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J. Biol. Chem., Vol. 277, Issue 2, 1493-1499, January 11, 2002
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,,,,, and¶
From the Berlex Biosciences Inc., Richmond,
California 94804 and the § Cleveland Clinic Foundation,
Cleveland, Ohio 44195
Received for publication, September 17, 2001
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Type I interferons (IFNs) are cytokines that play
a central role in mediating antiviral, antiproliferative, and
immunomodulatory activities in virtually all cells. These activities
are entirely dependent on the interaction of IFNs with their particular
cell surface receptor. In this report, we identify two specific
tyrosine residues located within the cytoplasmic domain of
IFNAR2c that are obligatory for IFN-dependent
signaling. Various IFNAR2c tyrosine mutants were expressed in a human
lung fibroscarcoma cell line lacking IFNAR2c (U5A). Stable clones
expressing these mutants were analyzed for their ability to induce
STAT1 and STAT2 activation, ISGF3 transcriptional complex formation,
gene expression, and cell growth regulation in response to
stimulation with type I IFNs. The replacement of all seven
cytoplasmic tyrosine residues of IFNAR2c with phenylalanine
resulted in a receptor unable to respond to IFN stimulation.
Substitution of single tyrosines at amino acid residue 269, 316, 318, 337, or 512 with phenylalanine had no effect on
IFN-dependent signaling, suggesting that no single tyrosine
is essential for IFN receptor-mediated signaling. In addition, IFNAR2c
retaining five proximal tyrosines residues (269, 306, 316, 318, and
337) or either two distal tyrosine residues (411 or 512) continued to
be responsive to IFN stimulation. Surprisingly, the presence of only a
single tyrosine at either position 337 or 512 was sufficient to restore
a complete IFN response. These results indicate that
IFN-dependent signaling proceeds through the redundant
usage of two tyrosine residues in the cytoplasmic domain of IFNAR2c.
Many cell surface receptors contain tyrosine residues that are
phosphorylated upon ligand-induced receptor activation. These phosphorylated tyrosines are specifically recognized by Src homology domains (SH2 or SH3) that are present in many transcription factors, phosphatases, and other mediators of downstream signaling (1). The
binding of SH2 or SH3 domains to phosphorylated tyrosines is, in many
cases, an absolute requirement for the activation of transcription
factors (2). In addition, these phosphotyrosine residues are essential
mediators of the interactions of specific phosphatases with certain
cell surface receptors (3-7). For some receptors, the mutation or
deletion of specific tyrosines completely abrogates
ligand-dependent signaling, whereas other cell surface receptors, such as the erythropoietin receptor, do not appear to depend
upon any one particular tyrosine but instead are capable of utilizing
any one of several tyrosines for signaling (8). Overall, such
observations demonstrate the importance of tyrosine residues in
signaling and suggest that in certain pathways particular tyrosine
residues can mediate the activation of specific transcription factors
that may in turn induce the expression of a distinct subset of genes.
One example of how intracellular tyrosines are utilized in signaling is
the type I interferon (IFN)1
pathway. Human type I IFNs ( It is thought that the phosphorylation of tyrosine residues on
IFNAR1 and IFNAR2c plays a critical role in the activation of STAT1 or
STAT2. The proposed STAT2-binding site on IFNAR1 includes two
phosphorylated residues, Tyr466 and Tyr481
(20). STAT-binding sites on IFNAR2c have also been proposed from
studies using IFNAR2c truncation mutants expressed in human (21) or
mouse cell lines (22). In human cells, truncation of the cytoplasmic
domain of IFNAR2c to amino acid 417 produces a receptor that is unable
to mediate the phosphorylation of STAT1 or STAT2. Expression of a
specific tyrosine mutant of human IFNAR2c in mouse L929 cells suggests
that the tyrosine phosphorylation of at least one of the proximal
tyrosines (Tyr269, Tyr306, Tyr316,
Tyr318, and Tyr337) of IFNAR2c is required but
not sufficient by itself, for STAT2 activation (22). Thus, in both
human and mouse cells, IFN-dependent signaling requires
both the presence of specific tyrosines within the cytoplasmic domains
of the receptor chains and the presence of STATs competent to interact
with these domains.
To examine the role of tyrosines present in the cytoplasmic domain of
IFNAR2c, we have expressed tyrosine mutants of IFNAR2c in U5A cells,
which do not express IFNAR2c, and have analyzed their ability to
respond to IFN (23). This approach has allowed us to analyze, for the
first time, the functional importance of specific tyrosine residues of
IFNAR2c in a human cell background.
Cell Lines and Reagents--
All cell lines, unless otherwise
noted, were purchased from the American Type Culture Collection,
cultured in Dulbecco's modified Eagle's medium (Invitrogen)
supplemented with 10% (v/v) heat-inactivated fetal bovine serum, 2 mM L-glutamine, 1% penicillin and streptomycin (Invitrogen), and incubated at 37 °C in 5% CO2. U5A
cells were obtained as previously described (21). Human IFN Selection of U5A Cell Lines Stably Expressing IFNAR2c Tyrosine
Mutants--
The mutants analyzed in this study are shown in Fig. 1. A
vector expressing IFNAR2c and a form of IFNAR2c in which all the intracellular tyrosines were substituted with phenylalanine,
pZIFN Immunoprecipitation and Immunoblotting--
STAT1, STAT2, and
IFNAR2c were immunoprecipitated from each mutant cell line as
previously described (21). Briefly, 1 × 107 cells
were solubilized in lysis buffer (20 mM Tris-HCl, pH 7.5, 1% Nonidet P-40 (v/v), 150 mM sodium chloride, 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, and insoluble material was
removed by centrifugation. For immunoprecipitation, the indicated
antibodies were added to each sample, incubated overnight, mixed with
Protein-G agarose (Roche Molecular Biochemicals), and resolved by
SDS-PAGE (10% Tris-glycine). The proteins were transferred to
polyvinylidene difluoride membranes (Pro-Blot, Applied Biosystems,
Inc.), and the membrane was placed in blocking buffer (20 mM Tris-HCl, pH 7.5, 0.1% Tween 20 (v/v), 150 mM sodium chloride, 1 mM EDTA, 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) overnight at room
temperature, incubated with the appropriate antibody (1:1000 dilution),
and washed in blocking buffer. Following washing, the membrane was
incubated with a specific horseradish peroxidase-conjugated secondary
antibody (1:1000 dilution) (BIOSOURCE
International) for 1 h, washed in blocking buffer, and developed
using a chemiluminescent detection method (Pierce). To reprobe
immunoblots, membranes were incubated overnight in 0.01 M
sodium citrate, pH 3.0, washed in blocking buffer, and probed again
with the appropriate antibody.
Ligand Binding Assay--
EZ-LabelTM human IFN Electrophoretic Mobility Shift Assay--
Gel shift assays were
performed using the 32P-labeled double-stranded
IFN-stimulated response elements oligonucleotide
5'-GCTTCAGTTTCGGTTTCCCTTTCCCGAG-3' and its complement
(25). Briefly, the cells were stimulated with IFN RNase Protection Assay--
The cells were stimulated with
either human IFN Cell Growth Assay--
The cells were seeded (5 × 104 cells/well) in a 6-well 35-mm cell culture plate and
allowed to attach overnight. Complete medium alone or containing
IFN The cell surface expression of IFNAR2c for all the stable IFNAR2c
tyrosine mutant clones (Fig. 1) was
confirmed by FACS (Fig. 2A)
and radioligand binding (Fig. 2B). FACS and radioligand
binding analysis correlated closely with each other (Fig. 2) and
demonstrated that the cell surface expression levels of IFNAR2c on all
the clones analyzed was equal to or greater than that observed in the
parental HT 1080 cell line. The only cell line in which IFNAR2c could
not be detected on the cell surface was the U5A cell line that was
previously shown to lack IFNAR2c (Fig. 2).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
, 
, and 
) have been shown to
induce the expression of a large number of genes involved in regulating a variety of important biological responses, including antiviral, antiproliferative, and immunomodulatory activities. The mechanisms by
which type I IFNs initiate such a broad spectrum of biological activities is only beginning to emerge. Type I
IFN-dependent signaling requires both type I IFN receptor
chains, IFNAR1 (human type I interferon receptor chain 1) and
IFNAR2c (human type I interferon receptor chain 2) (9-12). Binding
of type I IFNs induces the assembly of these receptor chains, which
leads to the phosphorylation of tyrosine residues located in the
intracellular domain of each receptor chain. These tyrosine
phosphorylation events are thought to be carried out by the Janus
kinases TYK2 and JAK1, which are themselves activated by tyrosine
phosphorylation (13, 14). IFN receptor activation also leads to the
phosphorylation and activation of the transcription factors STAT1 and
STAT2. Additional transcription factors, such as STAT3 (15),
phosphatidylinositol 3-kinase (16), mitogen-activated protein kinase
(17), and CrkL/STAT5 (18, 19), also become activated in response
to IFN. It is clear that phosphorylation of the cytoplasmic tyrosines of IFNAR1 and IFNAR2c is involved in type I IFN signaling; however, the
role that specific tyrosines play in this signal transduction pathway is unclear.
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1b
(specific activity 2.6 × 107 units/mg), IFN2
(specific activity 3.0 × 108 units/mg), and IFNAR2c
antisera were obtained as previously described (24). Antibodies against
STAT1, STAT2, and phosphotyrosine were purchased from Santa Cruz
Biotechnology. Phycoerythrin-conjugated anti-mouse Fab antibodies were
purchased from Dako A/S (Glostrup, Denmark).
RLYF (7F) (21, 22), was kindly provided by Dr. Oscar
Colamonici (Department of Pharmacology, University of Chicago, Chicago,
IL). The 7F construct was subjected to site-directed mutagenesis to add
back single tyrosines creating plasmids F269Y, F306Y, F316Y, F318Y, and
F337Y. The 7F plasmid was also used as a PCR template and cloned into
the vector pCDEF1, kindly supplied by Dr. Jerome A. Langer (Department
of Molecular Genetics and Microbiology, University of Medicine and
Dentistry of New Jersey, Piscataway, NJ) using HindIII and
SpeI. This construct was then mutagenized using the
QuikChange site-directed mutagenesis kit (Stratagene) to add back
single tyrosines at positions 411 and 512. A double tyrosine add-back
at residues 411 and 512 (4Y5Y) was also constructed in this vector.
Plasmids containing single tyrosine to phenylalanine substitutions
(Y269F, Y316F, Y318F, Y337F, and Y512F) were kindly provided by
Dr. M. Rubinstein (Department of Molecular Genetics and Virology, The
Weizmann Institute of Science, Rehovot, Israel). The Y512F plasmid was
mutagenized to create the double tyrosine knock-out at residues 411 and
512 (4F5F). U5A cells (1 × 106 cells/35-mm well) were
transfected using Superfect (Qiagen) as previously described (21).
Multiple stable cell populations for each IFNAR2c mutant were selected
in growth medium containing G418 (1 mg/ml). After drug selection, cell
populations were selected for single clones expressing IFNAR2c by
fluorescence-activated cell sorting (FACS) and cloned by limiting
dilution (FACS VantageTM SE, Becton Dickinson Bioscience,
San Jose, CA). For FACS analysis, 1 × 107 cells were
incubated with a purified monoclonal antibody (27D11), recognizing the
extracellular domain of IFNAR2c, for 1 h at 4 °C, washed with
ice-cold phosphate-buffered saline and then incubated with a
phycoerythrin-conjugated anti-mouse Fab antibody (Dako, Glostrup, Denmark) for 1 h at 4 °C. The resulting
individual clones were then screened by FACS, and only clones having a
FACS signal equal to or greater than the parental HT1080 cells were
picked and expanded. IFNAR2c expression for all clones was further
confirmed by using a radioligand binding assay (see below).
2
(PBL Biomedical Laboratories) was labeled with
-33P-labeled ATP as previously described (21).
Radiolabeled IFN2 (278 pM; specific activity of 60-62
µCi/µg) was added to 5 × 105 cells and allowed to
bind for 90 min at room temperature before the unbound IFN was washed
away with cell culture medium. Nonspecific binding was determined by
adding a 100-fold excess of unlabeled IFN1b to compete for binding
during the 90-min incubation. The cells were lysed in
phosphate-buffered saline containing 1% SDS, and the
radioactivity was determined by scintillation counting.
2 (1000 units/106 cells) or IFN1b (1000 units/106
cells) for 30 min, and the cell pellets were collected and processed for electrophoretic mobility shift assay as previously described (25).
The resulting DNA-binding complexes were separated by electrophoresis
through a 6% polyacrylamide gel and analyzed by autoradiography.
2 (1000 units/106 cells) or IFN1b
(1000 units/106 cells) for 17 h before RNA was
isolated, and gene expression was analyzed by an RNase protection assay
as previously described (21, 26).
1b (1500 units/106 cells, IFN1b) was added every
day for the duration of the experiment. Cell growth was determined
using the Alamar Blue method as previously described (27). Alamar
BlueTM (BIOSOURCE International) dye
reduction was measured after incubation with cells for 40 min at
37 °C using a Millipore fluorescence measurement system Cyberfluor
2300 (
emission = 590; excitation = 530).
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
View larger version (47K):
[in a new window]
Fig. 1.
IFNAR2c tyrosine mutants expressed in U5A
cells. Schematic representation of the cytoplasmic domain of
IFNAR2c tyrosine mutants expressed in U5A cells. The seven cytoplasmic
tyrosines of IFNAR2c were specifically mutagenized to produce 16 different mutants that were stably expressed in U5A cells. The residue
number for each of the seven tyrosines is indicated followed by the
single-letter amino acid code for that residue. U5A cells expressing
the wild type IFNAR2c (R2c) and a form of IFNAR2c in which
all the tyrosines have been replaced with phenylalanine (7F)
are shown. Single tyrosine-to-phenylalanine substitutions at residues
269, 316, 318, 337, and 512 are shown along with the double
tyrosine-to-phenylalanine substitution mutant having only the distal
tyrosines, Tyr411 and Tyr512, replaced with
phenylalanine (4F5F). The single tyrosine add-back clones
are shown in the lower half of the figure along with the
double add-back clone where all the tyrosines were replaced with
phenylalanine except for Tyr411 and Tyr512
(4Y5Y).
View larger version (25K):
[in a new window]
Fig. 2.
IFNAR2c tyrosine mutants are expressed on the
surface of U5A cells. A, FACS analysis of U5A cells
expressing IFNAR2c tyrosine mutants. IFNAR2c was detected on the
surface of U5A cells expressing IFNAR2c tyrosine mutants using a
specific monoclonal antibody (27D11) that recognizes IFNAR2c as
described under "Materials and Methods." Shown on the y
axis is the normalized geometric mean of the fluorescent
signal. The geometric means were normalized to the geometric mean of
the fluorescence signal observed for U5A cells. The data are
representative of the means (± 15%) derived from multiple experiments
(n 
2). B, radioligand binding of
phosphorylated IFN2 to U5A cells expressing IFNAR2c tyrosine
mutants. Ligand binding was performed as described under "Materials
and Methods." Ligand binding is presented on the y
axis as the fold increase in binding over U5A cells. All
values were corrected for nonspecific binding (100-fold excess
unlabeled IFN1b). The values were calculated from a representative
mean (± 15%) derived from experiments performed in quadruplicate.
HT1080, parental cell line; U5A, U5A cells alone.
All other cell lines are as described for Fig. 1.
Using these clones, we analyzed STAT1 and STAT2 activation by
phosphotyrosine immunoblotting after 15 min of stimulation with either
IFN2 or IFN1b (1000 units/ml/107 cells). For all
clones in which only a single tyrosine had been substituted with
phenylalanine, STAT1 and STAT2 activation were efficiently induced by
both IFN2 and IFN1b (Fig.
3A). IFN-dependent STAT activation was also observed in the two cluster mutants of IFNAR2c, one having both the distal tyrosines at amino acid positions 411 and 512 substituted to phenylalanine (4F5F) and the other having
all five proximal tyrosines (Tyr269, Tyr306,
Tyr316, Tyr318, and Tyr337)
substituted by phenylalanine (4Y5Y) (Fig. 3B). However, STAT activation was not observed when all seven tyrosines were absent (clone
7F) (Fig. 3B). Next, IFNAR2c mutants in which each of
the seven tyrosines were individually added back were assessed for their ability to induce STAT activation in response to IFN stimulation (Fig. 3C). Surprisingly, a single tyrosine at either amino
acid position 337 or 512 (clones F337Y or F512Y, respectively)
was sufficient to mediate IFN-dependent activation of STAT1
and STAT2. All of the remaining single tyrosine add-back clones (269Y,
306Y, 316Y, 318Y and 411Y) were unable to sustain any detectable
IFN-dependent STAT activation.
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After receptor activation, STAT1 and STAT2 are phosphorylated and form
a complex with the DNA-binding protein p48 to make up the
IFN-stimulated growth factor complex, ISGF3. The ISGF3 transcription
factor then translocates to the nucleus and binds to IFN-stimulated
response elements, located within the promoter regions of
IFN-responsive genes, to induce gene expression. Therefore, it was
important to determine whether the IFN-dependent tyrosine phosphorylation of STAT1 and STAT2, mediated by the various IFNAR2c tyrosine mutants, resulted in the assembly of a functional ISGF3 (Fig.
4). Cell lines expressing IFNAR2c
tyrosine mutants were stimulated with 1000 units/106 cells
IFN1b or IFN2 for 30 min, cells were harvested, and ISGF3 complex
formation was determined as described under "Materials and
Methods." In all cases, formation of an ISGF3 complex was detected in
IFNAR2c tyrosine mutants that were previously shown (Fig. 3) to be
capable of inducing tyrosine phosphorylation of STAT1 and STAT2 in
response to IFN stimulation.
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IFN stimulation results in the expression of a plethora of genes that
mediate a variety of important biological effects. To further
characterize IFNAR2c tyrosine mutants, we investigated the differential
expression of a number of IFN-inducible genes using an RNase protection
assay. The I-TAC, ISG 54, and TRAIL genes2 are preferentially
induced by IFN1b, as compared with various IFN species, whereas
the ISG 6-16 gene was induced by either IFN (21, 26). Briefly, 1 × 106 cells were stimulated with 1000 units/ml of either
IFN1b or IFN, cells were harvested, RNA was isolated, and the
RNase protection assay was performed as described under "Materials
and Methods." Induction of I-TAC, ISG 54, ISG 6-16, and TRAIL by IFN
appeared to be unaffected by any of the single
tyrosine-to-phenylalanine substitutions (Fig.
5A). As expected, both the U5A
cells and the 7F clone did not demonstrate IFN-mediated gene
expression, whereas the 4F5F and 4Y5Y cluster mutants were fully
functional (Fig. 5B). Furthermore, single tyrosine
add-backs, except for 337Y and 512Y, failed to exhibit
IFN-inducible expression of I-TAC, ISG 54, and TRAIL. Surprisingly,
IFN-dependent stimulation of the 337Y and 512Y clones
resulted in the full expression of all of the genes analyzed.
IFN-inducible gene expression in 337Y and 512Y was at levels equal to
or greater than those observed for the parental HT1080 cells (Fig.
5C). It is also of interest to note that the expression of
ISG 6-16, especially in response to IFN1b, was not completely lost
in the single tyrosine add-back clones 269Y, 306Y, 316Y, and 318Y (Fig.
5C).
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The relative levels of gene expression did vary somewhat among the
different IFNAR2c tyrosine mutants with the highest level observed in a
cell line expressing wild type IFNAR2c (Fig. 5). Finally, the
preferential induction of I-TAC, ISG 54, and TRAIL by IFN1b that was
observed in the parental HT1080 cell line was still present in both the
337Y and 512Y clones.
IFNs have potent antiproliferative effects on many different cell
types. Therefore, to evaluate the IFNAR2c mutants in terms of a
functional response, we measured the IFN-dependent
antiproliferative effects for each clone (Fig.
6). The results are expressed as the
percentages of growth compared with untreated controls. Cell growth
rates were measured for up to 6 days using Alamar Blue, as previously
described (27). As expected, in U5A cells expressing wild type IFNAR2c,
all the single tyrosine knock-out mutants and the proximal and distal
cluster mutants were able to express clear antiproliferative effects in
response to IFN1b stimulation (Fig. 6). Among the single tyrosine
add-backs, only the 337Y and 512Y IFNAR2c clones were able to
elicit an antiproliferative effect in response to IFN stimulation. The
magnitude of the effect was equal to or greater that that observed for
HT1080 or U5A cells expressing wild type IFNAR2c (R2c). This result
further illustrates the importance of Tyr337 and
Tyr512 in IFN-dependent signaling and extends
this observation to include their roles in the mediation of important
biological effects such as antiproliferative responses.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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The regulation of many cellular activities is mediated through the control of phosphorylation and dephosphorylation events. Many different cytokine and growth factor receptors contain specific tyrosine residues within their cytoplasmic domains that are required for signaling. In the case of the type I IFN receptor, tyrosine phosphorylation events have been shown to involve the JAK-STAT signaling pathway (28). The role of phosphorylation and dephosphorylation events in type I IFN signaling appears to be tightly controlled by the interplay of a complex system of different tyrosine kinases and phosphatases, which is not yet understood.
In terms of IFN signaling, STAT1 and STAT2 play a major role in type I IFN signaling, although STAT3 (15), STAT4 (29), and STAT5 (19) can also be activated in response to IFN in some cells. In all cases, ligand-dependent activation of STATs require that they become phosphorylated on specific tyrosines located in the C-terminal portions of the proteins. The phosphorylation of these tyrosines involves the receptor-associated Janus kinases JAK1 and TYK2, which themselves become activated by tyrosine phosphorylation. Activation of JAK1 and TYK2 requires the heterodimeric assembly of the two type I IFN receptor chains, IFNAR1 and IFNAR2c. It is thought that one of the first phosphorylation events carried out by activated Janus kinases is the specific phosphorylation of tyrosine residues on IFNAR1 and IFNAR2c.
Because it is known that the Janus kinases can phosphorylate various STATs, other mechanisms must be involved that confer signaling specificity. One of these mechanisms may involve the specific phosphorylation of either IFN receptor chain. Indeed, a number of studies have identified important tyrosines located within the cytoplasmic domain of many cytokine and growth factor receptors. It has been shown that these tyrosines can mediate the specific docking of adapter proteins, STATs, and phosphatases to the receptors (30). Indeed, specific tyrosines in IFNAR1 have been suggested to be involved in the regulation of IFNAR1-associated phosphatase activity (31) as well as STAT2 binding (20). Further studies have suggested that STAT2 and STAT1 preassociate with IFNAR2c and that phosphorylation of Tyr466 on IFNAR1 acts as an additional docking site and/or activation step for STAT2 that leads to STAT1 activation and the subsequent formation of a STAT1-STAT2 heterodimer (32). Recent studies using mutants of IFNAR2c having a truncated (21, 22) or substituted (20, 33-35) cytoplasmic domain lend support to the hypothesis that the cytoplasmic tyrosines of IFNAR2c play a role in the activation of STAT1 and STAT2. Furthermore, such studies suggest that tyrosines located in the cytoplasmic domain of IFNAR2c play an important role in STAT activation leading to IFN-mediated gene activation and antiproliferative effects. This assumption is supported by recent studies carried out using mouse L929 cells expressing a tyrosine mutant of human IFNAR2c in which it was shown that tyrosines in the cytoplasmic domain of IFNAR2c are important for IFN-dependent activation of ISGF3 (22). However, other studies demonstrate that when a form of human IFNAR1, in which all the tyrosines are mutated to phenylalanines, was expressed in a mouse background, it retained its ability to respond to human IFN (36). This observation calls into question the essential role of IFNAR1 tyrosines in IFN signaling and explains the complicated nature of analyzing the effects of human receptor chains expressed in a mouse cell background.
A summary of the functional properties of all the IFNAR2c tyrosine mutants analyzed in the study is presented in Table I. We have shown that replacing all of the cytoplasmic tyrosines of IFNAR2c with phenylalanines results in a receptor unable to induce JAK-STAT-mediated signaling in response to IFN stimulation, thereby demonstrating the general importance of these tyrosines in type I IFN-dependent signaling. Tyrosine residues within the cytoplasmic domain of IFNAR2c are physically clustered into two groups, both proximal (Tyr269, Tyr306, Tyr316, Tyr318, and Tyr377) and distal (Tyr411 and Tyr512) to the cell membrane. Of the seven cytoplasmic tyrosines of IFNAR2c, Tyr306 and Tyr411 are not conserved across species and are therefore not likely to be crucial to signaling. Complete tyrosine-to-phenylalanine substitution of either tyrosine cluster (4F5F or 4Y5Y) resulted in clones fully responsive to IFN stimulation, suggesting that important tyrosines reside in both the proximal and distal tyrosine clusters. Furthermore, the single substitution of each of the remaining tyrosines-to-phenylalanine had no effect on IFN signaling, which also suggests that no single tyrosine is absolutely essential for IFN-dependent STAT activation, gene regulation, and biological effects.
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When single IFNAR2c tyrosine add-backs were analyzed for their ability to mediate IFN-dependent signaling, Tyr337 and Tyr512 were both capable of mediating signaling on their own. Surprisingly, only the individual substitution of these two tyrosines was able to restore STAT1 and STAT2 activation, ISGF3 complex formation, gene expression, and antiproliferative responses in U5A cells. Detailed studies are ongoing to determine the roles of Tyr337 and Tyr512 in IFN-dependent antiviral responses.
Previous studies using IFNAR2c truncation mutants expressed in U5A cells suggested that the distal region of IFNAR2c (amino acids 462-515) was not necessary for receptor function. However, truncation of IFNAR2c at amino acid residues 417 or 346 resulted in a completely inactive receptor (21). Both of these mutants contain Tyr337, which we have now shown is sufficient to mediate signaling in the context of an otherwise functional receptor. This apparent discrepancy could be explained by the fact that a deletion of the cytoplasmic domain of IFNAR2c might disrupt the tertiary structure around Tyr337 in such a way that it is no longer able to play a role in IFN-dependent signaling.
It is interesting to note that the induction by IFN1b of the ISG
6-16 gene could be observed at low levels in some IFNAR2c mutants
having only a single tyrosine. These clones show no detectable STAT1/STAT2 activation, ISGF3 formation, antiproliferative response, or
ISG 54, TRAIL, or I-TAC gene induction, suggesting that the IFN
receptor may utilize other means of gene induction independent of
JAK-STAT signaling.
The redundant usage of different tyrosines in the regulation of receptor-mediated signaling has been previously documented for other receptor systems. However, this is the first demonstration of a similar signaling mechanism in the type I IFN system. It remains unclear why these two specific tyrosines can mediate similar IFN-dependent signaling events. It is possible that the proximal location or microenvironment of Tyr337 and Tyr512 within the cytoplasmic domain of IFNAR2c presents these two residues as excellent substrates for JAK kinases, redundant docking sites for SH2 domain-containing proteins, or important docking sites for as yet unknown signaling components.
The tyrosine phosphorylation of IFNAR1 has also been demonstrated to be important in IFN-mediated signaling. The importance of the interaction between both phosphorylated IFN receptor chains with each other remains to be determined. The proposed interactions with other receptor chains, such as IFNGR2, also needs further investigation (37). It is likely that these interactions help to regulate the efficiency of IFN-mediated signaling and the mechanism by which proteins involved in signaling, such as STATs, phosphatases, adapter molecules, and docking proteins, interact with the assembled receptor complex. There is currently no human cell line available in which IFNAR1 is not expressed; therefore, most IFNAR1 studies have made use of human receptor chains expressed in mouse cells or by using competing phosphopeptides. Using human U5A cells that lack IFNAR2c but stably express specific tyrosine mutants of IFNAR2c, we have been able to demonstrate the direct role of Tyr337 and Tyr512 in IFN-dependent signaling. It is possible that phosphorylated tyrosines within either IFNAR2c or IFNAR1 interact with SH2 domains present in proteins, such as STATs, functionally bridging the two receptor chains. In the case of STATs, this interaction could then result in the presentation of the functionally important STAT1 and STAT2 tyrosines (Tyr701 and Tyr690, respectively) as high affinity substrates for either JAK1 or Tyk2. In this way, both IFNAR1 and IFNAR2c receptor chains would work in concert to achieve a maximum response to IFN stimulation. We conclude, based on this study and others, that the tyrosine residues critical for carrying out this function are Tyr466 on IFNAR1 and Tyr337 or Tyr512 on IFNAR2c.
In this report, we have focused on characterizing the role of tyrosine
phosphorylation in the JAK-STAT signaling pathway by using IFNAR2c
tyrosine mutants expressed in U5A cells. Further work is needed to
clarify the mechanism by which IFN-mediated signaling pathways utilize
specific phosphorylated tyrosines of either IFNAR1 or IFNAR2c to
mediate downstream signaling events. In addition to the JAK-STAT
signaling pathway, other proposed IFN-mediated signaling pathways that
need further study include the activation of the insulin receptor
substrate/phosphatidylinositol 3-kinase, Vav proto-oncogene,
CBL/Crk-protein family, and mitogen-activated protein kinase.
Furthermore, the regulation of phosphorylation events by phosphatases
such as SHP-1 or SHP-2, both of which contain SH2 domains, remains
unclear. The identification of two critical tyrosines in IFNAR2c that
are essential for efficient JAK-STAT signaling and the use of IFNAR2c
tyrosine mutants characterized within this study will help to further
elucidate the type I IFN signaling pathway.
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ACKNOWLEDGEMENTS |
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We thank the Cell Core Facility and the nucleotide sequencing groups at Berlex Biosciences for help with cell banking and nucleotide sequencing, Drs. Oscar Colamonici and Menachem Rubinstein for help in obtaining some of the plasmids containing IFNAR2c tyrosine mutants, Dr. Ian Kerr for making available the U5A cells, and Jerome Langer for providing the pcDEF1 vector. We thank Susan Harvey for help in performing FACS analysis, Tao Wei for performing the RNase protection assays, and Dean Harde for technical assistance.
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FOOTNOTES |
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* This work was supported in part by National Cancer Institute Grant POI 62220 (to G. R. S.) and by a grant (project 3) to R. M. R.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Dept. Immunology, Berlex Biosciences, 15049 San Pablo Ave., Richmond, CA 94804. Tel.: 510-669-4043; Fax: 510-669-4246; E-mail: ed_croze@berlex.com.
Published, JBC Papers in Press, October 26, 2001, DOI 10.1074/jbc.M108928200
2 T. C. Wagner, S. Velichko, D. Vogel, M. R. S. Rani, S. Leung, R. M. Ransohoff, G. R. Stark, H. D. Perez, and E. Croze, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: IFN, type I interferon; ISGF3, IFN-stimulated gene factor 3; STAT, signal transducer and activator of transcription; FACS, fluorescence-activated cell sorting.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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) are
shown. STAT1 and STAT2 activation was determined by phosphotyrosine
immunoblotting where a plus sign (+) indicates that both phosphorylated
STAT1 and STAT2 were detected after IFN-stimulation and a minus sign
(