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
Received for publication, December 16, 2000, and in revised form, January 30, 2001
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ABSTRACT |
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Interferons (IFNs) play critical roles in host
defense by modulating gene expression via activation of signal
transducer and activator of transcription (STAT) factors. IFN- 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- We recently reported that IFN activates another transcription factor,
NF- 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- Biological Reagents and Cell Culture--
Recombinant human
IFN- Transfection Conditions and Constructs--
High-efficiency,
transient transfection of cells (107) was accomplished by
electroporation (capacitance, 300 microfarads; 250 V) with 500 µg of
salmon sperm DNA and 20 µg 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. NF- Introduction of Phosphopeptides into Permeabilized
Cells--
Daudi cells were permeabilized with streptolysin O as
described previously (26). Phosphopeptides (5 µM)
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- I Akt in Vitro Kinase Assays--
At various times after IFN- 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 proteinase 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 alkaline-phosphatase-conjugated anti-digoxygenin and CDP-Star substrate (Boehringer Mannheim).
The Role of Tyrosine Motifs in the IFNAR1 Chain in IFN-promoted
NF- PI-3K Is Required for IFN-dependent NF-
To determine the functional importance of PI-3K in NF-
The activity of NF- 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 antibody-mediated cross-linking. In addition, a NF-
We examined whether expression of IFN Activates Akt, Which Is Required for IFN-dependent
NF-
To determine the importance of Akt for IFN-promoted NF- 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 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
signal-transducing 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- These results led us to examine the role of PI-3K in NF- 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, 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- 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- 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 IFN-dependent 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.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
, -
, 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 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).
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.
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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
(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 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.
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).
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 µg/ml soybean trypsin inhibitor, 5 µg/ml leupeptin, and 1.75 µg/ml benzamidine), and extracts were
frozen and stored at
80 °C (15). For EMSA, the nuclear extracts
were incubated with a 32P-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.
-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.
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).
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.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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.
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Fig. 1.
Effects of phosphopeptides on IFN-promoted
NF- B activation. A,
phosphopeptides (5 µM) corresponding to the amino
acids surrounding intracellular tyrosine residues of IFNAR1
(PY466, INY[PO4]VFFPSL;
PY481, IDEY[PO4]FSEQPL;
PY527, HKKY[PO4]SSQTSQ;
PY538, SGNY[PO4]SNEDES) or
nonphosphorylated peptide (NPY527) 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 IFN-promoted NF-
B activation, cells were treated
in the presence or absence of genistein (Gen; 100 µM) for 30 min before the addition of IFN-
.
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 IFN-induced 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).
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Fig. 2.
The role of PI-3K in
NF- B activation by
IFN-
/
.
A, EMSA with a 32P-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 32P-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.
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 PI-3K pathway
is distinct from the well-established IFN-stimulated response
element-dependent mechanism in regulating gene expression.
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.
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 µM (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
dose-dependent reduction in the antiviral action of IFN
(Fig. 3C), indicating that PI-3K is involved in the pathway
leading to antiviral activity by IFN.
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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.
p85 that blocks IFN-induced
NF-
B activation would sensitize Daudi cells to IFN-induced 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.
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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).
B Activation--
An important target of PI-3K is the
serine-threonine 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 ~5-fold 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.
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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 anti-Akt. 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 µM) 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).
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 IFN-promoted 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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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-
/
.
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 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.
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).
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.
/
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.
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ACKNOWLEDGEMENTS |
---|
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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FOOTNOTES |
---|
* 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. The 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: lpfeffer@utmem.edu.
Published, JBC Papers in Press, January 30, 2001, DOI 10.1074/jbc.M011006200
2 C. H. Yang, A. Murti, and L. M. Pfeffer, unpublished observations.
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ABBREVIATIONS |
---|
The abbreviations used are:
IFN, interferon;
CAT, chloramphenicol acetyltransferase;
EMSA, electrophoretic mobility
shift assay;
IB, inhibitor of
B;
NF-
B, nuclear factor
B;
PI-3K, phosphatidylinositol-3 kinase;
STAT, signal transducer and
activator of transcription;
PK, protein kinase.
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