THE JOURNAL OF BIOLOGICAL CHEMISTRY
© 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 276, No. 17, Issue of April 27, pp. 13756 –13761, 2001
Printed in U.S.A.
Interferon ␣/␤ Promotes Cell Survival by Activating Nuclear Factor
␬B through Phosphatidylinositol 3-Kinase and Akt*
Received for publication, December 16, 2000, and in revised form, January 30, 2001
Published, JBC Papers in Press, January 30, 2001, DOI 10.1074/jbc.M011006200
Chuan He Yang‡, Aruna Murti‡, Susan R. Pfeffer‡, Jong G. Kim‡, David B. Donner§, and
Lawrence M. Pfeffer‡¶
From the ‡Department of Pathology, University of Tennessee Health Science Center, Memphis, Tennessee 38163, and
§Department of Microbiology and Immunology, Walther Oncology Center, Indiana University School of Medicine,
Indianapolis, Indiana 46202
Interferons (IFNs) play critical roles in host defense
by modulating gene expression via activation of signal
transducer and activator of transcription (STAT) factors. IFN-␣/␤ also activates another transcription factor,
nuclear factor ␬B (NF-␬B), which protects cells against
apoptotic stimuli. NF-␬B activation requires the IFN-dependent association of STAT3 with the IFNAR1 chain of
the IFN receptor. IFN-dependent NF-␬B activation involves the sequential activation of a serine kinase cascade involving phosphatidylinositol 3-kinase (PI-3K)
and Akt. Whereas constitutively active PI-3K and Akt
induce NF-␬B activation, Ly294002 (a PI-3K inhibitor),
dominant-negative PI-3K, and kinase-dead Akt block IFNdependent NF-␬B activation. Moreover, dominant-negative PI-3K blocks IFN-promoted degradation of ␬Box ␣.
Ly294002, a dominant-negative PI-3K construct, and kinase-dead Akt block IFN-promoted cell survival, enhancing apoptotic cell death. Therefore, STAT3, PI-3K,
and Akt are components of an IFN signaling pathway
that promotes cell survival through NF-␬B activation.
Although discovered by virtue of their antiviral activity,
IFNs1 also have antiproliferative, antibacterial, antiprotozoal,
and immunomodulatory functions. These multifunctional cytokines are produced in response to infectious agents such as
viruses, mycoplasma, and bacteria, as well as in response to
noninfectious agents (growth factors, other cytokines, and double-stranded RNA). In addition, IFN has anticancer activity in
vivo and is clinically useful in the treatment of laryngeal and
genital papillomas, chronic viral hepatitis, and multiple sclerosis. Understanding the molecular basis of IFN action is very
important, taking into account the therapeutic potential of IFN
as well as its role as a model for understanding the function of
many cytokines.
All type I IFNs (IFN-␣, -␤, and -␻) bind to a common cell
surface receptor that is comprised of IFNAR1 and IFNAR2
chains (1–3). These IFNs activate the Jak1 and Tyk2 tyrosine
* This work was supported by National Institutes of Health Grants
CA73753 (to L. M. P.), CA73023 (to D. B. D.), and CA67891 (to D. B. D.)
and by funds from the Department of Pathology, University of Tennessee. The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. Section 1734 solely to
indicate this fact.
¶ To whom correspondence should be addressed. Tel.: 901-448-7020;
Fax: 901-448-6979; E-mail: [email protected].
1
The abbreviations used are: IFN, interferon; CAT, chloramphenicol
acetyltransferase; EMSA, electrophoretic mobility shift assay; I␬B, inhibitor of ␬B; NF-␬B, nuclear factor ␬B; PI-3K, phosphatidylinositol-3
kinase; STAT, signal transducer and activator of transcription; PK,
protein kinase.
kinases and generate cytoplasmic signals by the tyrosine phosphorylation of STAT proteins (4 – 6). IFN-activated STATs
(STAT1, STAT2, and STAT3) dimerize and translocate into the
nucleus to induce gene transcription. STAT proteins function
in the gene activation pathway induced by many other cytokines (7, 8). Therefore, the IFN-activated JAK/STAT signaling
pathway serves as a paradigm for understanding cytokine signal transduction in general. However, serine phosphorylation
events are also critical for the biological response to IFN-␣ (5,
9 –12). IFN-␣ activates PI-3K, an upstream element in a serine
kinase transduction cascade, by inducing the rapid tyrosine
phosphorylation of its regulatory 85-kDa (p85) subunit (13, 14).
Furthermore, the IFN-␣-dependent recruitment of PI-3K to the
IFNAR1 chain of the type I IFN receptor requires the tyrosine
phosphorylation of the STAT3 docking site on the intracellular
domain of IFNAR1 (14, 15).
We recently reported that IFN activates another transcription factor, NF-␬B (16). Under most circumstances, NF-␬B lies
dormant in the cytoplasm through the binding of I␬B inhibitory
proteins. Stimulating agents such as viruses, cytokines, and
lipopolysaccharides promote dissociation of inactive NF-␬B/I␬B
complexes, allowing NF-␬B to enter the nucleus and bind DNA.
NF-␬B binds to cis-acting ␬B sites in the promoters and enhancers of key cellular genes. Active, DNA-binding forms of
NF-␬B are dimeric complexes, composed of various combinations of members of the Rel/NF-␬B family of polypeptides (p50,
p52, c-Rel, v-Rel, RelA (p65), and RelB). In addition to regulating immune and inflammatory responses, NF-␬B suppresses
apoptosis (17–20). Mice that lack the RelA/p65 gene die embryonically from extensive apoptosis within the liver (17). Activation of NF-␬B by IFN protects cells from killing through the
apoptotic pathway (16). In contrast, inhibition of NF-␬B nuclear translocation and activation by the introduction of a mutant form of I␬B that acts as a super-repressor enhance apoptotic killing by IFN.
We evaluated the role that PI-3K and its downstream target,
the serine-threonine kinase Akt/PKB, play in IFN-signaling.
We identify an IFN signaling pathway that protects cells
against proapoptotic agents. IFN receptor signaling through
the STAT3 docking site on the IFNAR1 chain leads to NF-␬B
activation, which involves the sequential activation of PI-3K
and Akt. STAT3, PI-3K, and Akt are indispensable for IFN-dependent cell survival (antiapoptotic) signals generated through
NF-␬B.
MATERIALS AND METHODS
Biological Reagents and Cell Culture—Recombinant human IFN-␣
(IFNCon1), provided by Amgen, was assayed by protection against the
cytopathic effect of vesicular stomatitis virus on human fibroblasts,
using the National Institutes of Health human IFN-␣ standard for
reference. Anti-Rel and I␬B␣ antibodies were generously provided by
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This paper is available on line at http://www.jbc.org
IFN Activates NF-␬B by PI-3K and Akt
Dr. N. Rice (National Cancer Institute, Frederick, MD). Human Daudi
cells were maintained in static suspension cultures at 2–15 ⫻ 105
cells/ml in RPMI 1640 medium supplemented with 10% defined calf
serum (HyClone Laboratories, Logan, UT). For experiments, cells were
suspended at 0.5–1 ⫻ 108 cells/ml in medium before the addition of IFN
or other agents.
Transfection Conditions and Constructs—High-efficiency, transient
transfection of cells (107) was accomplished by electroporation (capacitance, 300 microfarads; 250 V) with 500 ␮g of salmon sperm DNA and
20 ␮g of plasmid DNA for each sample. Using a green fluorescent
protein construct, we found that transfection efficiency of Daudi cells is
⬎90%, as determined by confocal microscopy. ⌬p85 is a p85 mutant in
which 35 amino acids from residues 479 –513 are deleted, and 2 amino
acids (Ser-Arg) are inserted (21). p110* is a constitutively active mutant
of the p110 subunit of PI-3K (22). CA-Akt is a constitutively active
mutant of Akt in which the Src myristoylation sequence was added to
the NH2 terminus of Akt (23). KD-Akt is a kinase-dead mutant of Akt
in which a point mutation K197M was introduced at a site required for
kinase activity (23). The pUX-CAT 3XHLA␬B CAT reporter construct
contains three tandemly repeated copies of the NF-␬B site from the
HLA-B7 gene (24).
NF-␬B Activity Measurements—Nuclei were extracted with buffer
(20 mM Tris-HCl, pH 7.85, 250 mM sucrose, 0.4 M KCl, 1.1 mM MgCl2, 5
mM ␤-mercaptoethanol, 1 mM NaF, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 5 ␮g/ml soybean trypsin inhibitor, 5 ␮g/ml leupeptin, and 1.75 ␮g/ml benzamidine), and extracts were frozen and stored
at ⫺80 °C (15). For EMSA, the nuclear extracts were incubated with a
32
P-labeled ␬B probe (5⬘-AGTTGAGGGGACTTTCCCAGG-3⬘) derived
from a NF-␬B binding sequence in the immunoglobulin gene promoter
(25). To define the presence of specific Rel proteins, nuclear extracts
were preincubated with a 1:50 dilution of anti-Rel antibodies at 25 °C
for 0.5 h and then subjected to EMSA. Gels were quantitated by PhosphorImage autoradiography. For reporter gene assays, COS-7 cells
were transiently cotransfected by electroporation with the pUX-CAT
3XHLA␬B CAT reporter construct (24) and the appropriate expression
vector. After 48 h, the cells were treated with IFNCon1 (5,000 units/ml)
for 15 min and assayed for CAT activity. After thin-layer chromatography, radioactivity was measured by PhosphorImage autoradiography.
Introduction of Phosphopeptides into Permeabilized Cells—Daudi cells
were permeabilized with streptolysin O as described previously (26). Phosphopeptides (5 ␮M) corresponding to the amino acids surrounding intracellular tyrosine residues of IFNAR1 (Fig. 1A: PY466, INY[PO4]VFFPSL; PY481,
IDEY[PO4]FSEQPL; PY527, HKKY[PO4]SSQTSQ; PY538, SGNY[PO4]SNEDES) or nonphosphorylated peptide (NPY527) were introduced into
permeabilized cells. IFN-␣-treated (5,000 IU/ml; 15 min) cells were subjected to EMSA. For the sample in Fig. 1A marked none, IFN-treated cells
were permeabilized, but no peptide was introduced.
I␬B␣ Degradation—At various times after IFN-␣ treatment, 1 ⫻ 108
cells were lysed directly in Laemmli buffer, and equivalent amounts of
protein were subjected to SDS-polyacrylamide gel electrophoresis. Proteins were transferred to polyvinylidene difluoride membranes, immunoblotted with specific affinity-purified rabbit anti-I␬B␣, and visualized
by chemiluminescence with the ECL reagent (Amersham Pharmacia
Biotech).
Akt in Vitro Kinase Assays—At various times after IFN-␣ treatment,
1 ⫻ 107 cells were lysed and incubated overnight at 4 °C with sheep
anti-Akt (Upstate Biotechnology, Inc.) bound to protein G-Sepharose.
The immunoprecipitates were collected by centrifugation and subjected
to immune complex kinase assays performed with histone H2B as a
substrate (27). The reaction was stopped by the addition of 3⫻ Laemmli
buffer, and proteins were separated by 12% SDS-polyacrylamide gel
electrophoresis and transferred onto polyvinylidene difluoride membranes for autoradiography. Phosphorylated H2B was quantitated on a
PhosphorImager. The blot was probed with anti-Akt to quantitate protein levels.
Antiviral and Apoptosis Assays—To determine antiviral activity, cell
cultures (5 ⫻ 105 cells/ml) were preincubated overnight with IFNCon1,
followed by infection with vesicular stomatitis virus for 1.5 h at 0.1
plaque-forming units/cell. At 24 h after infection, the virus yield in the
medium was assayed by plaque formation on indicator Vero cells (28).
To determine apoptosis, cells were cytospun onto glass slides, fixed with
4% formaldehyde, permeabilized with 0.2% Triton X-100, and processed
for terminal deoxynucleotide transferase-mediated dUTP nick end labeling according to the manufacturer’s recommendations (Boehringer
Mannheim). Alternatively, lysates of control and IFN-treated (1,000
IU/ml; 24 h) cells were analyzed for apoptotic DNA by modification of a
chemiluminescence-based assay (29). In brief, cells (5 ⫻ 106) were lysed
in hypotonic buffer and sequentially digested with RNase and protein-
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ase K. Low molecular weight DNA was extracted and subjected to
nonisotopic labeling of 3⬘ ends with digoxygenin-11-dUTP and Taq
DNA polymerase. Labeled DNA was separated by electrophoresis on
1.6% agarose and transferred to nitrocellulose, and fragmented DNA
was visualized by chemiluminescence detection with alkalinephosphatase-conjugated anti-digoxygenin and CDP-Star substrate
(Boehringer Mannheim).
RESULTS
The Role of Tyrosine Motifs in the IFNAR1 Chain in IFNpromoted NF-␬B Activation—The IFNAR1 chain of the human
IFN-␣/␤ receptor acts as a species-specific transducer for type-1
IFN action when transfected into heterologous mouse cells (30,
31). Expression of the human IFNAR1 chain of the type I IFN
receptor in murine cells confers sensitivity to human IFN induction of NF-␬B activation, suggesting that this chain of the
receptor is critical to IFN-induced NF-␬B activation (16). To
determine whether intracellular tyrosine residues of IFNAR1
are involved in IFN-dependent NF-␬B activation, peptides corresponding to the amino acids surrounding the four intracellular tyrosine residues in the IFNAR1 chain were introduced into
streptolysin O-permeabilized Daudi cells. The permeabilized
cells were treated with IFN-␣ and assayed for NF-␬B activation. A phosphopeptide corresponding to Tyr527 (YSSQ motif)
blocked IFN-induced NF-␬B activation (Fig. 1). In contrast, the
nonphosphorylated Tyr527 peptide and phosphopeptides corresponding to Tyr466, Tyr481, and Tyr538 had no effect on IFN-dependent NF-␬B activation. These results indicate that NF-␬B
activation is directed through the tyrosine phosphorylation of
the conserved YSSQ motif in the IFNAR1 chain. This motif is
responsible for bringing PI-3K into a complex with the IFN
receptor (14). The general requirement for IFN-dependent tyrosine phosphorylation is demonstrated by the finding that the
tyrosine kinase inhibitor genistein blocks IFN-dependent
NF-␬B activation (Fig. 1B). Because the docking domain of
PI-3K on IFNAR1 was necessary for IFN-induced NF-␬B activation, we examined whether IFN-induced NF-␬B activation
involved PI-3K.
PI-3K Is Required for IFN-dependent NF-␬B Activation—To
determine whether PI-3K plays a role in IFN-dependent NF-␬B
activation, we transfected into Daudi cells a mutant p85 subunit (⌬p85) of PI-3K, which functions as a dominant-negative
inhibitor for PI-3K-mediated events (21), or a constitutively
active PI-3K (p110*, catalytic subunit of PI-3K). IFN treatment
of cells transfected with an empty vector induced a prominent
NF-␬B complex (Fig. 2A). Expression of ⌬p85 blocked IFNinduced NF-␬B activation, demonstrating that PI-3K is involved in NF-␬B activation by IFN. In contrast, p110* promoted NF-␬B activity with the NF-␬B complex comprised of
p50 and c-Rel because the complex was supershifted by antisera to either p50 or c-Rel. The p110*-induced NF-␬B complex
was indistinguishable from that promoted by IFN (Fig. 2B).
To determine the functional importance of PI-3K in NF-␬B
activation, cells were cotransfected with a NF-␬B-CAT reporter
plasmid and p110*, ⌬p85, or an empty vector and assayed for
CAT activity 2 days after transfection (Fig. 2C). COS-7 cells
were used for these assays because they are IFN-responsive in
reporter assays, whereas Daudi cells are not (16). Moreover,
COS-7 cells are IFN-responsive as demonstrated by NF-␬B
activation in gel shift assays (data not shown). p110* expression stimulated ␬B-dependent transcription ⬃6-fold as compared with that seen in cells transfected with empty vector.
Moreover, IFN stimulated ␬B-dependent transcription ⬃8-fold
in cells transfected with empty vector. In contrast, expression
of ⌬p85 suppressed IFN-activated ␬B-dependent transcription
by ⬎90%. ⌬p85 expression had no effect on IFN-stimulated
response element-dependent transcription (data not shown).
These results indicate that NF-␬B-activation by IFN via the
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IFN Activates NF-␬B by PI-3K and Akt
FIG. 1. Effects of phosphopeptides
on IFN-promoted NF-␬B activation.
A, phosphopeptides (5 ␮M) corresponding
to the amino acids surrounding intracellular tyrosine residues of IFNAR1
(PY466, INY[PO4]VFFPSL; PY481, IDEY[PO4]FSEQPL; PY527, HKKY[PO4]SSQTSQ; PY 538 , SGNY[PO 4 ]SNEDES) or
nonphosphorylated peptide (NPY 527 )
were added to streptolysin O-permeabilized Daudi cells. IFN-␣-treated (5,000
IU/ml; 15 min) cells were subjected to
EMSA. For the sample marked none,
IFN-treated cells were permeabilized, but
no peptide was introduced. B, to test the
role of tyrosine phosphorylation in IFNpromoted NF-␬B activation, cells were
treated in the presence or absence of
genistein (Gen; 100 ␮M) for 30 min before
the addition of IFN-␣.
FIG. 2. The role of PI-3K in NF-␬B
activation by IFN-␣/␤. A, EMSA with a
32
P-labeled ␬B probe on nuclear extracts
from control and IFN-treated Daudi cells
transiently transfected for 48 h with ⌬p85
or empty vector (EV). B, EMSA with a
32
P-labeled ␬B probe on nuclear extracts
from cells transiently transfected for 48 h
with p110* or empty vector (EV). Nuclear
extracts prepared from p110*-transfected
cells were preincubated with antisera
directed against specific Rel proteins. C,
NF-␬B-dependent reporter gene activity
in IFN-treated COS-7 cells transiently
cotransfected with the pUX-CAT
3XHLA␬B construct and ⌬p85, p110*, or
empty vector (EV). Data shown are the
average of three experiments (S.E. ⬍
15%), expressed relative to CAT activity
in cells transfected with empty vector. D,
Daudi cells were transiently transfected
for 48 h with ⌬p85 or empty vector. Cell
lysates from IFN-treated cells (5,000
IU/ml) were resolved by SDS-polyacrylamide gel electrophoresis, blotted onto
polyvinylidene difluoride membranes,
probed with anti-I␬B␣, and visualized by
enhanced chemiluminescence.
IFN Activates NF-␬B by PI-3K and Akt
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FIG. 3. The effects of the PI-3K inhibitor LY294002 on IFN-induced NF-␬B activation, antiviral activity, and cell survival. A, EMSA
on nuclear extracts from IFN-treated (5,000 IU/ml; 15 min) Daudi cells that were pretreated with LY294002 for 1 h. B, Daudi cells were pretreated
with LY294002 for 1 h before the addition of IFN (1,000 IU/ml) and assayed for apoptosis by terminal deoxynucleotide transferase-mediated dUTP
nick end labeling assays. The data represent the mean of three independent experiments in which at least 500 cells were scored for each variable
(S.E. ⬍ 10%). C, Daudi cells were pretreated with LY294002 for 1 h before the addition of IFN (1,000 IU/ml) and assayed for sensitivity to the
antiviral action of IFN. The results of two separate experiments were averaged (S.E. ⬍ 5%). The data are expressed as the effect of LY294002 on
IFN-treated Daudi cells relative to control cells at each LY294002 concentration.
PI-3K pathway is distinct from the well-established IFN-stimulated response element-dependent mechanism in regulating
gene expression.
The activity of NF-␬B is tightly controlled by inhibitory I␬B
proteins that bind to NF-␬B complexes and thus sequester
NF-␬B in the cytoplasm. Cytokines, such as interleukin 1 and
tumor necrosis factor, promote the serine phosphorylation of
I␬B and its polyubiquitination and proteosome-mediated degradation and thereby induce NF-␬B translocation to the nucleus. We reported previously that IFN induced a progressive
decrease in cellular levels of I␬B␣, indicating that IFN stimulated NF-␬B activation by promoting I␬B␣ degradation (16).
Moreover, I␬B␣ proteins with mutations or deletions of serine
phosphorylation sites function as super-repressors of IFN-induced NF-␬B activation. As shown in Fig. 2D, whereas IFN
promoted I␬B degradation in cells transfected with the empty
vector, expression of ⌬p85 blocked IFN-promoted I␬B␣ degradation. These results indicate that IFN-mediated NF-␬B activation is PI-3K-dependent.
PI-3K Is Required for IFN-mediated Cell Survival—IFN
antagonizes apoptosis by protecting cells against a variety of
proapoptotic stimuli, such as virus infection, and antibodymediated cross-linking. In addition, a NF-␬B-dependent
pathway protects cells against the apoptotic action of IFN
itself (16). To determine whether PI-3K plays a role in biological activities mediated by IFN, we examined the effects of
the selective PI-3K inhibitor LY294002 on IFN action in
Daudi cells. LY294002 produced a dose-dependent reduction
in IFN-induced NF-␬B activation (Fig. 3A). Moreover,
LY294002 increased IFN-induced apoptotic cell death with
an IC50 of ⬃1 ␮M (Fig. 3B) but did not induce apoptosis by
itself. A hallmark of the biological activities of IFN is its
ability to inhibit viral replication. LY294002 produced a dosedependent reduction in the antiviral action of IFN (Fig. 3C),
indicating that PI-3K is involved in the pathway leading to
antiviral activity by IFN.
We examined whether expression of ⌬p85 that blocks IFNinduced NF-␬B activation would sensitize Daudi cells to IFNinduced apoptosis. In empty vector-transfected cells, IFN only
slightly increased apoptosis (from 0.1% to 0.6%), as determined
by terminal deoxynucleotide transferase-mediated dUTP nick
end labeling assays. However, as shown in Fig. 4A, expression
of ⌬p85 markedly sensitized Daudi cells to IFN-induced death
(⬃50%). A prominent feature of apoptosis is the formation of
DNA ladders, which reflects DNA cleavage into discrete multimers of ⬃200 base pairs. When cell lysates of IFN-treated
Daudi cells expressing ⌬p85 were examined by a highly sensitive chemiluminescence-based DNA fragmentation assay, the
formation of the telltale DNA ladder was clearly evident when
compared with lysates of IFN-treated Daudi cells transfected
with an empty vector (Fig. 4B). These results indicate that the
PI-3K-dependent pathway leading to NF-␬B activation protects
cells against the proapoptotic action of IFN. Thus, IFN generates a strong cell survival signal through PI-3K.
IFN Activates Akt, Which Is Required for IFN-dependent
NF-␬B Activation—An important target of PI-3K is the serinethreonine kinase Akt/PKB (32, 33), which is known to promote
cell survival. Thus, we examined whether IFN activates Akt.
Akt was immunoprecipitated from lysates of Daudi cells and
assayed for enzymatic activity using histone H2B as a substrate. Although similar amounts of Akt were immunoprecipitated from IFN-treated cells and untreated cells, IFN rapidly
increased Akt enzymatic activity (within 5 min) with an ⬃5fold increase at 15 min (Fig. 5A). The IFN-dependent increase
in Akt activity was blocked by LY294002, demonstrating that
Akt activation is downstream of PI-3K.
To determine the importance of Akt for IFN-promoted NF-␬B
activation, we transfected into Daudi cells a catalytically inactive mutant of Akt called KD-Akt or a constitutively active Akt
mutant (CA-Akt). As shown in Fig. 5B, KD-Akt blocked IFNpromoted NF-␬B activation, as compared with IFN-treated
cells transfected with empty vector (EV). In contrast, CA-Akt
promoted NF-␬B activity in Daudi cells (Fig. 5C), and the
NF-␬B complex formed was indistinguishable from that promoted by IFN.
DISCUSSION
IFN elicits pleiotropic biological effects by regulating gene
expression through signals generated upon its binding to a
distinct surface receptor on target cells. Type I IFNs bind to a
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IFN Activates NF-␬B by PI-3K and Akt
FIG. 4. The role of PI-3K in IFN-␣/␤-promoted cell survival.
IFN-treated (1,000 IU/ml; 24 h) Daudi cells transiently transfected for
48 h with ⌬p85 or empty vector were analyzed for apoptosis by terminal
deoxynucleotide transferase-mediated dUTP nick end labeling assays
(A) or for apoptotic DNA by a chemiluminescence assay with a DNA
ladder provided for reference (B).
ubiquitously expressed cell surface receptor composed of the
IFNAR1 and IFNAR2 subunits (1, 3, 34, 35). Whereas IFNAR2
is the ligand-binding subunit, IFNAR1 acts as a species-specific
transducer for the actions of type I IFN (30, 31). We have
demonstrated previously that the STAT3 transcription factor
binds to the tyrosine-phosphorylated IFNAR1 chain through
its SH2 domain, and this binding is directed to a YSSQ motif.
This motif is perfectly conserved in the cytoplasmic tails of
IFNAR1 homologues from diverse species, suggesting that it
may play a critical role in type I IFN signaling by specifically
docking important SH2 domain-containing cytoplasmic proteins
(31, 36). A YXXQ motif in the cytosolic tail of the shared signaltransducing gp130 chain of the interleukin 6 receptor family is
required for cytokine-dependent STAT3 activation (37).
In the present study, we report that a phosphopeptide corresponding to the YSSQ motif blocked IFN-induced NF-␬B
activation. These results indicate that NF-␬B activation is directed through the tyrosine phosphorylation of the conserved
YSSQ motif in the IFNAR1 chain. This motif is responsible for
bringing PI-3K into a complex with STAT3 and the IFN receptor (14). The two SH2 domains of p85 mediate the association of
PI-3K with tyrosine-phosphorylated proteins containing a
pYXXM consensus sequence. This motif is perfectly conserved
in STAT3 homologues from several species. Transfection with a
Phe656-STAT3 point mutant (in which a Phe was substituted
for Tyr656 of the YXXM motif) blocks IFN-dependent NF-␬B
activation, but not IFN-stimulated gene factor 3 activation,
which depends on the formation of STAT1/STAT2 dimers.
Therefore, NF-␬B activation by IFN requires STAT3 phosphorylation at tyrosine residue 656 (a YXXM motif), i.e. the PI-3K
docking site. We recently showed that STAT3 is responsible for
bringing PI-3K into a complex with the IFN-␣/␤ receptor (14).
Expression of STAT3 in an IFN-resistant Daudi cell line complemented defective NF-␬B activation, as well as several other
signaling defects (25). These results suggest that STAT3 may
act upstream of PI-3K in NF-␬B activation by IFN-␣/␤.
These results led us to examine the role of PI-3K in NF-␬B
activation by IFN. By using constitutively active and dominant-negative mutant proteins and pharmacological inhibitors,
we have shown that PI-3K is necessary for NF-␬B activation by
IFN, as demonstrated in gel shift assays and in ␬B-dependent
gene reporter assays. Moreover, we found in preliminary studies that PI-3K plays a role in the up-regulation of genes by IFN
that are dependent on NF-␬B, such as MHC class I and IFN
FIG. 5. IFN activates Akt and the role of Akt in IFN-induced
NF-␬B activation. A, lysates from Daudi cells treated for the indicated
times with IFN were immunoprecipitated with anti-Akt. Immune complex kinase assays were performed with histone H2B as substrate. The
proteins were resolved by SDS-polyacrylamide gel electrophoresis, blotted onto polyvinylidene difluoride membranes, and probed with antiAkt. The relative (IFN-treated/control) enzymatic activity determined
by PhosphorImager analysis is presented below each lane. To test the
role of PI-3K, cells were incubated with LY294002 (Ly; 10 ␮M) for 60
min before the addition of IFN-␣. B, EMSA on nuclear extracts from
IFN-treated (5,000 IU/ml; 30 min) Daudi cells electroporated with kinase-dead Akt (KD-Akt) or empty vector (EV). C, EMSA on nuclear
extracts from Daudi cells electroporated with constitutively active Akt
or empty vector (EV).
regulatory factor 1.2 Cell survival induced by IFN after virus
infection in vivo may be due to both direct inhibition of viral
replication and protection against virus-induced apoptosis.
Thus, our results show that IFN utilizes PI-3K to augment
antiviral activity and to promote cell survival via NF-␬B activation. Because we show that the distinct actions of IFN on
viral replication, apoptosis, and cell survival can be modulated,
it may now become possible to enhance clinically useful IFN
actions or, alternatively, attenuate undesirable IFN actions
under specific pathological conditions.
Although there are several downstream targets of PI-3K, the
serine-threonine kinase Akt has attracted much attention because of its role in cell survival. Akt was discovered as the
product of the oncogene v-akt that transforms lymphoid cells
(33). Based on homology to the PKA and PKC family of protein
kinases, Akt was also named protein kinase B and RAC-PK
(32). The PI-3K/Akt pathway provides cell survival signals in
response to nerve growth factor, insulin-like growth factor 1,
2
C. H. Yang, A. Murti, and L. M. Pfeffer, unpublished observations.
IFN Activates NF-␬B by PI-3K and Akt
platelet-derived growth factor, interleukin 3, and the extracellular matrix (38). Akt apparently promotes cell survival by
phosphorylating multiple targets, including the Bcl-2 family
member BAD (39), the apoptosis-inducing enzyme caspase-9
(40), and the Forkhead transcription factor FKHRL1 that regulates Fas ligand gene expression (41). Our results show that
IFN activates Akt enzymatic activity and that kinase-dead Akt
blocks IFN-promoted NF-␬B activation, indicating that Akt is
important for IFN-promoted NF-␬B activation. Moreover, a
constitutively active Akt construct promotes NF-␬B activation.
To our knowledge, these results are the first to place Akt in an
IFN signaling pathway. Because we found that IFN activates
Akt, it will be important to establish which possible substrates
for Akt undergo IFN-dependent phosphorylation and determine their physiological significance in IFN-promoted cell survival. I␬B kinases are a likely substrate because Akt regulates
these kinases to activate NF-␬B in response to tumor necrosis
factor and platelet-derived growth factor (42, 43).
A STAT3-dependent pathway and a PI-3K/Akt-dependent
pathway are known to promote cell survival (38, 42– 47). Interleukin 6 generates cell survival signals to prevent apoptosis
through STAT3 activation (48). In the present report, we have
defined a mechanism by which IFN promotes cell survival by
showing that it activates NF-␬B through a PI-3K/Akt pathway.
This pathway apparently requires STAT3, which acts as an
adapter for PI-3K (14). Thus, STAT3, PI-3K, and Akt are all
components of the cell survival signaling pathway used by IFN.
It will be important to determine whether other cytokines
similarly generate cell survival signals by NF-␬B activation.
How STAT3 and PI-3K/Akt specifically relate to one another is
not yet known. Recent findings indicate that IFN activates
PI-3K through STAT3-independent pathways (49) and that
PI-3K is required for STAT3-dependent and STAT3-independent signaling (50, 51). Thus, STAT3 and PI-3K are complexly
related in IFN signal transduction.
IFN-␣/␤ promotes the survival of activated T cells (52), protects CD4⫹ cells from human immunodeficiency virus-induced
cell death (53), and protects lymphoblastoid cells from cell
death induced by virus infection or cross-linking of surface
immunoglobulins (16). The clinical efficacy of IFN in the treatment of cancer and viral diseases is often limited by its inability to efficiently induce cell death (54). In contrast, the therapeutic action of IFN-␤ in multiple sclerosis may reflect its
ability to protect neuronal cells against proapoptotic cytokines.
Our results suggest that the ability of IFN to promote apoptosis
is counterbalanced by the induction of potent cell survival
signals through signaling dependent on STAT3, PI-3K, and Akt
that leads to NF-␬B activation.
Acknowledgments—We thank N. Rice (National Cancer Institute,
Frederick, MD) for providing a panel of anti-Rel antibodies, E. Croze
(Berlex Biosciences, Richmond, CA) for providing phosphopeptides, G.
Murti (St. Jude Children’s Research Hospital, Memphis, TN) for help
with confocal microscopy, and M. Kasuga (Kobe University, Kobe, Japan), J. Vilcek (New York University, New York, NY), R. Roth (Stanford
University, Stanford, CA), and L. T. Williams (University of California,
San Diego, CA) for providing expression vectors.
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