From the Department of Pharmacology, University of
Illinois, Chicago, Illinois 60612 and the § Department of
Immunology, Berlex Biosciences, Richmond, California 94804.
Received for publication, January 4, 2001, and in revised form, April 6, 2001
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ABSTRACT |
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An obligatory step in the activation of
Signal Transducers and Activators
of Transcription (STATs) by cytokines is their docking to
specific receptors via phosphotyrosines. However, this model does not
address whether STATs pre-associate with their corresponding receptor
or exist free in the cytoplasm before receptor activation. In this
report, we demonstrate that pre-association of STAT1 with the receptor
is required for type I interferon (IFN) signaling. Interestingly, the interaction between the human type I IFN receptor and STAT1 is not direct but mediated by the adapter protein
receptor for activated protein kinase C (RACK1). Disruption of
the IFN Cytokines and interferons
(IFNs)1 bind to receptors of
the cytokine receptor superfamily (1-3), resulting in the activation of kinases of the Jak family and transcription factors designated STATs
or Signal Transducers and
Activators of Transcription (4-7). The
Jak-STAT pathway has evolved as the paradigm of cytokine and IFN
signaling (4-7). Although STAT can be activated by different cytokines
(i.e. STAT1 is activated by IFN STATs are recruited to distinct phosphotyrosines within the receptor
complex and then are phosphorylated, probably by Jaks, on the highly
conserved C-terminal tyrosines (i.e. tyrosine 701 of Stat1),
allowing the SH2 domain of one STAT to interact with the phosphorylated
tyrosine on another STAT to form homo- or heterodimers. STAT dimers
translocate to the nucleus, where they bind specific DNA elements
to activate or inhibit transcription of specific genes (reviewed in
Refs. 5 and 8).
One distinctive feature in the type I IFN system is that STAT2 is
pre-associated with IFN We have recently reported (13) that RACK1, originally described as a
Receptor for Activated C
Kinase We report here that RACK1 constitutively interacts with
non-phosphorylated STAT1 and functions as an adaptor between this factor and the long form of the Cell Lines, Reagents, and Antiviral Assays--
U-266 and Daudi
cells were grown in RPMI (Life Technologies, Inc.) supplemented with
10% (v/v) fetal bovine serum. Human IFN Immunoprecipitation and Immunoblotting--
U-266 or Daudi cells
(1 × 107 cells) were treated as indicated and then
lysed in lysis buffer (20 mM Tris-HCl, pH 6.6 containing 1% Nonidet P-40, 50 mM NaCl, 1 mM EDTA, 2.5%
glycerol (v/v), 1.0 mM sodium fluoride, 1.0 mM
sodium orthovanadate, 1.0 mM phenylmethylsulfonyl fluoride,
0.5 µg/ml leupeptin, and 5.0 µg/ml trypsin inhibitor) for 30 min at
4 °C. Immunoprecipitations were performed as previously described
(9). Proteins were transferred to polyvinylidene difluoride membranes,
immunoblotted with the indicated antibodies, and developed using a
chemiluminescent detection method (Pierce).
GST Fusion Proteins and Mammalian Expression Constructs--
The
different GST fusion proteins encoding different regions of the
cytoplasmic domain of IFN Study of the Adaptor Function of RACK1 Using a Wheat Germ in
Vitro Translation--
STAT1 and RACK1 were produced by a T7 wheat
germ in vitro transcription/translation kit (Promega)
following the manufacturer's procedure.
[35S]methionine-labeled STAT1 and RACK1 proteins alone or
in combination were incubated with GST- Expression of IFN RACK1 Specifically Associates with STAT1--
It has been
suggested that proteins containing WD repeats may serve as
scaffold or adaptor proteins (27). Because RACK1 interacts with a
region of IFN
To further characterize the RACK1-STAT interactions, a GST fusion
protein that encoded the full-length RACK1 protein was used to
determine whether other STAT proteins bind RACK1. Fig. 1B
shows that GST-RACK1, but not GST alone, binds to STAT1. However,
GST-RACK1 failed to bind STAT2, -3, -4, -5, or -6 (Fig. 1,
C-F), confirming that the interaction between RACK1 and
STAT1 is specific. Interestingly, GST- RACK1 Functions as an Adaptor between STAT1 and the
IFN
We next performed an alanine scan of this region to further define the
RACK1 and STAT1 binding sites. Although no individual mutation
completely abolished RACK1 or STAT1 binding to GST-
Although the interaction between IFN RACK1 Interacts Specifically with the
Non-phosphorylated Form of STAT1--
Although the experiments
presented above demonstrate that the non-activated forms of
IFN The Interaction between RACK1 and IFN
We next studied whether huIFN The results presented in this study demonstrate that the adaptor
protein RACK1 links STAT1 to the human type I IFN receptor. The
interaction between the receptor and RACK1 is required for activation
of STAT1 and the induction of an antiviral state by huIFN These findings raise the question whether a model in which STATs
pre-associate with cytokine receptors through adaptor proteins containing a WD motif also applies to other cytokine systems. It
has been recently reported that the WD motif-containing protein StIP (STAT3-Interacting Protein)
interacts with several STATs and JAKs (29). However, there are
differences between RACK1 and StIP. 1) StIP interacts with more
than one STAT and Jak, suggesting that it is not STAT or cytokine
specific, and 2) it has not been addressed whether StIP associates
directly with cytokine receptors. Nevertheless, it is tempting to
speculate that StIP and RACK1 are members of a novel family of proteins
involved in Jak-STAT signaling. The concept that other
STAT-specific adaptors may exist is also supported by reports
indicating that activation of STAT5 by growth hormone occurs in the
presence of growth hormone receptors devoid of all tyrosines (30,
31).
The alternative to a general model in which all STATs are
pre-associated with cytokine receptors is that the only system that requires such pre-association is the type I IFN pathway. In this scenario, the recruitment of STAT1 through RACK1 may reflect a more
stringent regulation of STAT activation due to the effect of type I
IFNs on cell proliferation, or may preclude the need for
phosphorylation of a specific tyrosine on the receptor for STAT1
activation. Unfortunately, we have not been able to address the latter
question, because activation of STAT1 is dependent on the previous
activation of STAT2 (11). However, addition of single tyrosines to
IFN Our data also suggest that once STAT1 is tyrosine-phosphorylated, it
can dissociate from RACK1 and the receptor complex. This is supported
by the finding that RACK1 interacts only with the inactive
(non-phosphorylated) form of STAT1 and that soon after type I IFN
stimulation STAT1 is almost exclusively detected in the nucleus,
whereas RACK1 remains in the cytoplasm. These data suggest that RACK1
should not be part of the ISGF3 or It should be pointed out that the region of IFN Finally, it should be pointed out that RACK1, as well as the
receptor-RACK1 interaction abolishes not only IFN
-induced
tyrosine phosphorylation of STAT1 but also activation of STAT2,
indicating that RACK1 plays a central role in early signaling through
the Jak-STAT pathway. These findings demonstrate the involvement of RACK1 in STAT1 activation and raise the possibility that other STATs
may pre-associate with cytokine receptors through similar adapter-STAT-mediated interactions.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
, IFN
, IL6, leukemia inhibitory factor, IL10, etc.), studies with knockout mice
clearly indicated that their function is well restricted to precise
systems. For example, STAT1 is only required for the physiological
functions of IFN
and IFN
(reviewed in Ref. 7).
R
L chain (9, 10). Activation of STAT2 in
response to type I IFNs (IFN
,
, or
) requires the presence of
this constitutive site and one or more of the five proximal tyrosines
of the
L chain (9). However, the mechanism for STAT1 activation by
type I IFNs has not been elucidated. It is known that activation of
STAT1 requires the previous activation of STAT2 (11), but it has not
been determined whether receptor tyrosines are also required for
activation. This is in clear contrast to the activation of STAT1 by
IFN
, which requires docking of STAT1 to a phosphorylated tyrosine on
the
chain of the receptor (12).
(14-16), constitutively interacts with the
long subunit of the type I IFN receptor (IFN
R
L/IFNAR2). RACK1 has
a molecular mass of 36,000 daltons and is composed of 7 WD repeats that resemble the structure of the
subunit of G proteins (G
) (17, 18). RACK1 also interacts with protein kinase C
, src (19),
integrins (20), PDE4D5 (21), and the
common
subunit of the granulocyte/macrophage colony-stimulating factor/IL3/IL5 receptors (22).
subunit of the IFN
R
(IFN
R
L). No interaction between RACK1 and other STAT factors was
detected. The interaction between IFN
R
L and RACK1 is critical for
normal STAT activation and IFN signaling. This is supported by the
finding that mutations in the RACK1 binding site of IFN
R
L, which
includes the Box 2 motif, impaired IFN
-induced tyrosine
phosphorylation of STAT1 and STAT2 and the development of the antiviral state.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
2 (specific activity,
2.2 × 108 units/mg) was a gift of Ronald Bordens
(Schering-Plough). The anti-phosphotyrosine antibody 4G10 was purchased
from Upstate Biotechnologies Inc., and the anti-RACK-1, anti-STAT1,
-STAT2, -STAT5, and -Jak1 monoclonal antibodies were purchased from
Transduction Laboratories, Inc. Polyclonal antibodies against STAT3,
STAT4, STAT5, and STAT6 were kindly provided by Drs. Evan Parganas and James Ihle (St. Jude Children's Research Hospital, Memphis, TN). The
anti-Stat1 and -Stat2 sera were kindly provided by Dr. A. Larner
(Cleveland Clinic, Cleveland, OH). Antiviral assays were performed as
previously described (23, 24).
R
L have been described previously (25).
For mapping of the RACK1 binding site of IFN
R
L, a combination of
two or three alanine mutations per construct was introduced in the
GST
L300-375 that contains the minimum region that binds RACK1. GST
fusion expression constructs with mutations of the RACK1 site were made
by polymerase chain reaction using the Quickchange kit (Stratagene).
All mutations were confirmed by sequencing. A GST fusion protein
encoding the full-length RACK1 (GST-RACK1) was produced by polymerase
chain reaction and subcloned into the pGEX-KG vector. GST fusion
proteins were produced in BL-21 cells as described previously (25).
Pull-down experiments and immunoblotting were performed using
the same procedure described above for immunoprecipitations.
L overnight, washed, and
analyzed by SDS-polyacrylamide gel electrophoresis as described for immunoprecipitations.
R
L Constructs in Mammalian
Cells--
Mammalian expression constructs with mutations of the RACK1
site of IFN
R
L (amino acids 302, 304, and 305) were made by
polymerase chain reaction using the Quickchange kit (Stratagene). All
mutations were confirmed by sequencing. Constructs were subcloned into
the pLXSN retroviral vector and transfected into the
2 packaging cell line, and retrovirus-containing supernatants were used to transduce LpR
cells (L-929 cells stably expressing the human
chain of the receptor; Ref. 25). Stable transfectants were selected in
medium containing G-418 (500 µg/ml) and hygromycin B (500 µg/ml),
and positive clones were screened by fluorescence-activated cell sorter
using the IFNaR
1 monoclonal antibody (26) that recognizes
IFN
R
L.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
R
L (amino acids 300-346) (13) that is required for
activation of STATs and the antiviral response (25), we hypothesized
that RACK1 could recruit STAT1 to the receptor complex. We first
performed coimmunoprecipitation experiments using an anti-RACK1
monoclonal antibody to test for an interaction between RACK1 and STAT1.
Fig. 1A shows that the
anti-RACK1 antibody coimmunoprecipitated STAT1 (upper panel,
lane 6) but not STAT3 (lower panel, lane
6) or STAT2 (data not shown) present in unstimulated U266 cell
lysates. This interaction is specific, because it cannot be detected by
non-immune IgM used as negative control (Fig. 1A, lane 5). The anti-STAT1 and -STAT3 antibodies also
coprecipitated STAT3 and STAT1, respectively, as previously reported
(28). However, the strong signal for STAT3 detected in anti-STAT1
immunoprecipitates (Fig. 1A, lower panel,
lane 2) may correspond in part to incomplete stripping of
the membrane after STAT1 immunoblotting. This result strongly suggests
that STAT1 specifically interacts with RACK1. It should be noticed,
however, that we have not consistently been able to
coimmunoprecipitate RACK1 using anti-STAT1 antibodies. One
possible explanation is that both antibodies recognize epitopes close
to the C-terminal part of the protein, where the RACK1 binding site may
be located, and therefore disrupt the interaction.
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Fig. 1.
RACK-1 interacts with STAT1.
A, U-266 (2 × 107
cells/immunoprecipitation (IP)) cell lysates were
immunoprecipitated with the indicated polyclonal antibodies against
STAT proteins (anti-STAT1, -STAT2, or -STAT3) or normal rabbit
(NR) serum. RACK1 was immunoprecipitated
using a specific anti-RACK1 monoclonal antibody or control monoclonal
antibody. Immunoblotting was sequentially performed using anti-STAT1,
-STAT3, and -STAT2 (data not shown) antibodies. B-F,
pull-downs with GST-RACK1 were performed to determine whether RACK1
interacts with different STATs. U-266 (B, C, and
E) or Daudi (F) cells were used as a source of
the indicated STAT proteins. STAT4 was produced by in vitro
translation (D). Immunoblotting was performed with the
indicated anti-STAT antibodies. WB, Western
blot.
L also bound STAT1 present in
cell lysates (Fig. 1B). This result differs from our
previous observation using STAT1 produced in wheat germ in
vitro translation systems, in which no interaction between
IFN
R
L and STAT1 was detected (9). One possible explanation is
that a protein such as RACK1 present in cell lysates, but absent in the
wheat germ in vitro translation system, may serve as an adapter between IFN
R
L and STAT1 (see below).
R--
We reasoned that if RACK1 links STAT1 to IFN
R
L,
deletions or mutations in IFN
R
L that decrease RACK1 binding
should also decrease the association of STAT1 with the receptor. To
test this hypothesis, we performed pull-down experiments using GST
fusion proteins containing different regions of the cytoplasmic domain of IFN
R
L. Fig. 2A,
top panel shows that GST fusion proteins encoding the entire
cytoplasmic domain (lane 7, GST
L-wt) and proteins
truncated at amino acids 462, 375, and 346 (lanes 3-5), but
not at amino acids 265-299 (lane 2), were also able to
interact with STAT1. Similarly, a GST fusion protein encoding amino
acids 300-515 (Fig. 2A, lane 6,
GST
L300-515) and therefore lacking the first 35 amino acids of the
cytoplasmic domain (265) also interacts with STAT1. The same
GST-
L fusion proteins that bound STAT1 also interacted with RACK1
(Fig. 2A, lower panel). This result indicates
that the minimal interaction domain for STAT1 corresponds to
amino acids 300-346 of IFN
R
L and is identical to the RACK1
binding site (Fig. 2A, lower panel and Ref.
13).
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Fig. 2.
RACK1 and STAT1 interact with the same region
of IFN R
L.
A, GST fusion proteins encoding the entire cytoplasmic
domain of IFN
R
L (amino acids 265-515, wild type
(WT)), an N-terminal deletion (300), or truncations at
residues 462, 375, 346, and 299 (25) were used to map the STAT1 and
RACK1 binding sites. GST alone and an anti-RACK1 antibody were used as
negative and positive controls, respectively. Lysates obtained from
U-266 cells were used as a source of STAT1 and RACK1. RACK1 and STAT1
were detected by immunoblotting with specific monoclonal antibodies
(Transduction Laboratories). The residues of IFN
R
L encoded by
each GST fusion protein are indicated. WB, Western
blot; CTRL, control. B, mapping of the RACK1
binding site within the 300-375 region of IFN
R
L. Upper
panel, pull-down experiments were performed with GST
L300-375
carrying combined mutations of the indicated amino acids to alanine.
U-266 cell lysates were used as a source of RACK1 and STAT1.
Precipitates were resolved using SDS-polyacrylamide gel
electrophoresis, transferred to polyvinylidene difluoride, and blotted
with anti-STAT1 and -RACK1 monoclonal antibodies. Lower
panel, the same GST fusion proteins used for the pull-down
experiment were analyzed by SDS-polyacrylamide gel electrophoresis and
stained with Coomassie Blue as a control. C, RACK-1
links STAT1 to IFN
R
L. [35S]Methionine-labeled RACK1
and STAT1 were produced alone (lanes 1-4 and
5-8, respectively) or in combination (lanes
9-12) using a T7 in vitro transcription/translation
kit. In vitro translated STAT1 and RACK1 were incubated with
GST or GST-
L or immunoprecipitated with anti-STAT1 and anti-RACK1
antibodies as positive controls. In independent experiments, low
amounts of STAT1 were coprecipitated by the anti-RACK1 antibody when
in vitro translated STAT1 was used as input. This is
probably due to some degree of interaction between STAT1 and the RACK1
homolog present in wheat germ, because RACK1 is relatively conserved
among species.
L, some mutations
produced a decrease in binding of STAT1 to IFN
R
L that paralleled
the decrease in binding of RACK1 to this receptor chain (Fig.
2B). The most intense reduction in binding was observed when
amino acids within the region 302-305 and 314-327 of IFN
R
L (Fig. 2B, lanes 2 and 6-8)
were mutated to alanine. The overlapping in RACK1 and STAT1 binding
sites strongly supports the concept that RACK1 functions as an adaptor
between IFN
R
L and STAT1.
R
L and STAT1 is detected in
cellular lysates (Figs. 1B and 2A), the
association between these proteins is not observed when STAT1 is
produced in wheat germ lysates (9). A possible explanation for this is
that RACK1 functions as an adaptor between IFN
R
L and STAT1 and
that the wheat germ homolog of RACK1 fails to interact with
IFN
R
L, STAT1, or both. Therefore, we assessed the ability of
GST-
L to bind STAT1 produced alone or together with RACK1 using a
wheat germ in vitro translation system. Fig. 2C
shows that GST-
L interacts with RACK1 but not STAT1 when these
proteins are produced separately (Fig. 2C, lanes
2 and 6). However, when RACK1 and STAT1 are in vitro translated together GST-
L pulls down STAT1 (Fig.
2C, lane 10). Thus, IFN
R
L and STAT1
interact only when RACK1 is present, strongly suggesting that RACK1
functions as an adaptor between IFN
R
L and STAT1.
R
L, RACK1, and STAT1 form a complex, they do not address
whether RACK1 interacts with the phosphorylated form of STAT1. This is
an important issue because once STAT1 is phosphorylated it must detach
from the receptor to form a DNA-binding complex in association with
STAT2 and p48. To address this issue, STAT1 phosphorylation was induced
by treating U-266 cells with IFN
for 15 min. Then, we assessed the
ability of GST-RACK1 and/or GST-IFN
R
L to associate with
tyrosine-phosphorylated STAT1, as determined by immunoblotting with the
anti-phosphotyrosine antibody 4G10. Immunoprecipitations with an
anti-STAT1 serum or GST alone were used as positive and negative
controls, respectively. The resultant precipitates were divided in
equal parts, resolved in separate gels, and immunoblotted using either
anti-phosphotyrosine or anti-STAT1 antibodies. Fig.
3A shows that neither GST-
L
nor GST-RACK1 can precipitate the tyrosine-phosphorylated fraction of
STAT1 after IFN
treatment (lower panel, lanes
6 and 7), but both bind non-phosphorylated STAT1 in
control cells (upper panel, lanes 2 and
3) as well as the non-phosphorylated fraction after IFN
treatment (upper panel, lanes 6 and
7). As expected, the anti-STAT1 antibody precipitates the
phosphorylated and non-phosphorylated forms of STAT1 (Fig.
3A, lanes 4 and 8). Identical
results were obtained in similar experiments in which the same membrane
was first immunoblotted with anti-phosphotyrosine and then anti-STAT1 antibodies and the converse (data not shown). These results demonstrate that RACK1 interacts only with the non-phosphorylated form of STAT1 and
support the concept that STAT1 dissociates from the IFN
R
L-RACK1
complex after becoming phosphorylated to form a DNA-binding complex.
This was further demonstrated by the finding that 20 min after IFN
treatment almost all STAT1 was localized to the nucleus (Fig.
3B, panel f), whereas RACK1 fluorescence increased and remained in the cytoplasm (panel e). In
untreated cells, RACK1 is detected in the cytoplasm, as previously
reported (13), whereas STAT1 was present in both cytoplasm and nucleus (Fig. 3B, panels c and d,
respectively). The specificity of the immunofluorescence procedure is
demonstrated by the complete lack of signal when normal rabbit serum
and anti-IgM were used as negative controls for STAT1 and RACK1 (Fig.
3B, panels a and b),
respectively.
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Fig. 3.
RACK1 interacts with
non-tyrosine-phosphorylated STAT1. A, U-266 cells were
treated with huIFN 2 or left untreated as described in the legend to
Fig. 1. Cell lysates were used for pull-downs with control GST
(lanes 1 and 5), GST-
L (lanes 2 and
6), GST-RACK1 (lanes 3 and 7), or
control anti-STAT1 serum (lanes 4 and 8).
Anti-STAT1 or anti-phosphotyrosine (pTyr) monoclonal
antibodies (upper and lower panels, respectively)
were used for immunoblotting. WB, Western blot.
B, the cellular localization of RACK1 (a,
c, and e) and STAT1 (b, d,
and f) in ECV304 endothelial cells treated with IFN
(e and f) or left untreated (a-d) was
studied using confocal microscopy as previously described (13). Alexa
568-labeled (red; a, c, and
d) anti-mouse IgG heavy and light chain and Alexa
488-labeled (green; b, d, and
f) goat anti-rabbit secondary antibodies were used to detect
RACK1 and STAT1, respectively. Non-immune IgM and normal rabbit serum
(NR) were used as negative controls (a and
b). Translocation of STAT1 was achieved by treatment with
huIFN
for 20 min (e and f). Nuclear staining
is indicated (arrows).
R
L Is Critical for
Activation of STAT1, STAT2, and the Antiviral Response--
To further
determine the importance of the interaction between IFN
R
L, RACK1,
and STAT1 in IFN
signaling, we expressed the human IFN
R
L chain
with mutations of the RACK1 binding site in mouse L-929 cells. Several
stable clones expressing the mutant IFN
R
L chain (designated
L
R1) were selected by fluorescence-activated cell sorter
analysis (Fig. 4). We next tested whether
disruption of the interaction between the receptor and RACK1 would
prevent the activation of STAT1. Human IFN
2 induced significantly
lower levels of STAT1 phosphorylation in cells expressing mutations of
the RACK1 binding site of IFN
R
L (Fig.
5A, lanes 3 and
6). Interestingly, tyrosine phosphorylation of STAT2 was
also reduced by the mutation of the RACK1 site (Fig. 5A,
lanes 3 and 6). Normal tyrosine phosphorylation
of STAT1 and STAT2 was detected when cells were treated with
murine IFN
4 (Fig. 5A, lanes 2 and 5), demonstrating that the STAT pathway was functional
when activated through the endogenous mouse receptor. The decrease in
tyrosine phosphorylation of STAT1 and STAT2 in response to huIFN
treatment was not due to a defect in kinase activation, because
tyrosine phosphorylation of Jak1 was normal (Fig. 5A,
lower panel). These results indicate that the interaction
between IFN
R
L and RACK1 is not only important for normal
activation of STAT1 but also for STAT2 tyrosine phosphorylation through
a mechanism that remains to be elucidated (see "Discussion").
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Fig. 4.
Expression of mutant
IFN R
L chain in L-929
cells. Expression of IFN
R
L chain containing a mutation of
the RACK1 binding site was assessed using the IFNaR
1
monoclonal antibody (dotted line) or IgG2a (negative
control, solid line) as indicated under "Materials and
Methods." L-929 cells expressing the wild type receptor
(
Lwt) were used as a positive control. The results of
three of the four clones generated are shown.
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Fig. 5.
Mutation of the RACK1 site on
IFN R
L impairs STAT
activation and the induction of an antiviral state. A,
cells stably transfected with IFN
R
L wild type
(
Lwt) or with mutations of the RACK1 binding site
(
L
R1, clones 21 (cl.21) and 24 (cl.24))
were treated with 1,000 units/ml IFN
2 (h
) or murine
IFN
4 (m
) or left untreated (control,
CT) at 37 °C for 15 min. Cell lysates were
immunoprecipitated with anti-STAT1 and -STAT2 (upper panel)
or anti-Jak1 sera (lower panel). Immunoblotting was first
performed with the anti-phosphotyrosine (pTyr) antibody 4G10
(upper panel), followed by stripping and reblotting with the
precipitating antibodies (lower panels; STAT1, STAT2, and
Jak1). IP, immunoprecipitation; WB, Western blot.
B, the ability of IFN to protect cells against the
cytopathic effect (CPE) of encephalomyocarditis
virus was determined using a standard antiviral assay. Cells were
pre-incubated with concentrations of IFNs ranging from 1-500 units/ml
in Dulbecco's modified Eagle's medium containing 2% fetal bovine
serum for 18 h. The medium was removed and replaced with medium
containing a dilution of encephalomyocarditis virus stock (1/10,000)
that killed 100% of the cells in 24 h. Cell viability was
determined 24 h later using
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.
mu, murine.
2 was able to elicit an antiviral
response in cells expressing a mutation of the RACK1 binding site of
IFN
R
L (Fig. 5B,
L
R1, clones 21 and 24). Fig.
5B shows that huIFN
2 induced significantly lower levels
of protection against encephalomyocarditis virus than did murine
IFN
4, which activates the endogenous murine receptor, in two
independent clones expressing mutations of the RACK1 site. The level of
protection detected was also lower than that induced by huIFN
2 in
cells expressing the wild type receptor. These results demonstrate that recruitment of RACK1 to the IFN
R complex is critical for the activation of STAT1 and STAT2 and for the induction of an antiviral state.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
. The
RACK1-mediated interaction between STAT1 and IFN
R
L and the direct
association of STAT2 with the distal part of the same chain (9, 10)
demonstrate that activation of the STAT pathway by type I IFNs requires
the pre-association of STAT factors with the receptor.
R
L constructs in which all tyrosines had been substituted for
alanines failed to affect STAT1 phosphorylation independently of STAT2
phosphorylation. This result raises the possibility that, unlike the
IFN
receptor, where phosphorylation of tyrosine 440 of the
chain
is critical for STAT1 phosphorylation (12, 32), tyrosine
phosphorylation of the IFN
R is not critical for STAT1 activation.
-activated factor complex
and does not translocate to the nucleus. This and our previous finding
that RACK1 is not tyrosine-phosphorylated (13) further support the
concept that RACK1 is an adaptor or scaffold protein important in
targeting specific signaling components such as STAT1 to the
appropriate subcellular compartment for their activation.
R
L that interacts
with RACK1 appears to overlap, at least in part, with the Box 2 domain.
It has been proposed that this motif could play a role in the
activation of Jak1 by the IL2R
chain (33). Our results suggest that
the interaction between RACK1 and the Box 2 motif could be important
for the recruitment of specific signaling proteins such as STAT1 and/or
for providing the appropriate receptor configuration that allows Jaks
to activate signaling components such as STATs. Unfortunately, the high
levels of endogenous RACK1 make it extremely difficult to express
potential dominant-negative mutants in which the interaction between
RACK1 and STAT1 has been disrupted. Nevertheless, these experiments are
important to determine whether the impaired tyrosine phosphorylation of
STAT1 and STAT2 observed when RACK1 cannot interact with the receptor
is due only to the failure to recruit STAT1 or to the fact that RACK1
may also recruit other signaling proteins. Either mechanism may explain the finding that RACK1 is also required for efficient phosphorylation of STAT2. Thus, the biological significance of RACK1 could go beyond
the recruitment of STAT1. This is also suggested by the finding that
the Box 2 motif is important in signaling by cytokine receptors in
which STAT1 is not required for biological activity.
-subunit of G-proteins, binds pleckstrin homology domains, the SH2
domain of src, and protein kinase C
, raising the possibility that
these or other proteins with similar motifs may be recruited by RACK1
to cytokine receptors. We are currently addressing the possibility that
RACK1 recruits ubiquitously expressed proteins activated by type I IFNs
such as insulin receptor substrate-phosphatidylinositol 3-kinase, Akt, and Fyn
(34-36)2 to the receptor.
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ACKNOWLEDGEMENTS |
---|
We especially thank Drs. Evan Parganas and James N. Ihle for the generous gift of different antibodies. We also thank Andrew Larner for the anti-STAT1 and -STAT2 sera.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant GM54709 (to O. C.).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.
¶ Researchers at the laboratories of both authors contributed equally to this article.
To whom correspondence should be addressed: Dept. of
Pharmacology, University of Illinois, 835 S. Wolcott Ave., M/C868 Rm. E403, Chicago, IL 60612. Tel.: 312-413-4113; Fax: 312-413-4140; E-mail: ocolamon@uic.edu.
Published, JBC Papers in Press, April 11, 2001, DOI 10.1074/jbc.M100087200
2 C. Prejean, T. Sarma, O. Kurnasov, O. Usacheva, B. Hemmings, L. Cantley, D. A. Fruman, L. A. Morrison, R. M. Buller, and O. R. Colamonici, submitted for publication.
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ABBREVIATIONS |
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The abbreviations used are:
IFN, interferon;
STAT, Signal Transducer and
Activator of Transcription;
IL, interleukin;
IFNR, IFN
receptor;
RACK1, receptor for activated protein kinase
C;
IFN
R
L, long form of the
subunit of the IFN
R;
GST, glutathione S-transferase;
hu, human;
StIP, STAT3-Interacting Protein.
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