Interferon
Activation of Raf-1 Is Jak1-dependent
and p21ras-independent*
Minoru
Sakatsume
,
Louis F.
Stancato
§,
Michael
David
,
Olli
Silvennoinen¶,
Pipsa
Saharinen¶,
Jacalyn
Pierce§,
Andrew C.
Larner
, and
David S.
Finbloom
From the
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 |
Signal transduction through the interferon
(IFN
) receptor involves the formation of a
ligand-dependent multimolecular association of receptor
chains (
and
), Janus tyrosine kinases (Jak1 and Jak2), and the
transcription factor (signal transducers and activators of
transcription 1
(STAT1
)) 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 IFN
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 IFN
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
IFN
activation of Raf-1 that is p21ras-independent.
 |
INTRODUCTION |
Interferon
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 1
(STAT1
)1 (1, 2). This
cascade is initiated by the ligand-induced tyrosine phosphorylation of
the IFN
receptor, Jak1, Jak2, and STAT1
(3).
Tyrosine-phosphorylated STAT1
homodimerizes, translocates to the
nucleus, and binds enhancers located in the promoters of IFN
-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 interferon
-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 IFN
(9). Therefore, both tyrosine and
Ser/Thr kinase activity can regulate IFN
-induced expression of
cellular genes.
IFN
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 |
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 IFN
or IFN
-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
IFN
, 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 IFN
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-rIFN
to Cells--
rIFN
was
radiolabeled to high specific activity using Bolton-Hunter reagent as
described (16). Cells were incubated with the radiolabeled IFN
for
2 h at 4 °C and assayed as described previously (16).
IFN
R
and
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
and
chain of the IFN
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 IFN
R
are as follows: IFN
R
(5
sense, 5
-GCAGAAGGAGTCTTACATGTGTGG-3
; 3
antisense,
5
-CTCTCTATTGGAGTCAGATGGCTG-3
) and IFN
R
(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 IFN
R, we performed southern
hybridization with specific cDNAs for IFN
R
and
.
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
-glycerophosphate, 0.2 mM sodium orthovanadate, 2 µM okadaic acid,
and 0.2%
-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 [
-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 IFN
. 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 IFN
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
E;
pRK5mJak1kd) were constructed as described (21). Cytomegalovirus-driven
p
-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 p
-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 |
Jak1 is required for IFN
activation in E1C3 cells (12) (Fig.
1A) as measured by the
inability of IFN
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 Fc
receptor I gene (15) (Fig.
1A, lanes 6-10). In contrast, parental HeLa cells (1D4) respond to IFN
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 IFN
to its receptor (23), we
measured the ability of radiolabeled IFN
to bind to its receptor on
the parental and E1C3 HeLa cells. The binding of
125I-rIFN
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 IFN
for the mutant cells. Since binding of IFN
measures essentially only the interaction between the ligand and the
chain of the IFN
receptor, intact binding parameters may exist,
yet there may be no
chain present and thereby no signaling through
the receptor (24). Moreover, the presence or absence of the
chain
has not been demonstrated in the E1C3 cells. To assure that there was
equal expression of both the
and
chain of the IFN
receptor,
we performed reverse transcriptase polymerase chain reaction
amplification of RNA from both cell types. Both the parental and
Jak1-deficient cells expressed
and
chain genes (Fig.
1C). Therefore, the lack of signal transduction through the
IFN
receptor in the Jak1-deficient cells was not the result of
alterations in the receptor (17).

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Fig. 1.
Interferon signal transduction in HeLa and
ID4 cells. A, incubation of Jak1-deficient HeLa cells with
IFN fails to stimulate GRR binding activity. Parental (ID4)
(lanes 1-5) and Jak1-deficient HeLa cells (E1C3)
(lanes 6-10) were treated with IFN (10 ng/ml) for the
indicated times, and GRR binding activity was assessed by
electrophoretic mobility shift assay. FcRF ,
IFN -induced GRR binding activity. B, saturation binding
of 125I-IFN to parental HeLa (1D4) and Jak1-negative
cells (E1C3). Cells were incubated with increasing amounts of
125I-rIFN , and the amount of bound and free IFN was
determined. r, IFN specifically bound in molecules/cell;
c, concentration of unbound IFN . Open circles,
1D4; closed circles, E1C3. C, messenger RNA
expression of (IFN R ) and (IFN R ) chains of the
IFN receptor. Reverse transcriptase-polymerase chain reaction
analysis was performed using specific primers for and chains of
IFN receptor. Glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) was used as an internal control.
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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 IFN
,
no MAPK activation was measurable in the E1C3 cells lacking Jak1 (data
not shown). We therefore evaluated whether IFN
could enhance Raf-1
activity and whether this was Jak1-dependent. Cells were
incubated with IFN
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-
]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 IFN
(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 IFN
(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 IFN
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). IFN
enhancement in kinase activity
of this Raf-1 should be independent of endogenous p21ras. In
the parental cells (2fTGH) IFN
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 IFN .
Parental cells (1D4) (lanes 1-3) or Jak1-deficient cells
(lanes 4-6) were treated with IFN , 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.
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Reconstitution of IFN
-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 IFN
-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 IFN
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 IFN
upon reconstitution. Reconstitution of R89L
Raf-1 activity in response to IFN
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 IFN
with an approximately 2-fold increase in Raf-1
activity. Therefore, two separate cell lines deficient in Jak1
responded to reconstitution with Jak1 for IFN
-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 -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.
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Since one of the major mechanisms for the activation of Raf-1 is
through p21ras, we measured the ability of IFN
to activate
p21ras as measured by the increase in p21ras-bound GTP.
Cells were labeled with 32Pi and then treated
for 15 min with IFN
. 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
IFN
, a marked increase occurred following treatment of cells with
EGF (Fig. 4). There was no measurable increase in p21ras activation in cells incubated with IFN
at
other time points (2.5, 5, and 10 min, data not shown). Therefore, the
activation of Raf-1 by IFN
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 IFN
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 IFN
-treated cells showed
no or very little enhancement (Table II).

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Fig. 4.
p21ras is not activated by
IFN . Cells were incubated with 32Pi and
then treated with either IFN 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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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
-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
-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
-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
-galactosidase transfectant (negative control). C,
lysates of cells transfected with cDNAs for each of the Jak1
kinases (wt and kd) and -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).
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DISCUSSION |
Activation of the Jak/STAT pathway by cytokines such as IFN
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 IFN
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 IFN
activates MAPK
activity and in some systems protein kinase C activity (7), our data
confirm and extend these observations by showing that IFN
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 IFN
-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 IFN
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
chain of the IFN
receptor), dimerizes upon binding
of IFN
(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
IFN
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).
IFN
-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 IFN
. It is of interest that treatment with IFN
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
IFN
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 IFN
to induce an anti-proliferative, differentiative, or apoptotic state. Cell specificity can also play a role in modulating IFN
-induced gene expression. This was recently demonstrated by studying the effects of IFN
on myocytes and microvascular
endothelial cells (42). Although IFN
can activate STAT1 in both
cells, only in the myocytes is MAPK activity and inducible nitric oxide synthetase gene expression observed in response to IFN
. 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 IFN
-induced genes depends upon
the activation of specific signaling cascades whose modulation allows
for multiple layers of control in mediating IFN
-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 2
receptor cDNA, Cris Marshall for the Raf-1
cDNA, and Genentech, Inc. for the interferon
.
 |
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.
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; rIFN
,
recombinant IFN
; IFN
R, IRN
receptor; MAP, mitogen-activated
protein; MAPK, MAP kinase; MEK, MAPK kinase; GRR, gamma response
region; Erk, extracellular-regulated kinase; EGF, epidermal growth
factor.
 |
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