From the Department of Pathology and the Irving
Comprehensive Cancer Center, Columbia University, College of
Physicians and Surgeons, New York, New York 10032 and the ¶ Public
Health Research Institute, New York, New York 10016
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ABSTRACT |
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Interferons and cytokines modulate gene
expression via a simple, direct signaling pathway containing receptors,
JAK tyrosine kinases, and STAT transcription factors. The
interferon- pathway is a model for these cascades. Two receptors,
IFNaR1 and IFNaR2, associate exclusively in a constitutive manner with
two JAK proteins, TYK2 and JAK1, respectively. Defining the molecular
interface between JAK proteins and their receptors is critical to
understanding the signaling pathway and may contribute to the
development of novel therapeutics. This report defines the IFNaR1
interaction domain on TYK2. In vitro binding studies
demonstrate that the amino-terminal half of TYK2, which is ~600 amino
acids long and contains JAK homology (JH) domains 3-7, comprises the
maximal binding domain for IFNaR1. A fragment containing amino acids
171-601 (JH3-6) also binds IFNaR1, but with reduced affinity.
Glutathione S-transferase-TYK2 fusion proteins
approximating either the JH6 or JH3 domain affinity-precipitate IFNaR1,
suggesting that these are major sites of interaction within the larger
binding domain. TYK2 amino acids 1-601 act in a dominant manner to
inhibit the transcription of an interferon-
-dependent
reporter gene, presumably by displacing endogenous TYK2 from the
receptor. This same fragment inhibits
interferon-
-dependent tyrosine phosphorylation of TYK2, STAT1, and STAT2.
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INTRODUCTION |
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Tyrosine kinase activation is a common mechanism for triggering
eukaryotic signaling pathways. Receptor-type tyrosine kinases, which
have extracellular ligand-binding domains and intracellular kinase
domains, are activated following ligand-induced dimerization or
oligomerization (1-3). The ligands for these receptors include a
variety of growth factors, hormones, and cytokines. The
interferon/cytokine family of receptors is a second class of receptors,
which bind a similar spectrum of ligands and also appear to be
activated following dimerization or higher order clustering (4, 5). However, the latter receptors do not contain intrinsic tyrosine kinase
domains, but rather associate in a noncovalent, constitutive manner
with various members of the JAK kinase family (6). The receptor-JAK
complex appears to function completely analogously to a receptor-type
tyrosine kinase. In the case of the interferon- (IFN-
)1 receptor, two
subunits have been identified (IFNaR1 (7) and IFNaR2 (8)), and these
are known to bind exclusively to two JAK kinases (TYK2 and JAK1,
respectively) (8-10). Following IFN-
binding, the JAK proteins are
activated (11-14), and two STAT proteins (STAT1 and STAT2) are
tyrosine-phosphorylated, heterodimerize, translocate to the
nucleus, and stimulate transcription of genes containing the
interferon-stimulated gene response element (ISRE) (15). A variety of
studies demonstrate that both TYK2 and JAK1 are required for IFN-
signaling (14, 16, 17).
The JAK kinases have a unique structure among non-receptor tyrosine kinases. In addition to a carboxyl-terminal tyrosine kinase domain, these proteins contain a set of six other regions of homology known as JAK homology (JH) domains (18), which are common to all members (JAK1 (19), JAK2 (18), JAK3 (20), TYK2 (21), and the Drosophila hopscotch gene (22)). Adjacent to the bona fide kinase domain is a kinase-like domain, which apparently lacks catalytic activity and has no clear function. The amino-terminal half of the JAK proteins is composed of five shared domains (JH3-7), which are not found in any other known proteins. Neither Src homology 2 or 3 nor pleckstrin homology domains are present in the JAK proteins.
The interferon/cytokine receptor cytoplasmic tails function as
multipurpose docking proteins. In addition to sites for the binding of
JAK tyrosine kinases, these receptors often contain tyrosine residues
that are inducibly phosphorylated following ligand binding. Some of
these phosphorylated sites bind Src homology 2 domains of signaling
molecules, most notably the STAT transcription factors (4). For
example, we have identified a binding site for STAT2 centered around
tyrosine 466 of the IFNaR1 subunit of the interferon- receptor (23).
We have previously characterized the TYK2-binding site on IFNaR1 in
detail (9, 11, 24). The minimal binding domain is an ~33-amino acid
juxtamembrane region, overlapping the box 1 and box 2 sequences of
IFNaR1 (24). Nearly every phylogenetically conserved residue in the
region is required for binding. However, the proline-rich box 1 sequence, which is not well conserved in IFNaR1, appears to play only a minor role in the TYK2-IFNaR1 interaction. In contrast, it has been
reported that box 1 is a critical determinant of the binding domain in the cytoplasmic portion of other cytokine receptors (25, 26).
Our results have also demonstrated that the TYK2-IFNaR1 interaction is
absolutely required for signaling (24). In this report, we complement
our previous characterization of the TYK2 interaction domain on IFNaR1
by identifying the regions of TYK2 that bind to IFNaR1. Our results
show that most, if not all, of the amino-terminal half of TYK2 is
required for this interaction.
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MATERIALS AND METHODS |
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Reagents--
Interferon-2 was a gift of J. Sepinwall (Hoffmann-La Roche). Polyclonal rabbit antiserum against TYK2
has been described previously (11). The following monoclonal antibodies
(mAbs) were obtained from commercial suppliers: 4G10, against
phosphotyrosine (Upstate Biotechnology, Inc.); anti-glutathione
S-transferase (GST) (Santa Cruz Biotechnology, Inc.), and
anti-TYK2 (Transduction Laboratories). A mAb against the influenza
hemagglutinin epitope (HA) (27) was from J. Kitajewski (Columbia
University, New York, NY), a polyclonal antibody against CD4 was from
R. Sweet (Smith-Kline-Beecham, King of Prussia, PA), and polyclonal
rabbit antisera against STAT1 and STAT2 (15) were provided by C. Schindler (Columbia University). All DNA-manipulating enzymes were
purchased from New England Biolabs Inc.
Plasmid Constructs-- Plasmids encoding GST-TYK2 fusion proteins were prepared by digesting the TYK2 cDNA with the appropriate restriction enzymes (illustrated in Fig. 3); blunting the ends as required; and ligating into the SmaI site of pGEX-1, -2T, or -3X (Pharmacia Biotech Inc.). GST-IFNaR1 fusion proteins have been described previously (9, 24). To express TYK2 in mammalian cells, the full-length cDNA (21) and subfragments, prepared by restriction digestion as illustrated in Fig. 1, were blunted to allow ligation into a version of the pMT2T expression vector (28) containing PstI, EcoRI, NotI, and SalI cloning sites. Stop codons in all three reading frames are contained in the expression vector. In the case of the two double truncation constructs (which span amino acids 171-601 and 263-601, respectively, as illustrated in Fig. 1), a cassette containing an HA tag and the 15 amino acids found at the amino terminus of TYK2 was amplified by polymerase chain reaction from a previously described construct (29) and ligated upstream of the corresponding TYK2 fragments. The CD4-IFNaR1 chimera has been previously described (30).
Cell Culture, Infection, and Transfection--
U2OS cells were
from American Type Culture Collection, and 293T cells were from H. Young (Columbia University). Cells were grown in Dulbecco's modified
Eagle's medium plus 10% fetal calf serum, split 1:10 (293T) or 1:7
(U2OS) from a newly confluent plate the day prior to transfection, and
subsequently transfected as described (31) with calcium phosphate
precipitates containing 10 µg of plasmid DNA/10-cm dish (see below
for cotransfection protocol for luciferase assay). Two days
post-transfection, cells were lysed as described previously (29). For
the reporter gene experiments, U2OS cells were transfected with 5 µg
of the -galactosidase expression plasmid, 5 µg of the
ISRE-luciferase plasmid, and 10 µg of the expression vector (16).
Binding Assays-- Lysates from transfected cells were incubated at 4 °C with GST fusion proteins, purified as described (23), already bound to the glutathione beads (Sigma) for 2-4 h. The complexes were recovered by centrifugation, washed three to four times, and then subjected to SDS-polyacrylamide gel electrophoresis in preparation for immunoblotting (see below).
Immunoprecipitation and Immunoblotting-- Cells were lysed in 1% Nonidet P-40, 150 mM NaCl, 50 mM Tris-HCl (pH 7.4), 2 mM phenylmethylsulfonyl fluoride, 0.2 unit/ml aprotinin, 1 mM sodium orthovanadate, 100 mM NaF, and 5 mM ZnCl2, and the proteins of interest were immunoprecipitated with a 1:50 to 1:200 dilution of the appropriate antibodies for 2-4 h on ice. The immunoprecipitates were collected on protein A beads, washed, fractionated by SDS-polyacrylamide gel electrophoresis, and transferred to a nitrocellulose membrane. The membrane was sequentially reacted with a 1:1000 to 1:3000 dilution of the primary antibody, a 1:10,000 dilution of the appropriate peroxidase-conjugated anti-IgG secondary antibody (Sigma), and LumiGloTM chemiluminescence reagent (Kirkegaard & Perry Laboratories, Inc.) and then exposed to film.
Luciferase Reporter Assays--
Thirty to thirty-six h after
transfection, quadruplicate cultures were either treated with 3000 units of IFN-2/ml for 18 h at 37 °C or left
untreated, washed twice with cold phosphate-buffered saline (137 mM NaCl, 2.7 mM KCl, 4.3 mM
Na2HPO4, and 1.4 mM
KH2PO4 (pH 7.4)), lysed in the manufacturer's
buffer (Promega), and rapidly frozen on dry ice. Lysates were thawed;
cellular debris was pelleted by centrifugation; and luciferase activity
was measured following the manufacturer's protocol (Promega), whereas
-galactosidase activity was measured as described (32). To determine
-fold induction, normalized activities were determined by dividing the luciferase activity by the
-galactosidase activity for each sample. Then, the normalized activity for the treated sample was divided by the
normalized activity for the companion untreated sample.
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RESULTS |
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Truncations Demarcate the IFNaR1-binding Domain on TYK2-- We have previously demonstrated that the TYK2-IFNaR1 interaction can be faithfully reproduced in vitro (9, 24). Therefore, to characterize the region of TYK2 required for binding to IFNaR1, we performed a series of analogous binding assays. Fig. 1 illustrates the TYK2 constructs used in the first set of binding experiments. These fragments were transiently expressed at high levels in 293T cells; the lysates were affinity-precipitated with GST fusion proteins containing all or part of the cytoplasmic domain of IFNaR1; and the resulting complexes were immunoblotted. Fig. 2A shows that a set of progressive carboxyl-terminal truncations were expressed at similar levels, but only the truncations terminating at amino acids 601 and 876 were precipitated as efficiently as full-length TYK2. The faint band observed in the other lanes was also seen in control experiments employing GST alone as an affinity precipitation reagent (data not shown). Probing the lower part of the same immunoblot with an anti-GST mAb demonstrated equal recovery of the fusion protein in each lane (data not shown).
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GST-TYK2 Fusion Proteins Bind IFNaR1-- To confirm the data in Fig. 2 and to further characterize the interaction domain, a second set of in vitro binding assays were performed using GST fusion proteins (Fig. 3A) corresponding approximately to individual TYK2 JH domains as well as various portions of the amino-terminal half of the protein. These GST-TYK2 fusion proteins were used to affinity-precipitate lysates from 293T cells transfected with a construct encoding a chimeric receptor consisting of the CD4 extracellular domain and the IFNaR1 cytoplasmic domain. This chimera co-immunoprecipitates endogenous TYK2 (24) and can activate TYK2 when the CD4 extracellular domains are dimerized by antibody cross-linking (30). We employed the chimera because CD4 antiserum is a sensitive reagent for immunoblotting (33).
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Amino-terminal Fragments of TYK2 Inhibit
IFN--dependent Reporter Gene Transcription--
To
demonstrate that the interaction domain identified in vitro
is relevant in vivo, we assayed the ability of various TYK2 fragments to inhibit IFN-
-dependent reporter gene
activity. Previously, we have shown that overexpression of
kinase-deficient TYK2 in U2OS cells can block the
IFN-
-dependent expression of a luciferase gene under the
control of an ISRE and a minimal promoter (16). Presumably, this
dominant-negative effect is caused by the displacement of endogenous
TYK2 from the receptor by exogenous, overexpressed, kinase-deficient
TYK2, thereby preventing receptor-mediated TYK2 activation and
subsequent STAT phosphorylation.
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Amino-terminal Fragments of TYK2 Inhibit Tyrosine Phosphorylation of TYK2 and STAT Proteins-- We hypothesized that the inhibition of ISRE-controlled luciferase activity resulted from displacement of endogenous TYK2 from the receptor. As a consequence of such displacement, tyrosine phosphorylation of TYK2 and both STAT1 and STAT2 should be inhibited. To test this, 293T cells were transfected with a subset of the constructs used in Fig. 4, and tyrosine phosphorylation was assayed. Because 293T cells can be transfected at very high efficiencies (>90%), an effector gene can be overexpressed in nearly every cell in a given culture. Thus, it is possible to determine the dominant inhibitory effect of the products of such transfected effector genes on endogenous proteins, as we have demonstrated previously (16, 23, 24). We did not employ 293T cells in the reporter gene induction experiments in Fig. 4, however, because E1A overexpression has been shown to inhibit STAT2 transactivation (34).
As expected, IFN-
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DISCUSSION |
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Since cytokines regulate the growth, differentiation, and effector function of a wide variety of cells, characterizing the interface between cytokine receptors and JAK family kinases is important for understanding the biology of these cells and may eventually aid in the design of new drugs. Toward these ends, we have begun to define the interaction domains on both IFNaR1 (9, 11, 24) and TYK2. This report demonstrates that the TYK2 domain spans the amino-terminal half of the protein. Specifically, in vitro binding of TYK2 truncation constructs to a GST fusion protein encoding the cytoplasmic domain of IFNaR1 reveals that maximal binding requires amino acids 1-601 of TYK2 and that weaker binding can still be detected with a construct encoding amino acids 171-601 (Fig. 2, A and B). These constructs correspond to the JH3-7 and JH3-6 domains of TYK2, respectively. Binding of the fragment containing amino acids 1-601 to the IFNaR1 cytoplasmic domain was disrupted by IFNaR1 mutations previously shown to abolish the ability of IFNaR1 to bind TYK2 and to mediate cell signaling (24) (Fig. 2C). A second set of in vitro binding studies (Fig. 3), using GST fusion proteins corresponding to TYK2 JH domains, indicate that the JH3 and JH6 domains are the major binding sites within the IFNaR1-binding domain. Although variable levels of fusion protein expression compromise our ability to precisely quantitate binding, the data indicate that the intervening domains (JH4 and JH5) bind the IFNaR1 cytoplasmic domain poorly. Longer constructs containing most or all of the JH3-7 domains appeared to bind IFNaR1 more avidly than the single JH3 and JH6 domains. Finally, two sets of dominant inhibitory experiments (Figs. 4 and 5) demonstrate that overexpression of the binding domain within the amino-terminal half of TYK2 effectively blocks both ISRE-driven reporter gene expression and tyrosine phosphorylation of TYK2, STAT1, and STAT2, presumably by displacing endogenous TYK2 protein from the receptor complex in the transfected cells. Interestingly, the TYK2 construct corresponding to amino acids 171-601 can dominantly interfere with TYK2 and STAT tyrosine phosphorylation (Fig. 5B), but is ineffective in blocking reporter gene expression (Fig. 4). This suggests that under the transfection conditions employed, submaximal STAT phosphorylation might nonetheless be sufficient to induce maximal levels of reporter gene transcription. However, these results might also reflect differences in the two cell lines employed. Overall, our data show that the binding of IFNaR1 to TYK2 requires a 601-amino acid region of TYK2 containing the JH3-7 domains, and within this region, the JH3 and JH6 domains appear to be particularly important.
A number of cytokine receptor-binding domains within JAK family kinases
have now been characterized. Chen et al. (35) have recently
reviewed these data. In the case of JAK2, which associates with a wide
variety of receptors, binding to the growth hormone receptor appears to
require the entire amino-terminal half of the protein (JH3-7) (25). In
contrast, binding to the granulocyte-macrophage colony-stimulating
factor receptor requires only the JH6 and JH7 domains of JAK2, although
this binding is quite weak (36). JAK3 association with the common
-chain of the interleukin-2 receptor appears to also be mainly
mediated by the JH6 and JH7 domains, although the JH3-5 domains appear
to also play a role (35). Including our data on the TYK2-IFNaR1
interaction, there are two examples of two different classes of binding
sites: one involving primarily the JH6 and JH7 domains and a second
requiring the JH3-7 domains.
Assuming that all cytokine receptor-JAK complexes are similar in overall topology, Chen et al. (35) have attempted to reconcile the existing data by proposing the existence of two domains that mediate receptor binding by a given JAK protein. One domain would be in the JH6 and JH7 region, and a second would be somewhere in the JH3-5 region. One of these domains may act as a generic JAK-binding site, whereas the other could provide specificity for a given receptor. The pattern of binding observed for different receptor-JAK pairs would then reflect variation in the relative affinity of these two sites. In cases where the affinity of the two sites is similar for a receptor, then binding will appear to require most or all of the JH3-7 domains. In this scenario, the TYK2-IFNaR1 interaction would fall into this category. Our observation that the entire JH3-7 region is required for strong binding but that the individual JH3 and JH6 domains show some independent binding would be consistent with such a model. Characterization of additional receptor-JAK pairs, identification of the specific JAK residues required for binding, and the eventual solution of the three-dimensional structure of one or more receptor-JAK complexes will be needed to prove this model.
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ACKNOWLEDGEMENTS |
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We thank J. Sepinwall, C. Schindler, J. Kitajewski, R. Sweet, and H. Young for reagents and cells.
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FOOTNOTES |
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* This work was supported by United States Public Health Service Grant CA56862 (to J. J. K.).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.
§ Present address: Johns Hopkins Oncology Center, 424 North Bond St., Baltimore, MD 21231.
To whom correspondence should be addressed: Dept. of
Pathology, Columbia University, College of Physicians and Surgeons, 630 West 168th St., New York, NY 10032. Tel.: 212-305-2646; Fax:
212-305-5498; E-mail: jjk5{at}columbia.edu.
1
The abbreviations used are: IFN-,
interferon-
; ISRE, interferon-stimulated gene response element; JH,
JAK homology; mAb, monoclonal antibody; GST, glutathione
S-transferase; HA, influenza hemagglutinin epitope.
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REFERENCES |
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