From the
Interferon-
The Type I interferons (IFN
Several proteins are substrates for
IFN
Many growth factor
receptors, including those for epidermal growth factor and
platelet-derived growth factor, associate directly through their
autophosphorylation sites with a common set of signaling proteins that
contain SH2 domains, including the phosphatidylinositol (PI) 3`-kinase,
Grb-2, SH-PTP2, phospholipase C
We performed experiments in which the patterns of tyrosine
phosphorylation induced by IFN
IRS-1 plays a central role in the signal transduction of
insulin and interleukin-4, and its signaling functions appear to be
essential for the mitogenic effects of these ligands(19) .
Recent evidence has suggested that IRS-1 may also be involved in growth
hormone signaling (26). The protein contains many potential tyrosine
phosphorylation sites in various hydrophobic contexts(16) .
These tyrosine residues play a dual role as substrates for upstream
tyrosine kinases and as specific docking sites for downstream SH2
proteins. At least eight tyrosine residues in IRS-1 undergo
phosphorylation by the activated insulin receptor, including residues
460, 608, 628, 939, and 987, which are in YXXM/YMXM
motifs and bind to p85 which activates the PI 3`-kinase(20) .
Three other motifs are also phosphorylated by the insulin receptor,
including Y
We currently think that the insulin
receptor tyrosine kinase regulates the insulin-dependent
phosphorylation of IRS proteins, whereas the receptors for IL-4 and
IFN
The shared use of the IRS signaling pathway
establishes a common link between apparently distinct signaling systems
for insulin, IGF-1, IL-4, and IFN
We thank Drs. Michael Brunda (Hoffman-La Roche) and
Paul Trotta (Schering-Plough) for providing us with IFN
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(IFN
) induces rapid tyrosine
phosphorylation of the insulin receptor substrate-1 (IRS-1), a docking
protein with multiple tyrosine phosphorylation sites that bind to the
Src homology 2 (SH2) domains of various signaling proteins. During
IFN
stimulation, the p85 regulatory subunit of the
phosphatidylinositol 3`-kinase binds via its SH2 domains to
tyrosine-phosphorylated IRS-1, and phosphatidylinositol 3`-kinase
activity is detected in association with IRS-1. Thus, IFN
responses occur by activation of the IRS signaling system, which it
shares with insulin, insulin-like growth factor-1, and interleukin-4.
, IFN
, and IFN
)
(
)exert a variety of biological effects on normal and
neoplastic cells that include antiviral and antiproliferative
activities(1) . Immediately after IFN
stimulation, several
signaling proteins in the receptor complex become
tyrosine-phosphorylated, including the
and
subunits of the
Type I IFN receptor (2, 3) and the Tyk-2 and Jak-1
tyrosine kinases(4, 5, 6, 7) . Both
kinases associate with components of the Type I IFN
receptor(7, 8) , and their activation early in the
IFN
signaling cascade is presumed to regulate tyrosine
phosphorylation of various downstream signaling molecules. Expression
of Jak-1 and Tyk-2 rescues an IFN
response in certain insensitive
cell lines, suggesting that these tyrosine kinases or related members
of the Janus family are essential for IFN
action(4, 9) .
-dependent tyrosine kinase activity. In response to IFN
treatment of cells, the Stat-2, Stat-1
, and Stat-1
components
of the transcriptional activator ISGF3
are rapidly phosphorylated
on tyrosine and associate with a 48-kDa protein (ISGF3
) to form an
active complex(10, 11, 12) . This complex
translocates to the nucleus and initiates gene transcription during
binding to interferon-stimulated response elements (10-13). In
addition, the vav proto-oncogene product
(p95
) is tyrosine-phosphorylated during IFN
stimulation (14); however, its precise role in the signal transduction
of IFN
remains to be determined. The involvement of multiple
pathways in IFN
signaling is consistent with its pleiotropic
biological effects on cells and tissues.
, and Ras-GAP(15) . The
receptors for insulin, IGF-1, and IL-4, however, do not strongly
associate with most SH2 proteins known to be involved in their
signaling pathways(16) . Instead, they stimulate tyrosine
phosphorylation of docking proteins in the IRS signaling family,
notably IRS-1, which binds directly to various SH2
proteins(16, 17, 18, 19) . We report
that IFN
also engages the IRS signaling system, as evidenced by
the rapid IFN
-dependent tyrosine phosphorylation of IRS-1 in
several hematopoietic cell lines. We also demonstrate that the p85
regulatory subunit of the PI 3`-kinase binds via its SH2 domains the
IFN
-induced phosphorylated form of IRS-1, and that PI 3`-kinase
activity can be detected in association with IRS-1.
Cells and Reagents
The U-266 (human myeloma),
Daudi (lymphoblastoid), and MOLT-4 (acute T-cell leukemia) cell lines
were grown in RPMI 1640 (Life Technologies, Inc.) supplemented with 10%
(v/v) fetal bovine serum (Life Technologies, Inc.) or 10% (v/v) defined
calf serum (Hyclone Laboratories) and antibiotics. Human recombinant
IFN2 was provided by Hoffmann-La Roche and Schering-Plough. The
antiphosphotyrosine monoclonal antibody (4G-10) and the monoclonal
antibody against the regulatory subunit of PI 3`-kinase were obtained
from Upstate Biotechnology (Lake Placid, NY) and were used for
immunoblotting. Nonimmune rabbit IgG was obtained from Sigma. The
polyclonal
IRS-1
antibody was raised against a GST
fusion protein of a region of rat IRS-1 (amino acid residues
1-135) containing its pleckstrin homology domain. An antibody
(
IRS-1
) against a synthetic peptide
(TYASINFQKQPEDRQ), corresponding to a sequence present in the C
terminus of IRS-1, was raised in rabbits. The polyclonal antibodies
against recombinant rat IRS-1 and the p85 regulatory subunit of PI
3`-kinase have been described elsewhwere (20) and were used for
immunoprecipitations. A polyclonal antibody against Stat-2 (p113,
186-199) has been described elsewhere(21) .
Immunoprecipitations and Immunoblotting
Cells were
stimulated with the indicated amounts of IFN or insulin for the
indicated periods of time. After stimulation, the cells were rapidly
centrifuged and lysed in a phosphorylation lysis buffer (0.5-1%
Triton X-100, 150 mM NaCl, 200 µM sodium
orthovanadate, 10 mM sodium pyrophosphate, 100 mM sodium fluoride, 1 mM EDTA, 50 mM Hepes, 1.5
mM magnesium chloride, 10% glycerol, 1 mM
phenylmethylsulfonyl fluoride, and 10 µg/ml aprotinin). Cell
lysates were immunoprecipitated with the indicated antibodies and,
after five washes with phosphorylation lysis buffer containing 0.1%
Triton X-100, were analyzed by SDS-PAGE and transferred onto
nitrocellulose (Schleicher and Schuell) or polyvinylidene difluoride
(Immobilon) filters, and the residual binding sites on the filters were
blocked by incubating with TBST (10 mM Tris, pH 8.0, 150
mM NaCl, 0.05% Tween 20, 10% bovine serum albumin) for
1-3 h at room temperature or overnight at 4 °C. The filters
were subsequently incubated with the antiphosphotyrosine monoclonal
antibody and developed using an enhanced chemiluminesence (ECL) kit
following the manufacturer's procedure (Amersham).
Preparation of Glutathione S-Transferase Fusion
Proteins
Glutathione S-transferase fusion proteins
containing the nSH2 and cSH2 domains of PI 3`-kinase (22) were
purified from transformed Escherichia coli bacteria which were
induced with 1 mM
isopropyl-1-thio--D-galactopyranoside as described
previously(23) . Briefly, after 2 h of additional growth,
bacteria were lysed by sonication in phosphate-buffered saline (PBS),
pH 7.0. Lysed bacteria were spun for 25 min at 14,000 rpm at 4 °C,
and the supernatant was immobilized on glutathione-Sepharose beads
(Pharmacia) and used for binding assays.
Assays for PI 3`-Kinase Activity
PI 3`-kinase
activity assays on IRS-1
immunoprecipitates obtained
from IFN
- or insulin-stimulated cells were performed as described
previously(24) . Briefly, after IFN
or insulin stimulation,
serum-starved Daudi cells were washed once with ice-cold PBS and twice
with 20 mM Tris, pH 7.5, containing 137 mM NaCl, 1
mM MgCl
, and 100 µM
Na
VO
(Buffer A). The cells were lysed in 1 ml
of Buffer A containing 1% Nonidet P-40, 10% glycerol, and 0.35 mg/ml
phenylmethylsulfonyl fluoride, and insoluble material was removed by
centrifugation. Cell lysates were immunoprecipitated with the indicated
antibodies, and immunoprecipitates were washed in PBS with 1% Nonidet
P-40 and 100 µM Na
VO
(three
times), 100 mM Tris, pH 7.5, containing 500 mM LiCl
and 100 µM Na
VO
(three times), and 10 mM Tris, pH 7.5, containing 100
mM NaCl, 1 mM EDTA, and 100 µM Na
VO
(twice). The pellets were resuspended
in 50 µl of 10 mM Tris, pH 7.5, containing 100 mM NaCl and 1 mM EDTA. 10 µl of 100 mM
MnCl
and 10 µl of phosphatidylinositol (2
µg/µl) sonicated in 10 mM Tris, pH 7.5, 1 mM EGTA were added to each pellet. The reaction was started by the
addition of 10 µl of 440 µM ATP containing 30 µCi
of [
P]ATP. After incubation for 10 min at 22
°C, the reaction was stopped by the addition of 20 µl of 8 N HCl and 160 µl of CHCl
:methanol (1:1). The
samples were centrifuged, and the lower organic phase was removed and
applied to a silica gel TLC plate which was coated with 1% potassium
oxalate. TLC plates were developed in
CHCl
:CH
OH:H
O:NH
OH
(60:47:11.3:2), dried, and visualized and quantitated on a Molecular
Dynamics PhosphorImager.
or insulin in hematopoietic cells
were studied. We noticed that both IFN
and insulin stimulate
tyrosine phosphorylation of a common 170-kDa protein in U-266 cells (Fig. 1). The molecular mass of this protein was similar to the
mass of the insulin receptor substrate-1(17) , suggesting that
IFN
uses an IRS signaling protein to mediate certain biologic
responses.
Figure 1:
Patterns of tyrosine phosphorylation
induced by IFN or insulin in U-266 cells. Antiphosphotyrosine
immunoblots. A, cells were incubated in the presence or
absence of 10
units/ml IFN
for 5 min at 37 °C as
indicated. B, cells were either not stimulated (lane
1) or stimulated with 1 µM insulin for either 5 min (lane 2) or 30 min (lane 3) at 37 °C as
indicated. Equal amounts of protein from total cell lysates (100
µg) were analyzed by SDS-PAGE.
To determine if IRS-1 is involved in IFN signaling,
lysates from control or stimulated U-266 or Daudi cells were
immunoprecipitated with polyclonal antibodies against IRS-1 and
immunoblotted with an antiphosphotyrosine antibody. Basal tyrosine
phosphorylation of a 170-kDa protein, corresponding to IRS-1, was
consistently detected in U-266 but not in Daudi cells. However,
IFN
and insulin strongly stimulated tyrosine phosphorylation of
IRS-1 in both cell lines (Fig. 2, A and B, and
data not shown). The IFN
-induced phosphorylation of IRS-1 was
rapid and transient, occurring within 1 min of treatment of Daudi or
MOLT-4 cells and diminishing after 90 min (Fig. 2C). It
was also dose-dependent, exhibiting a similar response pattern with the
phosphorylation of Stat proteins (Fig. 2D).
Figure 2:
Tyrosine phosphorylation of IRS-1 in
response to IFN and insulin. Antiphosphotyrosine immunoblots. A, serum-starved U-266 cells were treated with IFN
(10
units/ml) for 5 min at 37 °C as indicated, and cell
lysates were immunoprecipitated with
IRS-1
,
IRS-1
, or normal rabbit serum (RS) as
indicated. B, Daudi cells were incubated in the presence or
absence of IFN
(10
units/ml) or insulin (1
µM) for 3 min at 37 °C. Cell lysates were
immunoprecipitated with either nonimmune RIgG or a polyclonal antibody
against baculovirus-generated rat IRS-1 as indicated. C, time
course of tyrosine phosphorylation of IRS-1. Cells were stimulated with
10
units/ml IFN
for the indicated time points, and
cell lysates were immunoprecipitated with either nonimmune RIgG or an
antibody against recombinant rat IRS-1 as indicated. D,
serum-starved U-266 cells were treated for 30 min at 37 °C with the
indicated amounts of IFN
, and cell lysates were immunoprecipitated
with either
IRS-1
(lanes 1-4) or with
a polyclonal antibody against Stat-2 (lanes
5-8).
After
tyrosine phosphorylation, IRS-1 binds to several SH2 proteins,
including the 85-kDa regulatory subunit (p85) of the PI
3`-kinase(20) . To determine whether IRS-1 binds p85 during
IFN stimulation, immunoprecipitates obtained with an anti-p85
antibody from Daudi cell lysates were immunoblotted with
antiphosphotyrosine (4G-10). Following IFN
or insulin treatment of
the cells, a 170-kDa tyrosine-phosphorylated protein was detected in
the anti-p85 immunoprecipitates. This phosphoprotein co-migrated with
IRS-1 immunoprecipitated directly with
IRS-1
(Fig. 3A). Moreover, anti-IRS-1 immunoprecipitates
contained p85
only after IFN
or insulin stimulation (Fig. 3B). The tyrosine-phosphorylated form of IRS-1
from IFN
- or insulin-stimulated cells also bound to a GST fusion
protein containing the N-terminal SH2 domain of p85 (Fig. 3C and 3D). Similar results were obtained using a GST fusion
protein containing the C-terminal SH2 domain of p85.
(
)Thus, IFN
stimulates the association of IRS-1
with the PI 3`-kinase, and this interaction most likely requires the
SH2 domains in p85(22) .
Figure 3:
Association of IRS-1 with the p85
regulatory subunit of PI 3`-kinase in Daudi cells. A,
antiphosphotyrosine immunoblot. Cells (4 10
/lane)
were incubated for 5 min at 37 °C in the presence or absence of
IFN
(10
units/ml) or insulin (1 µM) as
indicated, and cell lysates were immunoprecipitated with normal RIgG,
p85, or
IRS-1
, as indicated. B, cells
(5
10
/lane) were incubated in the presence or
absence of IFN
or insulin for 7 min at 37 °C as indicated, and
cell lysates were immunoprecipitated with
IRS-1
,
normal RIgG, or
p85 as indicated and immunoblotted with a
monoclonal antibody against p85
. C, cells (9
10
/lane) were stimulated with IFN
or insulin for 5 min
at 37 °C as indicated, and cell lysates were incubated for 3 h at 4
°C with either GST alone or a GST fusion protein containing the
N-terminal SH2 domain of p85
, both of which were bound to
glutathione-Sepharose beads. Bound proteins were separated by SDS-PAGE
and immunoblotted with antiphosphotyrosine. D, cells (1.5
10
/lane) were incubated for 3 min with IFN
as
indicated, and cell lysates were bound to either GST alone or a
GST-nSH2 fusion protein. Bound proteins were analyzed by SDS-PAGE and
immunoblotted with
IRS-1
.
The PI 3`-kinase appears to play an
important role in various biological responses and is activated by many
growth factors and cytokines(25) . Its activation by insulin
occurs during association with tyrosine-phosphorylated
IRS-1(20) . To determine whether PI 3`-kinase activity is
detected in association with IRS-1 during IFN stimulation, PI
3`-kinase assays were carried out on
IRS-1 immunoprecipitates.
Before stimulation, a basal level of PI 3`-kinase activity was detected
in the
IRS-1 immunoprecipitates, which was equivalent to the
nonspecific activity detected in immunoprecipitates with preimmune
rabbit serum (Fig. 4). IFN
or insulin stimulated the
association of PI 3`-kinase activity with IRS-1 that was
immunoprecipitated specifically with
IRS-1
(Fig. 4). It is likely that the PI 3`-kinase is activated
by IFN
during its association with IRS-1.
Figure 4:
Association of PI 3`-kinase activity with
IRS-1 during IFN or insulin stimulation in Daudi cells.
Serum-starved cells were treated for 5 min in the absence or presence
of IFN
(10
units/ml) or insulin (100 nM) as
indicated, cell lysates were immunoprecipitated with
IRS-1
or preimmune serum, and immunoprecipitates were assayed in
triplicate for PI 3`-kinase activity. The lower spots in the
TLC plate represent the origin, and the upper spots represent
the phosphorylated phosphatidylinositol substrate. Data were plotted as
the average -fold activation ± S.E. for triplicate
determinations.
Considerable progress
has been made in our understanding of IFN signaling between the
plasma membrane and nucleus. The Jak-Stat pathway provides a plausible
mechanism for the regulated assembly of ISGF-3, which regulates
expression of genes containing the IFN-stimulated response
elements(13) . The molecular mechanism used by IFN
to
regulate other signaling pathways, however, is poorly understood. Our
finding that IFN
stimulates tyrosine phosphorylation of IRS-1
suggests the existence of a pathway for the regulated engagement of
additional SH2 signaling proteins during IFN
stimulation. It will
be important to identify other SH2 proteins engaged by IRS-1 in
response to IFN
, as well as in response to IFN
and IFN
,
which also induce its phosphorylation on tyrosine residues.
VNI, which binds Grb-2, and
Y
IDL and Y
ASI which bind
SH-PTP2(27) . Studies to determine the motifs of IRS-1 that are
tyrosine-phosphorylated in response to IFN
should provide valuable
information on the mechanisms by which the signaling specificity is
established through the common use of this protein by different
cytokines and growth factors.
use Janus family tyrosine kinases to accomplish such
phosphorylation; however, direct evidence that IRS-1 acts as a
substrate for Janus kinases during IFN
stimulation remains to be
obtained. Many receptors activate the Janus family of tyrosine kinases,
but most of them do not phosphorylate IRS-1. The identity of the
elements responsible for this selectivity are unknown. Interestingly,
the receptors for insulin, IGF-1, and IL-4 contain a common amino acid
sequence motif, LxxxxNPxYxss, which appears
to contribute to the interaction of these receptors with
IRS-1(28) ; however, this sequence motif is not found in the
cloned components of the Type I IFN receptor(8, 29) ,
suggesting that a different motif may be involved or another subunit
remains to be found.
. Recently, Larner et al.(30) suggested that IL-4 attenuates the transcriptional
activation of IFN-induced cellular gene expression in monocytes and
related cell lines. Although the mechanism of this phenomenon is
unknown, our results raise the possibility of an antagonism between
IFN
and IL-4 occurring through the common use of proteins in the
IRS signaling system. If this hypothesis is correct, then insulin and
IGF-1 may also influence IFN
signaling. Additional work in this
area should reveal important relationships between these apparently
distinct factors and others as well.
.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.