Activation of Raf-1 by Interferon γ and Oncostatin M Requires Expression of the Stat1 Transcription Factor*

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 Stat1α, 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 Stat1α is required for maximal transcriptional activation of early response genes by interferon γ (IFNγ) as well as the antiviral and antigrowth actions of this cytokine. Incubation of cells with either IFNγ 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 Stat1α or Stat2. Unexpectedly, incubation of Stat1-deficient, but not Stat2-deficient cells with IFNγ 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 IFNγ. Stat1-negative cells reconstituted with either Stat1α or Stat1α with a point mutation in the site where it is serine-phosphorylated displayed normal activation of Raf-1 by IFNγ and OSM. However, activation of Raf-1 was not observed in lines that expressed Stat1α containing a mutation in its tyrosine phosphorylation site or in its SH2 domain. These results provide the first example of a novel role of Stat1α 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.

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)(4)(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 Rasdependent (9) while IFN␣/␤ and IFN␥ activation of Raf-1 seems to be p21 ras -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.

MATERIALS AND METHODS
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 g/ml) and 20 units/ml interleukin-2 at 37°C for 72 h in RPMI 1640 ϩ 10% fetal calf serum. Cells were then washed in RPMI 1640 and maintained in RPMI 1640 ϩ 2% fetal calf serum for 18 h at 37°C prior to incubation with IFN␥.
Raf-1 Assay-Cells were transfected with 3 g of R89LRaf-1 plasmid and 1 g 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 g 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 125 I-labeled goat antimouse IgG, following mouse monoclonal anti-Raf-1 blotting.
Immunoprecipitations-PBLs (30 ϫ 10 6 ) were incubated with or without IFN␥, pelleted, and washed with ice-cold phosphate-buffered * 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. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
saline. The cells were solubilized in lysis buffer (120 l) containing 300 mM NaCl, 50 mM Tris (pH7.4), 1 mM sodium orthovanadate, 25 mM NaF, 1 mM EDTA, 10 g/ml leupeptin, 10 g/ml aprotinin, 25 M p-nitrophenyl guanidinobenzoate, and 0.5% Nonidet P-40. The lysate was incubated on ice for 10 min, centrifuged at 14,000 ϫ 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.

RESULTS
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 p21 ras (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 32 P 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 Ϯ 0.7-fold and OSM 2.0 Ϯ 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 IFNstimulated gene expression (13). Although the U6A cell line showed stimulation of R89LRaf-1 activity by IFN␥ (3.4 Ϯ 0.5fold) and OSM (2.0 Ϯ 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).
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 ligandindependent 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.
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.   FIG. 1. IFN␥ and OSM do not activate Raf-1 kinase in cells that do not express Stat1. Cells were transfected using DEAE-dextran with 3 g of R89LRaf-1 plasmid ϩ 1 g of SV40 large T-antigen plasmid. 48 h post-transfection, cells were serum-starved for 2 h, and cell lysates were prepared (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) from either untreated cells (lanes 1, 5, and 9), cells incubated for 5 min with 20 ng/ml IFN␥ (lanes 2, 6, and 10), or 0.1 ng/ml OSM (lanes 3, 7, and 11) or pervanadate (P-V) (lanes 4, 8, and 12). Cell extracts were incubated with monoclonal antibody 9E10, which recognizes the myc epitope tag. Immunoprecipitates were assayed for Raf-1 kinase activity using the coupled Raf kinase assay (11). Data was plotted as a fold increase in Raf-1 activity over the untreated control. The data shown are representative of one of three separate experiments. In the three experiments performed, IFN␥ stimulated Raf-1 activity 3.6 Ϯ 0.7-fold over untreated wild type cells, and OSM stimulated Raf  1-3), S727A Stat1 (lanes 4-6), Y701F (lanes 7-9), or SH2 mutant Stat1 (lanes 10-12) were transfected with 3 g of R89LRaf1 ϩ 1 g of SV40 large T-antigen plasmid and treated and lysed as described in Fig. 1. Myc epitope-tagged R89LRaf-1 was immunoprecipitated and assayed for kinase activity. The fold stimulation of Raf-1 kinase activity on S727A Stat1 U3A cells was 2.6 Ϯ 0.3-fold with IFN␥ treatment and 2.7 Ϯ 0.4-fold with OSM treament. Cells reconstituted with Y701F or the SH2-mutated Stat1 showed no significant increase in either IFN␥-or OSM-stimulated Raf-1 activity compared with untreated cells.
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.
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 p21 ras , 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).
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  (bottom panel). B and C, PBLs were incubated with IFN␥ as in A, and cellular extracts were prepared and incubated with Jak1 antiserum (lanes 1 and 2) or nonspecific antiserum (lanes 3 and 4). The resulting blots were probed for the presence of either Raf-1 (B) or Stat1 (C). 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.

DISCUSSION
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.