assessed by competition with
the phosphorylated
-PDGFR tail. Similar results were obtained when
residue +5 Pro was substituted by Lys. However, a 100-fold
decrease in affinity was observed when the +4 Leu residue was
substituted for Ser instead of Ala (data not shown).
The results
presented here suggest that the control of SH2 domain binding
specificity is more complex than previously supposed. By manipulating
the sequence of the residues COOH-terminal to the PLC-1-binding
site in the tail of the
-PDGFR, we have been able to change a site
that is initially specific for PLC-
1 and does not recognize PI3K,
to a site with mixed binding specificity that complexes with both
PLC-
1 and PI3K. Further alteration creates a site that is specific
for PI3K, and does not bind stably to PLC-
1 (Table 2).
Binding of PI3K appears especially sensitive to the presence of a Met
in the +3 position, while PLC-
1-binding is also modulated by
the +4 and +5 residues. The data presented here suggest that
the natural PI3K-binding site at Tyr
of the
-PDGF
receptor has two elements involved in specificity. The residues at
+1 and +3, especially the +3 Met, induce high affinity
binding to PI3K. In contrast, the residues at +4 and +5 do
not appear of particular importance for PI3K binding, but contribute to
specificity by repelling other SH2-containing proteins that might
otherwise bind this site, such as PLC-
1. It is of interest in this
regard that IRS-1, which upon phosphorylation binds PI3K but not
PLC-
1(39) , has nine YXXM motifs of which four
have Ser at the +4 position(40) . None of these sites has
a +4 Leu. The two preferential PI3K-binding sites on IRS-1, at
Tyr
and Tyr
(41) , have Ser and Asp
at the +4 position, respectively. These results suggest a means by
which SH2-binding sites can be tailored to be either specific, as seen
in the
-PDGFR, or promiscuous, as in the Met receptor.