(Received for publication, October 2, 1995; and in revised form, January 26, 1996)
From the
Irs-proteins link the receptors for insulin/IGF-1, growth
hormones, and several interleukins and interferons to signaling
proteins that contain Src homology-2 (SH2). To identify new
Irs-1-binding proteins, we screened a mouse embryo expression library
with recombinant [P]Irs-1, which revealed a
specific association between p59
and Irs-1. The
SH2 domain in p59
bound to phosphorylated
Tyr
and Tyr
, which are located in
YXX(L/I) motifs. Mutation of p59
at the
COOH-terminal tyrosine phosphorylation site (Tyr
)
enhanced its binding to Irs-1 during insulin stimulation. Binding
experiments with various SH2 proteins revealed that Grb-2 was largely
excluded from Irs-1 complexes containing p59
,
whereas Grb-2 and p85 occurred in the same Irs-1 complex. By comparison
with the insulin receptor, p59
kinase
phosphorylated a unique cohort of tyrosine residues in Irs-1. These
results outline a role for p59
or other related
Src-kinases during insulin and cytokine signaling.
Irs proteins are important elements in signal transduction by
the receptors for insulin, IGF-1, various interleukins (interleukin-4,
-9, -13, and -15), interferons (interferons a and b), and growth
hormones (1, 2, 3, 4, 5, 6, 7) .
After tyrosine phosphorylation, Irs-1 provides a common interface
between the activated receptor and various downstream signaling
proteins containing Src homology-2 (SH2) ()domains,
including phosphatidylinositol 3-kinase, p55
,
Grb-2, SH-PTP2, Nck, and possibly
Crk(8, 9, 10, 11, 12, 13, 14) .
Moreover, Irs-1 associates with the integrin a
b
and SV40 T-antigen, which may play roles in growth regulation or
transformation(15, 16) . As a consequence of docking
to these and possibly other proteins, Irs-1 mediates multiple
downstream signals including the direct activation of
phosphatidylinositol 3-kinase and SH-PTP2, the indirect stimulation of
MAP kinase and p70
, and other events that
regulate gene expression, inhibit apoptosis, and stimulate mitogenesis,
chemotaxis, and glucose
transport(10, 17, 18, 19, 20) .
We utilized an expression screening approach based on the CORT
technique to identify new proteins that bind to tyrosine phosphorylated
Irs-1(9, 21) . The Src family tyrosine kinase,
p59, was isolated from a mouse embryo library
with [
P]Irs-1. p59
contains an SH3 and an SH2 domain at its NH
terminus and a catalytic domain at its COOH
terminus(22) . Both SH2 and SH3 domains are essential for
regulation of the enzymatic activity, because deletion of either domain
leads to constitutive activation of the kinase and an increased
transformation
potential(23, 24, 25, 26) . An
important role for p59
during signal
transduction by tyrosine kinase receptors has been implicated in a
number of systems: the p59
associates with
middle T-antigen(27, 28) , the ligand-stimulated PDGF
and colony-stimulating factor-1 receptors(29, 30) ,
and B and T cell antigen receptors during antigen
stimulation(31, 32) . In these systems,
p59
may participate in the stimulation of cell
proliferation. Inhibition of p59
function by
microinjection of antibodies against p59
or DNA
constructs encoding dominant negative proteins reduces the mitogenic
response of fibroblasts to PDGF, suggesting that p59
is an important signaling element(33) . Moreover,
disruption of the p59
gene in mice causes a
signaling defect in mature thymocytes(34, 35) . The
Fyn
mice also exhibit specific neurologic
deficits such as impaired long term potentiation, spatial learning,
hippocampal development(36) , impaired ability to initiate
suckling reflex(37) , and defective myelination(33) ,
suggesting a general role for p59
during
development.
In this paper, we demonstrate that Irs-1 binds
p59in vitro and in vivo. The
SH2 domain from p59
specifically binds to the
Tyr(P)
and Tyr(P)
in Irs-1 located in
YXX(L/I) motifs and may compete for with Grb-2 for association
with Irs-1. Thus, p59
may play an important role
in Irs-1-mediated signaling during insulin/IGF-1 or cytokine signaling.
Twenty 15-cm plates representing 500,000 plaques were overlaid with
nitrocellulose filters (Millipore, HATF) that were impregnated with 10
mM isopropyl-b-D-thiogalactopyranoside (BRL)
and incubated for 10 h at 37 °C. The filters were removed, briefly
washed at room temperature with TNT buffer (10 mM Tris-HCl, pH
8.0, 150 mM NaCl, 0.05% Tween 20), and then incubated in TNT
buffer containing 5% Carnation instant dry milk for 6 h. The filters
were incubated overnight at 4 °C with
[P]Irs-1 (50 mg/ml), and then washed 3 times at
room temperature with 10 mM Tris-HCl (pH 8.0), containing 150 mM NaCl and 0.01% Tween 20. The dry filters were exposed at -70
°C for 24 h to Kodak XAR-5 film with an intensifying screen.
Fifteen of the 30 primary positive plaques remained positive during two
rounds of screening with [
P]Irs-1. The cDNA
inserts in pBluescript were prepared by in vivo excision
according to the manufacturer's instructions (Stratagene).
Immunocomplexes were prepared from cell extracts by incubation with
various rabbit polyclonal antibodies and detected by immunoblotting as
described previously(19) . The antibody against Irs-1
(Irs-1) was raised against the last 12 residues in the COOH
terminus;
SH-PTP2 was raised against NH
-SH2 domain of
SH-PTP2;
Grb2 was purchased from Santa Cruz, and
p85 was from
UBI. Normal rabbit IgG was used as a control antibody.
In other experiments, cell
lysates were prepared from insulin-stimulated or unstimulated
CHO and CHO
/Irs-1 cells in 20 mM Tris-HCl (pH 7.5) containing 137 mM NaCl, 100 mM NaF, 1 mM MgCl
, 1 mM CaCl
, 200 mM sodium orthovanadate, 0.4 mM phenylmethylsulfonyl fluoride, 50 µg/ml aprotinin, 50
µg/ml leupeptin, 10% glycerol, and 1% Nonidet P-40 (Calbiochem).
The extracts were clarified by centrifugation at 100,000
g for 1 h at 4 °C. The supernatants were incubated with the
GSTSH2
for 2 h, washed with extraction buffer, and
analyzed by immunoblotting(14, 19) .
Figure 1:
The association of p59 with Irs-1 in vitro. A, Irs-1 was labeled
with [
-
P]ATP by insulin receptor and
incubated with GST fusion proteins (1 mg of SH3
SH2
and GST, and 0.5 mg of GST-Fyn) as
indicated. The complexes were precipitated with glutathione-Sepharose,
washed, and analyzed by SDS-PAGE on a 7.5% SDS gel. B, cell
lysates were prepared from insulin stimulated (+) and
unstimulated(-) CHO
/Irs-1 cells and incubated with
an increasing amount of GST-SH2
. The GST fusion
protein was then precipitated by glutathione-Sepharose, washed, and
separated on 7.5% SDS-PAGE. Irs-1 was detected by immunoblotting with
Irs-1.
Figure 2:
The association of p59 with Irs-1 in vivo. Cell extracts prepared from
CHO, CHO
, CHO/Irs-1, or CHO
/Irs-1 cells
before or after expression of murine Fyn
were incubated
without or with 100 nM insulin for 5 min. Cell extracts were
then incubated with various antibodies including
Fyn (A and C) or
Irs-1 (B and D). The
immuncomplexes were resolved by SDS-PAGE and immunoblotted with
Irs-1 (A),
PY (B), or
Fyn (C and D) and detected with
[
I]protein A. These results are representative
of two experiments.
Insulin stimulated tyrosine phosphorylation of Irs-1 in each cell
line as measured in Irs-1 immunoprecipitated by immunoblotting
with aPY (Fig. 2B); the highest level of Irs-1
phosphorylation occurred in the CHO
/Irs-1 cells as
described previously (Fig. 2B). The overexpression of
p59
had no reproducible effect on Irs-1 tyrosine
phosphorylation.
Specific immunoprecipitates of Irs-1 were examined
for p59 association before and after insulin stimulation.
Endogenous p59
associated poorly with Irs-1 in CHO,
CHO
, and CHO/Irs-1 (Fig. 2D); however,
endogenous p59
associated with Irs-1 in
CHO
/Irs-1 cells during insulin stimulation (Fig. 2D, lane n). These results suggest that
the association of Fyn with Irs-1 was relatively weak and required a
high level of Irs-1 phosphorylation in order to be detected (Fig. 2B). Consistent with this hypothesis,
overexpression of murine Fyn
increases the
insulin-stimulated association of murine Fyn
with Irs-1
in all of the cell lines, as observed by aFyn immunoblots of
Irs-1
immunoprecipitates (Fig. 2D, lanes d, h, l, and p). Insulin-stimulated association
of Fyn with Irs-1 was greatest in the CHO
/Irs-1 cells
expressing the Fyn
, where it was also strong enough to
be observed in
Fyn immunoprecipitates by
Irs-1 blots (Fig. 2A, lane p). The basal association of
Fyn
may reflect the basal phosphorylation of Irs-1 in
these cells. Thus, insulin promoted the association of p59
with Irs-1 in intact cells, consistent with our in vitro experiments.
Figure 3:
Binding of Irs-1 phosphopeptides to
GST-SH2. GST-SH2
or GST
alone were incubated with Irs-1 tryptic phosphopeptides and
precipitated with glutathione-Sepharose. Supernatants and elutes from
the precipitates were resolved by reverse-phase on the HPLC, and the
radioactivity was measured with an on-line radiodetection system
(Packard). These results are representative of two independent
experiments.
Figure 4:
Formation of Irs-1 complexes. Cell lysates
prepared from insulin-stimulated or unstimulated CHO/Irs-1
cells overexpressing either p59
or the
COOH-terminal mutant p59
. Before and after insulin
stimulation (100 nM), cell extracts were incubated with the
indicated antibodies, and proteins were immunoblotted with indicated
antibodies. These results are representative of two independent
experiments.
Figure 5:
Insulin-stimulated p59 tyrosine kinase activity. Cell lysates from
insulin-stimulated or unstimulated CHO cells overexpressing murine
Fyn
were incubated with
Fyn or
FLAG monoclonal
antibody. The immuncomplexes were isolated on protein A-Sepharose, and
kinase assays were performed in immuncomplexes with denatured enolase
as a substrate. Autophosphorylated p59
and
phosphorylated enolase are indicated. These results are representative
of two independent experiments.
In order to assess if Irs-1 is
phosphorylated by p59 kinase, an in vitro kinase
assay was performed to examine the phosphorylation of recombinant Irs-1
by GST-Fyn (Fig. 6A). The phosphorylation of Irs-1 by
GST-Fyn was comparable with the phosphorylation of an equimolar amount
of enolase; phosphoamino acid analysis of phosphorylated Irs-1 showed
that the phosphorylation was exclusive on tyrosine (Fig. 6B). The phosphorylation of Irs-1 was not due to
kinase contamination during preparation of GST fusion protein, because
neither substrate was phosphorylated by GST-SH3
or GST
alone (Fig. 6A, lanes a, b, and e). The pattern of tyrosine phosphorylation sites in Irs-1 was
different during incubation with the insulin receptor or GST-Fyn (Fig. 6C). Based on our previous
assignments(14) , both the insulin receptor and Fyn
phosphorylated Tyr
, whereas the phosphorylation of
Tyr
was much weaker by p59
(Fig. 6C). Many other differences occurred in the
phosphopeptides map, although the exact sites involved were not
identified. Tyrosine phosphorylation of Irs-1 in the intact cells was
not detectably increased by overexpression of Fyn
,
probably owing to the low stoichiometry of the association, as
described previously by the PDGF receptor(29) .
Figure 6:
In vitro phosphorylation of Irs-1
by p59. A, Irs-1 or enolase was
phosphorylated by GST, GST-Fyn, or GST-SH3
for 1
h and separated on 7.5% SDS-PAGE, and phosphorylation of Irs-1 was
measured by autoradiography. These results are the representative of
three independent experiments. B, phosphorylated Irs-1 bands
were excised, and amino acid analyses were performed as described under
``Experimental Procedures.'' C, Irs-1 were
phosphorylated by insulin receptor or GST-Fyn in the presence of
[
-
P]ATP, separated on SDS-PAGE, and
transferred to nitrocellulose membrane. Phosphorylated Irs-1 were
excised and eluted from membrane by digestion with trypsin. Tryptic
phosphopeptides were analyzed on HPLC, and radioactivity was detected
with an on-line radiodetection system (Packard). This results are
representative of two independent
experiments.
The p59 tyrosine kinase associates with Irs-1 in vitro and in vivo during insulin stimulation,
suggesting that it may contribute to the signal mediated by Irs-1 in
certain cellular backgrounds. The p59
may also be
involved in signaling from other receptors that engage Irs-1 if
Tyr
and Tyr
are phosphorylated by
activated kinases in the receptor complex. Although p59
is activated during association with several growth factor
receptors(30, 43, 44) , we did not detect
activation during insulin stimulation. However, we cannot role out the
possibility that the portion of p59
associated with Irs-1
was activated but is impossible to detect in a large pool of unbound
p59
(44) . The p59
recruited into
the Irs-1 signaling complex may mediate a second phase of tyrosine
phosphorylation of Irs-1 or the phosphorylation of other proteins in
the Irs-1 complex. Because other members of the Src kinase family have
similar SH2 domains, these kinases may also associate with Irs-1 and
contribute to the overall response in various cellular backgrounds.
The SH2 domain of p59 selectively binds to two
phosphorylation sites in Irs-1, EY
VNI and
NY
IDL, which are different from the consensus sequence
YEEI for the SH2 domain of p59
determined by
phosphopeptide library screening(21) . Both binding sites have
hydrophilic residues at +1 and +3 position, which are
consistent with the PDGF receptor binding sites for Src, including
DY
VPM(45) , EY
IYV, and
IY
VDP(29) . Interestingly, Tyr
and
Tyr
also bind Grb-2 and SH-PTP2,
respectively(14) . In fact, p59
and Grb-2 are not
found in the same Irs-1 complex, indicating that a competition may
exist in the cell for the binding of these two proteins to the
activated Irs-1. The formation of unique Irs-1 signaling complexes may
be a general mechanism to produce specific signals. This will become
clearer as we identify more SH2 proteins that engage phosphorylated Irs
proteins under various conditions.
In vitro kinase assays
reveal that Irs-1 is a good substrate for p59. Some of
the tyrosine phosphorylation sites on Irs-1 are shared by both the
insulin receptor and Fyn, whereas others are different. However,
overexpression of Fyn does not lead to a significant phosphorylation of
Irs-1 in resting cells. Even during insulin stimulation, the
phosphorylation of Irs-1 in CHO cells is not significantly enhanced by
overexpressing murine p59
, suggesting that the insulin
receptor, not p59
, is the major tyrosine kinase that
phosphorylates Irs-1. However, it is possible that p59
phosphorylates tyrosine residues in a small subset of Irs-1
molecules that are not detected by
PY immunoblotting.
The
phosphorylation of Irs-1 by p59 may play an important
role in nontyrosine kinase receptors. Cross-linking of the B-cell
antigen receptor in primary B cells and mature B cell lines causes the
phosphorylation of Irs-1 (data not shown). Because p59
is
a known component of this signaling complex, it may be involved in the
phosphorylation of Irs-1.
p60 from nontransformed
fibroblasts is normally phosphorylated extensively at
Tyr
, which is highly conserved among the Src kinase
family (Tyr
in Lck and Tyr
in
p59
)(22, 46) . Phosphorylation of this
tyrosine residue, mediated by p50
, down-regulates
tyrosine kinase catalytic activity and biological functions of the
Src-related kinases(31, 32, 46) . This
inhibition involves an intramolecular association between the SH2
domain and the COOH-terminal phosphotyrosine
residue(22, 41) . The removal of Tyr
from p59
enhances its binding to Irs-1 during
insulin stimulation, suggesting that Tyr
may compete with
Irs-1 for binding to the SH2 domain. Thus, the binding of wild type
p59
to Irs-1 should stimulate its tyrosine kinase by
displacing the COOH terminus. Although we were unable to document this
increase, future experiments along this line of investigation should be
explored and could reveal a subset of Irs-1/Fyn complexes that mediate
a specific biological response.
The association of p59 with the PDGF or colony-stimulating factor-1 receptors may
contribute to the mitogenic response(29, 30) .
Currently, we do not know the biological effect of p59
during its association with Irs-1. Insulin-stimulated DNA
synthesis appears normal in cells overexpressing p59
and
Irs-1 (data not shown). However, the endogenous level of p59
in CHO cells is relatively high, and possibly sufficient for a
maximal response. A reduction of the p59
levels may help
address this question. However, deletion of the COOH terminus of Irs-1,
or substitution of Tyr
and Tyr
with
phenylalanine has no effect of Irs-mediated and insulin-stimulated
mitogenesis in 32D myeloid cells, suggesting that Irs-1/Fyn complexes
are not involved. (
)
In summary, p59 takes a
place next to other SH2 proteins as an Irs-1-associated enzyme that may
contribute to the full Irs-1 signal. The differential association of
p59
with Irs-1 complexes supports the idea that Irs-1
signaling complexes are not homogenous, and different elements in the
complex may influence the aggregate signal. Much work remains to
identify other novel proteins that associate with Irs-1 and Irs-2 (47) and to determine the pattern of tyrosine phosphorylation
that occurs during activation of various receptors, which will
influence the ultimate biological signal.