From the Laboratory of Cellular and Molecular
Biology, NCI, National Institutes of Health, Bethesda, Maryland 20892, the § Laboratory of Experimental Immunology, NCI, National
Institutes of Health, Frederick, Maryland 21702, and the
¶ Division of Cytokine Biology, Center for Biologics Evaluation
and Research, Bethesda, Maryland 20892
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ABSTRACT |
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A primary signaling cascade
responsible for the expression of cytokine-stimulated immediate early
genes involves the activation of the Jak/Stat pathway. In addition to
being tyrosine-phosphorylated, several signal transducers and
activators of transcription (Stats), including Stat1, Stat3, and
Stat4, are phosphorylated on a conserved serine residue, which is a
consensus phosphorylation site for mitogen-activated protein kinases
(MAPKs). Serine phosphorylation of Stat1
is required for maximal
transcriptional activation of early response genes by interferon
(IFN
) as well as the antiviral and antigrowth actions of this
cytokine. Incubation of cells with either IFN
or oncostatin M (OSM)
activates Raf-1, a serine/threonine kinase responsible for the ultimate
activation of p42 MAPK. To examine whether any of the signaling
components that are required for activation of the Jak/Stat pathway are
also necessary for activation of Raf-1 by IFNs and OSM, we examined
activation of Raf-1 in cell lines that are deficient in either Stat1
or Stat2. Unexpectedly, incubation of Stat1-deficient, but not
Stat2-deficient cells with IFN
or OSM for 5 min displayed no
increase in Raf-1 activity. In peripheral blood lymphocytes Raf-1 was
associated with Stat1, and this interaction was disrupted after
incubation of cells with IFN
. Stat1-negative cells reconstituted
with either Stat1
or Stat1
with a point mutation in the site
where it is serine-phosphorylated displayed normal activation of Raf-1
by IFN
and OSM. However, activation of Raf-1 was not observed in lines that expressed Stat1
containing a mutation in its tyrosine phosphorylation site or in its SH2 domain. These results provide the
first example of a novel role of Stat1
not as a transcription factor, but as a protein which may function to scaffold signaling components required for activation of the distinct Raf/MEK/MAPK signaling cascade.
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INTRODUCTION |
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Activation of the mitogen-activated protein kinase
(MAPK)1 signaling cascade is
a key regulator of cell proliferation, differentiation, and development
(1, 2). This process involves a cascade of enzymes initiated by the Raf
family of serine/threonine protein kinases of which Raf-1 is the best
characterized member. Recent studies from this laboratory and others
clearly demonstrated that the Jak/Stat and Raf/MEK/MAPK signaling
cascades are intimately linked (3-5). A serine residue located at
amino acid 727 in Stat1 was shown to be phosphorylated in response
to IFN
treatment. Mutation of this site decreased activation of
several IFN
-stimulated, Stat-regulated genes (6) and also abrogated
the antiviral and antiproliferative effects of IFN
(7, 8). Serine
727 in Stat1
is conserved in Stat3 and Stat4 and is an ideal site
for proline-directed serine kinases such as MAP kinases. IFNs and OSM
also rapidly stimulate p42 MAP kinase activity, and the kinase itself
has been shown to associate with the
chain of the IFN
receptor,
the gp130 subunit of the OSM receptor, and Stat1
(Ref. 3 and data
not shown). In addition, expression of mutated forms of p42 MAPK, such
that they have no enzymatic activity, inhibits IFN
/
- and
IFN
-stimulated luciferase reporter constructs containing either GAS
or ISRE enhancers (3).
The mechanisms that control Raf activation are poorly understood.
Activation of Raf by most mitogens and OSM is Ras-dependent (9) while IFN/
and IFN
activation of Raf-1 seems to be
p21ras-independent (4, 10). Activation of Raf-1 by these
cytokines does not occur in the absence of Jak1 (4, 10). Furthermore, constitutively active Jak1 expressed in COS cells results in elevated Raf-1 activity (4). In the studies presented here, we observed that
expression of the Stat1 transcription factor also is required for
IFN
and OSM stimulation of Raf-1 kinase activity.
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MATERIALS AND METHODS |
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Cells--
2fTGH cells were maintained as adherent cultures in
Dulbecco's modified Eagle's medium (Life Technologies, Inc.)
supplemented with 10% fetal bovine serum (Hyclone). For Raf-1 assays,
cells were placed in serum-free media for 2 h prior to cytokine
treatment. Peripheral blood lymphocytes were isolated from normal
donors and incubated with PHA (1 µg/ml) and 20 units/ml interleukin-2 at 37 °C for 72 h in RPMI 1640 + 10% fetal calf serum. Cells
were then washed in RPMI 1640 and maintained in RPMI 1640 + 2% fetal calf serum for 18 h at 37 °C prior to incubation with
IFN.
Raf-1 Assay--
Cells were transfected with 3 µg of R89LRaf-1
plasmid and 1 µg of SV40 T-antigen plasmid using DEAE-dextran (4).
The SV40 T-antigen plasmid was included in the transfection to increase the expression of Raf-1 (4). 48 h post-transfection, lysates were
prepared from either untreated cells or cells incubated for 5 min with
IFN (20 ng/ml) or OSM (0.1 ng/ml). Cells were solubilized in lysis
buffer (10 mM HEPES, pH 7.4, 1% Triton X-100, 300 mM NaCl, 1 mM EGTA, 10 mM
-glycerophosphate, 1 mM sodium orthovanadate, and 1 mM phenylmethylsulfonyl fluoride). Cell extracts were
incubated with 1 µg of monoclonal antibody 9E10, which recognizes the
myc epitope tag (GGEQKLISEEDL) followed by adsorption to protein
G-Sepharose. Immunoprecipitates were assayed for Raf-1 kinase activity
as described (4). For the calculations of Raf-1 activity, the amount of Raf-1 protein was determined by probing the membrane with
125I-labeled goat anti-mouse IgG, following mouse
monoclonal anti-Raf-1 blotting.
Immunoprecipitations--
PBLs (30 × 106) were
incubated with or without IFN, pelleted, and washed with ice-cold
phosphate-buffered saline. The cells were solubilized in lysis buffer
(120 µl) containing 300 mM NaCl, 50 mM Tris
(pH7.4), 1 mM sodium orthovanadate, 25 mM NaF,
1 mM EDTA, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 25 µM p-nitrophenyl guanidinobenzoate, and 0.5%
Nonidet P-40. The lysate was incubated on ice for 10 min, centrifuged
at 14,000 × g for 10 min, and the supernatant was
incubated with anti-Raf-1 antibody (Santa Cruz Biotechnology) or
anti-Jak1 antibody (Upstate Biotechnology) followed by incubation with
protein G-Sepharose at 4 °C for 1 h. The protein G beads were
washed three times with lysis buffer that contained 0.1% Nonidet P-40,
prior to suspending in SDS sample buffer. Proteins were separated by
SDS-PAGE, and the resulting immunoblots were probed with either Stat1
antibody or Raf-1 antibody.
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RESULTS |
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The availability of cell lines which are deficient in specific
components required for IFN activation of the Jak/Stat pathway allowed
us to examine whether the Stat proteins might regulate Raf-1 activation
by IFN and OSM. OSM, which uses the gp130 receptor chain in a manner
similar to the cytokines interleukin-6, leukemia inhibitory factor, and
ciliary neurotrophic factor, has been shown to stimulate tyrosine
phosphorylation of both Stat1 and Stat3 while IFN
activates
primarily Stat1. Wild type 2fTGH cells express a mutated,
constitutively active p21ras (6), leading to elevated Raf-1
activity which does not allow for analysis of cytokine-regulated
endogenous Raf-1 activity. We therefore transfected cells with a
CAAX and myc epitope-tagged Raf-1, which contains a mutation
in the Ras binding domain of Raf-1 (R89LRaf-1) (12). The kinase
activity of this protein is independent of Ras. Transfected R89LRaf-1
was immunoprecipitated from 2fTGH cells with an antibody against the
myc epitope tag, and Raf-1 activity was assayed by measuring
incorporation of 32P into kinase-inactive MAPK, which
reflects the relative activity of the immunoprecipitated Raf-1. As
shown previously (4) and in Fig. 1,
myc-tagged Raf-1 transfected into 2fTGH cells showed enhanced activity
when cells were exposed to IFN
or OSM. Although a representative
experiment is displayed in Fig. 1, an average of three experiments
demonstrated that IFN
stimulated the transfected Raf-1 kinase
activity 3.6 ± 0.7-fold and OSM 2.0 ± 0.3-fold. R89LRaf-1 was also transfected in Stat1-deficient U3A cells and Stat2-deficient U6A cells. Both of these cell lines were derived from 2fTGH cells and
selected for defects in IFN-stimulated gene expression (13). Although
the U6A cell line showed stimulation of R89LRaf-1 activity by IFN
(3.4 ± 0.5-fold) and OSM (2.0 ± 0.2-fold) comparable to the
parental 2fTGH cells (lanes 9-11), U3A
cells, which do not express Stat1, showed no activation of Raf-1 by
either cytokine (lanes 5-7). Similar
results were seen with IFN
/
activation of Raf-1 (data not
shown).
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To determine whether there was a global defect in U3A cells with regard to regulation of Raf-1 kinase, we incubated cells with pervanadate, which stimulates Raf-1 activity in a ligand-independent manner. There was no statistically significant difference in pervanadate-stimulated Raf-1 activity between all three cell lines (Fig. 1, lanes 4, 8, and 12).
Because U3A cells were selected by chemical mutagenesis for a defect in
IFN activation of immediate early genes, we wanted to ensure that no
mutation other than their failure to express Stat1 accounts for their
inability to support IFN and OSM activation of R89LRaf-1. Therefore,
stable cell lines derived from U3A cells, which were transfected with
Stat1
, were analyzed for IFN
and OSM activation of transfected
R89LRaf-1 (Fig. 2). When wild type Stat1
was expressed in U3A cells, IFN
and OSM activation of Raf-1
was equivalent to the parental 2fTGH cell line.
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There are several conserved domains in the Stat family of transcription
factors including the sites of tyrosine phosphorylation, the SH2
domains, and the serine phosphorylation sites in Stat1, -3, and -4 (14). To determine whether these conserved motifs were required for IFN
and OSM activation of Raf-1, U3A cells were selected with the
appropriate mutation. These cell lines were transfected with R89LRaf-1
to analyze for its activation as a result of treatment of cells with
IFN or OSM (Fig. 2). Substitution of serine 727 with alanine in the
transfected Stat1
did not alter the ability of IFN
or OSM to
stimulate Raf-1 activity (compare lanes
1-6). Expression of Stat1
with a mutation in
its SH2 domain or tyrosine 701, which is phosphorylated as a result of
incubation of cells with cytokines, showed no activation of Raf-1
(lanes 7-12). Incubation of these
cells with IFN
gave similar results (data not shown). All
reconstituted lines showed about the same levels of expression of
Stat1
as 2fTGH cells (data not shown).
Stat3, like Stat1, contains a serine in its carboxyl terminus which is
phosphorylated as a result of incubation of cells with a variety of
cytokines. Although activation of Stat3 by IFNs is not seen in 2fTGH
cells (6), incubation of cells including 2fTGH cells with cytokines
signaling through the gp130 receptor subunit stimulate robust tyrosine
phosphorylation of Stat3. We therefore wanted to determine whether
Stat1 was required for OSM activation of both Raf-1 and tyrosine
phosphorylation of other Stats or whether Stat1 was selectively needed
for activation of Raf-1. 2fTGH cells and U3A cells were incubated with
OSM for 5 or 15 min, and cellular extracts were prepared and
immunoprecipitated with Stat1 or Stat3 antisera. Cells were also
incubated with IFN under the same conditions as an internal control
for tyrosine phosphorylation of Stat1. Immunopellets were resolved by
SDS-PAGE, and the resulting blots were probed with antiphosphotyrosine
antibody (Fig. 3). In 2fTGH cells both
IFN
and OSM stimulated about the same degree of tyrosine
phosphorylation of Stat1 while only OSM activated Stat3. Incubation of
U3A cells with OSM also resulted in strong tyrosine phosphorylation of
Stat3, similar to that seen in parental cells. These results clearly
indicate that while Stat1 is required for OSM activation of Raf-1, its
expression has no influence on the ability of this cytokine to
stimulate tyrosine phosphorylation of Stat3. It thus appears that the
regulatory function of Stat1 is specific for activation of the Raf-1.
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Although expression of Stat1 was clearly required for IFN and OSM
stimulated Raf-1 activity, we were not able to detect a reproducible
association between these two proteins in cell extracts derived from
2fTGH cells (data not shown). However, a stable association of these
proteins may not occur because these cells express mutated p21ras, which results in constitutively activated Raf-1 (5). To
determine whether an association between Raf-1 and Stat1 might exist in nontransformed cells we used primary PBLs isolated from normal human
donors. PBLs were incubated with phytohemagglutinin for 72 h to
stimulate proliferation of T cells as well as to render them sensitive
to treatment with IFN
(15). Cells were washed and incubated in
medium with 2% fetal calf serum for 18 h prior to the addition of
IFN
. Cellular extracts were prepared from untreated and
IFN
-treated PBLs and immunoprecipitated with Raf-1 antiserum (Fig.
4A) and resolved proteins
transferred to Immobilon. Blots were probed for the presence of Stat1
(upper panel). Stat1 is constitutively associated
with Raf-1 in PBLs (lane 1) while nonimmune
rabbit serum failed to detect significant amounts of either Stat1 or
Raf-1 (lane 4). Incubation of PBLs with IFN
for as little as 1 min resulted in the loss of Raf-1 association with Stat1 (lanes 2 and 3). Similar results
were observed with respect to IFN
/
-stimulated dissociation of
Raf-1 and Stat1 in PBLs (data not shown). Reprobing the blot for the
presence of Raf-1 indicated that it was present at the same
concentrations in all samples (lanes
1-3, lower panel). Under
similar conditions we have not been able to detect Stat3 or Stat5
associated with Raf-1 (data not shown).
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Although we have been able to detect IFN-stimulated activation of
Erk2 in primary PBLs, activation of Raf-1 has been inconsistent. In
HeLa cells a constitutive association of Jak1 and Raf-1 has been
observed using the same Raf-1 antiserum used in Fig. 4A
(10). To determine whether Raf-1 and Jak1 interact with each other in primary PBLs, cells were incubated with or without IFN
for 5 min and
cellular extracts were immunoprecipitated with Jak1 antiserum (lanes 1 and 2) or nonspecific
antiserum (lanes 3 and 4). The immunoprecipitates were resolved by SDS-PAGE, and the membranes were
probed for either the presence of Raf-1 (Fig. 4B) or Stat1 (Fig. 4C). Similar to HeLa cells, a specific association
between Jak1 and Raf-1 can be detected in primary PBLs. Although the
amount of Raf-1 associated with Jak1 is modest in this experiment, if Raf-1 antiserum is used instead of Jak1 antiserum the degree of association between these proteins appears to be enhanced (data not
shown). We could also detect a specific association between Jak1 and
Stat1 in PBLs (Fig. 4C). Similar to interactions between Jak1 and Raf-1, the association between Jak1 and Stat1 also is not
altered by incubation of cells with IFN
(compare lanes
1 and 2). In summary, these data support the
notion that Stat1 is an integral component of the signaling complex
required for activation of Raf-1 by OSM, IFN
, IFN
/
, and
possibly by other cytokines.
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DISCUSSION |
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The role of Stats as key components in cytokine-stimulated immediate early genes is well documented. Tyrosine phosphorylation of these proteins is required for their ability to bind DNA while serine phosphorylation of Stat1 and Stat3 is required for them to function optimally as transcriptional activators. The role that IFN- and OSM-stimulated Raf kinase serves in the biological actions of these cytokines is being investigated. Recent studies in this laboratory indicate that the antiproliferative actions of IFNs are absent in cells derived from mouse embryos with targeted deletions of the Raf kinases (data not shown). Stat1 may therefore function in a regulatory loop which allows coordinate activation of both the Jak/Stat and Raf/MAPK signaling cascades.
Our results indicate that in addition to its role as a transcription
factor, Stat1 also is essential for stimulation of Raf-1 kinase
activity by several cytokines including OSM, IFN, and IFN
/
(data not shown). The detailed mechanisms by which Stat1 controls IFN-
and OSM-stimulated activation of Raf-1 are being examined. However, it
is clear that other Stat proteins such as Stat2 and Stat3 cannot
substitute for Stat1 in stimulating Raf-1 kinase activity. The fact
that tyrosine 701 in Stat1 (as well as a functional SH2 domain), which
is phosphorylated as a consequence of cytokine treatment of cells, is
required for IFNs and OSM to stimulate Raf-1 activity suggests that
Stat1 may function as a docking protein to permit recruitment of Raf-1
into signaling complexes, containing Jaks, p42 MAP kinase,
phosphatidylinositol 3-kinase, and other key regulatory enzymes which
modulate both the Raf/MEK/MAPK and Jak/Stat signaling cascades (3,
16-19). Stat1 can also be detected in immunoprecipitates of Raf-1 in
PBLs, and this association is absent after incubation of cells with IFN
(Fig. 4). These results imply that the mechanisms by which Raf-1 binds
to Stat1 (either directly or indirectly) and the role that Stat1 plays
in IFN activation of Raf-1 are distinct.
Incubation of cells with either IFN or OSM for only 5 min stimulates
Raf-1 activity. This finding suggests that Stat1 does not function as a
transcription factor in this process in the sense that it needs to be
tyrosine-phosphorylated, translocate to the nucleus, and stimulate the
expression of an early response gene(s) whose protein product(s) are
required for IFN and OSM stimulation of Raf-1. However, it has been
recently demonstrated that Stat1 can affect the constitutive expression
of caspase RNAs by a mechanism that does not require that the protein
be tyrosine-phosphorylated (20). It is therefore conceivable that the
constitutive expression of Stat1 is affecting the expression of a
protein which is required for stimulation of Raf-1 by IFNs, OSM, and
possibly other cytokines. The fact that vanadate treatment of either
wild type or Stat1-negative cells stimulates Raf-1 kinase activation to
the same extent argues that the components needed to stimulate Raf-1 by
a non-receptor-mediated process are not dependent on the expression of
Stat1.
As far as we are aware, this is the first example of a protein which not only directly or indirectly regulates RNA polymerase II activity, but also regulates the enzymatic activity of a cytoplasmic kinase that has important functions in cell proliferation, development, and differentiation.
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ACKNOWLEDGEMENTS |
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We thank Bristol Myers Squibb for generously providing the oncostatin M. We thank Drs. George Stark, Ian Kerr, James Darnell, and Curt Horvath for generously providing the cell lines used in these studies, and David Finbloom and Ana Gamero for their critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant CA77366 (to A. C. L.).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: Dept. of
Immunology, Cleveland Clinic Research Foundation, 9500 Euclid Ave.,
Cleveland, OH 44195. Tel.: 216-445-9045: Fax: 216-444-8372;
E-mail: larnera{at}cesmtp.ccf.org.
1 The abbreviations used are: MAPK, mitogen-activated protein kinase; IFN, interferon; OSM, oncostatin M; PHA, phytohemagglutinin; PAGE, polyacrylamide gel electrophoresis; PBL, peripheral blood lymphocyte.
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REFERENCES |
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