(Received for publication, October 19, 1995; and in revised form, February 9, 1996)
From the
Based on the reports of the activation of the transcription
factor known as STAT3 (for signal transducers and activators of
transcription) or APRF (for acute phase response factor) by various
cytokines, we investigated the possible role of STAT3 in type I
interferon (IFN) receptor signaling. We show that STAT3 undergoes
IFNdependent tyrosine phosphorylation and IFN
treatment
induces protein-DNA complexes that contain STAT3. In addition, STAT3
associates with the IFNAR-1 chain of the type I receptor in a tyrosine
phosphorylation-dependent manner upon IFN
addition. The binding of
STAT3 to the IFNAR-1 chain occurs through a direct interaction between
the SH2 domain-containing portion of STAT3 and the
tyrosine-phosphorylated IFNAR-1 chain. Furthermore,
tyrosine-phosphorylated STAT3 bound to the IFNAR-1 chain also undergoes
a secondary modification involving serine phosphorylation. This
phosphorylation event is apparently mediated by protein kinase C, since
it was blocked by low concentrations of the protein kinase inhibitor
H-7. The biological relevance of IFN activation of STAT3 is further
illustrated by the finding that STAT3 is not activated by IFN in a cell
line resistant to the antiviral and antiproliferative actions of
IFN
but in which other components of the JAK-STAT pathway are
activated by IFN
.
Cytokines are multifunctional mediators of the growth and
differentiation of hematopoietic, lymphopoietic, and neural systems.
They exert their effects through specific surface receptors expressed
on target cells, triggering biological effects through the activation
of specific gene transcription. One approach to the understanding of
the molecular basis of transcriptional activation by cytokines is the
identification of cis-responsive elements within genes and the
transcription factors responsive to cytokine signals. For example, the
type I interferons (IFNs), ()IFN
and IFN
, induce
the transcription of the early IFN-stimulated gene (ISG) gene
family(1) . IFNs are cytokines that have profound effects on
cells, including antiviral protection, inhibition of the proliferation
of normal and transformed cells, and modulation of the immune system.
IFN signaling to the cell nucleus involves the tyrosine phosphorylation
of STAT (signal transducers and activators of transcription) proteins.
IFN-activated STAT1 and STAT2 translocate to the nucleus, where they
recognize the conserved IFN stimulus response element (ISRE) within the
promoter of ISGs, which is both necessary and sufficient for ISG
transcription(2, 3) . Central to the type I
IFN-activated pathway are two non-receptor protein tyrosine kinases,
JAK1 and TYK2, which apparently mediate the tyrosine phosphorylation of
IFN receptor chains and STATs(4, 5) .
The
intracellular domains of type I IFN receptor chains contain conserved
motifs that likely function in transmembrane
signaling(6, 7) . The ligand-induced tyrosine
phosphorylation of STAT transcription factors is one of the events most
proximal to cytokine-dependent JAK activation(2, 3) .
Recent studies indicate that many cytokines, including type I IFNs,
induce tyrosine phosphorylation of the STAT3 transcription
factor(8, 9) , also known as the acute phase response
factor (APRF) involved in acute phase gene expression. We previously
proposed that the intracellular domain of the IFNAR-1 chain plays a
critical role in type I IFN signaling by specifically docking important
SH2 domain-containing cytoplasmic proteins(6) . Therefore, we
investigated the possible role of STAT3 in IFN signaling through
the IFNAR-1 chain.
The type I IFN receptor apparently consists of
multiple glycoprotein subunits(6, 10, 11) .
The cDNAs coding for two subunits, the IFNAR-1 and IFNAR-2 chains, have
recently been
cloned(12, 13, 14, 15) . We show
that STAT3 associates with the IFNAR-1 chain in a tyrosine
phosphorylation-dependent manner after exposure of cells to IFN. The
binding of STAT3 to the IFNAR-1 chain can occur through a direct
interaction between the SH2 domain-containing portion of STAT3 and
tyrosine-phosphorylated IFNAR-1 chain. The biological significance of
IFN activation of STAT3 is further borne out by the finding that STAT3
is the only signaling molecule in the JAK-STAT pathway not activated by
IFN in an IFN
-resistant cell line.
For
precipitation with GST fusion proteins, lysates from control or
IFN-treated Daudi cells were precipitated with STAT3-GST or GST bound
to glutathione-agarose beads. The precipitated proteins were resolved
by SDS-PAGE (7.5%), blotted onto PVDF membranes, and probed with
IFNAR-1. For blotting with GST fusion proteins,
IFNAR-1
immunoprecipitates resolved by SDS-PAGE were probed with STAT3-GST or
GST (purified after elution with glutathione from agarose beads). The
IFNAR-1 chain was visualized by ECL using a hamster
GST mAb and a
goat anti-hamster IgG horseradish peroxidase conjugate (Southern
Biotechnology Associates).
Figure 1:
The presence of STAT-related proteins
in ISRE and SIE protein-DNA complexes induced by IFN. A,
nuclear extracts were prepared from control and IFN-treated (5,000
IU/ml) IFN-sensitive Daudi cells and then subjected to EMSA with a P-labeled ISRE or SIE probe in the absence or presence of
a 50-fold excess of unlabeled oligonucleotide probes. In addition, one
set of nuclear extracts from IFN-treated cells was preincubated with
STAT3 prior to EMSA analysis. The positions of ISGF complexes and
the SIF complexes are indicated. B, nuclear extracts from
IFN-treated (5,000 IU/ml) Daudi cells were incubated with normal rabbit
serum (NRS),
STAT1,
STAT2, or
STAT3 prior to
EMSA analysis with a
P-labeled SIE
probe.
Figure 2:
Coprecipitation of tyrosine-phosphorylated
STAT3 with the IFNAR-1 chain in IFN-sensitive Daudi cells. A,
lysates prepared from control or IFN-treated (5,000 IU/ml) cells were
immunoprecipitated with either IFNAR-1,
Tyr(P) (
pTyr) or
STAT3. The proteins were resolved by
SDS-PAGE, blotted onto PVDF membranes, and probed with
STAT3. IP, immunoprecipitate; WB, Western blot. B,
to show serine phosphorylation of STAT3, cells were treated with H-7 at
varying concentrations (0-100 µM) or staurosporine (st, 300 nM) for 30 min prior to IFN
addition.
Lysates prepared from IFN-treated (5,000 IU/ml; 15 min) cells were
precipitated with
IFNAR-1 and blotted with
STAT3. The faster
and slower migrating forms of STAT3 are indicated on the figure as STAT3
and STAT3
,
respectively.
We
have previously shown that the IFNAR-1 chain undergoes IFN-dependent
tyrosine phosphorylation(6) . In addition, several
tyrosine-phosphorylated proteins coprecipitate with the IFNAR-1 chain.
To determine if STAT3 coprecipitated with IFNAR-1, lysates from control
and IFN-treated Daudi cells were precipitated with IFNAR-1 and
analyzed by blotting with
STAT3 (Fig. 2). Although similar
amounts of IFNAR-1 chain were precipitated by
IFNAR-1 (data not
shown), only
IFNAR-1 precipitates from IFN-treated cells contained
STAT3 protein (Fig. 2). Furthermore, blotting with
Tyr(P)
indicated that the kinetics of tyrosine phosphorylation of IFNAR-1 and
STAT3 were remarkably similar. IFN-dependent coprecipitation of STAT3
with the IFNAR-1 chain was also observed in HeLa epithelioid carcinoma
cells and U-266 lymphoblastoid cells (data not shown). As observed in
STAT3 precipitates, the STAT3 band precipitated by
IFNAR-1
resolved as a doublet. This result directly places the kinase
responsible for the secondary modification of STAT3 (serine
phosphorylation) in close proximity to the IFNAR-1 chain of the
receptor.
Figure 3:
Precipitation and direct blotting of the
tyrosine-phosphorylated IFNAR-1 chain in IFN-sensitive Daudi cells by
the STAT3-GST fusion protein. A, lysates from control or
IFN-treated (5,000 IU/ml; 15 min) cells were precipitated with
STAT3-GST, GST, or by Tyr(P) (
pTyr). The
precipitated proteins were resolved by SDS-PAGE, blotted onto PVDF
membranes, and probed with
IFNAR-1. B, lysates prepared
from control or IFN-treated (5,000 IU/ml; 15 min) cells were
precipitated with
IFNAR-1. The proteins were resolved by SDS-PAGE,
blotted onto PVDF membranes, and probed with STAT3-GST or GST. To test
for the role of tyrosine phosphorylation of the IFNAR-1 chain in its
interaction with STAT3, cells were treated in the presence or absence
of genistein (gen, 100 µM) for 30 min prior to
IFN
addition.
Figure 4:
The failure of IFN to activate STAT3
in IFN-resistant Daudi cells. A, lysates prepared from control
or IFN-treated (5,000 IU/ml) cells were precipitated with
STAT3.
The proteins were resolved by SDS-PAGE, blotted onto PVDF membranes,
and probed with
Tyr(P) (pTyr) or
STAT3. B,
nuclear extracts from Daudi cells treated with IFN
(5,000 IU/ml)
were preincubated with
STAT1,
STAT2, or
STAT3 and then
subjected to EMSA with a
P-labeled SIE probe. The
positions of SIF complexes formed in extracts from IFN-sensitive cells
are presented for reference. gen,
genistein.
The binding of cytokines to cell surface receptors on target
cells induces the transcription of specific sets of genes. This new
gene expression involves the cytokine-induced tyrosine phosphorylation
of specific subsets of STAT transcription factors and the formation of
phosphorylation-dependent STAT complexes. The mechanism by which a
cytokine and its cognate receptor selectively activate only certain
STATs is poorly understood, but several lines of evidence indicate that
it involves intermediate phosphotyrosine- and SH2 domain-dependent
complexes between the receptor chain and specific STAT factors. For
example, IFN induces the tyrosine phosphorylation of the IFNGR-1
chain of the multisubunit IFN
receptor as well as of
STAT1(37) . A functionally critical tyrosine residue in the
membrane distal region of the IFNGR-1 chain is involved in STAT1
activation. Furthermore, phosphopeptides corresponding to this region
interact with STAT1 and block its activation. These results were the
first to show that a specific tyrosine-based activation motif (TBM) in
the cytosolic tail of an IFN receptor subunit dictated specific STAT
activation. The results reported herein establish that IFN
activates STAT3 directly through an interaction between the
tyrosine-phosphorylated IFNAR-1 chain and the SH2 domain-containing
half of STAT3. It is likely that STAT3 becomes tyrosine-phosphorylated
by the receptor-associated JAK1 or TYK2 kinases. Most importantly, we
demonstrate a direct association of a STAT with a cytokine receptor
chain and thus provide a mechanism whereby cytokine receptors dictate
signaling specificity.
The IFNAR-1 chain of the type I IFN receptor undergoes ligand-dependent tyrosine phosphorylation and plays a crucial role in signal transduction(6, 38, 39) . The cytoplasmic tails of the mouse, human, and bovine IFNAR-1 chains contain a perfectly conserved membrane distal amino acid motif, KYSSQTSQDSGNYSNE(6, 7) . We previously proposed that this TBM plays a critical role in the signaling of type I IFN through its receptor by specifically docking important SH2 domain-containing cytoplasmic proteins(6) . Recently, it was reported that a TBM of YXXQ in the cytosolic tail of the shared signal-transducing gp130 chain of the IL6 receptor family is required for cytokine-dependent STAT3 activation(40) . Thus, it is possible that the conserved YSSQ motif of the cytosolic tail of the IFNAR-1 chain may also serve as a docking site for STAT3. We are presently investigating whether this motif in fact is the STAT3 docking site on the IFNAR-1 chain.
STAT3 also undergoes serine
phosphorylation(25, 26) , a modification that was
blocked in Daudi cells by serine kinase inhibitors (H-7 and
staurosporine) in IFN-treated cells. Thus, we can place both serine and
tyrosine protein kinases in the vicinity of the type I IFN receptor and
in early transmembrane signaling events. Furthermore, the IC for inhibitory action of H-7 on IFN-induced STAT3 serine
phosphorylation suggests that PKC may mediate the serine
phosphorylation of STAT3. We have previously shown that, although Daudi
cells express PKC
, -
, -
, -
, -
, and -
,
IFN
selectively activates PKC
and
PKC
(27) .
Thus, it is tempting to suggest that
either one or both IFN-activated PKC subspecies may mediate the
phosphorylation of STAT3. Recently, it has been shown that the serine
phosphorylation of STAT1 and STAT3 is necessary for maximal activation
of transcription(26) .
Finally, although much attention has
been focused on the role of STAT1 and STAT2 in IFN action, our
results suggest that STAT3 is also involved in the biological actions
of IFN. We previously reported that IFN
rapidly induces ISG
transcription in IFN-sensitive and IFN-resistant Daudi
cells(17, 28) . However, while ISG transcription
persists at high levels in the IFN-sensitive Daudi line, the activation
of ISG transcription is only transient in IFN-resistant Daudi cells.
IFN-resistant Daudi cells undergo the normal activation of other
components of the JAK-STAT pathway, as determined by the
ligand-dependent tyrosine phosphorylation of STAT1, STAT2, JAK1, and
TYK2.
It is of note that the ligand-dependent tyrosine
phosphorylation of the IFNAR-1 chain was reduced in IFN-resistant Daudi
cells when compared with IFN-sensitive cells. In the present report we
show that, although IFN activates STAT3 in IFN-sensitive Daudi cells,
STAT3 was not activated in the IFN-resistant line when assayed by STAT3
tyrosine phosphorylation or by formation of STAT3-containing
protein-DNA complexes in gel supershift assays. We provide evidence for
a direct phosphotyrosine-dependent interaction between the IFNAR-1
chain of the human type I interferon receptor and the SH2
domain-containing portion of STAT3. This association is required for
the formation of functional STAT3-containing transcription factors in
response to interferon-
.