From the Section of Hematology/Oncology, University
of Illinois at Chicago, and West Side Veterans Affairs Hospital,
Chicago, Illinois 60607, the § Department of Pathology,
University of Tennessee, Memphis, Tennessee 38163, the
¶ Department of Cancer Biology, The Cleveland Clinic Foundation
Research Institute, Cleveland, Ohio 44195, the
Department of
Medical Genetics and Microbiology, University of Toronto, Toronto,
Ontario M5S 1A8, Canada, and the ** Cancer Biology Program, Division of
Hematology-Oncology, Department of Medicine, Beth Israel Hospital,
Boston, Massachusetts 02215
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ABSTRACT |
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Expression of human and long form of the
(
L) subunits of type I interferon receptor (IFN-R)
in mouse cells is sufficient to activate the Jak-Stat pathway and to
elicit an antiviral state in response to human IFN
2 and IFN
. We
demonstrate herein, however, that these cells respond to the
antiproliferative effects of murine IFN
but not human type I
IFNs. These results suggest that an unknown species-specific component
is required for the antiproliferative effect of human type I IFNs. The
absence of this component can be complemented by expressing the human
L chain truncated at amino acid 346. Thus, the distal
region of
L appears to function as a negative regulator
of the growth inhibitory effects of type I IFNs. Further studies
looking for possible targets of the
L regulatory domain
demonstrated that this region associates with a tyrosine phosphatase.
These results suggest that a protein associated with the negative
regulatory domain of
L, likely a tyrosine phosphatase, plays a role in regulating the growth inhibitory effects of human type
I IFNs.
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INTRODUCTION |
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The most prominent effects of type I interferons
(IFN)1 are the antiviral and
antiproliferative actions (1). These effects are mediated through
binding to the type I interferon receptor (IFN-R or IFNR), which is
composed of two subunits termed
, or IFNAR1, and
, or IFNAR2
(2-10). The genes encoding the different subunits of the type I IFN-R
are clustered in the q22.1 region of human chromosome 21 (6, 7,
11-16). This region also harbors an orphan class II cytokine receptor,
the CRFB-4 gene, which is encoded on human chromosome 21 between the
genes for the
chain of the IFN
R and the
subunit of the
IFN
R (10, 17). Expression of the human
and long form of the
chain (
L) subunits in mouse L-929 cells fully
reconstitutes the activation of the Jak-Stat pathway and the induction
of an antiviral state in response to HuIFN
2 and HuIFN
(9).
Furthermore, only the first 82 amino acids of the cytoplasmic domain of
the
L chain are required to activate the Jak-Stat
pathway and induce the antiviral effect in response to IFN
2
(18).
The and
subunits of the type I IFN-R associate with protein
tyrosine kinases of the Jak family (4, 8, 18). The
subunit
interacts with Tyk2 (4, 19, 20) while the cytoplasmic domain of the
L contains a docking site for Jak1 (18). Binding of type
I IFNs to their receptor triggers rapid tyrosine phosphorylation of
Tyk2 and Jak1 kinases, type I IFN-R subunits (21-25), and Stat factors
(reviewed in Refs. 26-28). Regulation of tyrosine kinase activity is
mediated in most cytokine systems by protein tyrosine phosphatases
(PTPs). For example, SHP1 (also named SHP, SHPTP1, HCP; PTP1C, Ref.
29), a predominantly hematopoietic tyrosine phosphatase that regulates
the activity of the erythropoietin and IL-3 systems (30-35), has also
been implicated in IFN
signaling in hematopoietic cells (36, 37).
However, the role of SHP1 in other cell types is not clear since this
PTP is mainly expressed in hematopoietic cells, whereas the IFN system
functions in almost all, if not all, cell lineages.
Mouse L-929 cells that coexpress wild-type human and
subunits
respond to the antiviral effects of human type I IFNs, demonstrating the presence of functional human type I IFN-R (9). We therefore decided
to test these cells for their ability to respond to the antiproliferative effects of type I IFNs. Human IFN
and IFN
induced only a minimal antiproliferative response, whereas murine type
I IFNs produced a marked inhibition of cell proliferation. These data
indicate that (i) induction of the antiproliferative and antiviral
responses occurs through partially divergent pathways and (ii) that a
novel species-specific signaling component is required, in addition to
the
and
L chains, for the growth inhibitory effect.
Surprisingly, the antiproliferative response was observed in cells that
express the human
L chain truncated at amino acid 346. Thus, the distal part of
L apparently contains a
negative regulatory domain that controls the growth inhibitory effects of type I IFNs. Further characterization of this negative regulatory domain revealed that it interacts with a PTP that appears to be distinct from SHP1 and SHP2. The data herein suggest that a novel species-specific component is required for the growth inhibitory effect
and that this effect is regulated by a distal region corresponding to
amino acids 346-417 on the
subunit.
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MATERIALS AND METHODS |
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IFNs and Antibodies--
Human recombinant IFN2 was
kindly provided by Drs. M. Brunda (Hoffmann-La Roche) and
Paul Trotta (Schering-Plough). The anti-phosphotyrosine antibody (4G10) was obtained from UBI (Lake Placid, NY). The polyclonal antibodies against the
(
511-557, IFNaRC-1),
S (IFNaRC-2), and
L
(
L265-515, and
L375-515) subunits of
the type I IFN-R, as well as the rabbit sera anti-Tyk2 have been
previously described (4, 38, 39). The anti-SHP1 and -SHP2 sera used for
immunoprecipitations and Western blotting were purchased from UBI,
Santa Cruz Biotechnologies, and Transduction Laboratories. The
anti-JAK1 sera was kindly provided by Dr. J. N. Ihle (St. Jude's
Children's Hospital, Memphis, TN).
Constructs and Expression of the Human Type I IFN-R Subunits in
Mouse L-929 Cells--
Mouse L-929 cell lines coexpressing different
constructs of the human and
L chains
LpZR
L, LpRZ
L462,
LpRZ
L417, LpRZ
L346, and
LpZR
L300 were described previously (9, 18). The L-929 transfectants stably coexpressing mutations of tyrosine 466 and truncation at amino acid 511 of the
subunit (designated as
Y1F511) with either wild type or truncation 346 of the
L subunit (LpZR
Y1F(511)
Lwt and LpZR
Y1F(511)
L346, respectively), as
well as coexpressing wild-type
chain and
L subunit
carrying a mutation of tyrosine 411 to phenylalanine, are described
elsewhere.2
Cell Proliferation Assays-- Cell proliferation was assessed by performing MTT assays (7, 40) and cell counts after treatment with the indicated amount of human and mouse IFNs. Briefly, cells were seeded at 6,000 cells/well in 24-well plates in a final volume of 0.6 ml and treated with the indicated concentrations of IFNs. The numbers of cells per well were determined by trypsinization and counting of duplicate wells in a hemocytometer. Experiments were performed at least twice with two independent clones carrying the same mutation.
Immunoblotting-- Cells were treated with different concentrations of the indicated IFNs for 15 min, rapidly centrifuged at 2000 × g for 30 s in an Eppendorf microfuge, and subsequently solubilized in lysis buffer. Immunoprecipitation and immunoblotting were performed as described previously (4).
Phosphatase Assays--
For protein phosphatase assays, cells
expressing wild-type chain and the
L subunit
truncated at the indicated amino acids were treated with or without
IFN
2 for 10 min at 37 °C and lysed in lysis buffer as described
previously (4). The
L subunit was precipitated using a
polyclonal serum raised against a GST fusion protein encoding the
entire cytoplasmic domain (
L265-515) (39),
immunoprecipitates were washed three times in cold phosphatase buffer
to remove phosphatase inhibitors. The phosphatase activity of the
immunocomplexes was determined using pNPP (Sigma) as a substrate. The
phosphatase assay was carried out at 37 °C for 0.5 h in 50 µl
of reaction mixture (100 mM HEPES, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM
dithiothreitol, 10 mM pNPP). The reaction was terminated by
adding 950 µl of 1 M NaOH. The reaction product, p-nitrophenolate, was quantified by measuring absorbance at
405 nm.
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RESULTS |
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Expression of the Human and
L Subunits of the
Type I IFN-R Is Not Sufficient to Reconstitute the Antiproliferative
Response--
Mouse L-929 cells transfected with the human
and
L subunits, LpZR
L.10, activate the
Jak-Stat pathway and are highly responsive to the antiviral effects of
HuIFN
2 and HuIFN
(9). To characterize the antiproliferative
effect of type I IFNs in these cells, we first performed MTT
proliferation assays using L-929 cells stably transfected with
wild-type human
and
L subunits
(LpZR
L.10). Fig.
1A shows that high doses of
HuIFN
2 (100,000 units/ml) had little effect on cell proliferation.
However, treatment with MuIFN
or MuIFN
at doses between 1,000 and 10,000 units/ml induced a significant antiproliferative effect. To
confirm the results observed with MTT assays, we performed similar
experiments in which cell numbers were assessed. Fig. 1B
shows that treatment of LpZR
L.10 cells (Fig.
1B,
L cells) with MuIFN
reduced cell
proliferation more than 95% over a period of 6 days, whereas HuIFN
2
produced a minimal response (20%, from 545,000 cells/well to 445,000 cells/well in control and HuIFN
2-treated cells, respectively).
Similar results were observed when MuIFN
and HuIFN
were used
(data not shown). These results indicate that the endogenous mouse type
I IFN-R expressed in L-929 cells can trigger a complete
antiproliferative effect in response to mouse IFNs. Thus, in contrast
to the antiviral effect, reconstitution of the human receptor with the
and
L chains in these cells is not sufficient to
trigger an antiproliferative effect in response to human type I IFNs,
suggesting that an additional human signaling component is required for
this effect.
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Deletion of a Negative Regulatory Region of the L
Chain Promotes the Growth Inhibitory Effects of Human Type I
IFNs--
Since deletion of the distal region of the
chain
produces an increase in HLA class I response induced by human IFN
2
(48), we wished to determine whether the antiproliferative response could be induced by human IFNs in L-929 cells that express
and
L subunits with deletions of various regions of their
cytoplasmic domains. Cells that express the
L chain
truncated at amino acid 417 are more responsive to MuIFN
than to
HuIFN
2 (Fig. 2A,
panel A). These results parallel our findings for L-929 or
A9+21 cells, which express wild-type receptor subunits (Fig. 1,
B and C). By direct contrast, L-929 cells
coexpressing both the wild-type
and
L truncated at
amino acid 346 (Fig. 2, panels B,
L346.2 cells), responded to the growth inhibitory effects of human type I
IFNs. Truncation of the cytoplasmic domain of
L at amino
acid 300, which removes the Jak1 binding site (18), abolished the antiproliferative response to human type I IFNs but did not affect the
antiproliferative response to MuIFN
(Fig. 2C),
demonstrating that the mouse signaling machinery is intact in these
cells. We also studied L-929 transfectants expressing wild-type
L and an
chain with a deletion of the negative
regulatory domain (truncation at amino acid 511 and tyrosine 466 mutated to phenylalanine; Fig. 2, panel D,
Y1F
L.11 cells) (48). These cells showed a
significant response to MuIFN
, whereas human type I IFNs induced
only a partial antiproliferative effect, which was similar to that
observed in cells expressing wild-type receptors. This result suggests that the negative regulatory region of the
subunit does not control
cell proliferation (48). However, L-929 cells coexpressing the same
mutations of the
subunit and
L truncated at amino acid 346 (Fig. 2, panel E,
Y1F511
L346.3) were extremely sensitive to
the antiproliferative effect of human and mouse type I IFNs. Similar
results were obtained with two independent clones carrying the same
mutation (data not shown). These data strongly suggest that a region
corresponding to amino acids 346-417 in the
L chain contains a negative regulatory domain and may be a possible target for
mouse regulatory proteins. Thus, removal of this negative regulatory
domain appears to complement the absence of an unknown species-specific
component required for the antiproliferative effect (see
"Discussion").
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The L Subunit of the Type I IFN-R Associates with a
Phosphatase--
Tyrosine phosphorylation plays a central role in IFN
and cytokine signaling. Therefore, to determine if the negative
regulatory domain of
L was associated with a PTP, we
performed in vitro phosphatase assays after
immunoprecipitation with anti-
L sera. As a source of
L chain, we used cell lysates obtained from mouse L-929
cells cotransfected with the wild-type
subunit and truncations of
the
L chain at amino acids 346, 417, or 462, respectively. Cells were treated with IFN
2 for 10 min, cell lysates
were immunoprecipitated with an antibody that recognizes all truncated
forms of the
L chain, and in vitro
phosphatase assays were performed on the immunoprecipitates. Fig.
3 shows that significant phosphatase
activity is associated with
L462 and
L417, but not with
L346 after IFN
2
treatment, indicating that the 346-417 region of
L
associates with a PTP. Since the increase in phosphatase activity
associated with the 346-417 region of
L is observed
only after IFN
treatment, we could not elucidate whether the
L-associated PTP is recruited to the receptor complex
after IFN
stimulation or is constitutively associated with the
L chain and activated by IFN
2 stimulation.
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Deletion of the Negative Regulatory Domain of the L
Subunit at Amino Acid 346 Results in Strong and Prolonged Tyrosine
Phosphorylation of Jak1--
We next sought to test if deletion of the
346-417 region of the
L chain and, consequently,
removal of the phosphatase interaction site had an effect on tyrosine
phosphorylation. We performed time course and dose response experiments
with mouse L-929 cells stably cotransfected with wild-type
chain
and
L truncated at amino acids 346 or 417, respectively.
Fig. 4A shows that more
intense phosphorylation of Jak1 was observed at lower doses of IFN
2
in cells expressing the
L subunit truncated at amino
acid 346, as compared with the
L chain truncated at
residue 417. Moreover, deletions distal to amino acid 346, but not at
amino acid 417, prolonged the period of time that Jak1 was
phosphorylated (Fig. 4B). The increase in tyrosine
phosphorylation observed was not due to different amounts of
immunoprecipitated Jak1 protein since stripping and reblotting of the
same membranes with an anti-Jak1 mAb showed a similar amount of Jak1 in
all lanes (Fig. 4, A and B, lower
panels). Two independent clones expressing
L
truncated at amino acid 346,
L346.2 and
L346.4, produced equivalent results. The
intensity of Tyk2 phosphorylation, however, was unaffected by
truncation of
L at residue 346 as revealed by
immunoprecipitation experiments with anti-Tyk2 sera followed by
immunoblotting with antiphosphotyrosine antibodies (data not shown).
Altogether, these data suggest that the 346-417 region of
L interacts with a phosphatase that regulates Jak1
phosphorylation.
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Mutation of Tyrosine 411 in the L Chain of Type I
IFN-R Does Not Alter the Response of Mouse Cells to Human Type I
IFNs--
It has been previously reported that SH2-containing
phosphatases (SHP1 and SHP2) interact with the
subunit of type I
IFNR (37, 49). Since the negative regulatory region of
L
contains only one tyrosine (Tyr-411), which (if phosphorylated) may
serve as docking site for SH2-containing tyrosine phosphatases, we
studied the effect of a phenylalanine mutation of tyrosine 411 on cell proliferation. Panel F (Fig. 2) shows that mutation of
tyrosine 411 to phenylalanine does not reconstitute the
antiproliferative effect of type I IFNs. Similar results were also
obtained with different clones carrying the same mutations (data not
shown). Therefore, tyrosine 411 is not critical for induction of the
negative regulatory effect and is presumably not a docking site for
SH2-containing phosphatases.
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DISCUSSION |
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The type I IFNs have multiple biological actions, including
antiviral and antiproliferative effects, which are the most prominent of these cellular responses (1). Binding of human type I IFNs to mouse
L-929 cells that coexpress human and
L chains is
sufficient to trigger activation of the Jak-Stat pathway and to produce
a full antiviral response, which demonstrates the presence of
functional human type I IFN-R subunits (9). We therefore tested these cells for their ability to respond to the antiproliferative actions of
human type I IFNs. Although these cells were able to respond to the
full growth inhibitory effects of MuIFN
, only a minimal response
was observed for human type I IFNs. Thus, the antiproliferative pathway
is intact in these cells but not fully activated via the human
receptors. Similar results were also observed for different clones of
mouse A9+21 cells that carry several copies of human chromosome 21, which is thought to contain the type I IFN-R cluster (7, 11-16,
41-45) and an uncharacterized signaling component (46, 47).
Altogether, these data indicate that (i) induction of the
antiproliferative and antiviral responses occurs through partially
divergent pathways and (ii) that a novel species-specific signaling
component is required, in addition to the
and
L
chains, for the growth inhibitory effect.
It has been reported that elements of the cytoplasmic domains of
cytokine receptors have a negative regulatory role in signaling. For
example, removal of the docking site for SHP1 in EPO-R results in
hypersensitivity to EPO and prolonged phosphorylation of Jak2 (31).
Similarly, deletion of the distal region of the subunit of the
IFN
R results in increased sensitivity to induction of HLA class I
antigens by IFN
2 (48). Correspondingly, removal of the distal region
(346-417) of the cytoplasmic domain of
L resulted in a
gain in response to the growth inhibitory effects of IFN
, indicating
that this region encodes a negative regulatory domain. The effect of
the negative regulatory domain of
L is specific, as
demonstrated by the finding that deletion of a homologous region of the
chain did not have an effect on cell proliferation (Fig. 2,
Y1F511
L cells). The negative regulatory domain of
L interacts with a PTP as indicated by detection of
phosphatase activity associated with the 346-417 region of
L and the finding that deletion of this region resulted
in prolonged phosphorylation of Jak1 in mouse cells. Mutation of the
only tyrosine in the negative regulatory domain (Tyr-411) did not have
the same effect on the antiproliferative response as deletion of the
346-417 region, indicating that the putative phosphatase is not docked
to
L through an SH2 domain. Moreover,
immunoprecipitation with antibodies against the
and
L chains, and pull-down experiments with GST fusion proteins encoding the cytoplasmic domain of the
and
L chains failed to precipitate SHP1 or SHP2 (data not
shown). Taken together, these data indicate that the 346-417 region of
L functions as a negative regulator for the antiproliferative effect
of IFNs, possibly by recruiting a regulatory phosphatase through an
SH2-independent mechanism. Consistently with the finding in mouse
cells, immunoprecipitations with anti-
L antibodies also
revealed phosphatase activity specifically associated with the
L subunit expressed in human cells (data not shown). It
should be noted, however, that in human cells no conclusive data have
been obtained using GST
L fusion proteins.
Since mouse IFNs completely inhibit proliferation of cells expressing
human and
L chains, we can conclude that the mouse receptor couples the signal induced by murine IFNs with the
intracellular proteins responsible for antiproliferative pathway in
these cells. Consequently, the inability of LpZR
L and
A9+21 cells to respond to the growth inhibitory effect of human IFNs
centers the defect at the level of the human receptor. One possibility
is that the intracellular domains of the human
and/or
L chains, which are responsible for activation of the
antiproliferative pathway, are not homologous to their murine
counterparts. However, this possibility is highly unlikely, based on
two primary observations. First, deletion of the negative regulatory
domain of
L allows a full human IFN
-induced
antiproliferative effect, indicating that the human receptor subunits
are capable of interacting with the appropriate mouse signaling
proteins. Second, these transfectants respond to the antiviral effect
of IFNs. Another possibility is that the missing species-specific
component corresponds to a third receptor subunit. In this scenario,
the antiproliferative response requires the assembly of a receptor
composed of three subunits; in this complex, the third receptor subunit
regulates the activity of a protein associated with the negative
regulatory domain of
L, presumably a PTP. If, in fact, a
phosphatase is involved, the third receptor subunit may delay
activation of the PTP or releases the PTP from the complex, resulting
in prolonged activation of Jak1 and generation of the antiproliferative
response. Thus, deletion of the negative regulatory domain of
L has the same outcome as activating the third receptor
subunit, blocking the action of the PTP or an unknown protein that
associates with this region.
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ACKNOWLEDGEMENTS |
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We thank Dr. J. N. Ihle for providing the Jak1 antisera.
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FOOTNOTES |
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* This work was supported by National lnstitutes of Health Grants CA73381 (to L. C. P.) and GM54709 (to O. R. C.), and by Grants 96-24 (to L. C. P.) and DB-74554 (to T. Y.) from the American Cancer Society.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. of Pathology,
University of Tennessee, 899 Madison Ave. M-576, Memphis, TN 38163. Tel.: 901-448-6173; Fax: 901-448-6979; E-mail:
OColamonici{at}utmem1.utmem.edu.
1
The abbreviations used are: IFN, interferon;
IFN-R, interferon receptor; PTP, protein tyrosine phosphatase;
L, long form of the
chain; HuIFN, human interferon;
MuIFN, mouse interferon; MTT,
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; GST,
glutathione S-transferase; pNPP, p-nitrophenyl
phosphate.
2 Domanski and Colamonici, manuscript in preparation.
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REFERENCES |
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