(Received for publication, August 16, 1995; and in revised form, September 12, 1995)
From the
PTB domains are non-Src homology 2 (SH2) phosphotyrosine binding
domains originally described in the receptor tyrosine kinase substrate,
Shc. By serial truncation, we show that a 174-residue region of Shc p52
(33-206) has full PTB activity. We also show that a 173-residue
region of insulin receptor substrate-1 (IRS-1; residues 144-316)
has related PTB activity. In vitro both domains bind directly
to activated insulin receptors. Binding is abrogated by substitution of
Tyr-960 and selectively inhibited by phosphopeptides containing
NPXY sequences. Phosphopeptide assays developed to compare PTB
domain specificities show that the Shc PTB domain binds with highest
affinity to XN
pY
motifs derived from middle T (mT), TrkA, ErbB4, or epidermal growth
factor receptors (
= hydrophobic,
=
-turn forming); the IRS-1 PTB domain does not bind with this
motif. In contrast, both the Shc and IRS-1 PTB domains bind
XXN
pY
sequences derived from insulin and interleukin 4 receptors, although
specificities vary in detail. Shc and IRS-1 are phosphorylated by
distinct but overlapping sets of receptor-linked tyrosine kinases.
These differences may be accounted for by the inherent specificities of
their respective PTB domains.
Insulin binding to the insulin receptor activates it as a
substrate kinase, leading to tyrosine phosphorylation of at least two
cytoplasmic proteins, IRS-1 and
Shc(1, 2) . IRS-1 is phosphorylated at many tyrosine
positions(3) , whereas Shc is phosphorylated predominantly at
one site in cells(4) . Since SH2 domain proteins bind
specifically with phosphotyrosyl sites in
proteins(5, 6) , IRS-1 is capable of multiple
interactions with SH2 proteins, including phosphatidylinositol
3-kinase, the phosphatase SH-PTP2, and Grb2, a linker protein upstream
of Ras. In contrast, when Shc is phosphorylated in cells, it interacts
primarily with Grb2(7) .
The phosphotyrosine binding (PTB)
domain (also called PID or SAIN domain) was recently found to provide a
mechanism for protein binding with phosphotyrosyl sequences, distinct
from SH2 domains(8, 9, 10, 11) .
Perhaps related to the phosphorylation of Shc by many tyrosine kinases,
in addition to the insulin receptor, its PTB domain appears to interact
with multiple phosphotyrosyl
proteins(8, 9, 10, 11, 12) .
The specificity of the Shc PTB domain can be analyzed by methods
analogous to those used previously for SH2 domains. The Shc PTB domain
binds with turn-forming motifs frequently containing
phosphorylated NPXY
sequences(13, 14, 15) , in contrast with SH2
domains that bind extended phosphopeptide sequences carboxyl-terminal
to phosphotyrosine (pTyr)(5, 6) . Since efficient
IRS-1 phosphorylation in cells also depends on the phosphorylation of a
turn-forming NPXY motif in insulin
receptors(16) , IRS-1 might contain a related PTB domain (even
though IRS-1 and Shc show no extended sequence homology). In yeast
two-hybrid experiments, the amino-terminal
500 residues of IRS-1
direct an interaction between the insulin receptor and IRS-1 that is
functionally related to Shc PTB domain interactions (11, 17) . The recent cloning of the IRS-2 gene
revealed two regions of deduced protein sequence homology with
IRS-1(18) , suggesting that one might function as a PTB domain.
We now show that a 174-residue region from the amino terminus of
human Shc (33-206) and a 173-residue region from human IRS-1
(144-316) bind similarly with activated insulin receptors. While
both domains bind -turn forming motifs amino-terminal to pTyr,
their specificities differ in detail.
Figure 1:
Demarcation of functional PTB domains.
The domain structures of IRS-1 (A) and Shc p52 (B)
are compared. Human IRS-1 contains a PH domain, a PTB domain, and
multiple sites of tyrosine phosphorylation (P). Human Shc
contains a PTB domain, an SH2 domain, and at least one in vivo phosphorylation site. The indicated sequences from IRS-1 and Shc
were expressed and assayed for peptide binding. C, assays
shown were conducted by combining the insulin receptor-related sequence I-LYASSNPApYLSASDV and GST/IRS-1 fusion proteins
1-134, 108-516, and 144-316; an example of similar
studies with Shc proteins was previously reported (13) . In panels A and B, under binding, +++
indicates high affinity, + is 20-50 fold lower affinity, and
- is no apparent binding.
Experiments with the yeast two-hybrid system have suggested that the amino-terminal one-third of IRS-1 might contain a functionally related domain(11, 17) . Therefore, GST/IRS-1 fusion proteins expressed in E. coli were constructed for use in binding experiments with intact insulin receptors and peptides derived from sequences surrounding insulin receptor Tyr-960 and IL4 receptor Tyr-497 (both NPXY motifs). IRS-1(4-516) and IRS-1(108-516) proteins exhibited binding in the peptide assay (Fig. 1, A and C). Since the former protein contains an intact PH domain, and the latter does not, the IRS-1 PH domain appears to be irrelevant for this interaction. An independently expressed PH domain (1-134) shows no function in this assay. We were interested in further delimiting the domain, although since IRS-1 and Shc share negligible sequence homology, it was not possible to align putative PTB domains. Comparisons between rat IRS-1 and IRS-2 proteins reveal only two regions of deduced sequence homology: their PH domains and an additional region carboxyl-terminal to it(18) . The second region of human IRS-1 (residues 144-316) expressed as a GST fusion protein binds in the peptide assays (Fig. 1, A and C) and with the intact insulin receptor (Fig. 2).
Figure 2: Interactions between PTB domains and intact insulin receptors. A, equivalent amounts of partially-purified native insulin receptors were stimulated with insulin and precipitated in the absence or presence of 5 µg of IRS-1(144-316) or Shc(1-238) PTB domain fusion proteins immobilized on glutathione-agarose beads. B, equivalent amounts of wt insulin receptors were stimulated or not with insulin and precipitated with the PTB domains in the presence or absence of 10 mM phosphotyrosine (pTyr). C, native (wt) or mutated (Y960F) insulin receptors were insulin-stimulated, autophosphorylated, and precipitated with the PTB domains. In the experiment shown, slightly over half the amount of Y960F receptor was present, compared to wild type. D, equivalent amounts of native insulin receptors were stimulated with insulin and precipitated in the absence or presence of 1.0 mM phosphopeptides mT pY250/8, IL4 pY497/11, or IR pY960/15, or the unphosphorylated peptide IR Y960/15. In all cases, PTB domain-bound proteins were separated by SDS-polyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride membranes, and detected by Western blotting with anti-insulin receptor antibodies.
Tyr-960 of the insulin receptor is within an
NPXY motif, and related sequences have recently been shown to
be important for interactions with the Shc PTB
domain(8, 13, 14, 15) . However, a
hydrophobic residue at the Xaa position is also
important for Shc PTB domain binding(12, 13) , and
this is lacking in the insulin receptor. (
)Peptide
competition assays were used to compare sequence requirements for
binding by the Shc and IRS-1 PTB domains. At 1.0 mM concentrations, NPXY phosphopeptides derived from the mT
antigen (mT pY250/8) and IL4 (IL4R pY497/11) and insulin receptors (IR
pY960/15) all blocked the association between the insulin receptor and
the Shc PTB domain (Fig. 2D). Identical studies with
the IRS-1 PTB domain showed inhibition with the IL4 and insulin
receptor sequences but no effect by the mT peptide. No inhibition with
either PTB domain occurred with unphosphorylated peptide controls.
Figure 3:
Competition assays with Shc and IRS-1 PTB
domains. A, GST/Shc(1-238), peptide I-LLSNPTpYSVMRSK, and varying concentrations of the
indicated peptides were combined and assayed as described under
``Materials and Methods.'' B,
GST/IRS-1(144-316),
I-LYASSNPApYLSASDV, and varying
concentrations of the same peptides were assayed in an identical
fashion.
Previously we showed that the hexapeptide, mT-derived sequence
LSNPTpY binds the Shc PTB domain with high affinity(13) .
Phosphorylation is required, whereas substitution of
Leu, Asn
, Pro
,
or Thr
with Ala significantly reduced affinity.
These findings suggested that: (i) a hexapeptide sequence is sufficient
for high affinity interaction, (ii) a hydrophobic residue at
Xaa
is important, (iii) the N and P positions of the
NPXY motif are critical, and (iv) the residue at the X position influences affinity. Similar modes of binding with the
Shc PTB domain are observed for the TrkA (IENPQpYFS), ErbB4
(AKKAFDNPDpYWN), and EGF receptor (HSTAVGNPEpYLN) sequences (Table 1). All have hydrophobic residues at -5 positions
relative to pTyr. We now show that the IRS-1 PTB domain does not bind
with the mT, TrkA, Erb4, or EGF receptor peptides (Table 1).
However, both IRS-1 and Shc PTB domains bind directly with insulin
and IL4 receptor sequences ( Fig. 2and Fig. 3), which
lack hydrophobic residues at Xaa (Table 1).
Therefore, the insulin and IL4 receptor sequences were used to compare
binding specificities (the corresponding sequence of the IGF-1 receptor
was felt to be too similar to that of the insulin receptor to warrant
independent analysis). Phosphorylation of tyrosine is crucial for
binding both peptide sequences (Table 1), consistent with the
requirements for insulin activation and Tyr-960 phosphorylation
exhibited for PTB interactions with the intact insulin receptors (Fig. 2). Removal of residues from the amino termini of the
insulin and IL4 receptor peptide sequences revealed the importance of
residues at the Xaa
and Xaa
positions, relative to pTyr, for binding with both PTB domains.
Carboxyl-terminal truncations were tolerated, providing pTyr (amide)
remains (data not shown).
Substitution studies were conducted with
both sequences to further probe binding mechanisms. Ala substitutions
of hydrophobic residues at the Xaa and
Xaa
positions diminished binding with both PTB
domains. Moreover, Ile
Ala within the IL4
receptor reduced binding with both domains, whereas Ala
Val substitution of the insulin receptor peptide increased
binding affinity. These findings suggest that hydrophobic side chains
at all three positions influence binding. Substitutions at the
Xaa
and Xaa
positions of the
insulin and IL4 receptor sequences were generally tolerated.
Substitution of Asn
within the NPXY motif
abrogates binding by both PTB domains. For the Shc PTB domain,
Pro
substitution leads to reduced but not abolished
binding; for the IRS-1 PTB domain, substitution of Pro
with Ala abolishes binding.
Substitutions at Xaa (the X of NPXY) are particularly interesting.
Glu
Ala substitution in the insulin receptor
sequence leads to 30-fold gain of function for binding with
the IRS-1 PTB domain (ID
= 6.5 µM).
The IL4 receptor contains Ala
naturally, and the
corresponding peptide binds with high affinity (ID
= 6.2 µM), equivalent to that of the
Ala-substituted insulin receptor sequence. Consistent with these
findings, Ala
Glu substitution in the IL4
receptor sequence leads to >50-fold loss of affinity for the IRS-1
PTB domain. Although the effects are in the same direction for
interactions with the Shc PTB domain, their magnitudes are much
smaller. These studies imply that biological systems may have evolved a
means of using this position for selectively modulating affinities of
PTB domain interactions. Along with its PTB domain, the PH domain of
IRS-1 may participate in insulin receptor-catalyzed substrate
phosphorylation (26) , and a proper balance of affinities for
both domains may be required for normal signaling.
Using all
available data, consensus sequences for PTB domain binding can be
constructed. The Shc PTB domain binds with highest affinity with
sequences having a hydrophobic residue at the Xaa position and an NPXpY motif (this study and (12) and (13) ). Asn
within the
NPXY motif appears to be critical, whereas Pro
can be substituted (e.g. Ala in the Ala scan or Leu of
c-ErbB2; (14) ). Ala
appears to be preferred
over Glu
(Table 1), and in combination the
residues within this region should be able to adopt a
-turn(13) . The consensus motif:
-X-N-
-
-pY, where
is hydrophobic (Leu, Ile, Val, Phe) and
are
-turn
forming residues, is present in the polyoma virus mT antigen, TrkA,
TrkB, TrkC, ErbB2, ErbB3, ErbB4, and the IL2 and EGF receptors.
However, if Xaa
is not hydrophobic, as in the
insulin, IL4, and IGF-1 receptors, then the Shc PTB domain will bind
with sequences having hydrophobic Xaa
,
Xaa
, and Xaa
residues in the
motif
-
-
-X-X-N-
-
-pY.
The IRS-1 PTB domain binds with the insulin and IL4 receptor
peptides, but not mT, TrkA, ErbB4, or EGF receptor peptides, to imply
that the isolated domains faithfully recapitulate biological substrate
specificities. Some combination of hydrophobic residues at
Xaa, Xaa
, and Xaa
positions are important, Asn
and
Pro
are crucial for high affinity binding, and
substitutions at Xaa
modulate affinity. Thus the
IRS-1 PTB domain binds preferentially with the sequence
-
-
-X-X-N-
-
-pY,
where
sites are generally hydrophobic and
indicates a
-turn-forming residue (
= P;
A > E). The PTB domain specificities are summarized
below