(Received for publication, July 11, 1995; and in revised form, August 25, 1995)
From the
Binding of interferon- (IFN
) to the multisubunit type
I IFN receptor (IFNR) induces activation of the Tyk-2 and Jak-1 kinases
and tyrosine phosphorylation of multiple signaling elements, including
the Stat proteins that form the ISGF3
complex. Although Jak
kinases are required for IFN
-dependent activation of Stats, the
mechanisms by which Stats interact with these kinases are not known. We
report that Stat-2 associates with
subunit of the
type I IFN receptor in an interferon-dependent manner. This association
is rapid, occurring within 1 min of interferon treatment of cells, and
is inducible by various type I (
,
,
) but not type II
(
) IFNs. The kinetics of Stat-2-IFNR association are similar to
the kinetics of phosphorylation of Stat-2, suggesting that during its
binding to the type I IFNR, Stat-2 acts as a substrate for
interferon-dependent tyrosine kinase activity. These findings support
the hypothesis that the type I IFNR acts as an adaptor, linking Stat
proteins to Jak kinases. Interaction of Stat-2 with the
subunit of the type I IFNR may be a critical signaling event,
required for the formation of the ISGF3
complex and downstream
transcription of interferon-stimulated genes.
In order for type I interferons to exert their pleiotropic
biological effects on cells and tissues, binding to the type I IFN ()receptor (IFNR) is required (1) . Previous studies
have established that the type I IFNR has a multisubunit
structure(2, 3, 4, 5, 6) .
In affinity cross-linking studies of
I-IFN
to the
type I IFNR,
I-IFN
2-IFNR complexes with approximate
molecular masses of 130-140 kDa (
subunit), 110-120
kDa (
subunit), 210-230 kDa (that appears to result from an
association of the
and
subunits), and less prominent
complexes of 75 and 180 kDa (most likely an association of the
subunit with the 75-kDa complex) are
detected(2, 3, 4, 5, 6, 7) .
The variant type I IFNR, expressed in some myelomonocytic cell lines,
is characterized by lack of expression of the 110-120- and
210-kDa complexes and the presence of
I-IFN
2-IFNR
complexes of 130-140 kDa (
subunit), 75 kDa, and 180 kDa
(association of the
subunit with the 75-kDa
complex)(2, 6) . The cloning of the genes encoding two
subunits of the type I IFNR has been reported(8, 9) .
The subunit cloned by Uzéet al.(8) has been shown to correspond to the previously
described
subunit of the receptor(10) . The relative
molecular mass of the
subunit appears to exhibit slight
variations in different cell lines, ranging from 110 to 135
kDa(2, 3, 4, 5, 6, 11, 12) ,
possibly due to differential glycosylation of the protein(4) .
The subunit cloned by Novick et al.(9) has been
reported to encode for a 51-kDa protein. Domanski et al.(7) have recently cloned a cDNA that encodes a 100-kDa
form of the type I IFNR. This receptor form and the one cloned by
Novick et al.(9) have identical extracellular and
transmembrane domains and the first 15 amino acids of the cytoplasmic
domain but differ in the rest of the cytoplasmic region(7) . In
the current study, we used antibodies generated against the receptor
subunits cloned by Uzéet al.(8) and by Novick et al.(9) to further
characterize the structure of the type I IFNR and its interactions with
other signaling molecules. To avoid confusion in the terminology of the
different subunits and to be consistent with the terminology used by
other groups(7) , we will refer to the product of the gene
cloned by Uzéet al.(8) as the
subunit of the type I IFN receptor, the product of the gene
cloned by Novick et al.(9) as the
subunit of the type I IFN receptor, and the product of the gene
cloned by Domanski et al.(7) as the
subunit of the type I IFNR. Our findings demonstrate that during
type I IFN stimulation, the transcriptional activator Stat-2 associates
with the
subunit of the type I IFNR, providing direct
evidence for an interaction of this member of the Stat family of
proteins with a specific component of the type I IFNR.
We initially sought to determine the specificity of the
antibodies raised against the different type I IFN receptor subunits.
We performed experiments in which I-IFN
2 was
cross-linked to its receptor on human cells, and after cell lysis, the
lysates were immunoprecipitated with the IFN
RC-1 or IFN
RC-2
antibodies and analyzed by SDS-PAGE. Fig. 1A shows such
an experiment using the U-266 human myeloma cell line. The IFN
RC-1
antibody immunoprecipitates the
subunit of the receptor, which
migrates as a doublet at 130-140 kDa (Fig. 1A).
It also co-immunoprecipitates an associated high molecular weight
complex (HMWC-1) migrating at approximately 210-230 kDa, which
most likely results from an association of the
subunit with the
100-kDa form of the
subunit (5, 6) (Fig. 1A). The IFN
RC-2
antibody immunoprecipitates a 70-75-kDa complex (Fig. 1A), corresponding to the
subunit of the type I IFNR (the expected M
of the
subunit in affinity cross-linking
studies is approximately 71 kDa when the M
of the
cross-linked IFN
2 molecule is taken into account). This antibody
also co-immunoprecipitates a 130-140-kDa doublet corresponding to
the
subunit, but it does not co-immunoprecipitate the HMWC-1
complex (Fig. 1A). HMWC-1, the 130-140-kDa (
subunit), and 70-75 kDa (
subunit) complexes
were also detectable when total cell lysates from affinity cross-linked
cells were analyzed in parallel (Fig. 1A). A
110-120-kDa complex seen in total lysates from affinity
cross-linked cells, which corresponds to the 100-kDa
subunit
(
subunit)(5) , could not be detected by any
of the anti-receptor antibodies studied here. A complex at
approximately 180 kDa (HMWC-2) was also seen in total cell lysates and
was weakly immunoprecipitated by both the IFN
RC-1 and IFN
RC-2
antibodies. Similar results were obtained when the Molt-4 human cell
line was studied, except that the receptor complexes migrated slightly
slower in these cells (approximately 15-20-kDa difference), a
finding consistent with the reported variations in the mobility of type
I IFNR components in different cell lines(11) . Taken
altogether, the results of the affinity cross-linking experiments
strongly suggested that two distinct forms of the type I IFN receptor
are co-expressed on the surface of human cells. The form of the
receptor immunoprecipitated by the IFN
RC-2 antibody is consistent
with the previously described variant form of the type I
IFNR(2, 6) . Further studies are required, however, to
characterize the exact interactions between different receptor subunits
and to establish that the form precipitated by the IFN
RC-2
antibody corresponds to the previously described variant receptor
form(2, 6) .
Figure 1:
Immunoprecipitation of two distinct
type I IFN receptor complexes by the IFNRC-1 and IFN
RC-2
antibodies. A,
I-IFN
2 was affinity
cross-linked to its receptor in U-266 cells, the cells were lysed, and
cell lysates were either analyzed directly by SDS-PAGE (lane
1) or immunoprecipitated with IFN
RC-1 (lane 2) or
IFN
RC-2 (lane 3) or preimmune rabbit serum (lane
4) prior to SDS-PAGE analysis. The gel was dried, and bands were
visualized by autoradiography. A band at 110-120 kDa could be
distinguished in lane 1 on shorter exposure of the
autoradiogram (data not shown). B, Molt-4 cell lysates
obtained after affinity cross-linking of
I-IFN
2 to
its receptor were immunoprecipitated with IFN
RC-1 (lane
1) or IFN
RC-2 (lane 2) or preimmune rabbit serum (lane 3) prior to SDS-PAGE
analysis.
We subsequently performed studies in
which cells were treated with IFN and cell lysates were
immunoprecipitated with the anti-receptor antibodies, analyzed by
SDS-PAGE, and immunoblotted with anti-phosphotyrosine. Fig. 2, A and B, shows that the
subunit of the receptor
is tyrosine-phosphorylated in response to IFN
treatment of cells,
in agreement with our previous findings using a monoclonal antibody
against this subunit(14, 15) . In addition, the
IFN
RC-1 antibody co-immunoprecipitated an interferon-dependent
tyrosine-phosphorylated protein with an M
of 135
kDa, corresponding to the phosphorylated form of the tyrosine kinase
Tyk-2(16) . Immunoblotting of anti-IFN
RC-1
immunoprecipitates with a monoclonal anti-Tyk-2 antibody demonstrated
that Tyk-2 is associated with the
subunit of the type I IFNR
prior to and after IFN
stimulation (Fig. 2C),
confirming the findings of a previous study (16) that had
established an association of the
subunit with Tyk-2. Tyk-2 was
not detectable in immunoprecipitates obtained with the
anti-IFN
RC-2 antibody (Fig. 2C), suggesting that
this kinase does not associate with the
subunit of
the receptor. Fig. 3A shows an experiment in which cell
lysates from IFN
-treated cells were immunoprecipitated with the
IFN
RC-2 antibody and immunoblotted with anti-phosphotyrosine. A
band corresponding to the 51-kDa
subunit could not be
detected in such immunoblots, perhaps because it co-migrates with the
heavy chain of rabbit immunoglobulin. Also no bands migrating at 102
kDa that would correspond to a phosphorylated receptor dimer were
detectable. A 113-kDa tyrosine-phosphorylated protein, however, was
clearly co-immunoprecipitated by this antibody upon treatment of cells
with IFN
. As the M
of this protein was
identical to the M
of the transcriptional
activator Stat-2, we sought to determine whether it corresponds to
Stat-2. Fig. 3B shows an anti-Stat-2 immunoblot on
immunoprecipitates obtained with the IFN
RC-1 or IFN
RC-2
antibodies. Stat-2 is not present in IFN
RC-1 immunoprecipitates,
but it is clearly detectable in IFN
RC-2 immunoprecipitates from
IFN
-treated cells. Thus, Stat-2 appears to specifically associate
with the
but not the
subunit of the type I
IFNR. The kinetics of the association of Stat-2 with the
subunit were subsequently studied. Fig. 4A shows
an experiment in which Daudi cells were treated for different times
with IFN
, and after cell lysis, the lysates were
immunoprecipitated with the IFN
RC-2 antibody and immunoblotted
with
Stat-2. IFN
-dependent association of Stat-2 with the
subunit was detectable within 1 min of treatment of
cells; the signal peaked at 5-30 min and decreased, although it
was still clearly detectable after 90 min of IFN
treatment. When
the time course of phosphorylation of the
subunit-associated form of Stat-2 was studied, we noticed that
the signal peaked at 5-30 min and diminished by 90 min of
IFN
treatment (Fig. 4B). When the tyrosine
phosphorylation of Stat-2 directly immunoprecipitated by an
Stat-2
antibody was studied, the signal was more intense at all times but also
declined at 90 min (Fig. 4B).
Figure 2:
Association of the tyrosine kinase Tyk-2
with the subunit of the type I IFNR. Molt-4 cells (5.4
10
/lane) (A) or U-266 cells (1.3
10
/lane) (B) were treated for 5 min at 37
°C with 10
units/ml IFN
as indicated, the cells
were lysed, and cell lysates were immunoprecipitated with IFN
RC-1 (lanes 1 and 2) or normal rabbit serum (RS) (lane 3) and immunoblotted with anti-phosphotyrosine (
PTyr). C, anti-Tyk-2 immunoblot. Molt-4 cells
(1.45
10
/lane) were treated with 10
units/ml IFN
for 5 min at 37 °C as indicated, the cells
were lysed, and lysates were precleared with non-immune rabbit
immunoglobulin and immunoprecipitated with the IFN
RC-1 (lanes
1 and 2) or IFN
RC-2 (lanes 3 and 4) antibodies.
Figure 3:
IFN-dependent association of Stat-2
with the
but not the
subunit of the type I
IFNR. A, anti-phosphotyrosine (
PTyr) immunoblot.
Daudi cells (2.9
10
/lane) were treated
with 2
10
units/ml IFN
for 5 min as indicated,
and cell lysates were immunoprecipitated with the IFN
RC-2 antibody (lanes 1 and 2). B,
Stat-2 immunoblot.
U-266 cells (4.2
10
/lane) were treated for
5 min with 10
units/ml IFN
as indicated, and cell
lysates were immunoprecipitated with the IFN
RC-1 antibody (lanes 1 and 2) or the IFN
RC-2 antibody (lanes 3 and 4) or preimmune rabbit serum (PIRS) (lane 5).
Figure 4:
Kinetics of the association of Stat-2 with
the subunit of the type I IFNR in Daudi cells. A, cells were treated with 10
units/ml IFN
for the indicated time periods at 37 °C, and cell lysates were
immunoprecipitated with the IFN
RC-2 antibody (lanes
1-5) or preimmune rabbit serum (lane 6) or an
antibody against Stat-2 (lane 7) and immunoblotted with an
Stat-2 antibody. B, cells were treated with 10
units/ml IFN
for the indicated times at 37 °C, and cell
lysates were immunoprecipitated with preimmune rabbit serum (PIRS,
lane 1) or the IFN
RC-2 antibody (lanes 2-6) or
an antibody against Stat-2 (lanes 7-11) and
immunoblotted with anti-phosphotyrosine (
PTyr). A weak
band corresponding to Stat-2 could be detected at 90 min in the
IFN
RC-2 immunoprecipitates (lane 6) after longer exposure
of the same blot (data not shown).
We have previously
shown that different type I IFNs induce tyrosine phosphorylation of a
common set of signaling proteins, including the and
(100
kDa) subunits of the type I IFNR(14, 15) , the Tyk-2
and Jak-1 kinases(15) , Stat-2 and Stat-1(15) ,
p95
(17) , and insulin receptor substrate (IRS)
proteins (18). (
)These data have suggested that all type I
IFNs activate common signaling cascades. However, differences among the
signaling pathways of different type I IFNs also exist, as suggested by
our finding that IFN
selectively phosphorylates p100, a protein
that associates with the
subunit of the type I IFNR(15) .
To determine whether different IFNs induce an association of Stat-2
with the
subunit, Daudi cell lysates were
immunoprecipitated with the IFN
RC-2 antibody and immunoblotted
with anti-phosphotyrosine or
Stat-2. Association of the
phosphorylated form of Stat-2 with the
subunit was
clearly inducible during treatment of cells with IFN
or IFN
(Fig. 5, A and B). In contrast, IFN
failed to induce such an association (Fig. 5, A and B), a finding consistent with the lack of involvement of
Stat-2 in IFN
signaling(19) . Interestingly, no protein of
the size of the IFN
-specific p100 protein was seen in IFN
RC-2
immunoprecipitates (Fig. 5A), suggesting that this
protein specifically associates with the
but not the
subunit. On the other hand, p100 was clearly detectable in
IFN
RC-1 immunoprecipitates from IFN
-treated cells, (
)in agreement with our original observation(15) .
Figure 5:
Association of Stat-2 with the subunit of the type I IFNR during treatment with different
interferons. A, Daudi cells were treated with 2
10
units/ml of the indicated interferons for 5 min at 37
°C, and cell lysates were immunoprecipitated with the IFN
RC-2
antibody (lanes 1-5) and immunoblotted with
anti-phosphotyrosine (
PTyr). B, the blot shown
in A was stripped and reblotted with an antibody against
Stat-2.
Significant progress has been made recently on our understanding of
the mechanisms of activation of the ISGF3 transcriptional activator
during IFN treatment of target cells. During IFN
stimulation,
three Stat proteins (Stat-2, Stat-1
, and Stat-1
) are
phosphorylated on tyrosine and associate with a 48-kDa protein
(ISGF3
) to form an active
complex(19, 20, 21, 22) . This
complex translocates to the nucleus and activates gene transcription
during binding to interferon-stimulated response elements(19) .
The exact mechanisms, however, by which Stat proteins interact with
IFN-dependent Jak kinases to act as substrates for their kinase
activity remain unknown.
In the current report we present evidence
that Stat-2 specifically associates with the subunit of the type I IFN
receptor cloned by Novick et al.(9) ( subunit). This association is rapid, is induced by various type I
but not type II IFNs, and has similar kinetics with the
IFN
-induced phosphorylation of Stat-2. A previous study (10) had suggested that Stat-2 may associate with the the type
I IFN receptor, as evidenced by the weak co-precipitation of
I-IFN
2 cross-linked complexes by an
Stat-2
antibody. By using this methodology (affinity cross-linking), however,
it is not possible to distinguish the specific receptor component that
interacts with Stat-2 nor is it possible determine whether such an
association is IFN
-dependent. The results of our studies
demonstrate that the association of Stat-2 with the type I IFNR is
IFN
-dependent, occurs specifically with the
but
not the
subunit, and appears to be of relatively high
stoichiometry as evidenced by the intensity of the detected signal.
Our findings also provide some hints on the kinase activity
responsible for Stat-2 phosphorylation. Colamonici et al.(10, 16) have reported that the subunit of
the receptor forms a complex with the tyrosine kinase Tyk-2, a finding
confirmed by us using the IFN
RC-1 antibody. Novick et al.(9) used an antibody that apparently detects both forms of
the
subunit (51 and 102 kDa) and were able to demonstrate an
association with the tyrosine kinase Jak-1. As Stat-2 appears to
interact specifically with the
but not the
subunit of the receptor, it is tempting to hypothesize that Stat-2 acts
as a specific substrate for Jak-1 but not Tyk-2. Furthermore, as the
Stat-2-IFNR association is IFN
-dependent, it is possible that it
involves binding of the SH2 domain of Stat-2 to the
subunit of the type I IFNR. Such a model for an interaction of
Stat-2 with the type I IFNR would be also consistent with the findings
of a recent study that demonstrated that the SH2 domain of Stat-2 is
the determinant of signaling specificity, while Tyk-2 is not
specifically required for Stat-2 phosphorylation(23) . It
remains to be determined whether Stat-1 also utilizes components of the
type I IFNR for its interaction with Jaks. Interestingly, a recent
study has demonstrated that phosphorylation of Stat-2 is required for
activation of Stat-1, but not vice versa, suggesting that one
binding site necessary for activation of Stat-1 may be the
phosphotyrosine of Stat-2 itself(24) . Taken together with our
data, these findings raise the possibility that binding of Stat-2 to
the
subunit of the type I IFNR is the critical event
required for the formation of the ISGF3
complex and downstream
transcription of ISGs.