Interferongamma Activation of Raf-1 Is Jak1-dependent and p21ras-independent*

Minoru SakatsumeDagger , Louis F. StancatoDagger §, Michael DavidDagger , Olli Silvennoinen, Pipsa Saharinen, Jacalyn Pierce§, Andrew C. LarnerDagger , and David S. FinbloomDagger par

From the Dagger  Division of Cytokine Biology, Center for Biologics Research and Evaluation, Food and Drug Administration and § Laboratory of Cellular and Molecular Biology, NCI, National Institutes of Health, Bethesda, Maryland 20892 and  Institute of Medical Technology, University of Tampere, Tampere, Finland

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

Signal transduction through the interferongamma (IFNgamma ) receptor involves the formation of a ligand-dependent multimolecular association of receptor chains (alpha  and beta ), Janus tyrosine kinases (Jak1 and Jak2), and the transcription factor (signal transducers and activators of transcription 1alpha (STAT1alpha )) in addition to activation of mitogen-activated protein kinases (MAPK). Interactions between components of the Jak/STAT cascade and the p21ras/Raf-1/MAPK cascade are unexplored. Treatment of HeLa cells with IFNgamma resulted in the rapid and transient activation of Raf-1 and MAPK. Parallel activation of cells resulted in essentially no enhancement of p21ras activation despite marked enhancement after treatment with epidermal growth factor. In HeLa (E1C3) and fibrosarcoma (U4A) cell lines, both of which are deficient in Jak1 kinase, Raf-1 activation by IFNgamma was absent. Reconstitution of Raf-1 activity was observed only with kinase active Jak1 in both cell lines. In COS cells, transient expression of wild type or kinase-inactive Jak1 coimmunoprecipitated with Raf-1, but activation of Raf-1 activity was only observed in cells expressing kinase-active Jak1. These observations suggest that a kinase-active Jak1 is required for IFNgamma activation of Raf-1 that is p21ras-independent.

    INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Interferongamma transcriptionally induces the expression of early response genes through the activation of the Janus tyrosine kinases 1 and 2 (Jak) and the latent transcription factor, signal transducers and activators of transcription 1alpha (STAT1alpha )1 (1, 2). This cascade is initiated by the ligand-induced tyrosine phosphorylation of the IFNgamma receptor, Jak1, Jak2, and STAT1alpha (3). Tyrosine-phosphorylated STAT1alpha homodimerizes, translocates to the nucleus, and binds enhancers located in the promoters of IFNgamma -sensitive early response genes. In addition to the tyrosine phosphorylation of components of this pathway, data suggest that serine/threonine (Ser/Thr) phosphorylation also plays a role in transcriptional regulation of these genes (4-6). Evidence for the role of Ser/Thr kinases in interferongamma -induced responses derive from studies showing a slow (60-120 min) activation of both MAP kinase (MAPK) and protein kinase C (7), differential modulation of the expression of early response genes by Ser/Thr phosphatase inhibitors (8), and a novel Ser/Thr kinase that mediates some of the antiproliferative effects of IFNgamma (9). Therefore, both tyrosine and Ser/Thr kinase activity can regulate IFNgamma -induced expression of cellular genes.

IFNgamma primarily activates STAT1 as the main driving force for gene expression. Although it has been suggested that MAPK activation may drive serine phosphorylation of STAT1, the relationship, if any, between activation of Jaks and MAPK is undefined. MAPK activation occurs as a result of activation of both MEK, a dual specific kinase, and Raf-1, a Ser/Thr kinase. Moreover, tyrosine phosphorylation of Raf-1 appears to modulate enzymatic activity and subcellular localization (10). Whereas activation of Raf-1 by many growth factors and cytokines is p21ras-dependent, evidence exists that Raf-1 activity can also be stimulated independent of its interaction with p21ras (11). To explore the potential relationship between those two signal transduction cascades, we used cell lines that lack Jak1 activity. We go on to show that Jak1 is required for Raf-1 activation and that this activity occurs independent of p21ras.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Cells-- The parental HeLa and E1C3 Jak1-deficient cell lines have been described (12), as have the parental 2fTGH human fibrosarcoma cell line and the Jak1-deficient U4A cell line (13). The Jak1-deficient cells do not support IFNalpha or IFNgamma -induced gene expression. Cells were maintained as adherent cultures in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% fetal bovine serum (Hyclone). For the MAPK, Raf-1, and p21ras assays, cells were placed in 1% fetal bovine serum 48 h before the experiment. For the 24-36 h immediately before cytokine treatment, the cells were cultured in serum-free media.

Electrophoretic Mobility Shift Assay-- After treatment with IFNgamma , cells were solubilized with cold whole cell extraction buffer (1 mM MgCl2, 20 mM Hepes, pH 7.0, 10 mM KCl, 300 mM NaCl, 0.5 mM dithiothreitol, 0.1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 1 mM vanadate, and 20% glycerol). DNA-binding proteins were assayed as described previously (14). Briefly, 10 µg of protein were incubated in binding buffer with the 32P-labeled oligonucleotide probe consisting of the double-stranded IFNgamma activation sequence (GAS) referred to as the gamma response region (GRR) (5'-AGCATGTTTCAAGGATTTGAGATGTATTTCCCAGAAAAG-3') of the promoter of the Fcgr1 gene (15). The sample was then applied to a 6% nondenaturing polyacrylamide gel to separate free probe from probe bound to protein.

Binding of 125I-rIFNgamma to Cells-- rIFNgamma was radiolabeled to high specific activity using Bolton-Hunter reagent as described (16). Cells were incubated with the radiolabeled IFNgamma for 2 h at 4 °C and assayed as described previously (16).

IFNgamma R alpha  and beta  Chain Gene Expression-- RNA was isolated by the RNAzol method as per the manufacturer's instructions. cDNAs were prepared from the RNA by reverse transcriptase, and then the amount of DNA encoding for the alpha  and beta  chain of the IFNgamma receptor was determined by PCR using primers specific for each gene as described previously (17). The reverse transcription and PCR were performed with the GeneAmp RNA polymerase chain reaction kit (Perkin-Elmer) according to the manufacturer's protocol. The reaction was performed in a DNA thermal cycler (Perkin-Elmer) for 30-35 cycles: 1 min of denaturation at 95 °C and 1 min of annealing and extension at 60 °C following 2 min of an initial denaturation step at 95 °C. The primers used for the detection of IFNgamma Rbeta are as follows: IFNgamma Ralpha (5' sense, 5'-GCAGAAGGAGTCTTACATGTGTGG-3'; 3' antisense, 5'-CTCTCTATTGGAGTCAGATGGCTG-3') and IFNgamma Rbeta (5' sense, 5'-AATGTGACTGTCGGGCCTCCAGAA-3'; 3' antisense, 5'-CTCTAAGATGGGCTGAGTTGGGTC-3'). An aliquot (10 µl) of amplified products was applied to a 2% agarose gel electrophoresis and stained by ethidium bromide. To ensure whether polymerase chain reaction bands were specific for IFNgamma R, we performed southern hybridization with specific cDNAs for IFNgamma Ralpha and beta .

MAPK (Erk2/p42) Assay-- MAPK assays were performed as described using an anti-Erk2 antibody (TR10) (kindly provided by Dr. Michael Weber, University of Virginia) (6).

Raf-1 Assay-- Nontransfected cells were maintained in serum-free conditions for 24-36 h before the initiation of the assay. This was required for an optimal decrease in basal Raf-1 kinase activity. For cells that underwent transfection with Jak1 constructs, the time of starvation was limited to only 2 h, since optimal Jak1 expression occurred at about 14 h after transfection. Cells were then solubilized in lysis buffer (150 mM NaCl, 25 mM Hepes, pH 7.3, 1 mM sodium orthovanadate, 1% Triton-X, 0.5% octylglucoside, 0.03% deoxycholate, 0.02% SDS, protease inhibitors, and 0.5 mM dithiothreitol), and the assay was carried out as described (18). The lysate was incubated on ice for 10 min and centrifuged at 14,000 × g for 10 min, and the supernatant was incubated with anti-Raf-1 antibody (polyclonal; Santa Cruz Biotechnology) followed by incubation with protein G-Sepharose at 4 °C for 1 h. The immunoprecipitates were washed twice with lysis buffer, and the kinase reaction was carried out at 30 °C for 10 min in kinase buffer (0.2 mM ATP, 30 mM MgCl2, 2 mM MnCl2, 40 mM sodium beta -glycerophosphate, 0.2 mM sodium orthovanadate, 2 µM okadaic acid, and 0.2% beta -mercaptoethanol) with 1 µg of purified recombinant MEK1 added as substrate. When the assay was carried out without the addition of MEK1, there was no measurable increase in MAPK phosphorylation. After MEK1 activation, 15 µCi of [gamma -32P]ATP and 1 µg of kinase-defective (K52R) Erk was added as substrate for an additional 2 min. The reaction was terminated by the addition of sample buffer and boiled for 5 min, and the proteins were separated by SDS-polyacrylamide gel electrophoresis. The gel was then transferred to a polyvinylidene difluoride membrane on which the amount of radiolabeled K52R Erk was quantitated by a PhosphorImager. The amount of Raf-1 protein on the same membrane was determined by probing the membrane with mouse monoclonal anti-Raf-1 followed by 125I-labeled goat anti-mouse IgG. Data are presented as -fold increase, as explained for the MAPK assay. In 2fTGH and U4A cells, a myc epitope-tagged Raf-1 (R89LRaf-1), mutated in the p21ras binding site and containing a p21ras membrane localization motif (CAAX), was used in a p21ras-independent Raf-1 assay (10). Cells were transfected with R89LRaf-1 plasmid using DEAE dextran. Forty-eight h post-transfection, lysates were prepared from either untreated cells or cells incubated for 5 min with IFNgamma . Cell extracts were prepared and incubated with monoclonal antibody 9E10, which recognizes the myc epitope tag (GGEQKLISEEDL). Immunoprecipitates were assayed for Raf-1 kinase activity as described above.

Ras Activation Assay-- The activation state of p21ras was performed as described elsewhere (19, 20). 100-mm dishes of HeLa cells were labeled with 1mCi/ml [32P]orthophosphate for 4 h at 37 °C in phosphate-free Dulbecco's modified Eagle's medium. The cells were then treated with IFNgamma or EGF for 15 min at 37 °C and lysed in 0.8 ml of lysis buffer (50 mM Hepes, 1% Triton X-100, 100 mM NaCl, 5 mM MgCl2, 1 mg/ml bovine serum albumin, 1 mM phenylmethylsulfonyl fluoride, 5 µg/ml leupeptin, 5 µg/ml aprotinin, 1 mM vanadate, and 1:10 dilution of culture supernatant of hybridoma-producing anti-Ras monoclonal antibody (Y13-259). After centrifugation at 10,000 × g at 4 °C for 10 min, 0.2 ml of detergent mixture (0.5% deoxycholate, 0.5% SDS, 0.5 M NaCl) and 20 µl of Protein G-Sepharose (Pharmacia Biotech Inc.) were added. The samples were incubated by rocking for 2 h at 4 °C and then washed extensively with wash buffer (50 mM Hepes, 0.5 M NaCl, 5 mM MgCl2, 0.1% Triton X-100, and 0.05% SDS). The immunoprecipitates were eluted by heating at 85 °C for 3 min in the elution buffer (0.075 M KH2PO4, pH 3.4, 5 mM EDTA, 0.5 mM GTP, and 0.5 mM GDP). After a brief centrifugation, 8 µl of supernatant was spotted onto a polyethyleneimine-cellulose thin layer chromatography plate. After drying, the plates was washed briefly in water and air dried before developing in 0.65 M KH2PO4, pH 3.4, for 75 min. Radioactivity was visualized by autoradiography and quantitated by PhosphorImager.

Expression Vectors and Transfections-- The full-length cytomegalovirus-driven wild type murine Jak1 cDNA (pRK5mJak1wt) and the kinase-negative form (ATP binding site K right-arrow E; pRK5mJak1kd) were constructed as described (21). Cytomegalovirus-driven pbeta -galactosidase was used as a control. Transfection of cDNAs was performed by electroporation of cells (1 × 107 cells/ml) in phosphate-buffered saline (300 V/cm, 800 microfarads; Cell-Porator; Life Technologies). Immunoblotting for detection of expressed Jak1 showed that 9-14 h of incubation after electroporation gave maximal expression of Jak1 protein.

Tac Selection-- Cytomegalovirus-driven interleukin 2 receptor (Tac) plasmid (provided by Dr. Bruce Howard, NIH, Bethesda, MD) was cotransfected with pRK5mJak1wt, pRK5mJak1kd, or pbeta -galactosidase into the Jak1-negative mutant HeLa cells (E1C3) by electroporation. After 14 h of incubation in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, cells were incubated with magnetic beads (DynaBeads) conjugated with anti-Tac monoclonal antibody (provided by Dr. T. Waldmann, NIH, Bethesda) in medium S (phosphate-buffered saline supplemented with 4 mM EGTA, 100 µg/ml chondroitin sulfate, 10 mM Hepes, 0.5 M MgCl2, 0.5 M MgSO4, 8 mg/ml nonfat dry milk, and 8 mg/ml bovine serum albumin) for 15 min at 37 °C. After incubation, the mixture of cells and beads was treated with 0.25% trypsin, 0.5 mM EDTA for 2 min followed by the addition of trypsin inhibitor (Sigma). Tac-positive cells that bound to magnetic beads were separated from Tac-negative cells by magnetic field. This separation procedure was repeated three times. Selected Tac-positive cells were washed extensively by Dulbecco's modified Eagle's medium and used for further experiments.

Association of p21ras with Raf-1-- 1D4 cells were solubilized as described above for the Raf-1 assay, and the cell lysates were immunoprecipitated with an antibody against p21ras (Quality Biotech Inc., Camden, NJ) (22). The immunoprecipitates were then analyzed for Raf-1 activity and were expressed as -fold increase for each experiment, where original data represent fluorescent readings from a PhosphorImager.

Statistical Analysis-- Each experiment was done three or four times independently, and data presented are the mean ± S.E. normalized to the intra-experimental control (**, p < 0.01; *, p < 0.05), except for Fig. 7 in which data of different treatment groups were pooled. Statistical significance was calculated by paired or unpaired t test.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Jak1 is required for IFNgamma activation in E1C3 cells (12) (Fig. 1A) as measured by the inability of IFNgamma to activate STAT1-dependent DNA binding to a GAS-like element referred to as the GRR, which is located in the promoter of the high affinity Fcgamma receptor I gene (15) (Fig. 1A, lanes 6-10). In contrast, parental HeLa cells (1D4) respond to IFNgamma by a rapid and intense activation of DNA binding activity (Fig. 1, lanes 1-5). Since the lack of Tyk2 was shown to affect binding of IFNalpha to its receptor (23), we measured the ability of radiolabeled IFNgamma to bind to its receptor on the parental and E1C3 HeLa cells. The binding of 125I-rIFNgamma was similar in both the parental and Jak1-deficient cell lines (Fig. 1B). Both the maximal number of receptors (approximately 1,400/cell) and the concentration at half-saturation (2 × 10-10 M) were identical and consistent with the absence of any alteration in the affinity of IFNgamma for the mutant cells. Since binding of IFNgamma measures essentially only the interaction between the ligand and the alpha  chain of the IFNgamma receptor, intact binding parameters may exist, yet there may be no beta  chain present and thereby no signaling through the receptor (24). Moreover, the presence or absence of the beta  chain has not been demonstrated in the E1C3 cells. To assure that there was equal expression of both the alpha and beta  chain of the IFNgamma receptor, we performed reverse transcriptase polymerase chain reaction amplification of RNA from both cell types. Both the parental and Jak1-deficient cells expressed alpha and beta  chain genes (Fig. 1C). Therefore, the lack of signal transduction through the IFNgamma receptor in the Jak1-deficient cells was not the result of alterations in the receptor (17).


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Fig. 1.   Interferongamma signal transduction in HeLa and ID4 cells. A, incubation of Jak1-deficient HeLa cells with IFNgamma fails to stimulate GRR binding activity. Parental (ID4) (lanes 1-5) and Jak1-deficient HeLa cells (E1C3) (lanes 6-10) were treated with IFNgamma (10 ng/ml) for the indicated times, and GRR binding activity was assessed by electrophoretic mobility shift assay. FcRFgamma , IFNgamma -induced GRR binding activity. B, saturation binding of 125I-IFNgamma to parental HeLa (1D4) and Jak1-negative cells (E1C3). Cells were incubated with increasing amounts of 125I-rIFNgamma , and the amount of bound and free IFNgamma was determined. r, IFNgamma specifically bound in molecules/cell; c, concentration of unbound IFNgamma . Open circles, 1D4; closed circles, E1C3. C, messenger RNA expression of alpha  (IFNgamma Ralpha ) and beta  (IFNgamma Rbeta ) chains of the IFNgamma receptor. Reverse transcriptase-polymerase chain reaction analysis was performed using specific primers for alpha  and beta  chains of IFNgamma receptor. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control.

Increased enzymatic activity of Raf-1 is the primary stimulus for MEK activation, which is responsible for the phosphorylation of MAPK. Although HeLa cells demonstrated MAPK activation in response to IFNgamma , no MAPK activation was measurable in the E1C3 cells lacking Jak1 (data not shown). We therefore evaluated whether IFNgamma could enhance Raf-1 activity and whether this was Jak1-dependent. Cells were incubated with IFNgamma and solubilized, and the extracts were incubated with anti-Raf-1 antibodies. The amount of Raf-1 in both the 1D4 and E1C3 cells were comparable, as determined by immunoblotting of cell extracts (data not shown). The Raf-1 immunoprecipitates were analyzed for enzymatic activity by incubating in the presence of ATP and purified recombinant MEK1, yielding an activated MEK1. [32P-gamma ]ATP was then added in the presence of an enzymatically inactive form of Erk2 (p42MAPK). The assay measures the ability of Erk2 to be phosphorylated by MEK1. In parental cells, there was a marked increase in the phosphorylation of Erk by Raf-1 in response to IFNgamma (Fig. 2, A, 1D4, and B, lanes 1-3). Raf-1 kinase activity was maximal (greater than 3-fold enhancement) after 5 min and then decreased to base-line levels after 15 min (Fig. 2A, 1D4). Base-line Raf-1 activity was similar in both cells lines, as determined by the extent of basal phosphorylation of Erk (Fig. 2B, lanes 1 and 4). In the Jak1-deficient cells (E1C3), there was no induction of Raf-1 activity (Fig. 2A). An increase in tyrosine phosphorylation of Raf-1 occurred in response to IFNgamma (Fig. 2C, lanes 1-3) only in the parental cells. Maximal tyrosine phosphorylation of Raf-1 occurred after 15 min, although detectable increases in phosphorylation were evident after 5 min. In contrast, there was no increase in Raf-1 phosphorylation in response to IFNgamma in the Jak1-deficient cell line (Fig. 2C, lanes 4-6). There was equal loading of Raf-1 protein in all lanes (Fig. 2C, anti-Raf-1 immunoblot, lanes 1-6) for both cells, 1D4 and E1C3. When E1C3 cells were treated with EGF, there was a 6.0-fold enhancement of Raf-1 activity (Table I). We also examined Raf-1 activation in the 2fTGH cell line, which is the parental cell line for the U4A cells that lack Jak1. Since the 2fTGH cells express a constitutively activated p21ras, they were transfected with a myc epitope-tagged Raf-1, which contains a mutation in the p21ras binding domain (R89LRaf-1) (10). IFNgamma enhancement in kinase activity of this Raf-1 should be independent of endogenous p21ras. In the parental cells (2fTGH) IFNgamma treatment resulted in a 2.1 ± 0.42 (mean ± S.D., n = 2)-fold increase in Raf-1 activity, as determined by measuring activation of R89L-Raf-1 (see "Experimental Procedures"), whereas there was no increased activity in the U4A cells (0.45 ± 0.7, mean ± S.D., n = 2).


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Fig. 2.   Activation of Raf-1 kinase by IFNgamma . Parental cells (1D4) (lanes 1-3) or Jak1-deficient cells (lanes 4-6) were treated with IFNgamma , and the cell lysates were assayed for Raf-1 kinase activity. The immunoprecipitates were also analyzed by SDS-polyacrylamide gel electrophoresis followed by immunoblotting. A, the bar graph presents the data as -fold increase of normalized Raf-1 activity over the value of untreated control (zero time point). B, the panel represents the phosphorylation of mutated Erk2 as a substrate of MEK1 in the kinase reaction. Lanes 1 and 4, 2 and 5, and 3 and 6 represent 0, 5, and 15 min, respectively. C, the upper panel represents the membrane probed with anti-phosphotyrosine (anti-PY). The lower panel represents the membrane reprobed with anti-Raf-1. The asterisks represent differences at a p < 0.05 compared with control. IP, immunoprecipitated.

                              
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Table I
Reconstitution of Raf-1 activation in Jak1-deficient cells with wild type Jak1
°p < 0.05, compared to no treatment, paired t test.

Reconstitution of IFNgamma -induced Raf-1 activity was carried out in both the E1C3 cells that were transfected with Jak1 along with Tac and T Ag as a cell selection system and the U4A cell lines, which were transfected with both Jak1 and myc epitope-tagged Raf-1. There was a 1.5-fold increase in Raf-1 activity in E1C3 extracts that were prepared from IFNgamma -treated cells transfected with kinase-active Jak1 that was statistically significant (p < 0.05, paired t test) when compared with control cells transfected with Tac and T Ag alone (Table I). EGF treatment of cells, which was used as a control for the assay, revealed a 6.0 ± 1.7-fold increase in Raf-1 activation. The amount of transfected Jak1 in the E1C3 cells compared with endogenous Jak1 in the parental cells is shown in Fig. 3. There is less Jak1 available in the E1C3 cells compared with the 1D4 wild type cells (Fig. 3, upper panel, lane 1 versus lane 3). This may account for the relatively modest IFNgamma activation of Raf-1 in these transfected cells. To reconfirm the ability of Jak1 to facilitate the activation of Raf-1 in E1C3 cells, we examined the ability of U4A cells (see above) to respond to stimulation with IFNgamma upon reconstitution. Reconstitution of R89L Raf-1 activity in response to IFNgamma occurred following transfection with the wild type Jak1 to levels observed in the parental cell line (Table I). Both the wild type 2fTGH and the transfected U4A cells responded to IFNgamma with an approximately 2-fold increase in Raf-1 activity. Therefore, two separate cell lines deficient in Jak1 responded to reconstitution with Jak1 for IFNgamma -induced activation of Raf-1.


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Fig. 3.   Jak1 expression in wild type and Jak1-deficient cells. Upper panel, 1D4 and E1C3 cells. Equal aliquots of cells from either 1D4 (wild type) or E1C3 transfected with a beta -galactose (gal) control or Jak1 plasmid were processed for immunoblot analysis and probed with an anti-Jak1 antibody. The membrane shown in the upper panel was processed by enhanced chemiluminescence. Lane 1 represents wild type cells. Lane 2 represents control cells transfected with the galactose plasmid. Lane 3 represents cells transfected with Jak1. Lower panel, 2fTGH and U4A cells. These cells were processed as described above. The immunoblot was processed with nitro blue tetrazolium chemistry. The lanes are as described above.

Since one of the major mechanisms for the activation of Raf-1 is through p21ras, we measured the ability of IFNgamma to activate p21ras as measured by the increase in p21ras-bound GTP. Cells were labeled with 32Pi and then treated for 15 min with IFNgamma . Cell extracts were made, p21ras was immunoprecipitated, and the GTP loading of p21ras was measured by thin layer chromatography. Whereas there was no evidence for increased GTP-bound p21ras after treatment of cells with IFNgamma , a marked increase occurred following treatment of cells with EGF (Fig. 4). There was no measurable increase in p21ras activation in cells incubated with IFNgamma at other time points (2.5, 5, and 10 min, data not shown). Therefore, the activation of Raf-1 by IFNgamma appears to occur at a time when there is no measurable activation of p21ras. Since it is known that GTP-bound p21ras binds Raf-1 (25), we next examined Raf-1 activity bound to p21ras in HeLa cells treated with IFNgamma or EGF. Treatment of HeLa cells with EGF resulted in a 6-fold increase in Raf-1 activity bound to immunoprecipitated p21ras (Table II). This enhancement was nearly identical to the 6.5-fold increase in EGF-stimulated GTP binding of p21ras (Fig. 4). Under the same conditions, the p21ras activity associated with p21ras in IFNgamma -treated cells showed no or very little enhancement (Table II).


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Fig. 4.   p21ras is not activated by IFNgamma . Cells were incubated with 32Pi and then treated with either IFNgamma or EGF for 15 min. p21ras activation state was performed as described. Radioactivity of GTP and GDP was quantitated and shown as GTP/GDP × 100.

                              
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Table II
Raf1 activation when coimmunoprecipitated with p21ras

To obtain more information regarding the ability of the Jak kinases to associate with and activate Raf-1, we performed coimmunoprecipitation experiments in HeLa cells and in COS cells transfected with either wild type or kinase-inactive Jak1. There was constitutive association of Raf-1 with Jak1 in HeLa cells (Fig. 5A, lanes 4-6). Previous studies have indicated that Jaks expressed in COS cells are constitutively activated and will tyrosine-phosphorylate STATs in the absence of IFNs. To determine the role of Jak1 stimulation on Raf-1 activity without ligand receptor interaction, COS cells were transfected with wild type or kinase-inactive Jak1. In COS cells transfected with wild type (wt) Jak1, activated Raf-1 enzyme activity measured approximately 2-fold (Fig. 5B) over a beta -galactosidase control transfection. In contrast, transfection with a kinase-inactive (kd) construct resulted in no enhancement of Raf-1 activity (Fig. 5B). When Raf-1 was immunoprecipitated and the immunoblot membrane was probed for Jak1, both wild type and kinase-inactive Jak1 were observed to be associated with Raf-1 (Fig. 4C, lanes 5 and 6). There was no association of Jak1 with Raf-1 when either the beta -galactosidase cDNA was transfected (Fig. 5C, lane 4) or when control normal rabbit IgG was used for the immunoprecipitations (Fig. 4C, lanes 1-3). Only in cells transfected with kinase-active Jak1 was tyrosine-phosphorylated Jak1 associated with Raf-1 (Fig. 4C, lower panel, lane 5 versus lane 6). This supports the contention that kinaseactive Jak1 must be present for Raf-1 to be activated as observed in Fig. 4B.


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Fig. 5.   Jak1 associates with endogenous Raf-1 in HeLa and activates Raf-1 in COS cells. A, HeLa cells were solubilized, Raf-1 was immunoprecipitated (IP), and the immunoprecipitates were analyzed by immunoblotting with anti-Jak1 or anti-Raf-1. Lanes 1-2, nIgG, normal rabbit IgG; lanes 4-6, anti-Raf-1. B, constructs of kinase-active Jak1 (wt), kinase-inactive/dead (kd) Jak1 and beta -galactosidase genes were transiently transfected in COS cells by electroporation. After 48 h, cells were lysed and subjected to Raf-1 kinase assay after immunoprecipitation with anti-Raf-1 antibody. Data are shown as -fold increase of Raf-1 activity over that of the beta -galactosidase transfectant (negative control). C, lysates of cells transfected with cDNAs for each of the Jak1 kinases (wt and kd) and beta -galactosidase (control) were subjected to immunoprecipitation with normal rabbit serum (nIgG) (lanes 1-3) or anti-Raf-1 (lanes 4-6). The immunoblots were then probed with anti-Jak1 (upper panel) or anti-phosphotyrosine (lower panel).

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Activation of the Jak/STAT pathway by cytokines such as IFNgamma allows not only for the induction of early response genes such as IRF1 and the high affinity Fcgr1, but also for genes that are delayed in their expression, such as human major histocompatibility class II antigens. A deficiency in any individual component of the pathway leads to disruption of the entire signaling cascade. On the other hand, under certain circumstances cytokine-induced proliferation and/or differentiation may require more than just activation of STAT proteins (26, 27). These findings suggest that other signaling pathways may operate in parallel or integral to the Jak/STAT pathway to enable full phenotypic responses of cells exposed to extracellular ligands. In this report, we explored the relationship between two pathways utilized by cytokines to activate cells: the Jak/STAT and the p21ras/Raf-1/MEK/MAPK pathway. We show that treatment of cells with IFNgamma leads to rapid activation of Raf-1 in a p21ras-independent manner. In cells lacking Jak1, no activation of Raf-1 occurs. Therefore, Jak1 appears to interact with this cascade at least at the level of Raf-1.

Although previous reports have determined that IFNgamma activates MAPK activity and in some systems protein kinase C activity (7), our data confirm and extend these observations by showing that IFNgamma can directly activate Raf-1 that is dependent upon the presence of intact Jak1 activity but independent of p21ras activation. The lower activation of Raf-1 in the transfection experiments with the E1C3 cells may indeed be a function of the reduced ability to express Jak1 in these cells along with its increased lability. The need for Jak1 suggests that an association between Jak1 and Raf-1 may be necessary for activation of Raf-1. Since Jak2 is present in these cells and in U4A cells, Jak2 apparently cannot substitute for this function. This is in contrast to growth hormone signaling in embryonic kidney cells in which p21ras, Raf, and Jak2 (but not Jak1) are required for MAPK activation (28). The role, if any, tyrosine phosphorylation plays in the regulation of Raf-1 activity is incompletely understood. We have shown that IFNgamma -induced tyrosine phosphorylation of Raf-1 is maximal 15 min after treatment, whereas Raf-1 enzyme activity becomes maximal after 5 min and is substantially reduced by 15 min. Increased Raf-1 activity has been associated with or without tyrosine phosphorylation, depending upon the activating stimulus (29). Tyrosine phosphorylation can be observed and is thought to be involved in either membrane targeting or release from receptors (30-33). Furthermore, serine/threonine phosphorylation of Raf-1 plays both an activating role, as observed for ceramide-induced activation and phosphorylation on Thr-269 (34), and an inhibitory role, as observed for protein kinase A-induced inhibition and phosphorylation on Ser-612 (35).

Since we observe that both Jak1 and Raf-1 can be coimmunoprecipitated when Jak1 is overexpressed, this suggests a direct interaction between Jak1 and Raf-1, possibly at the level of the IFNgamma receptor. Dimerization of Raf-1 through a receptor-independent or -dependent mechanism is sufficient for Raf activation (36, 37). It is conceivable that a pool of Raf-1 associates with Jak1 (and thereby the alpha  chain of the IFNgamma receptor), dimerizes upon binding of IFNgamma (a bivalent homodimer), and becomes activated. However, this does not exclude the possibility that Jak1 may function through an intermediary kinase that may be responsible for the activation of Raf-1. Although it has been recently suggested that Jak2 can be coimmunoprecipitated with Raf-1 and p21ras, this was observed by overexpression in nonmammalian insect cells (38). However, the authors also comment on their inability to show co- immunoprecipitation of Jak2 and Raf-1 in mammalian cells treated with IFNgamma and fail to address the issue of p21ras-dependence of Raf-1 activation. The absence of p21ras activity for cytokine stimulation of Raf-1 has been well documented to occur for other cytokines (11). Although the use of dominant negative p21ras mutants can in some instances confirm this notion, transfections with these constructs have not yielded suitable results for a measurable dominant negative effect (data not shown).

IFNgamma -induced anti-viral activity requires the expression of Jak1 with intact kinase activity. No anti-viral activity is seen in the Jak1 kinase-inactive transfectants (39). One interpretation of these findings is that Jak1 activity is required for the activation of other systems in addition to just STAT activation (which occurs in a delayed fashion in the kinase-inactive transfectants). Our data reveal that kinase-active Jak1 may be required to obtain full Raf-1 activation and that possibly Raf-1 activity may be required for optimal anti-viral effects of IFNgamma . It is of interest that treatment with IFNgamma does not result in the activation of p21ras, since activated p21ras is known to inhibit phosphokinase R activity, an enzyme critical for anti-viral activity (40).

There have been several reports on the activation of protein kinase C, calcium fluxes, and Ser/Thr phosphorylation in mediating the effects of IFNgamma on a variety of cell types (41). These pathways appear to control activation of genes such as the major histocompatability complex class II antigens, enhancement of antigen presenting activity, and fas gene activation, which potentially play a role in the ability of IFNgamma to induce an anti-proliferative, differentiative, or apoptotic state. Cell specificity can also play a role in modulating IFNgamma -induced gene expression. This was recently demonstrated by studying the effects of IFNgamma on myocytes and microvascular endothelial cells (42). Although IFNgamma can activate STAT1 in both cells, only in the myocytes is MAPK activity and inducible nitric oxide synthetase gene expression observed in response to IFNgamma . The authors suggest that MAPK activation along with STAT1 activation was necessary for inducible nitric oxide synthetase gene expression in myocytes. Therefore, selective expression of IFNgamma -induced genes depends upon the activation of specific signaling cascades whose modulation allows for multiple layers of control in mediating IFNgamma -regulated responses.

    ACKNOWLEDGEMENTS

The authors thank Drs. Richard Flavell for the HeLa Jak1-deficient cells, George Stark and Ian Kerr for the fibrosarcoma Jak1-deficient cells, Michael Weber for the anti-MAPK antibody, Thomas Waldmann for anti-TAC antibody, Bruce Howard for the interleukin 2alpha receptor cDNA, Cris Marshall for the Raf-1 cDNA, and Genentech, Inc. for the interferongamma .

    FOOTNOTES

* 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.

par To whom correspondence should be addressed: FDA, Div. of Cytokine Biology, HFM-505, 29 Lincoln Dr., Bethesda, MD 20892-4555. Tel.: 301-827-1735; Fax: 301-402-1659; E-mail: Finbloom{at}A1.cber.fda.gov.

1 The abbreviations used are: STAT, signal transducers and activators of transcription; IFN, interferon; rIFNgamma , recombinant IFNgamma ; IFNgamma R, IRNgamma receptor; MAP, mitogen-activated protein; MAPK, MAP kinase; MEK, MAPK kinase; GRR, gamma response region; Erk, extracellular-regulated kinase; EGF, epidermal growth factor.

    REFERENCES
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Abstract
Introduction
Procedures
Results
Discussion
References

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