(Received for publication, December 6, 1994; and in revised form, May 19, 1995)
From the
Potential signaling substrates for the insulin-like growth
factor I (IGF-I) receptor are SH2 domain proteins including the p85
subunit of phosphatidylinositol 3-kinase, the tyrosine phosphatase Syp,
GTPase activating protein (GAP), and phospholipase C- (PLC-
).
In this study, we demonstrate an association between the IGF-I receptor
and p85, Syp, and GAP, but not with PLC-
in lysates of cells
overexpressing the human IGF-I receptor. We further investigated these
interactions using glutathione S-transferase (GST) fusion
proteins containing the amino-terminal SH2 domains of p85 or GAP, or
both SH2 domains of Syp or PLC-
to precipitate the IGF-I receptor
from purified receptor preparations and from whole cell lysates. p85-,
Syp-, and GAP-GSTs precipitated the IGF-I receptor, whereas the
PLC-
-GST did not. Using phosphopeptides corresponding to IGF-I
receptor phosphorylation sites, we determined that the p85- and Syp-GST
association with the IGF-I receptor could be inhibited by a
carboxyl-terminal peptide containing pY1316 and that the GAP-GST
association could be inhibited by a NPXY domain peptide. The
GAP-GST binding site was confirmed by showing that a mutant IGF-I
receptor with a deletion of the NPXY domain including tyrosine
950 was poorly precipitated by the GAP-GST. We conclude that p85 and
Syp may bind directly to the IGF-I receptor at tyrosine 1316, and that
GAP may bind to the IGF-I receptor at tyrosine 950. An association
between the IGF-I receptor and PLC-
was not evident. p85, Syp, and
GAP are potential modulators of IGF-I receptor signal transduction.
The insulin-like growth factor I (IGF-I) ()receptor
is a member of the large family of tyrosine kinase growth factor
receptors which undergo autophosphorylation upon ligand
binding(1, 2) . The activated receptor is then able to
transduce this signal intracellularly by interacting with specific
downstream protein substrates, ultimately resulting in hormonal effects
such as cellular growth and differentiation(3, 4) .
Autophosphorylation of the IGF-I receptor is followed immediately by
phosphorylation of insulin receptor substrate-1
(IRS-1)(5, 6, 7, 8) .
The
tyrosine phosphorylation of IRS-1 transforms this 185-kDa molecule into
a ``docking'' protein, facilitating its interaction with a
number of other signaling molecules containing Src homology 2 (SH2)
domains(9, 10) . SH2 domains are conserved regions of
100 amino acids that promote association with phosphorylated
tyrosines surrounded by specific amino acid motifs such as those found
in IRS-1 and activated growth factor
receptors(11, 12) . Although IRS-1 is clearly an
important signaling molecule for the insulin and IGF-I receptors (5, 6, 7, 8, 13, 14) ,
recent evidence gained from IRS-1 knockout mice has suggested that an
alternative signaling pathway(s) exists(15, 16) . We
hypothesize that one such pathway may result from the direct
association of potential signaling molecules with the IGF-I receptor.
Recently, Van Horn et al.(17) , demonstrated that the p85 subunit of phosphatidylinositol 3-kinase (PI 3-kinase) interacts directly with the insulin receptor, and we have localized this insulin receptor binding site to the YXXM motif at tyrosine 1322 (18) . Although direct association of p85 with the IGF-I receptor has not previously been demonstrated, PI 3-kinase activity increases after IGF-I stimulation. Furthermore, Yamamoto et al.(19) showed that PI 3-kinase activity associates with immobilized IGF-I receptors and can be competed away with increasing concentrations of a p85 fusion protein.
Syp (PTP1D, SHPTP2) is a protein tyrosine phosphatase that contains two SH2 domains. Syp is the homologue of corkscrew, an SH2 phosphatase in Drosophila known to enhance signal transduction of the receptor tyrosine kinase torso(20, 21, 22, 23) . Microinjection experiments indicate that Syp is involved in positive signal transduction of receptor kinases as inhibition of Syp leads to a decrease in insulin- and IGF-I-stimulated mitogenesis(24, 25) . Syp is known to bind to IRS-1(26) , but we and others have also demonstrated that a Syp-glutathione S-transferase (GST) fusion protein can associate directly with the insulin receptor at tyrosine 1322(18, 27) . It can also directly associate with the epidermal growth factor (28) and platelet-derived growth factor (29) receptors. It is not known if Syp can interact directly with the IGF-I receptor.
GTPase-activating protein (GAP) is
another SH2 domain protein involved in tyrosine kinase growth factor
receptor signal transduction. GAP inactivates ras by
stimulating the hydrolysis of GTP-ras, thereby functioning as
a negative regulator of mitogenic signal transduction(30) . GAP
associates directly with the activated insulin receptor (31) at
tyrosine 960(18) , but it is not known if GAP interacts with
the IGF-I receptor. Phospholipase C- (PLC-
) is another SH2
domain protein which is recruited directly to the activated epidermal
and platelet-derived growth factor receptors and is an important
mitogenic signaling molecule(32) . No previous association
between PLC-
and the insulin or IGF-I receptors has been reported.
In this study, we investigate the direct association of p85, Syp,
GAP, and PLC- with the IGF-I receptor and map the sites of
receptor interaction utilizing SH2 domain-containing fusion proteins
and phosphopeptides modeled after phosphotyrosine-containing domains of
the IGF-I receptor.
Figure 1:
Protein
identification. Monolayers of CHO were incubated at 37
°C for 1 min in the absence or presence of IGF-I, 100 ng/ml. A shows whole cell lysates analyzed by 7.5% SDS-PAGE, then
transferred to nitrocellulose and immunoblotted with an
antiphosphotyrosine antibody. Stimulated cell lysates were also
immunoprecipitated with an anti-IRS-1 antibody (B) or with an
anti-IGF-I receptor antibody (C) as described under
``Experimental Procedures.'' The precipitates were analyzed
by 7.5% SDS-PAGE, transferred to nitrocellulose, and immunoblotted with
an antiphosphotyrosine antibody. D, lanes 1-4,
depict an anti-GAP immunoblot. The first lane shows the total cell
lysate. The unstimulated sample in lane 2 and the
IGF-I-stimulated sample in lane 3 were first precipitated with
an antiphosphotyrosine antibody, then immunoblotted with an anti-GAP
antiserum. Lane 4 shows the antibody alone without cell lysate
immunoblotted with the anti-GAP antiserum. Lane 5 shows the
cell lysate both precipitated and immunoblotted with the
antiphosphotyrosine antibody.
Figure 2:
IGF-I receptor association with p85, Syp,
and GAP. Whole cell lysates from monolayers of CHO were
pretreated with 25 µM PAO for 5 min (lane 3 only), stimulated with IGF-I, 100 ng/ml, for 1 min at 37 °C,
then precipitated with an anti-IGF-I receptor antibody. After analysis
by 7.5% SDS-PAGE and transfer to nitrocellulose, the membranes were
immunoblotted with an anti-p85 antibody (lane 1), an anti-Syp
antibody (lane 2), an anti-GAP antiserum (lane 3), or
an anti-PLC-
antibody.
These data indicate that p85, Syp,
and GAP, but not PLC-, are precipitated along with the IGF-I
receptor. It is not clear whether these proteins are associating
directly with the IGF-I receptor or indirectly with other proteins
which interact with the IGF-I receptor such as IRS-1. To further
explore the possibility of direct IGF-I receptor-SH2 domain protein
interactions, we studied the ability of SH2 domain-GST fusion proteins
to interact directly with the IGF-I receptor.
Figure 3:
SH2 domain-GST precipitation of the IGF-I
receptor from whole cell lysates. Whole cell lysates from CHO stimulated with IGF-I, 100 ng/ml, for 1 min at 37 °C were
incubated with increasing concentrations of p85- (A), Syp- (B), GAP- (C), or PLC-
- (D) GST-fusion
proteins and glutathione-Sepharose for 90 min. The precipitates were
collected, analyzed by 7.5% SDS-PAGE, transferred to nitrocellulose,
and immunoblotted with an antiphosphotyrosine
antibody.
The IGF-I receptor
was not precipitated by the GAP-GST or by the PLC--GST (Fig.3, C and D). No phosphorylated proteins
were precipitated by the PLC-
-GST (Fig.3D), but
the GAP-GST was able to precipitate a 120-kDa, an 80-kDa, and a 62-kDa
phosphoprotein from whole cell lysates. A very low molecular mass
protein can be seen in the last two lanes of the Syp- and the
PLC-
-GST immunoblots. This band is the molecular mass of the added
fusion proteins. It may represent either the tyrosine phosphorylation
of the fusion proteins or nonspecific binding by the secondary
horseradish peroxidase antibody used in the detection assay.
Figure 4:
SH2 domain-GST precipitation of the IGF-I
receptor from purified receptor preparations. IGF-I receptors were
purified from CHO, stimulated with IGF-I, 100 ng/ml, and
activated in the presence of 50 µM ATP and 10 mM MnCl
. The samples were incubated with increasing
concentrations of p85- (A), Syp- (B), GAP- (C), or PLC-
- (D) GST-fusion proteins and
glutathione-Sepharose for 90 min at 4 °C. The precipitates were
collected, analyzed by 7.5% SDS-PAGE, transferred to nitrocellulose,
and immunoblotted with an antiphosphotyrosine
antibody.
Figure 5:
Precipitation of the IGF-I receptor from
whole cell lysates by the GAP-GST after PAO pretreatment. A,
whole cell monolayers of CHO were preincubated in the
absence or presence of 25 µM PAO for 5 min at 37 °C.
The cells were then stimulated in the absence or presence of IGF-I, 100
ng/ml, and warmed at 37 °C for 1 min. Cell lysates were incubated
with 1.25 µM GAP- (lanes 3 and 4) or
PLC-
-GST (lanes 5 and 6) and
glutathione-Sepharose for 90 min at 4 °C. The precipitates were
then analyzed by SDS-PAGE, transferred to nitrocellulose, and
immunoblotted with an antiphosphotyrosine antibody. Lanes 1 and 2 represent total cell lysates without and with IGF-I
stimulation. B, CHO
whole cell monolayers were
stimulated with IGF-I at 37 °C for 5 (lanes 1 and 3) and 60 min (lanes 2 and 4). The samples
were immunoprecipitated with an antiphosphotyrosine antibody,
fractionated by 7.5% SDS-PAGE, and then immunoblotted with an anti-GAP
antiserum.
To
determine if some of the increase in the phosphorylated protein band at
120 kDa was from tyrosine phosphorylation of GAP, whole cell monolayers
of CHO were preincubated with PAO, then immunoprecipitated
with the antiphosphotyrosine antibody and immunoblotted with an
anti-GAP antiserum. Fig.1D demonstrates that GAP is
not tyrosine-phosphorylated in whole cell lysates. However, after PAO
preincubation, tyrosine phosphorylation of GAP can be clearly detected (Fig.5B, lanes 1 and 2). In fact,
after 60 min of IGF-I stimulation in the presence of PAO, >90% of
the total cellular GAP protein is precipitated by the
antiphosphotyrosine antibody (Fig.5B, lanes 2 and 4). GAP can, therefore, become
tyrosine-phosphorylated in IGF-I-stimulated whole cells pretreated with
PAO.
Fig.6A shows
that p85-GST binding to the IGF-I receptor is inhibited by increasing
concentrations of the pY1316. No receptor is precipitated by the
p85-GST in the presence of 750 µM pY1316 (Fig.6A, lane 6). In contrast, neither the
triple tyrosine peptide nor the NPXY peptide was able to
inhibit the p85-GSTIGF-I receptor interaction at a concentration
of up to 500 µM.
Figure 6: IGF-I receptor binding to the p85-GST is inhibited by phosphopeptide pY1316. A, partially purified receptors were stimulated with IGF-I in the presence of ATP and were then incubated with a 1.0 µM concentration of the p85-GST, increasing concentrations of the phosphopeptide pY1316, and glutathione-Sepharose for 90 min at 4 °C. Precipitates were analyzed by 7.5% SDS-PAGE and immunoblotted with an antiphosphotyrosine antibody. B, this experiment was similar to that described in A, except that phosphopeptides pYpYpY (lanes 2-4) and RPXpY (lanes 5-7) were used instead of pY1316.
Figure 7: IGF-I receptor binding to the Syp-GST is inhibited by phosphopeptide pY1316. Partially purified receptors were stimulated with IGF-I in the presence of ATP and were then incubated with a 1.0 µM concentration of the Syp-GST, increasing concentrations of the phosphopeptide pY1316 (A) or pYpYpY and RPXpY (B), and glutathione-Sepharose for 90 min at 4 °C. Precipitates were analyzed by 7.5% SDS-PAGE and immunoblotted with an antiphosphotyrosine antibody.
Figure 8:
IGF-I receptor binding to the GAP-GST is
inhibited by phosphopeptide RPXpY, and the GAP-GST does not
bind to an NPXY deletion receptor mutant. A,
partially purified receptors were stimulated with IGF-I in the presence
of ATP and were then incubated with a 1.0 µM concentration
of the GAP-GST, increasing concentrations of the phosphopeptides pY1316 (lanes 2-4), pYpYpY (lanes 5-7), and
RPXpY (lanes 8-10), and glutathione-Sepharose
for 90 min at 4 °C. Precipitates were analyzed by 7.5% SDS-PAGE and
immunoblotted with an antiphosphotyrosine antibody. B,
partially purified receptors from
CHO cells were activated
as described above and were incubated with increasing concentrations of
the GAP-GST (lanes 1-3) or the p85-GST (lanes 4 and 5) and glutathione-Sepharose for 90 min at 4 °C.
Precipitates were fractionated by 7.5% SDS-PAGE and immunoblotted with
an antiphosphotyrosine antibody.
IRS-1 is known to be important in insulin and IGF-I receptor signal transduction. However, recent work in transgenic IRS-1 knockout mice has suggested the existence of IRS-1-independent, alternate signaling pathways(15, 16) . In this report, we provide evidence to suggest a direct interaction between p85, Syp, and GAP and the IGF-I receptor. We have also localized the probable sites of SH2 domain binding to the IGF-I receptor utilizing phosphopeptides derived from important autophosphorylation sites on the IGF-I receptor.
A monoclonal anti-IGF-I receptor antibody was able to precipitate
p85 from IGF-I-stimulated whole cell lysates prepared from a monolayer
of CHO. Although it cannot be definitively proven in these
experiments, this most likely represents a direct and independent
interaction between p85 and the IGF-I receptor, rather than an
interaction between p85 and IRS-1 in a binary IGF-I receptor
IRS-1
complex. Although the nature of the IGF-I receptor
IRS-1
interaction is poorly defined, it is quite evanescent. IRS-1 does not
co-precipitate with the IGF-I receptor when an IGF-I receptor antibody
is used(41) , nor does the IGF-I receptor co-precipitate with
IRS-1 when an IRS-1 antibody is used(8) . The inability to
detect IGF-I receptor
IRS-1 complexes in this fashion makes it
unlikely, but not impossible, that the presence of p85 in the
anti-IGF-I receptor precipitate results from an IGF-I
receptor
IRS-1
p85 interaction. We interpret these data to
suggest a direct interaction between p85 and the IGF-I receptor.
Backer et al.(42) have previously shown that p85
could associate with either IRS-1 or directly with the insulin
receptor, although as much as 70% of the total cellular p85 in their
studies was associated with IRS-1 in insulin-stimulated cells
suggesting that the p85IRS-1 interaction is the primary route by
which PI 3-kinase is activated upon insulin
stimulation(8, 9, 41, 42, 43, 44) .
However, transgenic mice with a knockout mutation of the IRS-1 gene are
not diabetic despite their inability to signal through IRS-1,
suggesting alternate pathways to PI 3-kinase activation including a
direct receptor
p85 interaction(15, 16) .
The
interaction of the IGF-I receptor with p85 either directly or via IRS-1
has been suggested previously. PI 3-kinase activity can associate with
immobilized IGF-I receptors and can be competed away with increasing
concentrations of a p85 fusion protein(19) . The activated
IGF-I receptor is also known to phosphorylate recombinant IRS protein in vitro on sites identical with those
phosphorylated by the insulin receptor, including those in the
YMXM motif known to bind
p85(8, 9, 26, 45, 46, 47) .
The preferred binding sequence for the SH2 domains of p85 is
pYMXM, and the methionine in the +3 position is critical
for p85 SH2 binding(26, 44) . We have previously
reported that the p85-GST containing the amino-terminal SH2 domain of
p85 can bind to the carboxyl terminus of the insulin receptor at the
binding motif pYTHM(18) . In this report, we
describe the direct binding of the p85-GST to the IGF-I receptor and
localize a binding site to a similar binding motif,
pY
AHM, in the carboxyl terminus. We cannot exclude the
possibility that IGF-I receptor phosphotyrosines other than those we
studied here may participate in p85 binding. Although IRS-1 and the
IGF-I receptor can both associate with p85 after IGF-I stimulation, the
relative contribution of each interaction to biological signaling
remains unknown.
Syp is an SH2-containing tyrosine phosphatase which
binds preferentially to phosphotyrosines in a
pY-hydrophobic-X-hydrophobic
motif(26, 27, 48, 49, 50) .
We describe here that an anti-IGF-I receptor antibody can precipitate
Syp. As described above for p85, we believe this suggests a direct
SypIGF-I receptor interaction rather than an IGF-I
receptor
IRS-1
Syp interaction because of the evanescent
nature of the IGF-I receptor
IRS-1 association. Furthermore, we
have also shown in vitro that a Syp-GST may bind directly to
the IGF-I receptor at phosphotyrosine 1316 in a pYAHM binding sequence.
We have not excluded the possibility that other receptor phosphotyrosyl
residues not studied may be involved in Syp binding as well. Recent
studies examining the crystal structures of amino-terminal SH2 domain
of Syp in separate complexes with two high affinity peptides
demonstrate the importance of a hydrophobic residue in the +5
position (50) . The IGF-I receptor has a hydrophobic amino
acid, glycine, in the +5 position from tyrosine 1316. The
biological relevance of the Syp
IGF-I receptor versus the
Syp
IRS-1 interaction has not been defined, but microinjection
studies have implicated Syp as a positive regulator of insulin- and
IGF-I-induced mitogenesis(24, 25) .
GAP is shown
here to associate with IGF-I receptors precipitated from whole cell
lysates prepared from monolayers of CHO pretreated with
phenylarsine oxide. A GAP-GST can associate with purified IGF-I
receptors in vitro and with IGF-I receptors from whole cell
lysates pretreated with phenylarsine oxide. Using phosphopeptides, we
have determined that the SH2 domain of the GAP-GST interacts with the
IGF-I receptor at tyrosine 950. The motif consists of a sequence with a
hydrophobic amino acid in the +1 and +3 positions and is
consistent with GAP binding sites previously described for the
platelet-derived growth factor (28, 51) and the
insulin receptor(18) .
It is surprising that GAP could be
precipitated along with the IGF-I receptor only from cells pretreated
with phenylarsine oxide. A possible explanation for this result is that
the degree of phosphorylation achieved after pretreatment with this
protein tyrosine phosphatase inhibitor is greater than in its absence.
Insulin receptors in a cell-free system, for example, phosphorylate to
a greater extent than do receptors in whole
cells(52, 53, 54) . We believe we were able
to increase the overall tyrosine phosphorylation of the IGF-I receptors
in whole cells by pretreatment with a phosphatase inhibitor, thereby
allowing detection of the IGF-I receptorGAP association. Pronk et al.(31) similarly found an association between the
insulin receptor and GAP in whole cell lysates only after pretreatment
with PAO.
Consistent with the association of GAP and the IGF-I
receptor, we have also found that IGF-I stimulation leads to tyrosine
phosphorylation of GAP protein. This IGF-I-stimulated GAP tyrosine
phosphorylation was only detectable in the presence of the phosphatase
inhibitor. In the untreated cell, this tyrosine phosphorylation may be
undetectable because of rapid dephosphorylation. These data further
indicate that GAP may interact with the IGF-I receptor in vivo under certain conditions and may be a component of the IGF-I
receptor signaling cascade. This interaction may be tightly regulated
by PAO-sensitive phosphatases. The IGF-I receptor does not associate
with PLC-, even in the presence of PAO or when the PLC-
-GST
is used at high concentrations.
In this report, we show that the IGF-I receptor may directly associate with SH2 domain proteins including p85, Syp, and GAP. We have mapped the IGF-I receptor binding site for p85 and Syp to tyrosine 1316 in the carboxyl terminus and the binding site for GAP to tyrosine 950 in the NPXY domain. The association between these proteins and the IGF-I receptor may provide an alternate mechanism to directly activate these signaling molecules.