(Received for publication, October 27, 1995)
From the
Binding of interferon (IFN
) to its receptor induces
activation of the Tyk-2 and Jak-1 tyrosine kinases and tyrosine
phosphorylation of multiple downstream signaling elements, including
the Stat components of the interferon-stimulated gene factor 3
(ISGF-3). IFN
also induces tyrosine phosphorylation of IRS-1, the
principle substrate of the insulin receptor. In this study we
demonstrate that various Type I IFNs rapidly stimulate tyrosine
phosphorylation of IRS-2. This is significant since IRS-2 is the major
IRS protein found in hematopoietic cells. The IFN
-induced
phosphorylated form of IRS-2 associates with the p85 regulatory subunit
of the phosphatidylinositol 3`-kinase, suggesting that this kinase
participates in an IFN
-signaling cascade downstream of IRS-2. We
also provide evidence for an interaction of IRS-2 with Tyk-2,
suggesting that Tyk-2 is the kinase that phosphorylates this protein
during IFN
stimulation. A conserved region in the pleckstrin
homology domain of IRS-2 may be required for the interaction of IRS-2
with Tyk-2, as shown by the selective binding of glutathione S-transferase (GST) fusion proteins containing the
IRS-2-IH1
or IRS-1-IH1
domains to Tyk-2 but
not other Janus kinases in vitro.
Type I interferons (IFNs) ()are pleiotropic cytokines
that exhibit multiple biological effects on normal and malignant cells,
including antiproliferative, antiviral, and immunomodulatory
activities(1) . Although the precise mechanisms by which Type I
IFNs exhibit their biological effects remain unknown, significant
advances have been made recently on our understanding of the early
events of signaling by the Type I IFN receptor (IFNR). Two kinases of
the Janus family, Tyk-2 and Jak-1, are associated with components of
the the Type I IFNR complex(2, 3) . IFN
stimulates tyrosine phosphorylation of the
and
subunits of
the Type I IFNR (4, 5, 6, 7) and
activation of Tyk-2 and
Jak-1(2, 8, 9, 10) . During IFN
stimulation, the Stat-2, Stat-1
, and Stat-1
components of the
transcriptional activator ISGF3
are rapidly phosphorylated on
tyrosine and associate with ISGF3
to form an active complex (11, 12, 13, 14) . This complex
translocates to the nucleus to initiate transcription of
interferon-stimulated genes during binding to interferon-stimulated
response elements(11, 12, 13, 14) .
Tyk-2 and Jak-1 are essential for this response, as
IFN
-insensitive cells are rescued by expression of Tyk-2 or
Jak-1(8, 15) .
Although the molecular link between
the Type I IFN receptor and ISGF3 provides an important paradigm
for interferon signaling, other signaling pathways may also be
involved. To our surprise, IFN
stimulates tyrosine phosphorylation
of IRS-1(16) . This is somewhat counterintuitive, since IRS-1
clearly mediates cell growth and metabolism during insulin/IGF-1 and
IL-4 stimulation(17) . Moreover, IRS-1 appears to mediate SV40
large T antigen transformation(18) . The IRS-1 signaling
function is best characterized during insulin stimulation, as it is
phosphorylated at multiple tyrosine residues which bind to the SH2
domains in PI 3`-kinase regulatory subunits
(p85
/p85
/p55
), Grb-2, Nck, Fyn, (
)and
SH-PTP2(19, 20, 21, 22) . IRS-1
associates with other proteins, including integrins
(
) (23) and 14-3-3 (
)proteins, and such associations may play an important role
in the biological action of these proteins. In addition to IL-4
receptors, a growing number of cytokine receptors mediate tyrosine
phosphorylation of IRS-1, including IL-9, IL-13, growth hormone, and
leukemia inhibitory
factor(18, 24, 25, 26, 27, 28, 29) .
As a consequence of these and other interactions, IRS-1 mediates
multiple downstream signals, including the direct activation of PI
3`-kinase and SH-PTP2, the indirect stimulation of mitogen-activated
protein kinase and p70
, and other events which regulate
gene expression and stimulate mitogenesis and glucose
transport(18, 23, 30, 31, 32, 33, 34) .
Interest in a putative ``IRS-2'' increased recently when
it was discovered that IRS-1 mice survive,
reproduce, and display only mild insulin
resistance(35, 36) . The mice are small during their
life, but display no other obvious phenotypic changes, including an
apparently normal immune response. (
)Many hematopoietic
cells do not contain IRS-1, but express the newly cloned isoform, IRS-2 (37) . In this study, we show that Type I IFNs stimulate
tyrosine phosphorylation of IRS-2 in several cell lines of diverse
hematopoietic origin, and that, after tyrosine phosphorylation, IRS-2
associates with the p85 regulatory subunit of the PI 3`-kinase.
Furthermore, we demonstrate that the pleckstrin homology (PH) domains
of IRS-2 and IRS-1 associate with the tyrosine kinase Tyk-2 in
vitro, suggesting that this motif in IRS-proteins mediates their
interaction with the Type I IFN receptor complex.
We investigated the tyrosine phosphorylation of IRS-2 during
IFN stimulation of various human hematopoietic cells. After
immunoprecipitation using specific anti-IRS-1 or anti-IRS-2 antibodies,
proteins were analyzed by SDS-PAGE and immunoblotted with an
antiphosphotyrosine monoclonal antibody. As shown
previously(14) , IFN
stimulated tyrosine phosphorylation
of IRS-1 in U-266 myeloma cells; it also stimulated the tyrosine
phosphorylation of IRS-2 (Fig. 1). The protein migrated slightly
above IRS-1, as predicted by its slightly larger size(37) .
Moreover, IFN
stimulated the phosphorylation of IRS-2 in the KG1
and KG1A acute myeloid leukemia cell lines (Fig. 1), which have
been shown previously to respond to the antiproliferative effect of
IFN
(41) .
Figure 1:
IFN induces
tyrosine phosphorylation of IRS-2. Antiphosphotyrosine imunoblots are
shown. Upper panel, serum-starved KG1 (4.8
10
/lane) or KG1A (2.8
10
/lane) cells
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
IRS-2 or
preimmune rabbit serum (PIRS) as indicated. Lower
panel, serum-starved U-266 cells (1.4
10
/lane)
were incubated for 5 min at 37 °C in the presence or absence of
IFN
(10
units/ml) as indicated, and cell lysates were
immunoprecipitated with
IRS-1
,
IRS-2, or normal
rabbit serum (NRS) as indicated.
The kinetics of IRS-2 phosphorylation was
examined in HEL and FDCP-2 cells. Treatment of HEL cells with human
IFN, or FDCP-2 cells with mouse IFN
/
, stimulated maximum
tyrosine phosphorylation of IRS-2 within 5 min (Fig. 2, A and B). The phosphorylation gradually declined and became
undetectable after 90 min, suggesting that the phosphorylation of IRS-2
is rapid and transient during IFN
stimulation. Similar time
courses for the phosphorylation of IRS-1 have been observed previously
during IFN
or insulin stimulation in other cell types. The
mechanism of dephosphorylation is unknown and may involve degradation
of IRS-2, activation of a phosphatase, or down-regulation of the
receptor.
Figure 2:
Kinetics of IFN-dependent
phosphorylation of IRS-2. Serum-starved HEL cells (2.8
10
/lane) (A) or FDCP-2 cells (2.5
10
/lane) (B) were treated with human IFN
or
mouse IFN
/
, respectively, for the indicated times at 37
°C. Cell lysates were immunoprecipitated with
IRS-2 (lanes
1-4) or normal rabbit serum (lane 5) and
immunoblotted with antiphosphotyrosine. C, serum-starved U-266
cells (2.1
10
/lane) were treated with IFN
for
the indicated times. Cell lysates were immunoprecipitated with either
IRS-2 (lanes 1-4) or
Tyk-2 (lanes
5-8) as indicated and immunoblotted with
antiphosphotyrosine.
The functional Type I IFN receptor complex is composed of
several components, including the Tyk-2 and Jak-1
kinases(2, 3) , which are responsible for signal
transmission. The time course of IRS-2 phosphorylation was studied in
parallel with the phosphorylation of Tyk-2 in U-266 cells. Tyk-2
migrates as a 135-kDa protein during SDS-PAGE. The IFN-induced
phosphorylation of Tyk-2 exhibited a similar time course with the
phosphorylation of IRS-2. Interestingly, a 135-kDa
tyrosine-phosphorylated protein that co-migrated with Tyk-2 was
co-immunoprecipitated by the
IRS-2 antibody in some experiments,
suggesting that Tyk-2 associates with IRS-2 (Fig. 2C).
However, this association was not observed consistently, possibly due
to low stoichiometry of such interactions; similar variable results
were reported previously for the association between insulin receptors
and IRS-1(42) . Apparently, the associations between receptor
complexes and IRS proteins are weak and/or transient, but sufficient to
mediate specific phosphorylation.
The mechanism of specific
recognition of IRS proteins by the Type I IFN receptor complex is
unknown. Two regions conserved in the NH terminus of IRS-1
and IRS-2, called the IH1
domain and IH2
domains, mediate the association with the insulin
receptor(37, 43) . The IH1
domain
resembles a pleckstrin homology domain (PH), whereas the IH2
domain is a phosphotyrosine binding domain(37) . The
IH1
domain is important for the sensitive interaction
between the insulin receptor and IRS-1, although the mechanism is
unknown(43) , while the IH2
domain binds to
phosphorylated LXXXXNPXYXSXS motifs
in the insulin and IL-4 receptors(44) ; however, the known
components of the Type I IFN receptor do not contain such
motifs(3, 45, 46) . To determine whether the
IH1
motif of IRS-2 mediates the interaction with the Type
I IFN receptor complex, a GST fusion protein containing the IH1
domain of IRS-2 was incubated with lysates from human cells.
Tyk-2 associated with the IRS-2/IH1
domain, suggesting
that this motif may provide a molecular link to the Type I IFN receptor
complex (Fig. 3, A and B). Similarly, a GST
fusion protein containing the IH1
domain of IRS-1
(IRS-1/IH1
) also bound to Tyk-2 (Fig. 3, C and D). In contrast, the GST-IRS-1/IH1
and
GST-IRS-2/IH1
fusion proteins did not bind to Jak-1
present in extracts from IFN
-stimulated cells (data not shown),
suggesting that this motif in IRS proteins interacts specifically with
Tyk-2 but not Jak-1. Additional studies using various mutations in the
IH1
domains will be needed to establish the significance
of this result in the biological context.
Figure 3:
Association of the pleckstrin homology
domains of IRS proteins with Tyk-2. A, U-266 cells (2.1
10
/lane) were treated with 10
units/ml
IFN
for 5 min at 37 °C as indicated, and cell lysates were
bound to either IRS-2/PH-GST or GST alone as indicated. Proteins were
analyzed by SDS-PAGE and immunoblotted with antiphosphotyrosine. B, the blot shown in A was stripped and reblotted
with a monoclonal antibody against Tyk-2. C, serum-starved
Daudi cells (5.8
10
/lane) were treated with
10
units/ml IFN
for 2 min at 37 °C as indicated,
and cell lysates were bound to either IRS-1/PH-GST or GST alone.
Proteins were analyzed by SDS-PAGE and immunoblotted with
antiphosphotyrosine. D, the blot shown in C was
stripped and reblotted with a monoclonal antibody against
Tyk-2.
In addition to IFN,
IFN
, and IFN
bind to the Type I IFN receptor and stimulate
tyrosine phosphorylation of Tyk-2, Jak-1, and Stats(5) . When
the tyrosine phosphorylation of IRS-2 in response to IFN
and
IFN
was studied, we found that both IFNs induce tyrosine
phosphorylation of IRS-2 (Fig. 4A). Similarly, IFN
and IFN
stimulated tyrosine phosphorylation of IRS-1 in Molt-4
cells (Fig. 4B and data not shown). In contrast,
IFN
binds to the Type II IFN receptor, which utilizes the Jak-1
and Jak-2 tyrosine kinases for signal
transduction(8, 9, 10, 14, 47) .
IFN
did not stimulate tyrosine phosphorylation of IRS-2 or IRS-1
in U-266 or Molt-4 cells, despite the fact that Jak-2 is expressed and
tyrosine-phosphorylated by IFN
in both cell lines (Fig. 4C and data not shown). Consistent with this
finding, binding studies with the GST-IRS-2-IH1
and
GST-IRS-2-IH1
fusion proteins demonstrated that these
motifs do not bind to the IFN
-regulated tyrosine kinase Jak-2 in vitro (Fig. 4, D and E, and data
not shown). Taken altogether, these findings suggest that Type I IFNs
utilize IRS proteins during signal transmission, and that IRS proteins
represent a point of diversity between the Type I and Type II IFN
receptors in hematopoietic cells.
Figure 4:
Tyrosine phosphorylation of IRS proteins
by IFN and IFN
but not IFN
. Antiphosphotyrosine
immunoblots are shown. A, serum-starved U-266 cells were
treated with 2
10
units/ml of the indicated IFNs
for 10 min at 37 °C, and cell lysates were immunoprecipitated with
IRS-2. B, serum starved Molt-4 cells were treated with 2
10
units/ml of the indicated interferons for 5 min
at 37 °C, and cell lysates were immunoprecipitated with
IRS-1
. C, U-266 cells were treated with
10
units/ml IFN
for 5 min at 37 °C, and cell
lysates were immunoprecipitated with an
Jak-2 polyclonal antibody
or normal rabbit serum (RS) as indicated. D, U-266
cells were treated with 10
units/ml IFN
for 5 min at
37 °C as indicated. Cell lysates were either immunoprecipitated
with an
Jak-2 antibody or were bound to GST-IRS2-PH as indicated
prior to SDS-PAGE analysis and immunoblotting with antiphosphotyrosine. E, U-266 cells were treated with 10
units/ml
IFN
for 5 min at 37 °C as indicated. Cell lysates were either
immunoprecipitated with an
Jak-2 antibody or were bound to
GST-IRS1-PH as indicated prior to SDS-PAGE analysis and immunoblotting
with antiphosphotyrosine.
We have previously established
that the p85 regulatory subunit of the PI 3`-kinase associates with
tyrosine-phosphorylated IRS-1 in an IFN-dependent
manner(16) . To determine whether IRS-2 associates with p85
during IFN
stimulation, lysates from IFN
-stimulated cells
were immunoprecipitated with an
p85 polyclonal antibody and
immunoblotted with antiphosphotyrosine. A 180-kDa phosphoprotein was
clearly detectable in the
p85 immunoprecipitates from
IFN
-stimulated cells and co-migrated with IRS-2 that was
immunoprecipitated directly with the
IRS-2 antibody (Fig. 5A). In experiments in which cell lysates were
immunoprecipitated with
IRS-2 and immunoblotted with a monoclonal
antibody against p85
, we noticed that p85
associates with
IRS-2 in an IFN
-dependent manner (Fig. 5B). Thus,
IRS-2, in addition to IRS-1, provides a link between the Type I IFN
receptor and the PI 3`-kinase in hematopoietic cells.
Figure 5:
IFN-dependent association of IRS-2
with the p85 regulatory subunit of the PI 3`-kinase. A,
serum-starved KG1 cells were incubated for 5 min at 37 °C in the
presence or absence of IFN
as indicated, and cell lysates were
immunoprecipitated with the indicated antibodies and immunoblotted with
antiphosphotyrosine. B, serum-starved KG1 cells were treated
for 5 min with IFN
or insulin as indicated, and cell lysates were
immunoprecipitated with the indicated antibodies and immunoblotted with
a monoclonal antibody against p85
.
Originally,
IRS-1 was thought to be a specific substrate for the insulin and IGF-1
receptors; however, a more complicated picture emerged with the
appreciation that other receptors engage and phosphorylate
IRS-1(16, 17, 24, 25, 26, 27, 28, 29) .
The purification and cloning of IRS-2 further complicates the matter as
the tyrosine phosphorylation motifs are not the same(37) .
Insulin, IL-4, and IFN stimulate tyrosine phosphorylation of IRS
proteins in hematopoietic cells; however, the biological effects of
these factors are not identical. Insulin/IGF-1 and IL-4 stimulate DNA
synthesis and growth of hematopoietic cells, and this response requires
IRS proteins(17, 37) , while IFN
inhibits cell
proliferation(1, 48) . Thus, phosphorylation of IRS
proteins may not always mediate the same signals. For example,
expression of IL-4 receptors and the
-chain of the IL-2 receptor
in L6 myoblasts mediates IRS-1 phosphorylation, but IL-4 does not
activate p21
or stimulate glucose uptake in this cell
background(49, 50) .
The PI 3`-kinase is activated
during association with IRS-1 in response to insulin stimulation.
Although monophosphopeptides mimic the ability of phosphorylated IRS-1
to activate the PI 3`-kinase, activation by a biphosphopeptide (based
on the amino acid sequence around Tyr and Tyr
in IRS-1) is more sensitive and reflects more closely the
activation by recombinant phosphorylated
IRS-1(38, 51) . The activity of the PI 3`-kinase
during association with IRS-1 may depend on the pattern of tyrosine
phosphorylation of the nine YXXM motifs. The sites of IRS-1
and IRS-2 tyrosine-phosphorylated during IFN
stimulation have not
been determined. Identification of such sites should provide valuable
information on the mechanisms of regulation of PI 3`-kinase activity
during IFN
stimulation and possibly provide an explanation for the
distinct characteristics of the ultimate biological responses between
IFNs and insulin.
The phosphorylation of IRS proteins is a common
event in the signaling pathways of the Type I IFNs. In contrast,
IFN does not stimulate phosphorylation of IRS proteins in U-266 or
Molt-4 cells; however, IFN
stimulates tyrosine phosphorylation of
IRS-1 in 3T3-F442A adipocytes, which correlates with tyrosine
phosphorylation of Jak-2 (26) . This contradicts our results in
the Molt-4 and U-266 hematopoietic cell lines studied here. Although
Jak-2 is clearly tyrosine-phosphorylated by IFN
in both cell
lines, it is possible that activation of Jak-2 alone is not sufficient
for tyrosine phosphorylation of IRS proteins, as we failed to observe
phosphorylation of IRS-1 or IRS-2. Tyk-2 but not Jak-2 associates with
the pleckstrin homology domains of IRS-1 and IRS-2 (IH1
)
which may mediate, at least partially, the recognition of the substrate
by the activated Type I IFN receptor in hematopoietic cells. It is
possible that the recognition of IRS-1 by the activated Type II IFN
receptor in 3T3-F442A cells requires an unknown signaling protein,
which is not present in hematopoietic cells.
The biological
consequences of tyrosine phosphorylation of IRS proteins by Type I IFNs
remain unknown. The specific signals required for the expression of the
antiproliferative effects of IFNs have not been identified. There is no
evidence available that the Stat pathway mediates this effect. However,
it is difficult to understand how IRS proteins are involved in the
generation of antiproliferative signals. It is possible that
differential phosphorylation of IRS proteins leads to the formation of
active versus inactive or entirely unique signaling complexes.
Studies to determine whether IRS-1 and/or IRS-2 expression in resistant
cells restores IFN sensitivity should provide interesting
information on the mechanisms of regulation of cell growth by the Type
I IFN receptor, as well as the specific functions mediated by each of
these proteins.