(Received for publication, April 19, 1995; and in revised form, May 23, 1995)
From the
Shc phosphorylation in cells following growth factor, insulin,
cytokine, and lymphocyte receptor activation leads to its association
with Grb2 and activation of Ras. In addition to being a cytoplasmic
substrate of tyrosine kinases, Shc contains an SH2 domain and a non-SH2
phosphotyrosine binding (PTB) domain. Here we show that the Shc PTB
domain, but not the SH2 domain, binds with high affinity (ID
Many cell surface receptors involved in the regulation of
growth, metabolism, and differentiation initiate their effects by
catalyzing the phosphorylation of tyrosine residues within their own
sequences and on additional cellular substrate proteins. The insulin,
PDGF, ( Shc proteins contain an SH2
domain and serve as substrates for tyrosine kinase-linked receptors (e.g.(1, 2, 3) ). Following
receptor activation, Shc is phosphorylated at Tyr
Figure 1:
Expressed
proteins and their capacity to bind with phosphopeptides. A,
the domain structure of p52 and p46 Shc proteins shows the relative
positions of its PTB, collagen homology (CH), and SH2 domains,
the alternative start sites, the known phosphorylation site (Y318), and terminal positions of proteins used in this study. B, binding assays were conducted with
We now show that the Shc PTB domain binds directly with a polyoma
virus mT antigen-derived phosphopeptide, an LXNPTpY motif
provides specificity for this interaction, and peptides corresponding
to this sequence adopt a
Since there are
additional forms of Shc in mammalian tissues, and one (p46) has a
shorter PTB domain due to alternative translational initiation at
Met
Peptide truncations and substitutions
were used to analyze determinants of Shc PTB domain binding specificity
in detail (Table 1). The 13-residue peptide LLSNPTpYSVMRSK was
designed to have six residues placed symmetrically on either side of
pTyr. While one residue (Leu
Phosphotyrosine itself binds weakly with the Shc PTB domain as it
does with SH2 domains. ( These data indicate that peptide binding with the Shc p52
PTB domain is closely related to SH2 domain binding in that (i)
phosphorylation serves as the on-off switch for recognition, (ii) the
surrounding sequence provides specificity for the interaction, (iii)
four to six or seven residues participate directly in the interaction,
and (iv) overall binding energies and relative contributions for pTyr versus surrounding residues are similar for the interactions.
However, the PTB domain shows a direct reversal in the orientation of
required peptide interactions: residues amino- but not
carboxyl-terminal to pTyr determine specificity. As a general feature
of SH2 domain mediated interactions, residues carboxyl-, but not
amino-, terminal to pTyr provide binding energy and thereby participate
in conferring specificity to the interaction (e.g.(15, 16, 17, 18, 19) ,
and 24). Results from the truncation studies show that six peptide
residues spanning pTyr When substitutions do not
perturb the structure, it is possible to assess side chain-specific
contributions to binding energy from alanine scanning and related
approaches. However, for PTB-binding ligands, this appears not to be
the case. Since the K
Figure 2:
Two-dimensional NOESY and ROESY spectra obtained for the
phosphorylated (Fig. 2, B and C) and
unphosphorylated (data not shown) sequences reveal a strong contact
between amide protons of Thr
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
≅ 1 µM) to phosphopeptides corresponding to the
sequence surrounding Tyr
of the polyoma virus middle T
(mT) antigen (LLSNPTpYSVMRSK). Truncation studies show that five
residues amino-terminal to tyrosine are required for high affinity
binding, whereas all residues carboxyl-terminal to tyrosine can be
deleted without loss of affinity. Substitution studies show that
tyrosine phosphorylation is required and residues at -5,
-3, -2, and -1 positions relative to pTyr are
important for this interaction.
H NMR studies demonstrate
that the phosphorylated mT antigen-derived sequence forms a stable
turn in solution, and correlations between structure and function
indicate that the
turn is important for PTB domain recognition.
These results show that PTB domains are functionally distinct from SH2
domains. Whereas SH2 domain binding specificity derives from peptide
sequences carboxyl-terminal to phosphotyrosine, the Shc PTB domain
gains specificity by interacting with
turn-forming sequences
amino-terminal to phosphotyrosine.
)EGF, and related receptors have intrinsic tyrosine
kinase activity and catalyze these phosphorylations directly.
Alternatively, lymphocyte, cytokine, and related receptors which lack
intrinsic tyrosine kinase activity associate noncovalently with
cytoplasmic tyrosine kinases to initiate their signaling events. In
each case, phosphorylated tyrosine residues within the receptors and/or
their substrates create docking sites for proteins with Src homology 2
(SH2) domains. SH2 domains are phosphotyrosine-binding modules
associated with a wide variety of cytoplasmic proteins that participate
in intracellular signal propagation.
and
associates with the Grb2/Sos complex to activate Ras (e.g.(4, 5, 6, 7) ). (
)However, in addition to serving as a Grb2 docking protein,
Shc proteins also associate with phosphoproteins including activated
EGF, PDGF, and insulin receptors, ErbB2, ErbB3, Trk, and the polyoma
virus middle T
antigen(1, 8, 9, 10, 11, 12, 13, 14) .
Since many of these phosphoproteins contain Asn-Pro-Xaa sequences
amino-terminal to phosphorylated tyrosines and Shc contains a
carboxyl-terminal SH2 domain (Fig. 1A), it has been
suggested that the Shc SH2 domain shows a preference for NPXY
motifs(8, 10, 11) . This would be unusual in
that alternative SH2 domains exhibit a high degree of specificity
toward residues carboxyl-terminal to pTyr (e.g.(15, 16, 17, 18, 19) ).
Although it has even been reported that the SH2 domain of Shc accounts
for its interactions with NPXY-containing phosphoproteins (10, 20) , we have consistently failed to observe
binding between the Shc SH2 domain and NPXY motifs. (
)The realization that Shc contains an alternative
phosphotyrosine binding (PTB) domain distinct from its SH2 domain (21, 22, 23) suggests that its PTB domain
might be responsible for Shc interactions with NPXY motifs.
I-radiolabeled peptide LLSNPTpYSVMRSK and GST/Shc fusion
proteins 1-392, 1-238, 1-229, 1-215,
1-206, 1-196, 46-229, 46-215, and
371-474. The thick gray line represents the averaged
initial slope for proteins 1-392, 1-238, 1-229,
1-215, and 1-206. C, competition assays using
GST/Shc(1-238), [
I]LLSNPTpYSVMRSK, and
varying concentrations of unlabeled peptide LSNPTpYSV, the
corresponding nonphosphorylated sequence, and a scrambled sequence
(NTSVSpYPL).
turn configuration in solution.
Therefore, although PTB and SH2 domains appear to be similar in many
respects, there are important functional distinctions between the
interactions that they mediate.
Recombinant Proteins and Synthetic
Peptides
Fragments of the human Shc p52 cDNA generated by
subcloning using the polymerase chain reaction were spliced into a
pGEX4T vector. Escherichia coli DH5 cells were
transformed with vectors encoding glutathione S-transferase/Shc fusion proteins GST/Shc(1-196),
GST/Shc(1-206), GST/Shc(1-215), GST/Shc(1-229),
GST/Shc(1-238), GST/Shc(46-215), GST/Shc(46-229),
GST/Shc(1-392) (PTB + CH domains), and
GST/Shc(371-474) (SH2 domain), where numbers in parentheses refer
to residues of Shc p52(1) . Proteins were expressed following
usual protocols and purified by affinity chromatography using
glutathione-agarose. Proteins were eluted from the washed beads with
glutathione or 8.0 M urea and dialyzed against 100 mM ammonium bicarbonate containing 1.0 mM dithiothreitol; no
difference in function was noted following elution by the two methods.
All isolated proteins were found to have >90% homogeneity and
appropriate sizes following separation by SDS-polyacrylamide gel
electrophoresis. Phosphopeptides were synthesized following a N
-Fmoc protecting group strategy and purified
by HPLC(17) .
Direct Binding Assays between Amino-terminal Shc Proteins
and Phosphopeptides
Peptide LLSNPTpYSVMRSK was radiolabeled with I-labeled Bolton-Hunter reagent and purified by HPLC (24) . To determine which of the fusion proteins would bind to
the radiolabeled peptide, 45 nCi (10
cpm) of
I-phosphopeptide and varying concentrations of the fusion
proteins were combined in 150 µl of assay buffer (20 mM Tris-HCl at pH 7.4, 250 mM NaCl, 0.1% bovine serum
albumin, and 10 mM dithiothreitol). Glutathione-agarose (50
µl of a 1:10 slurry, was added, the solutions were vortexed, and
the samples were incubated overnight at 22 °C with constant mixing.
Following centrifugation for 5 min at 12,000
g,
supernatant solutions were removed and
I radioactivity
associated with the pellets was determined. For competition assays,
sufficient Shc fusion protein (typically 8-10 µg of
GST/Shc(1-238)) to sequester
7% of the radiolabeled peptide
(45 nCi) and varying concentrations of unlabeled peptides were combined
in 150 µl of assay buffer. Samples were otherwise treated as
described above.
Solution Structures of mT NPXY Peptides
Peptides
(1.5 mM) were dissolved in either 0.5 ml of DO (pD
4.8, direct meter reading) or 0.7 ml of H
O (pH 4.5)
containing 50 mM KCl. Spectra were observed at 400 MHz and 4
°C using a Varian Unity-Plus spectrometer Double-quantum-correlated
spectroscopy (DQF-COSY), total correlation spectroscopy (TOCSY; mixing
time 55 ms), nuclear Overhauser enhancement spectroscopy (NOESY; mixing
times 200, 400, and 600 ms), and rotating-frame Overhauser enhancement
spectroscopy (ROESY; mixing times 80 and 160 ms) experiments were
performed. The water solvent resonance was attenuated by presaturation.
In the NOESY spectra, no evidence of spin diffusion was observed.
Resonance assignment was obtained by standard sequential methods. For
quantitative comparisons NOESY and ROESY spectra at different mixing
times were normalized relative to shared intraresidue cross-peaks (Asn
NH
and Tyr ortho-meta).
PTB Domain Delineation
Shc-related sequences
were expressed as GST fusion proteins to determine the functional
boundaries of the Shc PTB domain (Fig. 1, A and B). The fusion proteins were combined with glutathione-agarose
beads and the radiolabeled phosphopeptide. Specific binding was
observed for concentrations as low as 0.1-0.2 µM of
Shc proteins 1-206, 1-215, 1-229, 1-238, and
1-392. Increasing levels of binding were seen with protein
concentrations up to 2 µM, and plateaus in binding
were seen at higher concentrations, indicating that the mode of peptide
binding was similar with each of the five proteins. In contrast, even
the highest concentrations of Shc protein 1-196 showed no
detectable peptide binding to demarcate the carboxyl terminus of the
domain between residues 196 and 205 of Shc p52.
of p52, additional proteins initiated at Met
were prepared (46-215 and 46-229). These proteins did
exhibit peptide binding, although significantly higher protein
concentrations were required for the effect (Fig. 1B).
Similarly, the SH2 domain of Shc showed phosphopeptide binding,
although even greater protein concentrations were required. By
rearranging the equation used to describe bimolecular interactions, K
=
[RL]/[R][L], to
[RL]/[L] =
[R]K
, the
[RL]/[L] value can be represented by the ratio of
bound/free ligand and the slope of a line defined by
[RL]/[L] versus [R] represents K
. Using this approach, initial slopes of
the plots of bound/free versus [R] were used to
compare relative PTB domain affinities. Analyses of the 9 Shc proteins
reveal that (i) proteins 1-206, 1-215, 1-229,
1-238, and 1-392, having the same amino-terminal start
site, bind to the mT peptide with equivalent affinities, (ii) proteins
having the second start site (46-215 and 46-229) bind with
10-20-fold lower affinity, and (iii) the Shc SH2
domain(371-474) binds with about 50-fold lower relative affinity.
These findings agree qualitatively with published studies suggesting
that PTB domains from p52 and p46 have
function(21, 22) . However, to our knowledge this is
the first attempt to quantify relative binding affinities of
PTB-related proteins and the first to show reduced affinities for Shc
p46 related proteins.
PTB Domain Binding Specificity
Based on these
findings, an assay method for analyzing the peptide binding specificity
of the Shc PTB domain was developed using protein 1-238
(arbitrarily chosen). Mixtures of GST/Shc(1-238), radiolabeled
peptide LLSNPTpYSVMRSK, glutathione-agarose beads, and varying
concentrations of unlabeled peptides were equilibrated, and PTB-bound I-peptide was separated from free peptide by
centrifugation. The amount of
I-peptide associated with
the glutathione-agarose pellet varied with concentration and relative
affinity of competing ligand (Fig. 1C). In the example
shown, the phosphopeptide having sequence LSNPTpYSV competes for PTB
binding with an ID
value of
1 µM. In
contrast, an identical but unphosphorylated sequence and a scrambled,
phosphorylated sequence do not bind appreciably to the PTB domain at
peptide concentrations up to 1 mM, indicating that the
phosphorylation of tyrosine within a specific sequence context is
required for high affinity interactions between the Shc PTB domain and
cognate proteins or peptides.
) could be removed from
the amino terminus of this sequence with full retention of binding
affinity,
removal of a second residue
(Leu
) resulted in a reduction in affinity below
levels of assay detection. Results were obtained for a full series of
peptides having progressively fewer residues amino-terminal to pTyr,
although once the residue at the pTyr
position had
been deleted binding remained undetectable. A similar truncation series
was analyzed for the carboxyl terminus of the peptide. However, in this
case, all six residues could be removed from the peptide carboxyl
terminus without decreasing affinity. Slightly higher affinities
(0.6-1.3 µM) were consistently seen with the shorter
sequences, although additional studies will be required to determine
whether longer sequences have a negative influence on binding. The
amino and carboxyl truncation series suggested that six residues from
Leu
to pTyr were needed for a high affinity
interaction. This concept was tested further with the six-residue
peptide LSNPTpY(NH
) to show that the hexapeptide motif is
both necessary and sufficient for high affinity interactions.
)Neither phosphoserine nor
phosphothreonine bound the PTB domain, as noted previously for SH2
domains.
to pTyr participate in the
interaction. An alanine scan through the LSNPTpYSV sequence was
conducted to gather more information about the degree of involvement of
each residue. The Leu
substitution reduced binding
affinity 50-fold, consistent with the role suggested for this residue
by the truncation data. Substitution of Ser
had no
effect, indicating that this side chain may not participate directly.
Replacements of Asn
, Pro
, and
Thr
within the NPXY motif all had dramatic
effects on binding. Substitution of either Asn or Thr led to
>300-fold reductions in affinity, while Pro
Ala resulted in a
100-fold drop in affinity. Thus, substitutions of five of the six
residues within the LSNPTpY sequence significantly reduced binding
affinity. Surprisingly, substitution of Ser
also
reduced binding affinity 14-fold, even though deletion of the residue
had no effect. The sequence specificity exhibited by the Shc PTB domain
is underscored by binding studies with randomized (keeping pTyr at the
same relative position) and inverted sequences, where affinity in each
case was below levels of assay detection.
value is
approximately 10
M and
G = -RTlnK
,
G ≅ -9.3 kcal/mol for the interaction between the parent
mT peptide and the Shc PTB domain.
The effect of each
substitution can be approximated by the expression
(
G) =
-RTln(ID
/ID
),
such that a 100-fold drop in affinity translates to a loss of 2.6
kcal/mol in binding energy. The sum of the individual differences in
free energy resulting from each substitution in the alanine scan is
>16 kcal/mol. As this is much greater than the available binding
energy (-9.3 kcal/mol), at least some of the replacements may
have more global effects on peptide structure.
NPXpY Peptide Structure
We have analyzed the mT
antigen-derived peptides for inherent structure. Algorithms for
predicting secondary structure (25) suggest that the
unphosphorylated NPTY sequence has a high probability of adopting a
turn (data not shown). In fact, peptides corresponding to
NPXY internalization motifs of such proteins as the LDL,
transferrin, and insulin receptors do form relatively stable
turn
structures in solution(26, 27, 28) . The
structure of the mT antigen-derived sequence has not been studied
previously, nor has the general effect of phosphorylation on
turn
stability. The one-dimensional
H NMR spectra of peptide
LSNPTpYSV exhibits chemical shift dispersion characteristic of ordered
(non-random) structure (Fig. 2A). Each amide resonance
is individually resolved and not attenuated by solvent presaturation.
The spectra also exhibit evidence of a minor conformer (<10%) in
slow exchange on the NMR time scale, presumably due to the cis-proline isomerization. The unphosphorylated sequence
(LSNPTYSV) was analyzed under identical conditions (data not shown).
Chemical shifts of the predominant conformer were similar, with two
exceptions: (i) the ortho (2, 6) and meta (3, 5) aromatic resonances of tyrosine itself are
shifted downfield as expected (
0.10 and 0.33 ppm), and (ii)
a
-proton of Asn
is perturbed. The latter, a
transmitted effect, is consistent with the weakly polar amide-aromatic
(Asn-Tyr) interaction as previously postulated in the unphosphorylated
NPXY motif of the LDL receptor(26) .
H NMR spectra of peptide
LSNPTpYSV. A, one-dimensional spectrum, with amide resonance
assignments: 1, Ser
; 2,
Asn
; 3, Ser
; 4,
Thr
; 5, pTyr; 6,
Val
; 7 and 7`, Asn
NH
; 8, C
H pTyr; 8`,
C
H pTyr. Two-dimensional NOESY (B) and ROESY (C) spectra demonstrate inter-residue contacts. Cross-peaks
are labeled: a or c, Thr
/pTyr; b or d, pTyr/Ser
; e,
Ser
/Val
; and f,
Ser
/Asn
. The NOESY and ROESY
mixing times were 400 and 160 ms,
respectively.
and pTyr or Tyr that is
characteristic of a
turn. Since the intensity of the NOE between
Thr
and Tyr is essentially the same, whether or not
Tyr is phosphorylated, we conclude that the stability of the
turn
is unaffected by phosphorylation. Accentuation of this contact in the
NOESY relative to the ROESY spectrum suggests that local correlation
times (
) at the center of the turn are longer (i.e. more ordered) than at the ends of the peptide. This
inference is supported by the temperature dependence of the amide
proton resonances: with increasing temperature in the range 4-35
°C, amide resonances in unstructured peptides characteristically
exhibit progressive broadening and attenuation due to more rapid
exchange with solvent water. In the mT NPTY peptides, however, the
amide resonances of the turn itself (Asn
,
Thr
, pTyr, and Ser
) remain sharp,
whereas those of Ser
and Val
exhibit the expected attenuation. Protection from solvent
exchange is associated with retention of the
Thr
/pTyr NOE throughout this temperature range.
Together, these results demonstrate that the NPTY and NPTpY sequences
nucleate a stable
turn in solution. Alanine substitutions
generally diminish the stability of
turns (25) . Since
Ala substitutions within the
turn of the mT peptide have dramatic
effects on binding,
turn stability and PTB domain binding
affinity appear to function in parallel.
Conclusion
Previous sequence alignments showed
that the amino terminus of Shc represents a new class of
phosphotyrosine binding domain structurally distinct from SH2
domains(21, 22, 23) . We now show that the
Shc PTB domain binds a LXNPTpY sequence from the polyoma virus
middle T antigen in a fashion that is functionally distinct from SH2
domains, that particular positions within this sequence are critical to
the maintenance of a high affinity interaction, and that these
sequences tend to form turn structures in solution. Since many
proteins contain NPXY motifs, one might expect that each would
be recognized by the Shc PTB domain. However, we have also shown that
residues other than Asn and Pro are critical for high affinity
interactions, particularly Leu
and
Thr
, and the NPXY sequences from such
proteins as the insulin and EGF receptors, ErbB2, ErbB3, ErbB4, TrkA,
TrkB, and TrkC vary at these positions. In fact, in one case tested, an
insulin receptor-derived NPEpY peptide binds the Shc PTB domain with
low affinity (data not shown), in support of the notion that distinct
NPXpY motifs are recognized differentially, perhaps by
distinct PTB proteins yet to be described.
We thank D. N. M. Jones, Q. X. Hua, and K. Hallenga
for advice on NMR methods.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.