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(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
Insulin binding to the insulin receptor activates it as a
substrate kinase, leading to tyrosine phosphorylation of at least two
cytoplasmic proteins, IRS-1 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 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
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
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
Figure 3:
Competition assays with Shc and IRS-1 PTB
domains. A, GST/Shc(1-238), peptide
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
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 Substitution studies were conducted with
both sequences to further probe binding mechanisms. Ala substitutions
of hydrophobic residues at the Xaa Substitutions at Xaa 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 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
Volume 270,
Number 46,
Issue of November 17, 1995 pp. 27407-27410
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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.
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) . 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. -turn forming motifs amino-terminal to pTyr,
their specificities differ in detail.
Recombinant Proteins and Synthetic
Peptides
Fragments of the human IRS-1 and human Shc cDNAs were
subcloned into a pGEX-4T vector using polymerase chain reactions. Escherichia coli strains DH5
or XL-1 blue were
transformed with vectors encoding glutathione S-transferase
(GST) fusion proteins GST/IRS-1(4-516), GST/IRS-1(108-516),
GST/IRS-1(144-316), GST/Shc(1-196), GST/Shc(1-206),
GST/Shc(1-238), GST/Shc(1-474), GST/Shc(20-206),
GST/Shc(33-206), and GST/Shc(46-206), (13) , where
numbers in parentheses refer to residues of human IRS-1 (19) and human Shc p52(20) , respectively. Proteins
were expressed and isolated as usual using glutathione-agarose affinity
chromatography and elution with glutathione(13, 21) .
Phosphopeptides were synthesized and purified as
described(22) .Direct Binding between Native and Mutated Insulin
Receptors and IRS-1 and Shc Proteins
Wild-type insulin receptors
were isolated from transfected NIH-3T3 cells and purified by wheat germ
agglutinin affinity chromatography. Mutated Y960F receptors were
isolated following similar protocols from transfected Chinese hamster
ovary cells(23) . The receptors were autophosphorylated by
sequential incubation with 100 nM insulin (16 h) and 50
µM ATP plus 5 mM Mn as
described(24) . Phosphorylated receptors were incubated with
glutathione-agarose-bound fusion proteins (5 µg) in the presence
and absence of competing ligands. Bound proteins were eluted from the
glutathione-agarose, separated by SDS-polyacrylamide gel
electrophoresis, transferred to polyvinylidene difluoride membranes,
and identified by blotting with anti-insulin receptor antibodies.
Binding Assays between Phosphopeptides and IRS-1 and Shc
Proteins
PTB-phosphopeptide assays are operationally similar to
previously described assays for SH2 domain
interactions(13, 21, 22) . Phosphopeptides
derived from the sequence surrounding Tyr-960 of the insulin receptor
(LYASSNPEpYLSASDV or LYASSNPApYLSASDV) or Tyr-250 of the mouse polyoma
virus mT antigen (LLSNPTpYSVMRSK) were I-radiolabeled by
a lactoperoxidase method or with Bolton-Hunter reagent, respectively,
and purified by HPLC(21) . Appropriate amounts of the Shc- or
IRS-1-derived GST-fusion proteins (typically 8-10 µg), the
radiolabeled peptides (mT for the Shc PTB, insulin receptor peptides
for the IRS-1 PTB), varying concentrations of unlabeled peptides, and
glutathione-agarose beads were combined in 200 µl of assay buffer
(20 mM Tris-HCl at pH 7.4, 250 mM NaCl, 0.1% bovine
serum albumin, and 10 mM dithiothreitol)(13, 21) . The mixtures were
incubated overnight and radioactivity associated with the unwashed
glutathione-agarose was determined.
Delineation of Shc and IRS-1 PTB Domains
We
previously showed that the Shc PTB domain could be pared down to
residues 1-206 of human Shc p52 without loss of function. Further
truncation at the COOH terminus (1-196) led to complete loss of
function, whereas initiation at Met-46 rather than Met-1 (corresponding
to the start site of Shc p46) led to 20-fold reduction in binding
affinity (13) . We have since found that the Shc PTB domain can
be truncated at its amino terminus (20-206 and 33-206)
without loss of binding affinity (Fig. 1B). Therefore,
a stably folded, functional Shc PTB domain is encompassed by a maximum
of 174 residues (33-206).
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.
Shc and IRS-1 PTB Domain Interactions with the Insulin
Receptor
Insulin receptors phosphorylated in vitro were
precipitated by Shc(1-238) and IRS-1(144-316) PTB domain
GST-fusion proteins (Fig. 2). Precipitation of the receptor
occurred only in the presence of the fusion protein (Fig. 2A) and insulin activation (Fig. 2B). Moreover, both interactions were inhibited
by phosphotyrosine, consistent with both proteins being classified as
``phosphotyrosine binding domains'' (Fig. 2B). Although the insulin receptor contains at
least six sites for tyrosine autophosphorylation(25) ,
efficient endogenous substrate phosphorylation requires phosphorylation
specifically at Tyr-960(16) . When Tyr-960 is mutated to Phe,
the bulk of insulin receptor phosphorylation remains
intact(23) . We now show that the Tyr-960 Phe mutation
blocks association of the insulin receptor with both PTB domains (Fig. 2C) to demonstrate that this site interacts with
both domains.
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.Comparative PTB Domain Binding Specificities
To
analyze the determinants of IRS-1 PTB domain binding and compare IRS-1
and Shc PTB domain specificities, direct binding assays were developed
similar to those used previously for SH2
domains(13, 21, 22) . For example, the Shc
PTB domain binds the mT peptide with highest affinity (ID = 1.2 µM and IL4 and insulin receptor-derived
sequences with 25-32-fold lower relative affinities (Fig. 3). In contrast, the IRS-1 PTB domain binds the IL4
receptor sequence with highest affinity (ID
= 6.2
µM), the native insulin receptor sequence with
intermediate affinity (ID
= 170 µM),
and not at all with the mT-derived sequence. These findings clearly
demonstrate significant differences in binding specificities between
these domains. In the remainder of this study, we have analyzed
determinants of Shc versus IRS-1 PTB domain specificity in
detail, using native and modified peptide sequences derived from the mT
antigen and the insulin, IL4, TrkA, ErbB4, and EGF receptors.
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.
, 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).
(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).
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.
(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.
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. , 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
Conclusion
PTB domains of similar length and
function exist in Shc and IRS-1. Since the sequences of these domains
have low homology, it is not yet possible to know whether they
represent distinct or related elements of protein structure. The
binding interactions mediated by PTB domains of Shc and IRS-1 are
closely related to analogous SH2 domain interactions in that binding
affinities are similar, phosphorylation serves as the on-off switch,
and surrounding sequence provides specificity. However, both PTB
domains show a reversal in the orientation of required peptide
interactions compared to SH2 domains, since residues amino- but not
carboxyl-terminal to pTyr play a major role in determining specificity.
Peptide sequences recognized by the two PTB domains are similar to one
another, as both bind -turn forming NPXpY motifs.
However, their specificities also differ since the Shc PTB domain binds
with highest affinity to motifs having a hydrophobic Xaa residue, relative to pTyr, while both IRS-1 and Shc PTB domains
bind motifs having some combination of hydrophobic residues at
Xaa
, Xaa
, and
Xaa
positions. Differences in PTB domain specificity
may be useful in targeting particular pathways for drug design, as has
been proposed previously for SH2 domains. Distinct specificities of
these PTB domains correlate with and may account for some biological
differences between these cytoplasmic substrates of tyrosine
kinase-linked receptors.
))
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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 L. S. Lock, C. R. Maroun, M. A. Naujokas, and M. Park Distinct Recruitment and Function of Gab1 and Gab2 in Met Receptor-mediated Epithelial Morphogenesis Mol. Biol. Cell, June 1, 2002; 13(6): 2132 - 2146. [Abstract] [Full Text] [PDF]
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B. Emanuelli, P. Peraldi, C. Filloux, C. Chavey, K. Freidinger, D. J. Hilton, G. S. Hotamisligil, and E. Van Obberghen SOCS-3 Inhibits Insulin Signaling and Is Up-regulated in Response to Tumor Necrosis Factor-alpha in the Adipose Tissue of Obese Mice J. Biol. Chem., December 14, 2001; 276(51): 47944 - 47949. [Abstract] [Full Text] [PDF]
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 D. Le Roith and Y. Zick Recent Advances in Our Understanding of Insulin Action and Insulin Resistance Diabetes Care, March 1, 2001; 24(3): 588 - 597. [Abstract] [Full Text]
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Y. B. Choi, C. K. Kim, and Y. Yun Lad, an Adapter Protein Interacting with the SH2 Domain of p56lck, Is Required for T Cell Activation ,2 J. Immunol., November 15, 1999; 163(10): 5242 - 5249. [Abstract] [Full Text] [PDF]
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B. Urso, D. L. Cope, H. E. Kalloo-Hosein, A. C. Hayward, J. P. Whitehead, S. O'Rahilly, and K. Siddle Differences in Signaling Properties of the Cytoplasmic Domains of the Insulin Receptor and Insulin-like Growth Factor Receptor in 3T3-L1 Adipocytes J. Biol. Chem., October 22, 1999; 274(43): 30864 - 30873. [Abstract] [Full Text] [PDF]
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K. Paz, Y.-F. Liu, H. Shorer, R. Hemi, D. LeRoith, M. Quan, H. Kanety, R. Seger, and Y. Zick Phosphorylation of Insulin Receptor Substrate-1 (IRS-1) by Protein Kinase B Positively Regulates IRS-1 Function J. Biol. Chem., October 1, 1999; 274(40): 28816 - 28822. [Abstract] [Full Text] [PDF]
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M. Marin-Hincapie and R. S. Garofalo The Carboxyl Terminal Extension of the Drosophila Insulin Receptor Homologue Binds IRS-1 and Influences Cell Survival J. Biol. Chem., August 27, 1999; 274(35): 24987 - 24994. [Abstract] [Full Text] [PDF]
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ABSTRACT
