From the
Reversible assembly of intracellular signaling complexes is,
in some cases, mediated by direct binding of a Src homology 2 (SH2)
domain of one protein to a phosphotyrosine moiety of another protein
(Cantley, L. C., Auger, K. R., Carpenter, C. L., Duckworth, B.,
Graziani, A., Kapeller, R., and Soltoff, S.(1991) Cell 64,
281-302). Using a degenerate phosphotyrosine-containing peptide
library, we showed that individual SH2 domains recognize
phosphotyrosine in a specific sequence context to provide fidelity in
signaling (Songyang, Z., Shoelson, S. E., Chaudhuri, M., Gish, G.,
Pawson, T., Haser, W. G., King, F., Roberts, T., Ratnofsky, S.,
Lechleider, R. J., Neel, B. G., Birge, R. B., Fajardo, J. E., Chou, M.
M., Hanafusa, H., Schaffhausen, B., and Cantley, L. C.(1993) Cell 72, 767-778). Recently a second type of phosphotyrosine
interaction domain (PID) or phosphotyrosine-binding domain (PTB) was
discovered in the amino terminus of the SHC proto-oncoprotein
(Kavanaugh, W. M., and Williams, L.(1994) Science 266,
1862-1865; Blaikie, P., Immanuel, D., Wu, J., Li, N., Yajnik, V.,
and Margolis, B.(1994) J. Biol. Chem. 269, 32031-32034).
Here we demonstrate, using a phosphotyrosine peptide library, that the
SHC PID domain preferentially binds to the sequence
Asn-Pro-Xaa-phosphotyrosine. This motif is in agreement with sequences
at sites implicated in in vivo SHC binding. These results
indicate that while SH2 domains predominantly interact with specific
residues carboxyl-terminal of phosphotyrosine, the PID domain has high
specificity for residues amino-terminal of phosphotyrosine.
Protein-tyrosine kinases (PTKs)
Primary sequences adjacent to the
phosphotyrosine are required for specific SH2 domain
recognition(2, 7) . Using the oriented phosphopeptide
library technique, we showed that residues COOH-terminal to the
phosphotyrosine are critical for high affinity and specificity in SH2
domain binding(3, 4) . In many cases, the specificities
predicted by the peptide library technique have been confirmed by
mapping in vivo binding sites of SH2 domains, by affinity
analysis, and by structural
studies(8, 9, 10, 11, 12, 13, 14) .
While the original phosphopeptide libraries only varied amino acids
COOH-terminal of phosphotyrosine, studies from other laboratories
indicated that residues NH
A search of protein data bases for other NPXY
sequences revealed many potential candidates for SHC binding. The
NPXY motif was originally discovered as a code for protein
internalization (21). NMR studies have determined that this sequence
could form a tight turn structure(22) . Unfortunately, little
work has been done to tell how many of these sequences are
tyrosine-phosphorylated in vivo. An NPXY site on the
insulin receptor (Tyr-960) is known to be phosphorylated and is
critical for signaling(23) . The NPXY sites on the Trk
PTK family are very likely recognized by SHC, because they are similar
to the TrkA NPXY sequence (). While this paper
was in review, two groups reported that the PID domain rather than SH2
domain of SHC is responsible for binding the NPXY sites on the
EGF, nerve growth factor, and insulin
receptors(24, 25) .
Our finding that the PID domain
of SHC preferentially recognizes sequences NH
We thank Michael Berne (Tufts University) for peptide
sequencing.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)play a
crucial role in cellular proliferation and differentiation. Tyrosine
phosphorylation by PTKs creates binding sites for SH2 domains that
specifically recognize the phosphotyrosine moiety. Therefore, proteins
containing SH2 domains can be recruited to the site of phosphorylation
and signaling complexes can be formed in a phosphotyrosine-dependent
manner(1, 2) .
-terminal of phosphotyrosine are
critical for specific binding to certain SH2-containing
proteins(15) . For example, many groups have reported that SHC,
a SH2 domain-containing protein, binds to tyrosine-phosphorylated
NPXY motifs on several signal
molecules(16, 17, 18) . It was thought that the
SH2 domain of SHC mediated these
interactions(16, 17, 18) . In fact, many of the
sites mapped for in vivo SHC binding were in poor agreement
with the optimal motif we predicted for the SH2 domain of SHC using the
original peptide library(4) . A more degenerate library was
constructed in order to explore the relative importance of residues
both NH
- and COOH-terminal of the phosphotyrosine moiety
for SH2 domain recognition. While this work was in progress, a second
type of phosphotyrosine-binding domain, structurally distinct from SH2
domains, was discovered in the NH
terminus of SHC (PID or
PTB domain)(5, 6, 26) . Using the new peptide
library we show here that the SHC SH2 domain specificity is determined
mainly by residues COOH-terminal of the phosphotyrosine while the SHC
PID domain specificity is determined primarily by residues
NH
-terminal of phosphotyrosine.
GST Fusion Proteins
SH2 domains of p85, Nck,
SHC, and Crk were constructed as described
previously(3, 4) . GST-SHC PID is a GST fusion
containing the NH-terminal (PID) region of SHC
(1-209)(6) .
Peptide Libraries
The peptide library used for
these studies has the sequence GAXXXpYXXXKKK, where
pY indicates phosphotyrosine and X indicates any amino acid
except for Trp and Cys. This library was synthesized as described
previously(3) . The theoretical degeneracy of this peptide
library is 1834 million.
Peptide Affinity Purification and Data
Analysis
After incubating the degenerate peptide library (1 mg)
with 200-300 µg of bead-immobilized GST-SH2 or PID fusion
proteins, the beads were washed twice with phosphate-buffered saline.
The phosphopeptides bound were eluted using 20 mM phenyl
phosphate(3) . Peptide sequencing and data analysis were as
described previously(3) .
The Importance of Amino Acids NH
The
specificities of SH2 domains from SHC, Crk, Nck, and p85 were studied
using a peptide library with residues degenerate at positions both N-
and COOH-terminal to the phosphotyrosine. The GST-SH2 domains were used
to affinity-purify a subgroup of high affinity peptides. To determine
the preference of amino acids, the bound phosphopeptides were sequenced
and compared with the sequence of the peptides nonspecifically retained
on the GST beads and the sequence of the complete peptide library. The
data were normalized such that the sum of the quantity of all amino
acids at a degenerate position is 18 (the number of different amino
acids). Thus, a selectivity value greater than 1 indicates preference
for a given amino acid. The results are presented in .
These results were also compared with the selectivities obtained using
a peptide library in which only three amino acids COOH-terminal to the
phosphotyrosine are degenerate(3, 4) . As shown in , although the selectivities determined with the more
highly degenerate library are lower than those observed with the
original library, the optimal amino acids at positions COOH-terminal of
phosphotyrosine were similar with the two different peptide libraries.
However, in contrast to expectations from mutational analysis of SHC
and Nck binding sites(16, 17, 18, 19) ,
the SH2 domains of these proteins did not exhibit strong selectivity
for amino acids NH-terminal
to the Phosphotyrosine for SH2 Domain Specificity
-terminal of phosphotyrosine.
Interestingly, the Crk, Nck, and SHC SH2 domains showed a weak
selection for Tyr at the Tyr(P)-1 position. The p85, Crk, and SHC
domains also exhibited a weak selection for acidic amino acids at the
Tyr(P)
and Tyr(P)
positions.
NH
The fact that the SH2 domain of
SHC shows little selectivity for amino acids NH-terminal PID Domain of SHC Selects for
Peptides with a NPXpY Motif
-terminal of
phosphotyrosine raised the possibility that a different region of SHC
mediates binding of SHC to proteins with the NPXpY motif.
Recent studies have indicated the presence of a second domain (PID
domain) in the NH
terminus of SHC that can interact with
tyrosine-phosphorylated proteins(5, 6, 26) . The
PID domain appears to have no significant structural similarity to SH2
domains. We tested the specificity of the SHC PID domain using the
degenerate peptide library. This domain showed a strong selection for
peptides with Asn 3 residues NH
-terminal to the
phosphotyrosine (Fig. 1). The optimal motif selected is Asn-Pro-X-pY-Phe-X-Arg. In contrast to SH2
domains, the PID domain selectivity appears to be dominated by contact
with residues NH
-terminal of the phosphotyrosine moiety.
Since the sequence NPXY is found in the sites where SHC binds
to tyrosine-phosphorylated TrkA, ErbB3, and polyoma middle t
antigen(16, 17, 18) , binding to these proteins
is likely to be mediated by the PID domain rather than the SH2 domain
of SHC (). Consistent with our peptide library prediction
that Asn at Tyr(P)
and Pro at Tyr(P)
are important for binding to the PID domain, mutation of these 2
residues in polyoma middle t antigen and ErbB3 inhibited in vivo SHC association(16, 17) .
Figure 1:
The specificity of the PID domain of
SHC. A degenerate peptide library was used to study the specificity of
the SHC PID domain (see ``Materials and Methods''). The
peptides that bound to GST-SHC PID were purified and sequenced, and the
sequence of purified peptides was compared with that of the starting
peptide library. The data were normalized such that a value 1 or less
indicates no selectivity for a given amino acid (3). A, B, C, D, E, and F show,
respectively, the selectivity (average of two experiments) at the
Tyr(P), Tyr(P)
,
Tyr(P)
, Tyr(P)
, Tyr(P)
, and
Tyr(P)
positions. Amino acids are presented in one-letter
codes.
In addition to the
conserved Asn at the Tyr(P) and Pro at the
Tyr(P)
positions, some of SHC-binding sites also
have phenylalanine or hydrophobic amino acids at the Tyr(P)
position (), in agreement with the weak selection
for these residues in the peptide library result (Fig. 1).
However, in contrast to SH2 domains, which generally select for
peptides with hydrophobic amino acids at the Tyr(P)
position(3, 4) , the PID domain weakly selected
for hydrophilic amino acids at the 3-position with Arg being preferred.
Although none of the known SHC binding sites has Arg at the 3-position,
most have hydrophilic amino acids (). The sites on TrkA,
EGF receptor, and ErbB3 also have acidic residues at the
Tyr(P)
position. In polyoma middle t antigen, this
residue is replaced by Thr. In our peptide library experiments, we
didn't detect any selection at the Tyr(P)
position, indicating that this position is not critical for SHC
binding. Instead, the Tyr(P)
residue could be a
signal for tyrosine phosphorylation. We have recently determined the
substrate specificities of several protein-tyrosine kinases
(PTKs)(20) . All the receptor PTKs we studied were shown to
prefer acidic residues at the -1 position, while cytosolic PTKs
selected
-branch amino acids at the same position (20).
Consistently, all the NPXY sequences on the receptor PTKs,
TrkA, EGF receptor, and ErbB3, have acidic residues at the -1
positions and are thought to be autophosphorylation sites. In contrast,
the NPTY site on middle t antigen contains a
-branch amino acid at
the -1 position and is phosphorylated by cytosolic Src family
PTKs.
-terminal to
the phosphotyrosine provides a new insight into protein-protein
interactions mediated by tyrosine phosphorylation. It is clear now that
two different classes of domains are responsible for binding to
phosphotyrosine. While one class (the SH2 domains) primarily contacts
amino acids COOH-terminal to the phosphotyrosine, the other class (PID
domain family) contacts sequences NH
-terminal to the
phosphotyrosine. Several homologues of the SHC PID domain have been
identified, and it is likely that more will be
found(5, 25, 26) . The SHC protein now becomes a
very interesting adapter molecule. It is able to trap
tyrosine-phosphorylated proteins in two distinct ways, either through
its SH2 domain or its PID domain. In addition, phosphorylation of SHC
on tyrosine results in recruitment of GRB2 into the complex via the
GRB2 SH2 domain. Thus, the distinct specificities of the SH2 and PID
domains of SHC increase the number of proteins with which SHC can
interact in signaling.
Table: Comparison of phosphopeptide specificity of the
SHC PID domain to specificities of SH2 domains
Table: Sequences at
SHC binding sites
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.