(Received for publication, July 18, 1995; and in revised form, October 25, 1995)
From the
The phosphotyrosine binding (PTB) domain specifically binds to tyrosine-phosphorylated proteins, but differs in structure and mechanism of action from the SH2 domain family. We quantitated the affinity, specificity, and kinetics of the interaction of the SHC PTB domain with a sequence motif, asparagine-X-X-phosphotyrosine (NXX(pY)), found in several receptor tyrosine kinases and oncogenic proteins. PTB domain-mediated interaction with the NXX(pY) motif of c-ErbB2 was characterized by similar overall affinity but slower kinetics than that reported for SH2 domains. This suggested that unlike SH2 domains, PTB domains may not rapidly exchange among associated proteins. Furthermore, when directly and quantitatively compared, PTB domain binding specificity did not significantly overlap with a panel of seven SH2 domains. Thus, signaling pathways involving PTB and SH2 domain-mediated interactions can be regulated separately. Finally, our data define the minimal SHC PTB domain binding motif as NXX(pY), not NPX(pY) as suggested by other authors, and suggest a high affinity motif, hydrophobic residue-(D/E)-N-X-X-pY-(W/F), found in the Trk and ErbB receptor tyrosine kinase families. We conclude that PTB domains mediate specific protein-protein interactions independent from those mediated by SH2 domains.
Intracellular signaling by receptor tyrosine kinases is initiated by autophosphorylation of tyrosine residues on the intracellular domains of these molecules (for reviews, see (1) and (2) ). One of the most important consequences of autophosphorylation is the creation of binding sites on the receptors for proteins containing domains which specifically interact with tyrosine-phosphorylated targets. The first class of these protein domains to be recognized was the SH2 domain (for reviews, see (3) and (4) ). Genetic, biochemical and structural evidence has demonstrated that SH2 domains are functional protein motifs of approximately 100 residues which specifically bind to phosphotyrosine and one or more adjacent amino acids. SH2 domains mediate the assembly of a potentially large number of signaling complexes, within which downstream signaling molecules can be regulated.
Recently, a novel protein domain, the phosphotyrosine
binding (PTB) ()domain, has been described which also
specifically binds to the tyrosine-phosphorylated form of target
proteins. The PTB domain was first described as a 186-amino acid
segment in the NH
terminus of the signaling adapter protein
SHC(5) . The SHC PTB domain binds to autophosphorylated growth
factor receptors (6, 7, 8, 9, 10) and to
other unidentified tyrosine-phosphorylated proteins in growth
factor-stimulated cells(5) . The PTB domain of SHC has
functional similarities to the SH2 domain family, but differs in both
structure and mechanism of action. Like SH2 domains, PTB domains
specifically recognize phosphotyrosine, bind to short sequences, and
mediate the assembly of signaling complexes during tyrosine kinase
signaling. However, the SHC PTB domain has little sequence homology to
the SH2 domain family, has a different predicted secondary structure,
and does not contain a functional FLVRES sequence (5) , which
constitutes part of the phosphotyrosine binding pocket in SH2
domains(11) . The SHC PTB domain is likely to be a member of a
larger family of similar domains(5, 7, 12) .
Unlike SH2 domains, little is known about the binding
characteristics of PTB domains. Available data suggest that SH2 domains
and PTB domains may recognize different tyrosine-phosphorylated
sequences through different mechanisms. Although SH2 domain specificity
is principally determined by residues COOH-terminal to the
phosphotyrosine(13, 14, 15, 16, 17) ,
the PTB domain of SHC requires an asparagine NH-terminal to
the phosphotyrosine, within the motif NXX(pY) (where N is asparagine, X is any amino acid, and pY is phosphotyrosine)(6) .
Furthermore, peptide ligands bind SH2 domains in an extended
conformation; high affinity binding of peptide ligands to the SHC PTB
domain may require a specific conformation conferred by multiple
residues in the peptide(6) . From these studies, several
receptor tyrosine kinases have been predicted to contain binding sites
for the SHC PTB domain. The affinity, specificity, and kinetics of PTB
domain-mediated interactions have not been quantitatively determined.
These data are critical to understanding the relationship of PTB and
SH2 domain-mediated interactions during tyrosine kinase signaling. In
this report, we examined quantitatively the binding of the SHC PTB
domain to sequence motifs derived from growth factor receptors and
oncoproteins. This data strongly suggest that PTB domains mediate
specific protein-protein interactions during tyrosine kinase signaling.
The PTB
domain of SHC (residues 46-232) was expressed as a glutathione S-transferase fusion protein from baculovirus as described (5, 20) . For real-time kinetic analysis, the PTB
domain sequence was tagged with the Glu-Glu epitope (21) at the
NH terminus, cloned into the baculovirus expression vector
pAC13(22) , and expressed as described(5) . The
Glu-Glu-tagged PTB domain was purified by affinity chromatography with
a monoclonal anti-Glu-Glu antibody (23) , followed by
chromatography with HighTrap Q anion exchange resin (Pharmacia LKB
Biotech). The purity of this preparation was >95% as assessed by
SDS-polyacrylamide gel electrophoresis and silver staining.
Data processing was with BIAevaluation software (Pharmacia, version 2.1). Dissociation rate constants were measured in buffer flow according to the following equation,
where k is the dissociation rate constant, R
is the relative response (representing binding
of PTB domain to the peptide) at time t, and R
is the relative response at the starting time, t
. Generally, t
was
approximately 10 s, and t was approximately 70 s after the
start of the elution. Response during this time was linear, indicating
that a homogeneous 1:1 dissociation model is applicable (data not
shown). Association rate constants were calculated from the measured k
according to the following association type 1
model(26) ,
where R is the steady state response
level, k
is the association rate constant, and C is the molar concentration of PTB domain. Dissociation
constants (K
) were calculated by dividing k
by k
.
The results of a typical experiment are shown in Fig. 1. PTB domain protein bound to immobilized peptide
b-NL(pY)(pY) (Fig. 1), but did not bind significantly to
uncoated or streptavidin-coated chips in the absence of phosphorylated
peptide, nor to immobilized, nonphosphorylated peptide containing the
same amino acid sequence (not shown). These data confirm our earlier
report that PTB domain binding requires the presence of
phosphotyrosine. The rate of dissociation of PTB domain from the
immobilized b-NL(pY)(pY) peptide under conditions of buffer flow was
linear, with a rate constant (k) of 2.5 ±
0.21
10
s
. The calculated
association rate constant (k
) was 4.7 ±
0.56
10
M
s
. The dissociation constant (K
) for PTB domain was 53 nM. This value
is in agreement with our previous report of an IC
of
30-50 nM for this peptide in competition binding
experiments using GST-PTB domain and ErbB2 protein derived from SKBR3
cells (6) and with the half-maximal effective concentration
(EC
) determined by an ELISA system described below.
Figure 1: Real-time kinetic analysis of SHC PTB domain binding. Peptide b-NL(pY)(pY) (Table 1) was immobilized on a streptavidin-coated BIAcore chip, and the indicated concentrations of PTB domain protein were injected over the chip surface (first arrow) as described under ``Materials and Methods.'' At the end of injection (second arrow), dissociation rates were measured in buffer flow. Resonance signal, which reflects PTB domain binding to the peptide, is given in arbitrary resonance units (RU). A typical experiment is shown.
We have developed an ELISA assay in a
microtiter plate format for measuring binding of the SHC PTB domain to
peptide ligands (see ``Materials and Methods''). 12CA5, a
monoclonal antibody directed against the influenza HA epitope, was
adsorbed to microtiter plates and used to capture HA-tagged GST-PTB
domain fusion protein. Biotinylated peptide ligands were then incubated
with the immobilized PTB domain, the plates washed, and the presence of
bound peptide detected by addition of streptavidin-coupled alkaline
phosphatase and a colorimetric phosphatase substrate. No color
development above background was observed in the absence of 12CA5
antibody or PTB domain (Fig. 2A). Saturable binding was
observed with increasing concentrations of biotinylated peptide derived
from ErbB2 (b-NL(pY)(pY)), with half-maximal binding (EC)
at approximately 20-50 nM in multiple experiments (Fig. 2A). This value is consistent with the K
derived from real-time kinetic analysis of
approximately 53 nM (above) and with competition binding
experiments performed in solution (6) . No binding was observed
with biotinylated, nonphosphorylated peptides (not shown). Therefore,
the ELISA binding assay isa valid method for quantitating PTB
domain-mediated interactions.
Figure 2:
PTB
domain binding to NXX(pY) and SH2 domain recognition motifs.
Binding of a HA epitope-tagged GST-PTB domain fusion protein to
biotinylated, tyrosine-phosphorylated peptides was analyzed by an ELISA
assay, as described under ``Materials and Methods.'' A, binding of PTB domain to increasing concentrations of
peptide b-NL(pY)(pY) (closed circles). Controls included
assays performed in the absence of the capturing antibody, 12CA5 (open squares), or in the absence of PTB domain protein (open triangles). A representative experiment is shown. B, binding of PTB domain to peptides representing binding
sites for the SH2 domains of GAP (open squares), PLC- (open triangles), and p85 (open circles), compared
with peptide b-NL(pY)(pY) (closed
circles).
Although the b-NL(pY)(pY) peptide
bound with high affinity to the SHC PTB domain, biotinylated peptides
containing recognition sequences for SH2 domains from the 85-kDa
subunit of phosphatidylinositol 3`-kinase (b-p85), from GAP (b-GAP), or
from PLC- (b-PLC) did not bind with measurable affinity to the SHC
PTB domain in the same experiment (Fig. 2B). These same
peptides did bind to their respective SH2 domains in separate
experiments ( Fig. 3and data not shown). Therefore, under these
conditions, the PTB domain does not recognize these SH2 binding sites.
None of these peptides contains an Asn at position -3 relative to
the phosphotyrosine, although peptide b-PLC has an Asn at the -2
position. These data are consistent with our previous report that an
Asn at the -3 position is necessary for PTB domain binding and
show that an Asn at the -2 position is not sufficient for
binding.
Figure 3:
Binding of SH2 domains to the PTB domain
peptide ligand b-NL(pY)(pY). SH2 domain binding to peptide ligands was
analyzed by ELISA (A and B) or by a solution binding
assay (C and D) (see ``Materials and
Methods''). A, binding of the NH-terminal (open circles) and COOH-terminal (open triangles) p85
SH2 domains to the PTB domain ligand b-NL(pY)(pY), compared with
PTB-b-NL(pY)(pY) binding (closed circles). B, binding
of the SHC SH2 domain to b-NL(pY)(pY) (open circles) compared
with PTB-b-NL(pY)(pY) binding (closed circles). A
representative experiment is shown. C, binding of the
PLC-
NH
-terminal (PLCnSH2) and COOH-terminal (PLCcSH2) SH2 domains to 10 µM peptide
b-NL(pY)(pY) (PTB peptide) and 10 µM peptide
b-PLC-
(PLC peptide). D, binding of GAP
COOH-terminal SH2 domain to 2 µM peptide b-NL(pY)(pY) (PTB peptide) and 2 µM peptide b-GAP (GAP
peptide).
To examine the affinity of SH2 domains for the PTB domain
recognition sequence, we performed ELISA binding assays of HA-tagged
SH2 domains with the PTB peptide ligand b-NL(pY)(pY). The COOH- and
NH-terminal SH2 domains of p85 bound to peptide
b-NL(pY)(pY) with a half-maximal binding concentration at least 2
orders of magnitude greater than that of PTB domain for the same
peptide (Fig. 3A). However, a peptide containing the
recognition sequence for the p85 SH2 domain (b-p85, Table 1)
bound to p85 SH2 domains with a half-maximal concentration of 40 nM in the same experiment (data not shown). This affinity is similar
to that of PTB domain for peptide b-NL(pY)(pY) (Fig. 2).
Similarly, the affinity of the SHC SH2 domain was at least 1000 times
lower for peptide b-NL(pY)(pY) than the affinity of PTB domain for this
peptide (Fig. 3B).
To examine the binding of other
SH2 domains to this peptide, we used a solution binding assay described
previously(6) . TrpE-SH2 domain fusion protein and biotinylated
peptides were mixed in solution, immunoprecipitated with anti-TrpE
antibodies, washed, and bound peptide detected as described above. No
binding of the b-NL(pY)(pY) peptide was detected to either the
NH-terminal or the COOH-terminal SH2 domains of PLC-
,
even at relatively high concentrations of peptide (Fig. 3C). A peptide containing a PLC-
recognition
motif bound to the NH
-terminal SH2 domain of PLC-
in
the same experiment (Fig. 3C). Similar results were
obtained with the NH
-terminal and COOH-terminal SH2 domains
of GAP (Fig. 3D).
Under these assay conditions, peptides derived from ErbB2 and ErbB3
had the highest apparent affinity (Fig. 4). Peptides derived
from the nerve growth factor receptor (TrkA) competed for binding, but
with an approximately 10-fold lower affinity in this assay.
Polyomavirus middle T antigen-derived peptides had very low affinity,
while insulin receptor and epidermal growth factor receptor peptides
did not significantly compete for binding. By comparing the sequences
of the highest affinity peptides with the lowest (Table 1), a
consensus sequence for higher affinity binding is apparent,
(D/E)-N-X-X-pY-(W/F). Furthermore, all high affinity sequences
contained hydrophobic residues () in the -5 and -6
positions.
Figure 4: Binding of SHC PTB domain to SHC binding sites in receptor tyrosine kinases and oncoproteins. Peptides representing the SHC binding sites on ErbB2 (closed circles), ErbB3 (open circles), NGF receptor (Trk, open triangles), polyomavirus middle T antigen (MT, open squares), EGF receptor (EGFR, open diamonds) and insulin receptor (IR, closed squares) were compared in a competitive ELISA binding assay (see ``Materials and Methods''). Control (closed diamonds) represents no PTB domain protein in the assay.
Reports from this and other laboratories have demonstrated that PTB domains are functionally similar to SH2 domains. Both domains mediate the assembly of signaling complexes during tyrosine kinase signaling by specifically binding to tyrosine-phosphorylated proteins. One of the most important questions raised by this model is whether PTB domains mediate protein-protein interactions distinct from those of SH2 domains and therefore are potentially involved in separate signaling pathways. The data in this report strongly support the concept that PTB domains are novel mediators of signaling complex assembly which differ in mechanism and specificity from the SH2 domain family.
First, the
kinetics of the interaction of the SHC PTB domain interaction with the
ErbB2-derived NLX(pY) motif appear to be different from those reported
previously for typical SH2 domains. Binding of the p85 SH2 domain to
phosphotyrosine-containing peptides, performed under very similar
conditions using the same BIAcore device as in this study, has been
reported to occur with similar K but much faster
kinetics by several investigators. For example, the k
of the SH2 domains of the 85-kDa subunit of phosphatidylinositol
3`-kinase have been reported to range from 1.6-3.3
10
M
s
(25) to 3-40
10
M
s
(23) ,
100-10,000 times faster than our values for the SHC PTB domain.
Furthermore, the k
of these SH2 domains ranged
from 0.98 to 1.4
10
s
,
also almost 100 times faster than the SHC PTB domain. These very high
on and off rates for SH2 domains have been interpreted to suggest that
SH2 domain-containing proteins can rapidly exchange among associated
proteins. Rapid dissociation would also allow for rapid control of SH2
domain-mediated interactions, by continually exposing SH2 domain
binding sites to phosphorylation and dephosphorylation by regulatory
enzymes. Our data suggest that PTB domains do not rapidly exchange as
do SH2 domains and therefore may be regulated differently. Furthermore,
the differences in binding kinetics support the idea that PTB
domain-mediated interactions have a different structural basis for
phosphotyrosine recognition than the SH2 domains.
Second, our results demonstrate directly that the affinity of a panel of seven SH2 domains for the b-NL(pY)(pY) peptide was at least several orders of magnitude lower than the affinity of PTB domain for this peptide. Furthermore, the PTB domain does not recognize several SH2 domain target sequences. Therefore the specificities of these SH2 domains and the SHC PTB domain are significantly different. This suggests that in vivo PTB and SH2 domains mediate separate protein-protein interactions and therefore may be regulated independently. Consistent with this concept, the SH2 and PTB domains of SHC bind different proteins in Balb/c 3T3 cells(5) . PTB and SH2 domains may also cooperate in signal complex assembly by binding to separate sites on the same molecule.
Finally, we have identified a candidate high
affinity binding motif for the SHC PTB domain by quantitatively
comparing PTB binding to predicted target sequences derived from
various signaling proteins. This motif,
-(D/E)-N-X-X-pY-(W/F), is present in the Trk tyrosine
kinase receptor family, including Trk A (nerve growth factor receptor),
Trk B (BDNF receptor), and Trk C (NT-3 receptor); the ErbB family,
including ErbB2, ErbB3, and ErbB4; in torso, a receptor
tyrosine kinase from Drosophilia, and in dozens of other
proteins. These data are consistent with our previous report of the
effects of single amino acid substitutions of ErbB2-derived peptides on
binding to the SHC PTB domain(6) . In those studies, the
presence of an aspartic acid at the -4 position (relative to the
phosphotyrosine) and either a tryptophan or phenylalanine at the
+1 position conferred higher affinity.
These results do not exclude the possibility that the peptides with lower affinities, such as those derived from the EGF and insulin receptors, may represent PTB domain binding sites in vivo. The competitive binding assay was used to compare the relative affinities of these SHC binding sites for the PTB domain, but the true affinites are critically dependent on assay conditions and may be underestimated in Fig. 4. Furthermore, these sequences may confer higher affinity in the context of the intact protein. We have previously suggested that the conformation of the PTB domain binding site is important for high-affinity interaction(6) . Recent reports have demonstrated that the SHC PTB domain can interact with the insulin receptor (7) and with EGF receptor in vitro(8, 9) ; the affinities of these interactions were not determined.
It has been proposed by other authors that the SHC PTB domain consensus binding site is the motif asparagine-proline-X-phosphotyrosine (NPX(pY)) (7, 8, 9, 10, 27) . The data in this report confirm our earlier conclusion (6) that NPX(pY) is not the only motif recognized by the SHC PTB domain. The proline at the -2 position is not necessary for high affinity binding (Fig. 1, Fig. 2, and Fig. 4)(6, 10) . In quantitative assays, the only two residues which are absolutely required for binding are the asparagine at the -3 position and the phosphotyrosine(6) . In fact, when compared directly, most NPX(pY)-containing sequences bind with lower affinity than does the ErbB2-derived NLY(pY) motif (Fig. 4)(6) . Therefore, current data suggest that it is more appropriate to define the minimal SHC PTB domain binding motif as NXX(pY). The proline in the -2 position may serve to enhance affinity in some contexts, as do multiple other residues.
In conclusion, this report addresses several of the fundamental questions raised by the identification of PTB domains. First, we show directly that the specificity of the SHC PTB domain is different from that of a panel of SH2 domains and is likely to be different from most SH2 domains. Second, the SHC PTB domain binds to targets with similar affinity but different kinetics from SH2 domains. Finally, we have defined a high-affinity binding site for the SHC PTB domain which is likely to be important in intracellular signaling, particularly by the ErbB and Trk protein families of receptor tyrosine kinases. These results, taken together with previous reports, strongly support the hypothesis that PTB domains are specific and independent alternatives to SH2 domains as mediators of signaling complex assembly during tyrosine kinase signaling.