From the a Department of Pathology, University of Tennessee, Memphis, Tennessee 38163, the c Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Pushchino, Moscow Region, Russia, d Section of Hematology/Oncology, University of Illinois, Chicago, Illinois 60606, g Department of Medical Genetics and Microbiology, University of Toronto, Toronto, Ontario M5S 3E2, Canada, f Department of Pathology, New York University School of Medicine, New York, New York 10016, and the e Oncology Center, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21231
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ABSTRACT |
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The precise role of the different subunits
( The human type I interferon
(IFN)1 family is composed of
multiple subtypes of IFN In most cytokine systems tyrosine phosphorylation of the receptor
subunits was originally thought to be required for recruitment and
phosphorylation of the Stat factors by the receptor-Jak kinase complex.
This model holds true for some cytokine systems such as the IFN In the type I IFN system, the role of tyrosine phosphorylation of the
type I IFN-R in Stat binding and activation is not clear (19-21). For
example, certain cell lines expressing a variant form of the type I
receptor, which fails to phosphorylate the This report seeks to answer several related questions regarding the
mechanisms of Stat activation by type I IFNs. First, what receptor
domains are required for Stats 1 and 2 activation? Second, what is the
importance of tyrosine phosphorylation of the receptor subunits in
signaling? Third, does activation of Stat3 require tyrosine
phosphorylation of the Our data indicate the following. 1) Low levels of tyrosine
phosphorylation of Stat2 can be supported by the
phosphotyrosine-independent binding site on residues 404-462 of the
Cell Lines, IFNs, Antibodies, and Antiviral Assays--
Human
recombinant IFN GST Fusion Proteins--
Experiments were performed with GST
fusion proteins encoding the whole cytoplasmic domain of
Expression of Different Mutants of the Immunoblotting--
Cells were treated with different
concentrations of the indicated IFNs for 10 min and rapidly solubilized
in lysis buffer (20 mM Tris, pH 7.5, 150 mM
NaCl, 10 mM sodium pyrophosphate, 20 mM NaF, 1 mM EDTA, 1 mM MgCl2, 1 mM dithiothreitol, 0.5% Triton X-100, 10 µg/ml
leupeptin, 10 µg/ml aprotinin, 100 mM
phenylmethylsulfonyl fluoride, 200 µM sodium
orthovanadate). Lower concentrations of NaCl (50 mM) were
used for coprecipitation of Stat2 with the receptor. Immunoprecipitation and immunoblotting were performed as described previously (4).
Radioiodination of Type I IFNs and Affinity
Cross-linking--
Radioiodination of IFN Electrophoretic Mobility Shift Assay (EMSA)--
Nuclear
extracts were prepared as described by Ghislain et al. (29)
and analyzed by EMSA using end-labeled ISRE and m67SIE oligonucleotides
to detect ISGF3 and c-sis-inducible factor complexes, respectively.
Stat2 Interacts with the 404-462 Region of
To map the Stat2-docking site on
We next studied whether Stat2 interacts directly with Heterologous Expression in L-929 and MEF
Finally, to explore the role of Activation of the Stat Pathway in Transfectants Expressing IFN
We next studied transfectants carrying simultaneous mutations of two
Stat2 sites. Therefore, these transfectants would be important to
determine if the remaining Stat2 site is enough to support Stat2
tyrosine phosphorylation. Fig. 3 shows that L
In summary, induction of normal levels of Stat2 tyrosine
phosphorylation by IFN
We also studied the role of the different tyrosines in the receptor
complex on the activation of Stat3. Interestingly, tyrosine phosphorylation of Stat3 was not affected by the absence of tyrosines on the
To determine whether the levels of activation of Stat2 and Stat1
detected in cells with mutations of the different Stat2 sites correlated with the formation of ISGF3, we performed EMSA using an ISRE
probe. Analysis of transfectants expressing mutation of only one Stat2
site revealed that mutation of Tyr Mutation of the Stat-binding Sites Impairs the Induction of an
Antiviral State--
We next explored the ability of the different
transfectants to support an IFN-induced antiviral state. We have
previously reported that deletion of the region of
Murine type I IFNs also failed to induce tyrosine phosphorylation of
Stat1 in MEF It is evident that tyrosine phosphorylation of receptor subunits
plays an important role in transmembrane signaling by some cytokines,
i.e. interleukin 6 and IFN We sought to test the hypothesis that there are redundant Stat2-docking
sites present on the Interestingly, mutations of all the tyrosines in the cytoplasmic domain
of the Our results also indicate that the study of Stat-docking sites using
phosphorylated peptides alone, without stable expression of the mutant
receptors in the appropriate cell lines, may lead to ambiguous
conclusions (19-21). The high concentrations of phosphorylated peptides used in some cases may result in non-physiological
interactions between SH2 domains and phosphopeptides. Songyang et
al. (35, 36) indicated that although SH2 domains have higher
affinities for a specific sequence, they could interact with more than
one sequence C-terminal to the phosphorylated tyrosine. Therefore, the
concentrations of phosphorylated peptides used in most studies (in the
order of 100 µM) may not represent physiological
interactions, unless the interactions are confirmed by mutational
analysis and expression in mammalian cells. In this scenario, stable
expression of the It has been proposed that Jak1 and Tyk2 are activated by
transphosphorylation (37-39) after heterodimerization of the /IFNAR1 and
L/IFNAR2) of the type I interferon
receptor (IFN-R) in the activation of signal transducer and activator
of transcription (Stat) 1, Stat2, and Stat3 has not yet been
established. In this report we demonstrate that there are functionally
redundant phosphotyrosine-dependent and -independent
binding sites for Stat2 in the
and
subunits of the type I
IFN-R. Expression of a type I IFN-R containing only the constitutive
Stat2 site or the proximal tyrosines of
L, but not the
docking site on the
chain (Tyr466 and
Tyr481), supported low levels of Stat2 activation. However,
the presence of only one intact Stat2 site did not lead to induction of
interferon-stimulated gene factor 3 (ISGF3) or an antiviral state.
Normal levels of Stat2 tyrosine phosphorylation, induction of ISGF3,
and an antiviral effect always required the proximal tyrosines of
L and at least one of the other Stat2 sites
(Tyr
466, 481 or
L404-462). These data
suggest that a threshold of Stat2 tyrosine phosphorylation is required
for complete activation of ISGF3. Interestingly, a receptor in which
all tyrosines were mutated to phenylalanine shows normal Stat3
phosphorylation and low levels of activation of Stat1.
INTRODUCTION
Top
Abstract
Introduction
References
(IFN
1, IFN
2, etc.), IFN
, and
IFN
(1). The human type I IFN receptor (IFN-R) or IFN
R is
composed of at least two subunits, termed
and
(also designated
as IFNAR1 and IFNAR2, respectively). The
subunit has two
transmembrane forms
Short (
S) and
Long (
L), both of which can bind IFNs with low affinity (2). High affinity binding occurs when both
and
either form of
are coexpressed; however, in the absence of
, the
chain cannot bind IFN (reviewed in Ref. 3). Each chain also
associates with a specific Jak kinase that is required for signaling;
the
chain docks Tyk2 (4, 5), whereas the
L subunit
interacts with Jak1 (6). Oligomerization of the receptor subunits
occurs upon ligand binding and induces intra- and intermolecular
phosphorylation of the Jak kinases (reviewed in Refs. 7-10). The
activated kinases are then thought to phosphorylate the receptor chains
and Stats 1, 2, and 3. The Stat molecules then homo- or heterodimerize
and migrate to the nucleus where they stimulate transcription of the
IFN-inducible genes (10-13).
and
interleukin 6 systems, where specific tyrosines in the
and gp130
subunits were shown to be essential for recruitment and phosphorylation
of Stat1 and Stat3, respectively (14, 15). However, recent reports
indicate that not all cytokine systems require receptor phosphorylation
for Stat activation, i.e. growth hormone and
granulocyte-colony-stimulating growth factor receptors devoid of
tyrosines can still activate Stat proteins (16-18).
subunit, are still
capable of producing an antiviral and antiproliferative effect in
response to IFN
2 (22). Moreover, Gibbs et al. (23) showed
that expression of a human
chain devoid of tyrosines in mouse
L-929 cells did not impair formation of the Stat1- and Stat2-containing ISGF3 complex. However, it is worth mentioning that
the role of the
chain of the receptor in Stat activation has not
been explored.
chain? In addition to the previously reported docking site for Stat2 on the
chain (Tyr466
and Tyr481), we have defined two new
Stat2-binding sites in the
L chain as follows: a
phosphotyrosine-independent constitutive binding region located at
amino acids 404-462, and a phosphotyrosine-dependent docking site formed by one or more of the five tyrosines N-terminal to
amino acid 346 (Tyr269, Tyr306,
Tyr316, Tyr318, and Tyr337).
L chain and the phosphotyrosine-dependent docking site on the proximal tyrosines of this chain but not by Tyr466 and Tyr481 of the
chain. However,
formation of ISGF3 and the induction of an antiviral state require the
proximal tyrosines of
L and at least one of the
remaining Stat2 sites (Tyr466 and Tyr481 of the
chain or amino acids 404-462 of
L). 2) There
appears to be a threshold of Stat2 tyrosine phosphorylation required
for complete ISGF3 activation and induction of an antiviral state. This
threshold is reached when at least two Stat2-docking sites are present
in the receptor complex. 3) Normal levels of tyrosine phosphorylation
of Stat1 require activation of Stat2. However, low levels of Stat1
tyrosine phosphorylation can be achieved in a receptor devoid of
tyrosines and Stat2-binding sites. 4) The presence of functional
tyrosine-independent docking sites on the IFN
R suggests that
other cytokine systems, i.e. growth hormone, may rely on
this mechanism to activate some Stat factors. 5) Tyrosine phosphorylation of Stat3 does not appear to require tyrosine
phosphorylation of the
or
L subunits. Finally, we
explored whether the expression of only
L would support
tyrosine phosphorylation of Stat2. Our data indicate that
L alone can support low levels of tyrosine phosphorylation of Jak1 and Stat2 but not Tyk2 and Stat1.
MATERIALS AND METHODS
2 (huIFN
2), IFNCon1, and IFN
(murine and human)
were kindly provided by Drs. Paul Trotta (Schering-Plough), Lawrence
Blatt (Amgen Biologicals), and S. Goelz (Biogen), respectively. Murine
IFN
was purchased from Access Biomedicals (San Diego, CA). The
anti-phosphotyrosine antibody (4G10) was obtained from Upstate
Biotechnology Inc. (Lake Placid, NY), and the anti-Stat2 serum was from
Santa Cruz Biotechnology. Monoclonal antibodies against Jak1 and Stat1
were purchased from Transduction Laboratories (Lexington, KY). The
anti-Stat1 and -Stat2 sera were kindly provided by Dr. A. Larner
(Cleveland Clinic Foundation, Cleveland, OH), and the anti-Jak1 and
-Stat3 sera were a generous gift of J. N. Ihle (St. Jude's
Children's Hospital, Memphis, TN). The polyclonal and monoclonal
antibodies against Tyk2 and the type I IFN-R subunits were described
previously (4, 24, 25). The mouse fibrosarcoma L-929 and
human myeloma U-266 cells were obtained from ATCC. Mouse embryonal
fibroblast null for the
chain (MEF
/
) (26) were a
kind gift of Dr. M. Aguet (Swiss Institute for Experimental Cancer
Research). Antiviral assays were performed as described previously (27,
28).
S (GST
S amino acids 265-331) and
L (GST
L265-515), as well as C-terminal
truncations of the cytoplasmic domain of
L at aa 462, 375, 346, and 299 (GST
L265-462, GST
L265-375, GST
L265-346, and
GST
L265-299, respectively, see Ref. 6). The
GST
L300-515 was produced by deletion of an
NcoI restriction fragment (from the NcoI site in
the pGEX-KG cloning site to the NcoI site at position 959 of the
L chain) from the GST
L265-515
construct (6). The GST fusion proteins were produced in BL-21 cells
(Novagen) and purified by affinity chromatography on
glutathione-Sepharose (Amersham Pharmacia Biotech). Five to ten µg of
the indicated GST fusion proteins were used for precipitations.
and
L
Subunit of Type I IFN-R in Mouse L-929 and MEF
/
Cells--
These constructs were generated using a polymerase chain
reaction-based protocol (Quickchange, Stratagene). All mutations were
confirmed by sequencing. The
and
L chain constructs
were subcloned into the pRep4 and pZipNeoSV(X) vectors, respectively, and used for expression in L-929 and
MEF
/
cells. L-929 transfectants were
selected in medium containing G-418 (500 µg/ml) and hygromycin B (500 µg/ml), and MEF
/
transfectants were grown only in
hygromycin B (500 µg/ml). Table I is a summary of the transfectants
produced. Fig. 2 shows a schematic representation of the wild type
(A) and mutated forms (B-D, top panels) of the
receptor used in this study. We produced the following cell lines
carrying mutations in only one Stat2 site: 1) L
YF526
L clones .4 and .22, corresponding to L-929 cells expressing wild type
L and the
chain without the Stat2 sites (stop codon
at position 526 and phenylalanine substituted for tyrosines 466 and
481); 2) L
L346.4, L-929 cells expressing
wild
type and
L truncated at position 346 to delete the
distal constitutive Stat2 site of
L (6); 3) L
LYF
clones .1 and .7, L-929 expressing wild type
chain and
L devoid of tyrosines and thus eliminating the proximal
tyrosines that serve as a Stat2-docking site (see below). The following
cell lines contain mutations in two Stat2 sites: 1) L
YF526
L346
clones .3 and .5, L-929 cells expressing
L
truncated at amino acid 346 to delete the constitutive Stat2-binding
site, and substitution of the tyrosines on the
chain
(Tyr466 and Tyr481) for phenylalanines; 2)
L
L346YF.1, corresponding to L-929 cells expressing
wild type
chain and
L truncated at amino acid 346 and with substitution of the remaining tyrosines of
L to
phenylalanines in order to delete both the constitutive site (aa
404-462) and the five proximal tyrosines of the cytoplasmic domain
(Tyr269, Tyr306, Tyr316,
Tyr318, and Tyr337) that serve as a docking
site for Stat2 (see below); and 3) M
YF
LYF, corresponding to MEF
null for the murine
chain (MEF
/
) that express
human
and
L chains without tyrosines (
YF526 and
LYF, respectively), but maintaining the constitutive Stat2 site of
L. The latter constructs were expressed in
MEF
/
because they appear to be toxic for
L-929 cells for reasons that are not clear. The
L
YF
L346YF clones 2 and 4 and M
YF
L346YF clones 7 and 9, corresponding to L-929 and MEF
/
, express
mutations of all three Stat2 sites. Finally, the M
L.1 cell line
corresponds to MEF
/
transfected with wild type human
L.
2 and affinity
cross-linking were performed as described previously (24).
RESULTS
L--
To determine whether Stat2 was able to interact
with the
L chain in vivo, we performed
coimmunoprecipitation experiments followed by Western blotting with an
anti-Stat2 serum. Fig. 1A shows that a polyclonal serum against
L coprecipitates
low levels of Stat2 in control U-266 cell lysates (lane 3,
L375-515 serum). However, the interaction between
L and Stat2 appears to be enhanced after IFN
treatment, since the levels of Stat2 coprecipitated by the
anti-
L serum relative to those precipitated by the
anti-Stat2 antibody used as positive control are higher in IFN-treated
(lanes 7 and 9) than in control cells
(lanes 3 and 4). As expected, Stat2 is
coprecipitated by an anti-Stat1 serum after IFN
treatment
(lane 8). No interaction between Stat2 and the
chain was
detected before or after type I IFN treatment (lanes 2 and
6), probably because this interaction is transitory and weak
as previously reported (Ref. 19, see also "Discussion").
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Fig. 1.
Direct association of Stat2 with
L. A, lysates obtained
from IFN
-treated (lanes 5-9) or untreated (lanes
1-4) U-266 cells were immunoprecipitated with antibodies against
the
(
511-557) or
L (
L375-515)
chains. Anti-Stat1 (lane 8) and -Stat2 (lanes 4 and 9) antibodies and normal rabbit serum (NR,
lanes 1 and 5) were used as positive and negative
controls, respectively. The control and IFN-treated samples were
analyzed in different gels. This accounts for the apparent lower
amounts of Stat2 immunoprecipitated after IFN
treatment.
B, lysates from IFN
2-treated (lower panel) or
untreated (upper panel) U-266 cells were precipitated with
GST fusion proteins encoding wild type
L
(
L265-515) or the indicated deletions of the
cytoplasmic domain. Precipitations with GST alone and
GST
S (lanes 1 and 2) as well as an
anti-Stat2 serum were used as negative and positive controls,
respectively. The lower signal for Stat2 precipitated with the
GST
L265-462 from control cells (lane 3, top
panel) is due to lower amounts of the fusion protein present in
this lane. C, GST fusion proteins encoding entire
cytoplasmic domains of
S,
L,
, and
IL10R
(GST-B4) chains were used to precipitate
[35S]methionine-labeled Stat1 (lanes 1-7) and
Stat2 (lanes 8-14) produced by in vitro
transcription/translation using a wheat germ lectin kit. Anti-Stat1 and
-Stat2 antibodies were used as positive control. An aliquot equivalent
to the input used for each precipitation was run directly in the gels
(Lysate). The migration of Stat1 and Stat2 is
indicated.
L, we performed binding
experiments with GST fusion proteins encoding the wild type form or
deletions of the cytoplasmic domain of
L. As a source of
Stat2 protein we used lysates from IFN
2-treated and control U-266
cells. Fig. 1B shows that Stat2 associates with wild type
GST
L (lane 7) and
L truncated
at amino acid 462 (lanes 3 and 10). However, more
proximal deletions at amino acids 404, 375, 346, and 299 (lanes
4-6 and 11) completely abrogated binding. Deletion of
the first 45 amino acids of the cytoplasmic domain that include the Box
1 motif (2 and 6), did not affect the interaction with Stat2 (GST
L300-515, lane 8). It is worth
mentioning that
L truncated at amino acid 462 bound
Stat2 with slightly less efficiency than the full-length cytoplasmic
domain. This may reflect a minor conformational change in the
Stat2-binding site produced by the deletion of the last 53 aa of
L. These results map the constitutive Stat2 binding region to amino acids 404-462 of
L. Treatment with
IFN
2 did not increase the association of Stat2 with
GST
L as detected by coprecipitation (Fig.
1A). This is probably due to fact that the GST fusion
proteins are not phosphorylated, and the increase in binding of Stat2
to
L after IFN treatment is accounted for by an
interaction between one or more phosphorylated tyrosines of
L and the SH2 domain of Stat2 (see below). No binding of
Stat2 to
S or a GST control was detected.
L
using a cell-free system that lacks adaptor proteins and tyrosine kinases. Stat2 was produced using a wheat germ in vitro
transcription/translation system, labeled with
[35S]methionine, and precipitated with different GST
fusion proteins (lanes 8-14). Stat2 produced in the wheat
germ system was precipitated by GST
L and the anti-Stat2
serum (Fig. 1C, lanes 10 and 13). No binding of
Stat2 to GST control or GST fusion proteins encoding the
,
S, or the IL10R
(CRFB4, Ref. 30) subunits was
observed (Fig. 1C, lanes 8, 9, 11, and 12, respectively). In vitro translated Stat1 (Fig. 1C,
lanes 1-7) did not interact with GST
L, GST
, GST
S, or GST-CRFB4 (lanes 2-5). These
results indicate that the interaction between Stat2 and
L is direct, since Stat2 and the GST fusion proteins
were produced in cell-free systems (wheat germ lysates and bacteria,
respectively) which should not contain adaptor proteins.
/
of
Mutant Human
and
L Chains--
To determine the
role of the different Stat2-binding sites in IFN
signaling, we
developed stable transfectants expressing receptors containing single
or combined mutations of the Stat2 sites (Table
I). Fig. 2
shows a schematic representation of the wild type receptor
(A) and the different mutations expressed in mouse
L-929 or MEF
/
cells (Fig. 2, B-D,
upper panels). Fig. 2B and Table I show stable
transfectants expressing mutations of only one of the Stat2-binding sites: (i) L
YF
L lacking the tyrosine-docking sites on
the
chain (Tyr466 and Tyr481); (ii)
L
L346 cells that lack the constitutive Stat2-binding site on
L (aa 404-462) (6); and (iii) L
LYF cells miss the proximal tyrosines of the
L chain, which in the course
of this work were shown to serve as docking sites for Stat2 (see
below). We also produced transfectants in which different combinations of two Stat2-docking sites on the receptor chains have been mutated (Fig. 2C): (i) L
L346YF lacking the constitutive
docking site and the proximal tyrosines of
L; (ii)
L
YF
L346, lacking the constitutive site of
L (aa
404-462) and the tyrosines on the
chain (Tyr466 and
Tyr481); and (iii) M
YF
LYF, corresponding to
MEF
/
expressing human receptors in which all the
tyrosines were mutated to phenylalanine but maintaining the
constitutive Stat2-docking site on
L. For reasons that
are not clear, we could not isolate L-929 transfectants
expressing the latter receptor combination. In addition, we produced
L-929 (Fig. 2C, L
YF
L346YF) and
MEF
/
(data not shown) cell lines lacking all the
Stat2-docking sites. Cell-surface expression of the human receptor
subunits was studied by cross-linking 125I-IFN
2 to the
receptor, followed by immunoprecipitation with antibodies against the
human
and
L chains, as well as mouse
subunit.
Fig. 2, B and C, shows that the anti-human
subunit antibody IFNaR3 immunoprecipitated the appropriate form of the
chain, and in most cases a high molecular weight complex that corresponds to the association of the
and
L (31)
subunits in L-929 and MEF
/
transfectants
(Fig. 2, B and C, lanes 2, 5, 8, 11, 13, 17, and 19). The anti-
L serum detects the
L chain (wild type or truncated, lanes 3, 9, 12, 15, 18, and 20), the complex formed by
and
L, and coprecipitates the
chain as described
previously (31). The anti-mouse
subunit antibody failed to detect
any receptor component in all cell lines tested (lanes 4, 6, 7, 10, and 14) demonstrating that no interaction between
this mouse chain and human IFN
2 takes place in these transfectants.
The anti-human
511-557 serum that recognizes an epitope in the
human
chain between amino acids 526 and 557 (6), and therefore not
present in the
YF526 construct, fails to detect this mutated chain
(lanes 1 and 16).
Summary of stable transfectants
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Fig. 2.
Expression of and
L constructs in L-929
and MEF
/
cells. Expression of the different constructs was assessed by
cross-linking 125I-IFN
2 to the receptor followed by
immunoprecipitation with specific antibodies against the different
receptor subunits. A, diagram of the wild type receptor
subunits including sites for interaction with Jak kinases and Stat2.
B, L-929 transfectants expressing mutations of
only one Stat2 site (L
YF
L, L
L346,
and L
LYF). C, L-929 and
MEF
/
transfectants expressing receptor complexes
with mutations of two (L
L346YF, L
YF
L346, and
M
YF
LYF) and three (L
YF
L346YF)
Stat2-binding sites. The top panels (B and
C) show a schematic representation of the constructs
expressed in murine L-929 and MEF
[
/[
(transfectants whose designation starts with L or
M, respectively) cells. Bottom panels show
expression in mouse L-929 and MEF
/
cells
of human receptor subunits. One clone of the various transfectant lines
is shown as an example. The following antibodies were used for
immunoprecipitation:
511-557 (
511) and IFNaR3 (6), recognize
different epitopes on the
chain (24, 31);
L515,
recognizes epitopes between amino acids 300 and 515 of the
L chain (31); and an antiserum against the murine
chain (mu
). Migration of the
,
L (*),
and the association of the
and
L chains
(
+
L) is indicated. A complete characterization
of the L
L346 cell line was previously reported (6); therefore,
only immunoprecipitations with IFNaR3 and murine
subunit antibodies
are shown to demonstrate that 125I-IFN
2 does not
interact with the mouse
chain. The upper part shows the constructs
used for expression. D, expression of human
L
in MEF
/
cells. The electrophoretic mobility of
previously unidentified complexes is indicated (left panel,
arrowhead and 170). The dashed lines
correspond to molecular mass markers, 184, 116, and 84 kDa (from
top to bottom).
L in Stat and Jak
activation, we developed a MEF
/
cell line expressing
only the human
L chain (Fig. 2D,
M
L). Fig. 2D (lane 3) shows that
the
L antisera detects this subunit in cells expressing
L wild type, whereas the anti-mouse or -human
chain
antibodies (lanes 1 and 2, respectively) do not
detect any affinity cross-linking complexes in these cells.
Interestingly, a 170-kDa and a higher molecular weight complex are
detected in M
L cells (Fig. 2D, 170 and
arrow). The identity of these complexes is not clear, but
they may correspond to
chain dimers, cross-linking of
125I-IFN
2 to a surface protein in the proximity of the
receptor complex, or novel receptor components. It is worth mentioning that these complexes may have not been detected previously because they
show similar electrophoretic mobility as those formed by the
association of
and
L and
and
S.
R
Chains with Mutations of the Stat2-docking Sites--
We initially
studied stable transfectants expressing a single mutation of the
previously identified Stat2-binding sites, i.e. Tyr
466, 481 (19) and the constitutive site (20) on
L404-462 (Fig. 3,
L
YF
L and L
L346, lanes 1-6). We also studied
mutations of a Stat2-docking site on the proximal tyrosines of
L (Tyr269, Tyr306,
Tyr316, Tyr318, and Tyr337) whose
presence became evident in the course of these experiments (L
LYF,
lanes 7-9). In this context, transfectants lacking only one
Stat2-binding site in the receptor complex would be informative regarding the role of that particular site on Stat2 tyrosine
phosphorylation. HuIFN
2 (lanes 3 and 6) can
induce tyrosine phosphorylation of Stat2 (A) and Stat1
(B) in transfectants carrying mutations of only the
constitutive site of
L (
L404-462) or the
chain (Tyr466 and Tyr481). More
importantly, the levels of tyrosine phosphorylation were comparable
with those induced by murine IFN
(m
) used as positive control (lanes 2 and 5). However, mutation of the
tyrosines of
L (L
LYF, lanes 7-9)
significantly decreased huIFN
2-induced tyrosine phosphorylation of
Stat2 (compare human and murine IFNs in L
YF
L and
L
L346 with L
LYF, lanes
1-9). These data suggest that the tyrosines N-terminal to aa 346 of
L play a more important role in Stat2 tyrosine
phosphorylation than the remaining sites.
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Fig. 3.
Tyrosine phosphorylation of Stat1, Stat2, and
Stat3 in L-929 cells expressing mutant and
L chains.
A and B, cells expressing mutations of only one
(L
YF
L, L
L346, and
L
LYF cells, lanes 1-9), two
(L
YF
L346, M
YF
LYF, and L
L346YF,
lanes 10-17), or three (L
YF
L346YF,
lanes 18-20) Stat2-binding sites were stimulated with
muIFN
(lanes 2, 5, 8, 11, 16, and 19),
huIFN
2 (lanes 3, 6, 9, 12, 14, 17, and 20), or
left untreated (lanes 1, 4, 7, 10, 13, 15, and
18) at 37 °C for 10 min. Cell lysates were
immunoprecipitated with an anti-Stat2 (A), anti-Stat1
(B) sera, resolved by SDS-polyacrylamide gel
electrophoresis, and immunoblotted with an anti-phosphotyrosine
antibody (4G10, Blot, pTyr, upper panel).
Membranes were stripped and reprobed with an anti-Stat2 serum or
anti-Stat1 monoclonal antibody (A and B,
lower panels). C, a similar experiment as in
A and B, but lysates were immunoprecipitated with
an anti-Stat3 serum and immunoblotted with anti-phosphotyrosine. The
lower panel shows an immunoblotting with an anti-Stat3
antibody after stripping of the filters.
YF
L346 (lanes
10-12) containing only the docking site on the proximal tyrosines
of
L and M
YF
LYF cells (lanes 13 and
14) expressing only the constitutive Stat2-binding site on
the
chain can support low levels of tyrosine phosphorylation of
Stat2 in response to huIFN
2. By contrast, L
L346YF cells
(lanes 15-17) expressing only the
subunit-docking site
(Tyr466 and Tyr481) are unable to support
huIFN
2-induced Stat2 tyrosine phosphorylation. As expected, deletion
of all three Stat2 sites completely abolished tyrosine phosphorylation
of Stat2 (Fig. 3A, lanes 18-20) but allowed low levels of
tyrosine phosphorylation of Stat1 (Fig. 3B, lanes 18-20) in
response to huIFN
.
2 requires the proximal tyrosines of
L and at least one of the other Stat2 sites, the
constitutive site of
L or Tyr466 and
Tyr481 on the
chain. The proximal tyrosines and the
constitutive site of
L are probably the most important
sites since they alone can support low levels of tyrosine
phosphorylation, whereas Tyr466 and Tyr481 on
the
chain may play an accessory role in tyrosine phosphorylation of
Stat2. A decrease in tyrosine phosphorylation of Stat1 (Fig. 3B) was observed in transfectants that have impaired
tyrosine phosphorylation of Stat2 as previously reported (32). It is worth mentioning that huIFN
2 was at least as efficient as
muIFN
in inducing tyrosine phosphorylation of Jak1 and Tyk2 in
all transfectant cell lines (data not shown) indicating that the levels
of human receptor subunits expressed were sufficient to achieve full
activation of the system.
(Fig. 3C, lanes 1-3),
L
(lanes 4-6), or both (lanes 7-9) chains
strongly arguing against the requirement of tyrosine phosphorylation of
the receptor for Stat3 activation and the role of this factor as an
adaptor for PI3K (see "Discussion").
466, 481 (Fig.
4, lanes 1-3) or the
constitutive site in
L (see Ref. 6) did not affect ISGF3
induction by huIFN
2. On the contrary, mutation of the proximal
tyrosines of
L (Fig. 4, L
LYF, lane 5) significantly impaired the formation of ISGF3. Similarly, a significant decrease (Fig. 4, L
YF
L346, lane 8) or
complete abrogation of the induction of the ISGF3 complex in response
to huIFNs is observed in transfectants carrying mutations of two
(L
L346YF and M
YF
LYF, lanes 12 and 18, respectively) or three (L
YF
L346YF, lane 15)
Stat2-binding sites. Formation of the ISGF3 complex was blocked by an
excess of cold ISRE oligonucleotide (lane 10).
Interestingly, although low levels of tyrosine phosphorylation of Stat2
were observed in some cases (Fig. 2, A and B,
L
LYF, L
YF
L346, and M
YF
LYF cells), this was
not sufficient to produce significant amounts of ISGF3, suggesting that
a threshold of Stat2 tyrosine phosphorylation has to be reached to
obtain ISGF3 formation. The formation of Stat3 containing
c-sis-inducible factor complexes in L
YF
L
and L
YF
L346YF cells treated with human type I IFNs was not
affected (data not shown).
View larger version (16K):
[in a new window]
Fig. 4.
EMSA with an ISRE probe. Cell extracts
from different transfectants were treated as indicated and used for
EMSA with an ISRE probe. The migration of the ISGF3 complex is
indicated. In some experiments a preparation of huIFN Con1
(huCon, lane 2) that has similar biological
activity as IFN
2 was used. L-929 cells were used as
positive control. Formation of ISGF3 was also competed by a 50-fold
excess of unlabeled ISRE oligonucleotide.
L
containing the Stat2-binding site (
L346 cells) did not affect the
induction of an antiviral state by IFN
2 (6). Similarly, 50%
protection against the encephalomyocarditis virus was observed with 5 and 14 units/ml huIFN
2 and muIFN
, respectively, in
YF
L cells (Table II)
expressing mutation of the Stat2-docking sites (Tyr466 and
Tyr481) on the
chain. However, mutation of the
proximal tyrosines of
L (Table II,
L
LYF cells) or simultaneous mutation of two Stat2-binding sites (L
YF
L346, L
L346YF, M
YF
LYF, and
L
YF
L346YF cells) significantly impaired the antiviral
response to huIFN
2 but did not affect the antiviral effect of
muIFN
(except in the MEF
/
transfectants that are
null for the
chain and therefore unresponsive to muIFN
4). The
antiviral data correlate with the levels of tyrosine phosphorylation
and ISGF3 induction observed in these cells.
Antiviral response
L Is Sufficient to Support Tyrosine Phosphorylation
of Jak1 and Stat2 but Not Tyk2 or Stat1--
To investigate further
the role of
L in activation of Stat2, we studied the
induction of tyrosine phosphorylation of this factor through the
endogenous murine
L chain expressed in MEF cells null
for the
chain (MEF
/
) using muIFN
, and in
MEF
/
transfected with the human
L
chain using human type I IFNs. This system allows us to completely
eliminate any role that the
chain, human or murine, could play in
the activation of Stat2. Moreover, it allows us to determine if
L alone is sufficient to activate other proteins of the
Jak-Stat pathway. Fig. 5A
shows that stimulation of the murine
L chain with
recombinant muIFN
induces Jak1 tyrosine phosphorylation, although to
a slightly lower extent than in L-929 cells (lane
2 and 4). As expected, treatment of
MEF
/
cells with recombinant muIFN
4 or muIFN
(Fig. 5A, lanes 5-7) did not induce significant
tyrosine phosphorylation of Tyk2 above base-line levels. Similarly,
huIFN
2 induced tyrosine phosphorylation of Jak1 in
MEF
/
cells expressing the human
L
chain (M
L cells, lane 9) but failed to activate
Tyk2 (data not shown).
View larger version (44K):
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Fig. 5.
Activation of the Jak-Stat pathway through
the L chain. A,
lysates from MEF
/
(lanes 1, 2, and
5-7), L-929 (lanes 3 and
4), or M
L (lanes 8 and
9) cells stimulated with muIFN
(m
,
lanes 2, 4, and 7), muIFN
4 (lane
6), and huIFN
2 (h
, lane 9), or left
untreated (CT, lanes 1, 3 and 8) were
immunoprecipitated with an anti-Jak1 antiserum (lanes 1-4,
8, and 9) and immunoblotted with anti-phosphotyrosine
antibodies (upper panel). Filters were stripped and blotted
with an anti-Jak1 antibody (lower panel). Lanes
5-7 show a similar experiment performed with
MEF
/
, but immunoprecipitations were performed with
an anti-Tyk2 antiserum. B, a similar experiment as in
A in which lysates from MEF
/
(lanes
1-3), L-929 (lanes 4-6), or
M
L (lanes 7-9) were immunoprecipitated with
anti-Stat1 (lanes 1-6) and anti-Stat2 (lanes
7-9) antibodies. Upper and lower panels
correspond to anti-phosphotyrosine (pTyr) and Stat1 or Stat2
immunoblots, respectively.
/
(Fig. 5B, lanes 2 and
3). The experiments aimed to determine whether different
preparations of murine IFN,
or
, induced tyrosine
phosphorylation of Stat2 in MEF
/
cells were
inconclusive (data not shown). Similar experiments performed with
M
L cells, however, showed that human
L
alone can support at least low levels of Stat2 tyrosine phosphorylation (Fig. 5B, lanes 8 and 9). These results clearly
demonstrate that
L alone can support low levels of
activation of Jak1 and Stat2 but not tyrosine phosphorylation of Tyk2
and Stat1 which require the presence of the
chain.
DISCUSSION
(14, 15). More precisely, tyrosines in different receptor subunits serve as docking sites for
Stat factors that are recruited to the receptor complex where they
serve as targets for tyrosine kinases, probably of the Jak family. It
is worth noting, however, that in some cytokine systems, i.e. growth hormone and granulocyte-colony-stimulating
growth factor, activation of Stat factors appears to occur in the
absence of tyrosines in the receptor (16-18). In the type I IFN system there are contradicting data regarding the role of the receptor subunits in the activation of Stat factors. For example, one report indicated that Tyr466 and Tyr481 of the
chain served as docking sites only for Stat2 (19), whereas others
indicated that the same tyrosines were able to bind not only Stat2 but
also Stat1 and Stat3 (20). In addition, Gibbs et al. (23)
indicated that cells expressing
chains devoid of tyrosines have
normal levels of activation of ISGF3.
and
chains of the receptor. We first
mapped a constitutive docking site in
L (20).
Surprisingly, elimination of both the constitutive site of
L and Tyr466 and Tyr481 of the
chain decreased, but did not abolish, Stat2 activation. The
additional elimination of the five proximal tyrosines of
L was required to completely abrogate activation of
Stat2. Therefore, there are redundant, yet still distinct mechanisms of
docking for Stat2, i.e.
phosphotyrosine-dependent and -independent, in the
and
chains of the receptor. Our results clearly demonstrate that there
are three Stat2-docking sites in the receptor complex as follows:
(a) a constitutive, phosphotyrosine-independent site on
L (aa 404-462); (b) one or more of the
proximal tyrosines (Tyr269, Tyr306,
Tyr316, Tyr318, and Tyr337) also in
L; and (c) Tyr466 and
Tyr481 in the
chain. Interestingly, isolated expression
of either the constitutive site or proximal tyrosines of
L was enough to support low levels of tyrosine
phosphorylation of Stat2/Stat1 but not for induction of ISGF3 or an
antiviral response. A receptor complex containing only the
Stat2-docking site in the
chain was unable to phosphorylate Stat2
suggesting, contrary to previous reports (19, 20), that this site may
only play a secondary role in activation of Stat2. This is also
supported by the detection of ISGF3 induction in cells that do not
phosphorylate the
chain (22) and by the finding that Stat2 is not
coprecipitated with the
chain after IFN
or IFN
treatment
(Fig. 1A). The presence of the proximal tyrosines of
L and at least one other Stat2 site within the receptor
complex was required to obtain significant phosphorylation activation
of Stat2/1, ISGF3 induction, and an antiviral response. These results
suggest that the proximal tyrosines of
L are absolutely
required, but not sufficient, for full phosphorylation and activation
of Stat2. Moreover, a threshold of Stat2 tyrosine phosphorylation
should be reached to achieve full activation of this factor, leading to
efficient formation of the ISGF3 complex and an antiviral state. In
this respect, the activation of the type I IFN system appears to be
different from other cytokine systems in which the presence of only one
Stat-docking site is enough to support Stat-mediated DNA binding
activity (14, 15). These results also strongly argue against a
mechanism in which Stat2 is transferred from the constitutive docking
site on
L to a phosphorylated tyrosine on the
chain,
to be finally phosphorylated by a Jak kinase (20). It appears that, at
least in part, the redundancy of Stat2-docking sites serves as a safety
net to preserve the response of a system that serves as the first line
of defense against viral infections. A decrease in activation of Stat1
accompanied the diminished activation of Stat2 as previously reported
(32).
chain did not affect activation of Stat3. Moreover, tyrosine
phosphorylation of Stat3 was even detected at normal levels in a
receptor complex completely devoid of tyrosines. A previous report
suggested that tyrosines 527 and 538 of the
chain are required for
Stat3 phosphorylation and subsequent activation of PI3K (21). Several
lines of evidence argue against the role of phosphorylation of these
tyrosines in IFN signaling. First, all the evidence suggests that
Tyr466 and Tyr481, rather than
Tyr527 and Tyr538 are the targets for tyrosine
phosphorylation (4, 19). Therefore, even if Tyr527 is in a
region containing a consensus sequence for the SH2 domain of Stat3,
phosphorylation of this residue should occur for SH2 binding. Neither
the mutational analysis (Fig. 3 and Refs. 4, 19, and 23) nor the
in vivo data (22) provide evidence that Tyr527
and Tyr538 are phosphorylated. Second, mutation of
Tyr527 and Tyr538 or deletion of the region
that includes these tyrosines does not affect IFN
signaling (Fig. 3
and Ref. 23). Third, PI3K appears to be associated with and activated
through IRS1/2 rather than Stat3 (33,
34).2 Fourth,
L-929 cells expressing receptors without tyrosines and
L346 (L
YF
L346YF) have normal IFN
-induced
PI3K-mediated serine phosphorylation.2 Therefore, the
finding that activation of Stat3 persists after mutation of the two
proximal tyrosines and deletion of the distal region containing the
putative Stat3-docking site of the
chain strongly argues against a
role for tyrosine phosphorylation of this receptor subunit in Stat3
activation and the role of this factor as an adaptor.
chain with mutations of Tyr466 and
Tyr481 (Stat2-docking site) and deletion of the distal
tyrosines (Stat3-docking sites) reveals that the results with peptides
in permeabilized cells belied the direct effect expected from
disrupting the interaction of the
chain with Stat2 or Stat3 (19,
21).
and
chains of the receptor. It is clear that activation of both kinases
is required for IFN
2 signaling, although a residual response for
IFN
can be observed in the absence of Tyk2 (38-40). Our results clearly indicate that activation of Jak1 occurs in the complete absence
of
chain and Tyk2 activation. Moreover, this activation is
sufficient to support low levels of Stat2 phosphorylation but not Stat1
by both IFN
and IFN
. Activation of Jak1 could be explained by
homodimerization of two
L chains or by
heterodimerization with a novel receptor component. The finding of a
novel 170-kDa complex in affinity cross-linking experiments performed
with MEF
/
transfected with
L wild
type (Fig. 2D) suggest that there are novel receptor chains,
whereas the higher molecular weight complex may be indicative of
chain dimers. Alternatively, the
L chain alone may be
sufficient to activate Jak1. Whatever the mechanism involved, we did
not find differences in Jak1 or Stat2 activation by IFN
and IFN
as previously reported for mutants lacking Tyk2. This is not surprising
because the activation of this pathway by these IFNs relies on the
selective requirement by IFN
of the 417-462 region of
L (41).
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ACKNOWLEDGEMENTS |
---|
We thank Drs. Michell Aguet and James Ihle
for providing the MEF null for the chain and the anti-Jak1 and
Stat3 sera, respectively.
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FOOTNOTES |
---|
* This work was supported by National lnstitutes of Health Grants GM54709 (to O. R. C.) and CA73381 (to L. C. P).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.
b Both authors contributed equally to this work.
h To whom correspondence should be addressed: Dept. of Pharmacology, University of Illinois, 835 S. Wolcott, Rm. E403 (M/C 868), Chicago, IL 60612. Tel.: 312-413-4113; Fax: 312-996-1225; E-mail: ocolamon{at}uic.edu.
The abbreviations used are: IFN, interferon; Stat, signal transducer and activator of transcription; aa, amino acids; ISGF3, interferon-stimulated gene factor 3; GST, glutathione S-transferase; ISRE, interferon-stimulated response element; EMSA, electrophoretic mobility shift assay; hu, human; mu, murine; PI3K, phosphatidylinositol 3-kinase.
2 S. Uddin, O. R. Colamonici, and L. C. Platanias, manuscript in preparation.
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REFERENCES |
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