(Received for publication, February 27, 1995; and in revised form, May 11, 1995)
From the
Interferon
Cytokines such as interferons (IFN),
Incubation of cells with IFN
In order to ensure that the transfected IFN
Figure 1:
Saturation binding of
Figure 2:
Activation of GRR-binding proteins in
3-1-12 and T41 cells. Cells were exposed to either human IFN
Figure 3:
Activation of Jak kinases and STAT1
Figure 4:
Co-immunoprecipitation of Jak1 with the
IFN
Figure 5:
In vitro kinase assay of Jak1 and
Jak2 in IFN
Figure 6:
In vitro kinase assay of Jak1 and
Jak2 in IFN
Figure 7:
Association of tyrosine kinase activity
with anti-IFN
Figure 8:
A, biosynthetic labeling of the
IFN
Figure 9:
The
IFN
In order to ascertain
whether Jak2 preferentially associated with the IFN
Figure 10:
Jak2 co-immunoprecipitates with the
anti-IFN
Figure 11:
STAT1
Interferon
In untreated cells Jak1 is
pre-associated with the
The degree of activation of the Jak
kinase itself in cells unexposed to ligand varies depending upon the
cells utilized. In the IL-6/leukemia inhibitory factor studies there
was slight but perceptible in vitro kinase activity for Jak1
but not Jak2 in untreated cells (26) . In PHA-activated T cells
and factor-starved FWT-2 cells, there was kinase activity of Jak1 in
untreated cells that was enhanced after IL-2
treatment(28, 29) . In untreated MOLT
Most of the Jak/Tyk kinases have been shown to
interact with membrane proximal domains (Box1/Box 2 regions) of the
cytoplasmic portion of receptors that are present in many cytokine
receptors(30, 31) . However, it is clear that there is
differential binding of Jak/Tyk kinases to these regions. Jak1
primarily associates with the
(IFN
) induces the expression of early
response genes by tyrosine phosphorylation of Jak kinases and
transcription factors referred to as STAT proteins. The topology of the
IFN
receptor is partially understood and the relationship between
the
chain that binds the ligand and the
chain that is
required for signal transduction is undefined. In a cell line which
expresses only the human
chain, we show that these cells did not
activate Jak kinases or STAT proteins with human IFN
, even though
Jak1 co-immunoprecipitated with the
chain. In cells unexposed to
IFN
, Jak1 preferentially associated with the
chain, while
Jak2 associated with the
chain. There was evidence for Jak1
kinase activity in untreated cells. For Jak2, kinase activity was
IFN
-dependent. Although the
chain was
tyrosine-phosphorylated in response to ligand, we found no evidence for
tyrosine phosphorylation of the
chain. These data are consistent
with a model of the IFN
receptor in which Jak1 associates with the
chain, whereas Jak2 associates with the
chain. IFN
clusters at least two receptor units which results in the tyrosine
phosphorylation of Jak1 and Jak2, the activation of Jak2 kinase
activity, and the recruitment of STAT1
resulting in its activation
by tyrosine phosphorylation.
(
)interleukins, and growth factors transduce signals across
the cell membrane that result in the expression of early response
genes. This process occurs through the activation of transcription
factors, STAT proteins, (signal transducers and activators of
transcription) and the tyk2/Jak family of protein tyrosine
kinases(1, 2) . Whereas the STAT proteins possess SH2
and SH3 domains(3) , the Tyk/Jak kinases have two putative
catalytic domains (of which only the carboxyl one is active) but lack
SH2 or SH3 domains(4) . These kinases have been shown to be
associated with several cytokine receptors that require dimerization by
the ligand to initiate signal transduction. One such ligand is
interferon
(IFN
), a homodimeric molecule, that bivalently
binds to its receptor and activates the Jak/STAT pathway by clustering
its receptor. The IFN
receptor consists of a high affinity ligand
binding chain (
) (K
=
10
M
) that alone is incapable
of transducing a signal across the membrane (5) . Another
transmembrane protein (accessory factor) is required for signal
transduction, and through the use of somatic cell hybrid cell lines,
its gene has been determined to be located on chromosome
21(6) . The accessory factor/
chain interacts with the
ligand binding component (
chain) in a species specific
manner(7, 8) . The gene for the accessory factor
(
chain) has been cloned and when co-expressed with the ligand
binding
chain reconstitutes a functional receptor
unit(9, 10) . The cDNA for the
chain encodes a
transmembrane protein of 310 amino acids with six putative
glycosylation sites and an intracytoplasmic domain of 66 amino acids.
stimulates an immediate tyrosine
phosphorylation of the
chain(11, 12) . Although
several tyrosines within the cytoplasmic domain are phosphorylated, the
phosphorylation of tyrosine 440 is necessary for initiation of the
signaling cascade as determined by site-directed
mutagenesis(13) . Tyrosine phosphorylation of the
chain
occurs concomitantly with tyrosine phosphorylation of both Jak1 and
Jak2(12, 14, 15, 16) . Both kinases
form a multimolecular complex with the IFN
receptor immediately
after ligand binding(12) . STAT1
has been shown to bind to
a peptide of the IFN
receptor containing the phosphorylated
tyrosine 440(11) . The contribution of each chain of the
IFN
receptor (IFN
R) to the formation of this complex and the
relationship of kinase activity to ligand binding still needs to be
defined. We have carried out a series of experiments designed to assess
the relationship between the
and
chains of the receptor and
the Jak kinases in both cell lines, primary monocytes, and in somatic
cell hybrids that express either the human
chain or both the
human
and
chains of the receptor.
Cells
HeLa cells were maintained in stationary
flasks or adapted for growth in spinner flasks as described
previously(12) . Human peripheral blood monocytes were obtained
from volunteers by leukapheresis. The monocytes were purified from the
mononuclear cells by Ficoll-Hypaque (LSM, Organon Teknika, Durham, NC)
sedimentation followed by countercurrent centrifugal elutriation, were
>95% pure, and used without further culturing. The T41 cell line is
a mouse L929 fibroblast cell line stably transfected with the human
IFN receptor
chain. The 3-1-12 cell line is a mouse-man
somatic cell hybrid (WavR4d-F9-4a) retaining only chromosome 21 (17) and has been similarly transfected with the human IFN
receptor
chain. Chromosome 21 also encodes for the IFN
receptor, and the responsiveness of the 3-1-12 cell line to IFN
can be used to assess the ability of the cells to retain chromosome
21(18) .
Flow Cytometric Analysis
The ability of the T41
and 3-1-12 cell lines to respond to IFN or IFN
was monitored
by the enhancement of major histocompatibility class I antigen
expression. Cells were treated with IFN for 24 h and then incubated
with antibodies specific for major histocompatibility class I antigens
followed by incubation with fluorescein isothiocyanate-conjugated
secondary antibody. The cells were then analyzed by flow cytometric
analysis on a FACScan.
Binding of IFN
Recombinant human IFN to Somatic Cell Hybrid
Lines
(Genentech, Inc., S. San
Francisco, CA) was radiolabeled using Bolton-Hunter reagent (Amersham
Corp.) to high specific activity as described(19) . Increasing
concentrations of
I-rIFN
were incubated at 4 °C
for 2 h with cells, and a 200-fold excess of unlabeled IFN
was
used in parallel tubes to correct for nonspecific binding. Free
IFN
was separated from bound IFN
by centrifugation over a
phthalate oil cushion as described (19) . Molecules bound per
cell were calculated from the specific activity of the radiolabeled
IFN
.
DNA-binding Proteins
After treatment with
IFN, cells were washed with cold phosphate-buffered saline and
solubilized with cold whole cell extraction buffer (1 mM
MgCl
, 20 mM Hepes (pH 7.0), 10 mM KCl,
300 mM NaCl, 0.5 mM dithiothreitol, 0.1% Triton
X-100, 200 µM phenylmethylsulfonyl fluoride, 1 mM vanadate, and 20% glycerol). DNA-binding proteins were determined
by electrophoretic mobility shift assay as described
previously(20) . Briefly, 10 µg of protein were incubated
with the
P-labeled oligonucleotide probe consisting of
double-stranded GRR (5` AGCATGTTTCAAGGATTTGAGATGTATTTCCCAGAAAAG 3`) of
the promoter of the Fcgr1 gene in binding buffer(21) . The
sample was then applied to a 6% nondissociating polyacrylamide gel in
order to separate free probe from probe bound to protein.
Biosynthetic Labeling of Cells
Monocytes were
resuspended into medium deficient for methionine, cysteine, and
glutamine for 30 min at 37 °C. This medium was replaced with fresh
media deficient for the same amino acids containing 400 µCi of
Translabel ([S[methionine and
S-cysteine) (ICN, Costa Mesa, CA), and the cells continued
in incubation for 3 h. The cells were then washed, lysed, and the
extracts subjected to immunoprecipitation as described below.
Immunoprecipitations
Following treatment with
IFN, cells (2
10
) were solubilized in 1%
Triton X-100 (Pierce) in a buffer consisting of 0.15 M NaCl,
50 mM Tris (pH 8.0), 50 mM NaF, 5 mM sodium
pyrophosphate, 1 mM phenylmethylsulfonyl fluoride, 1 mM orthovanadate. The post-nuclear lysate was then precleared with a
suspension of protein G-agarose (Pharmacia Biotech Inc.) for 1 h at 4
°C. After separation of the protein G-agarose from the lysate by
centrifugation, the lysate was incubated with either normal rabbit
serum, rabbit anti-IFN
R, rabbit anti-Jak1 (Upstate Biotechnology,
Inc., Saranac Lake, NY), rabbit anti-Jak2 (Upstate Biotechnology,
Inc.), or rabbit anti-STAT1
(22) for 2-18 h at 4
°C. Two different anti-IFN
R antibodies were used. One is a
polyclonal antibody (anti-IFN
R) raised against purified
chain of the receptor(23) , and the other is a polyclonal
antibody (anti-AF-1) raised against a 30-amino acid peptide of the
cytoplasmic domain of the accessory factor/
chain (amino acids
307-337). This antibody was affinity purified on an
antigen-conjugated agarose column. Protein G-agarose was then added for
1 h at 4 °C to isolate the immune complexes. The protein G
conjugates were then washed in lysis buffer and boiled in SDS sample
buffer. For immunoprecipitations specifically designed to assess
protein-protein interactions, cells were lysed in the same buffer as
above except that digitonin was substituted for Triton X-100, and the
immune complexes were washed in 0.1% digitonin containing buffer. The
samples were then analyzed using 8% SDS-PAGE followed by
electrophoretic transfer to polyvinylidene difluoride membranes
(Immobilon-P, Millipore, Bedford, MA) as described(12) .
In Vitro Kinase Assays
Cells, untreated or treated
with IFN, were washed, solubilized, and the extracts subjected to
immunoprecipitation as described above. The immunoprecipitates were
then resuspended in kinase assay buffer (100 mM NaCl, 50
mM Hepes (pH 7.3), 0.1% Triton X-100, 6.25 mM MnCl
, 0.5 mM dithiothreitol). To this buffer
was added either 25 µM ATP and 5 µCi of
[
-
P]ATP when autoradiography was performed
or 2.5 mM ATP when anti-phosphotyrosine immunoblotting was
performed. The reaction mixture was incubated for 15 min at 30 °C
and the immunoprecipitates washed several times with lysis buffer prior
to the addition of SDS sample buffer. The samples were then subjected
to SDS-PAGE analysis followed by either autoradiography or
immunoblotting with anti-phosphotyrosine antibodies (PY20) and enhanced
chemiluminescent imaging.
IFN
The ability of the somatic cell hybrid 3-1-12 cell line to
retain chromosome 21 was assessed using IFN Signaling in the Somatic Cell Hybrid Cell
Lines
. For the T41 cells
there was no change in human IFN
-induced major histocompatibility
class I expression as measured by peak channel fluorescence (134 in
untreated cells versus 132 for IFN
treatment). This
finding was consistent with the fact that the L929 cells are murine in
origin and do not respond to human IFN
(these cells do respond to
murine IFN
). In contrast, the 3-1-12 cell line responded to human
IFN
by enhanced peak channel fluorescence from 124 to 141. These
data confirmed that chromosome 21 was present within this cell line.
R
chain was
adequately expressed, both the T41 and the 3-1-12 cell line were
examined for the surface expression of IFN
R by measuring the
binding of
I-rIFN
(Fig. 1). The
chain
alone is sufficient to impart an high affinity binding state. Both cell
lines expressed approximately similar numbers of binding sites: the T41
cells bound about 6000 molecules/cell, whereas the 3-1-12 cells bound
about 5000 molecules/cell. The concentration at half-maximal binding
for both cell lines was about 10
M, which
was consistent with a high affinity binding site. The affinity and
number of molecules per cell for these cell lines was similar to
previously reported values for other cells(19) .
I-IFN
to T41 and 3-1-12 cells. Cells were incubated
at 4 °C with increasing concentrations of
I-IFN
with or without a 200-fold molar excess of unlabeled IFN
. The
cells were then centrifuged through phthalate oils and the
radioactivity in the pellet (bound) and supernatant (free) were
measured. Data are represented as molecules (mol) of IFN
bound per cell. The curves represent specific binding of
I-IFN
to the cells. Open circles, T41
cells; closed circles, 3-1-12
cells.
The ability
of the two cell lines to signal was examined next by measuring the
formation of the IFN-activated GRR binding complex, FcRF
(Fig. 2). In T41 cells only murine IFN
stimulated the
formation of FcRF
(Fig. 2, lane 5). In contrast
both murine (Fig. 2, lane 2) and human IFN
(Fig. 2, lane 3) resulted in assembly of the FcRF
complex in 3-1-12 cells. That the intensity of the signal was
considerably less for human IFN
compared with murine IFN
in
the 3-1-12 cell line was probably the result of limited expression
and/or association of the
component with the
component of
the IFN
R. Since both the Jak1 and Jak2 tyrosine kinases have been
shown to be involved in the signal transduction pathway for IFN
,
we next examined whether the
component alone was sufficient for
the ligand-induced tyrosine phosphorylation of these tyrosine kinases.
Incubation of the 3-1-12 cell line with IFN
resulted in the
tyrosine phosphorylation of both Jak1 (Fig. 3A, lane 2)
and Jak2 (Fig. 3B, lane 2). In contrast, in those cells
expressing only the
component of the receptor, no tyrosine
phosphorylation of Jak1 (Fig. 3A, lane 4) or Jak2 (Fig. 3B, lane 4) was observed. This implied that the
chain alone in spite of being capable of binding ligand with high
affinity could not facilitate ligand induced tyrosine phosphorylation
of the Jak kinases. When these same cells were examined for the
tyrosine phosphorylation of STAT1
in response to IFN
, the
results were similar to that observed for the Jak kinases (Fig. 3C). Only in 3-1-12 cells where a functional
/
receptor unit existed was there human IFN
-induced
tyrosine phosphorylation of the STAT1
(Fig. 3C, lane 3
versus 6). T41 cells that were treated with murine IFN
exhibited ligand-induced tyrosine phosphorylation of Jak1 and Jak2
(data not shown) and STAT1
(Fig. 3C, lane 5).
There was no STAT1
tyrosine phosphorylation in response to human
IFN
in the T41 cell line (Fig. 3C, lane 6). Equal
amounts of immunoprecipitated protein (Jak1, Jak2, and STAT1
) were
loaded onto each lane (Fig. 3, A-C, lower panel).
(lanes 3 and 6) or murine IFN
(Genentech) (lanes 2 and 5) for 10 min at 37 °C and then
solubilized in whole cell extract buffer. Extracts were incubated with
P-GRR and DNA bound proteins were separated on 6%
nondissociating gels. The figure is an autoradiogram. FcRF
denotes the GRR binding complexes induced by IFN
. CTL, untreated control cells. m-IFN
, murine
IFN
. h-IFN
, human
IFN
.
in response to IFN
in 3-1-12 and T41 cells. Cells were exposed to
either murine or human IFN
for 10 min at 37 °C and then
solubilized in 1% Triton X-100 buffer. A, the extracts were
incubated with anti-Jak1 and analyzed by immunoblotting. The upper
panel represents the membrane probed with anti-phosphotyrosine
(PY20) and then developed using ECL. The lower panel represents the same membrane reprobed for Jak1 protein and
developed using colorimetric analysis. B, the extracts were
incubated with anti-Jak2 and analyzed as described in A. C, the extracts were exposed to anti-STAT1
and analyzed
as described in A. CTL, untreated control cells. m-IFN
, murine IFN
. h-IFN
, human
IFN
.
We have previously shown that the Jak1 kinase constitutively
associated with the IFNR and that ligand binding then resulted in
its tyrosine phosphorylation(12) . We examined whether the
chain or the
chain was required for this association. Both
the T41 and 3-1-12 cell lines were solubilized and subjected to
immunoprecipitation with anti-IFN
R
chain antibodies. The
immunoprecipitates were probed with anti-Jak1 antibodies (Fig. 4). Both the T41 (Fig. 4, lane 3, upper
panel) and the 3-1-12 cells (Fig. 4, lane 4, upper
panel) had Jak1 constitutively associated with the IFN
R
.
The IFN
R
protein was immunoprecipitated equally in both lanes (Fig. 4, lanes 3 and 4, lower panel). Since in
T41 cells only the
chain is present, these data suggested that
Jak1 preferentially associated with the
component. When a similar
experiment was attempted and probed with Jak2, no association was seen
in the T41 or the 3-1-12 cells even when these cells were treated with
IFN
(data not shown). Presumably, if Jak2 preferentially
associates with the
chain, the poor expression of this gene may
account for our inability to detect the association in the 3-1-12 cell
line. The lack of association in the T41 cells suggests that Jak2 does
not associate with the
chain.
R in T41 and 3-1-12 cells. Cells (untreated) were solubilized
in 1% digitonin buffer and the extracts incubated with
anti-IFN
R
(lanes 3 and 4) or normal rabbit
serum (NS) (lanes 1 and 2). The
immunoprecipitates were then split between two polyacrylamide gels and
analyzed by immunoblotting with anti-Jak1 antibodies (upper
panel) or with the immunoprecipitating antibody
(anti-IFN
R
) (lower panel). The immunoblots were
developed using ECL.
Jak Kinase Activity in Untreated and IFN
Since Jak1 associated with the IFN-treated
Cells
receptor in a
untreated cells, we assessed whether this kinase demonstrated catalytic
activity as measured by an in vitro kinase assay.
Immunoprecipitates prepared with anti-Jak1 and anti-Jak2 antisera were
assayed for kinase activity using either
[
-
P]ATP (Fig. 5) or unlabeled ATP
followed by phosphotyrosine detection by immunoblotting (Fig. 6). In cells untreated with IFN
there was a
substantial amount of Jak1 kinase activity as judged by
autophosphorylation of Jak1 (Fig. 5, lane 3, upper
panel). There was no increased activity after treatment with
IFN
(Fig. 5, lane 4, upper panel). In contrast
there was no observed Jak2 kinase activity in cells untreated with
IFN
. Only with IFN
treatment was Jak2 kinase activity
observed (Fig. 5, lane 6, upper panel). When the
kinased immunoprecipitated Jak1 was subjected to phosphoamino acid
analysis, essentially all of the incorporated radioactivity was on
tyrosine moieties (data not shown). Equal amounts of the
immunoprecipitated Jak kinases were loaded in each lane (Fig. 5, lanes 3-6, lower panel). When the experiment was
performed using unlabeled ATP in conjugation with phosphotyrosine
analysis by immunoblot, similar results were obtained (Fig. 6).
Jak1 demonstrated constitutive tyrosine kinase activity
(autophosphorylation) (Fig. 6A, lane 3) with no further
increased kinase activity after IFN
treatment (Fig. 6A,
lane 4). On the other hand, Jak2 (Fig. 6A, lane 5 and 6) demonstrated tyrosine kinase activity only after
cells had been stimulated with IFN
. With no ATP present in the
assay buffer only the endogenous tyrosine phosphorylation is observed
in response to IFN
(Fig. 6B, lanes 4 and 6).
-treated HeLa cells. Cells were either exposed to
IFN
(lanes 2, 4, and 6) or untreated (lanes
1, 3, and 5) and solubilized in 1% Triton X-100. The
extracts were then incubated with either normal rabbit serum (lanes
1 and 2), anti-Jak1 (lanes 3 and 4), or
anti-Jak2 (lanes 5 and 6). The immunoprecipitates
were washed and subjected to an in vitro kinase assay using
[
-
P]ATP and analyzed by SDS-PAGE followed
by electrophoretic transfer to a polyvinylidene difluoride membrane.
The upper panel is an autoradiogram of the membrane. The
membrane was then reprobed with the immunoprecipitating antibodies for
specific protein loaded onto each lane (lower panel). The
membrane was developed using colorimetric chemistry. CTL,
control untreated cells. NS, normal rabbit
serum.
-treated HeLa cells using anti-phosphotyrosine
immunoblotting. Cells were treated as described in the legend to Fig. 5. Immunoprecipitates were subjected to an in vitro kinase assay in the absence (B) or presence (A)
of ATP. A, in vitro kinase assay in the presence of
ATP. The upper panel represents the immunoblot probed with
anti-phosphotyrosine antibody (PY20). The lower panel represents the same membrane reprobed with the immunoprecipitating
antibody and developed using colorimetric chemistry. B, in
vitro kinase assay in the absence of ATP. The upper and lower panel are as described in A. The presence of
tyrosine-phosphorylated Jak1 and Jak2 in the upper panel represent endogenous tyrosine phosphorylation in IFN
-treated
cells. NS, normal rabbit serum. CTL, untreated
control cells. P-Tyr,
phosphotyrosine.
Since the IFNR
chain was rapidly
tyrosine-phosphorylated following ligand binding, we next determined
whether the kinase that was responsible for this was pre-associated
with the IFN
receptor. Cells were treated with or without IFN
and then solubilized in digitonin to maintain any protein-protein
interactions. The extracts were then immunoprecipitated with antibodies
directed against the IFN
R
chain. The immunoprecipitate was
then analyzed by an in vitro kinase reaction. In untreated
cells (Fig. 7A, lanes 1 and 2) there was
tyrosine phosphorylation of the IFN
R
chain (arrow)
in the presence of ATP (lane 2). For comparison, the
endogenous tyrosine phosphorylation of the IFN
R (arrow)
and Jak kinases (asterisk) was measured in response to
IFN
(Fig. 7A, lane 3). When the immunoprecipitates
from untreated cells were washed under more stringent conditions with
1% Triton X-100 prior to performing the in vitro kinase assay,
the ability to tyrosine-phosphorylate the IFN
R was lost (Fig. 7A, lane 8 versus 6). This suggested that the
tyrosine kinase that was responsible for tyrosine phosphorylation of
the IFN
R
chain was pre-associated with the IFN
R in a
receptor-kinase complex. Since Jak1 was shown to pre-associate with the
chain and be active in untreated cells by an in vitro kinase assay, Jak1 was a likely candidate responsible for the
tyrosine phosphorylation of the
chain of receptor. When the
anti-IFN
R
chain immunoprecipitates were probed for the
presence of Jak1, there was a loss of Jak1 in those cells washed with
1% Triton X-100 (Fig. 7B, lanes 5 and 6 versus 7 and 8) even though total IFN
R
protein was the
same (Fig. 7C, lanes 5-8).
R
immunoprecipitations. A, HeLa cells
were exposed to IFN
(lanes 3 and 4) or untreated (lanes 1, 2, 5-8) and solubilized in 1%
digitonin buffer. The extracts were then incubated with
anti-IFN
R
antibody. The immunoprecipitates were then either
washed in 0.1% digitonin buffer (lanes 1-6) or 1% Triton
X-100 buffer (lanes 7 and 8). The immunoprecipitates
were then subjected to an in vitro kinase assay with (lanes 2, 4, 6, and 8) or without (lanes 1, 3,
5, and 7) ATP. The immunoprecipitates were then analyzed
by immunoblotting with anti-phosphotyrosine antibodies. Lanes 1 and 2 represent a kinase activity co-immunoprecipitating
with the IFN
R in untreated cells. Lane 3 represents
endogenous tyrosine phosphorylation of the IFN
R
chain (arrowhead) and Jak kinases (asterisk) in
IFN
-treated cells. Lanes 5 and 6 are as
described for lanes 1 and 2. Lanes 7 and 8 represent the loss of kinase activity co-immunoprecipitating with
the IFN
R. B and C, the immunoblot of the
membrane represented in A (lanes 5-8) was
divided in two portions. The upper potion (B) was reprobed
with anti-Jak1 and developed using colorimetric chemistry. The lower
portion (C) was reprobed with anti-IFN
R
and
developed using colorimetric chemistry. CTL, untreated control
cells. P-Tyr, phosphotyrosine. IgH, immunoglobulin
heavy chain.
The IFN
In order to fully assess the role of the
IFNR
Chain and Signal
Transduction
R
chain, we raised polyclonal antibodies against a peptide
of the
chain consisting of amino acids 307-337 of the
intracytoplasmic domain. The reactivity of this affinity-purified
antibody was assessed on biosynthetically labeled human peripheral
blood monocytes. The IFN
R
chain and other associated chains
have been extensively studied in these cells by both radiolabeling and
biosynthetic labeling followed by immunoprecipitation (24) .
When monocytes were biosynthetically labeled and the digitonin lysates
subjected to immunoprecipitation with the anti-IFN
R
chain
(anti-AF-1), there was a prominent band at about 38 kDa (Fig. 8A, lane 2, arrow) that was not observed with
control antibodies (Fig. 8A, lane 1) or antibodies
directed against the IFN
R
chain (Fig. 8A, lane
3). A similar band was noted in cells solubilized in 1% Triton
X-100 (data not shown). The migration of the immunoprecipitated
IFN
R
chain as a 38-kDa protein is consistent with the
predicted amino acid sequence of 310 amino acids with some degree of
post-translational modification such as glycosylation. Under the
conditions utilized the antibodies against the
chain
co-immunoprecipitate the
chain, but antibodies against the
chain only precipitate the
chain and associated Jak kinases (Fig. 8A, lane 3 and Fig. 9, lane 6).
The ability of the anti-IFN
R
antisera to co-immunoprecipitate
the
chain of the receptor was directly measured by incubating
extracts prepared with digitonin with anti-IFN
R
and probing
the membrane with anti-IFN
R
(Fig. 8B). Under
these condition the IFN
R
chain co-immunoprecipitates with the
chain in cells untreated (Fig. 8B, lane 3) or
treated (Fig. 8B, lane 4) with IFN
.
R
chain. Monocytes were incubated with Translabel for 3 h,
washed, and solubilized. The extracts were then incubated with either
normal rabbit IgG (nIgG) (lane 1), affinity purified
anti-IFN
R
chain (anti-AF-1) (lane 2), or
anti-IFN
R
chain (lane 3). The arrow represents the 38-kDa IFN
R
chain. The figure is an
autoradiogram. B, the
and
chains of the IFN
R
are associated in untreated cells. HeLa cells were solubilized with 1%
digitonin buffer and the extract incubated with normal rabbit IgG (nIgG) (lanes 1 and 2) or
anti-IFN
R
(anti-AF-1) (lanes 3 and 4). The immunoprecipitates were then analyzed by
immunoblotting with anti-IFN
R
chain and developed using ECL. nIgG, normal rabbit IgG. anti-AF-1,
anti-IFN
R
chain. anti-IFN
R, anti-IFN
R
chain.
R
chain is not tyrosine-phosphorylated in response to
IFN
. HeLa cells were either exposed to IFN
(lanes 2,
4, and 6) or untreated (lanes 1, 3, and 5) and solubilized in 1% digitonin buffer. The extracts were
then incubated with either normal rabbit IgG (nIgG) (lanes
1 and 2), anti-IFN
R
chain (anti-AF-1) (lanes 3 and 4), or anti-IFN
R
chain
(anti-IFN
R) (lanes 5 and 6). The
immunoprecipitates were then analyzed by immunoblotting with
anti-phosphotyrosine antibodies (PY20). NS, represents
nonspecific bands.
In order to
examine whether or not the IFNR
chain like the
chain
undergoes tyrosine phosphorylation in response to the binding of
IFN
to the cells, we treated cells with IFN
and then
solubilized them in digitonin (Fig. 9). Using
anti-IFN
R
chain antibody it was evident that the
chain
of the receptor was clearly tyrosine-phosphorylated in response to
IFN
(Fig. 9, lane 5 versus 6). In addition, a band
migrating slightly slower than the IFN
R
chain at about 130
kDa represented the tyrosine phosphorylated Jak1/2 kinases. This has
been confirmed by reprobing the blots with anti-Jak kinase antibodies
(data not shown). In contrast, when the anti-IFN
R
chain
antibodies (anti-AF-1) were utilized, there was no observable tyrosine
phosphorylation of the
chain (Fig. 9, lane 3 versus
4). However, it was evident that the antibody was indeed
immunoprecipitating the receptor complex since the
tyrosine-phosphorylated
chain at 90 kDa and the Jak kinases at
130 kDa were present in the immunoprecipitate.
R
chain,
HeLa cells were either untreated or treated with IFN
and then
solubilized in digitonin. Immunoprecipitates with anti-IFN
R
were resolved by SDS-PAGE and then immunoblotted with anti-Jak2 (Fig. 10). Jak2 associated with the
chain in a
constitutive fashion (Fig. 10, lane 3). However, upon
IFN
signaling there was a consistent increase in the amount of
Jak2 that could be co-immunoprecipitated with the
chain,
suggesting that recruitment of Jak2 occurred during signal transduction
or that the affinity of Jak2 increased for the clustered IFN
R (Fig. 10, lane 4). When antibodies against Jak1 were
utilized to probe anti-IFN
R
immunoprecipitates, no
co-immunoprecipitation of Jak1 was detectable (data not shown),
suggesting either that there was no direct interaction between Jak1 and
the IFN
R
chain or that the anti-IFN
R
antibody
itself disrupted any weak interaction that may have occurred.
R
chain. HeLa cells were incubated with IFN
(lanes 2 and 4) or untreated (lanes 1 and 3) and solubilized in 1% digitonin buffer. The extracts were
then incubated with either normal rabbit serum (nIgG) (lanes 1 and 2) or anti-IFN
R
chain (anti-AF-1) (lanes 3 and 4) and analyzed by
immunoblotting with anti-Jak2 and developed using ECL. IgH,
immunoglobulin heavy chain. nIgG, normal rabbit serum. anti-AF-1, anti-IFN
R
chain.
Upon
activation by IFN there is rapid tyrosine phosphorylation of
STAT1
. In order to determine if STAT1
could be directly
detected within the receptor-Jak complex, we exposed cells to IFN
at 4 °C and then solubilized them in digitonin. The extracts were
incubated with anti-STAT1
antibodies and the membranes probed with
anti-phosphotyrosine. In cells treated with IFN
(Fig. 11, lane 4) there were two additional co-immunoprecipitating
tyrosine-phosphorylated bands that correspond to the IFN
R
chain and the Jak1/2 kinases both of which can also be shown to be
immunoprecipitated with the anti-IFN
R (lane 6).
is part of the
IFN
R
Jak complex. HeLa cells were either untreated (lanes
1, 3, and 5) or treated with IFN
(lanes 2,
4, and 6) for 10 min at 4 °C, solubilized in
digitonin buffer, and subjected to immunoprecipitation with either
normal rabbit serum (NRS) (lanes 1 and 2),
anti-STAT1
(lanes 3 and 4), or
anti-IFN
R
(anti-IFN
R) (lanes 5 and 6). The immunoprecipitates were analyzed by SDS-PAGE followed
by immunoblotting with anti-phosphotyrosine (P-Tyr) antibodies
(PY20) and developed using ECL.
activation of STAT1
requires both Jak1
and Jak2 as well as tyrosine phosphorylation of the
chain of the
IFN
receptor. Although it has been known that another component of
the receptor is necessary for signal transduction to occur, only
recently has the gene for this accessory factor (
chain) been
cloned and shown to encode for a transmembrane protein of 310 amino
acids with a 66-amino acid intracytoplasmic
domain(9, 10) . Nothing is known regarding the role of
this protein in the formation of the ligand induced multi-molecular
complex. In this report we show that both the
and
components are required to activate STAT proteins. In cells expressing
only the
chain, we show that Jak1 constitutively associates with
this component but does not become tyrosine-phosphorylated after
treatment with IFN
in spite of high affinity binding of the ligand
to the
chain. Only in cells expressing both the
and
component are Jak1 and Jak2 tyrosine-phosphorylated in response to
IFN
treatment. Jak1 pre-associates with the
component and
shows no increased association with the receptor after IFN
treatment. In contrast, Jak2 is pre-associated with the
component, but also is either recruited to the receptor after ligand
binding or its affinity for the
chain increases after ligand
binding. The data derived from the somatic cell hybrid cells suggest
that the association of Jak2 with the receptor
chain is an
absolute requirement for subsequent activation of STAT1
by
tyrosine phosphorylation. Although the
chain is
tyrosine-phosphorylated in IFN
-treated cells, we have been unable
to observe any ligand-dependent tyrosine phosphorylation of the
chain. Therefore, it is likely that this component may not directly
interact with the STAT proteins as has been proposed to occur for the
chain of the IFN
receptor (11) and the ligand
binding component of the IL-4 receptor(25) . We have also shown
that in digitonin-solubilized extracts the
and
components
can be co-immunoprecipitated in untreated cells, and therefore, it is
likely that dimerization of at least two
complexes
initiates signal transduction.
chain and, by in vitro kinase
assay, is enzymatically active as measured by autophosphorylation. When
the IFN
R
chain is immunoprecipitated and subjected to in
vitro kinase assay, the
chain along with the
co-immunoprecipitating Jak kinases are tyrosine-phosphorylated in cells
unexposed to IFN
. This suggests that the kinase responsible for
the tyrosine phosphorylation of the
chain in vivo is
pre-associated with the receptor and may be Jak1. Washing of the
anti-IFN
R
immunoprecipitate in 1% Triton X-100 results in
loss of the activity and loss of co-immunoprecipitating Jak1. We have
shown previously that under similar conditions in untreated cells no
detectable Jak2 is immunoprecipitated with the anti-IFN
R
chain (12) . This finding is similar to that observed with the
leukemia inhibitory factor receptor
chain or gp130 of the IL-6
receptor. Both Jak1 and Jak2 are pre-associated with the
components of the IL-6 and leukemia inhibitory factor receptor, and
upon immunoprecipitation of the receptors in Brij96 (a detergent with
properties similar to digitonin), in vitro tyrosine
phosphorylation of the
component is lost after a Nonidet P-40
wash (26, 27) .
cells,
there was in vitro tyrosine autophosphorylation of Jak1 that
did not increase after IL-2 exposure(29) . Therefore, varying
degrees of in vitro kinase activity for Jak1 exist in
untreated cells that may or may not be enhanced after ligand
stimulation. For receptors utilizing Jak1 and Jak2 or Jak3, it appears
that Jak1 rather than Jak2/3 tends to demonstrate more in vitro kinase activity in cells untreated with ligand. These data are
consistent with the concept that the initial reaction after clustering
of receptors by ligand may allow Jak1 access to substrate. Jak2
activation quickly follows and in addition is recruited to the receptor
or binds more tightly to the clustered complex(12) . It is at
this point that STAT1
can be shown to become part of the
receptor-Jak complex.
chain and Jak2 with the
chain
of the IFN
receptor. For the IL-2 receptor, Jak1 associates with
the serine-rich membrane proximal region of the IL-2R
, whereas
Jak3 associated with the COOH-terminal region of the
component, a
region mutated in X-linked severe combined immunodeficiency patients
who have altered T cell function(28, 29) . In cell
lines expressing all three Jak kinases, Jak2 did not associate with the
IL-2 receptor. The kinase-specific association to distinct receptor
components may be a mechanism for specificity of signal transduction
through the Jak/Tyk pathway. The interaction of Jak2 with the
chain of the IFN
R appears to be a requirement for IFN
signaling. This was also seen for the IL-2R
c/Jak3
association(28, 29) . From our previous studies and
the data in this report, it appears that Jak2 is required for the
tyrosine phosphorylation of STAT1
, whereas Jak1 is more closely
affiliated with the tyrosine phosphorylation of the IFN
R
chain. Therefore, in receptor systems that utilize paired Tyk/Jak
kinases, one of the kinases pre-associated with the receptor may
facilitate the tyrosine phosphorylation of one or more chains of the
receptor, whereas the other kinase may be more responsible for the
activation of STAT proteins.
, interferon
; PAGE, polyacrylamide gel electrophoresis; IL, interleukin.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.