From the Department of Pathology and Irving Cancer Center, Columbia University, College of Physicians & Surgeons, New York, New York 10032
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ABSTRACT |
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The interaction between Src-homology 2 domains
(SH2) domains and phosphorylated tyrosine residues serves a critical
role in intracellular signaling. In addition to the phosphotyrosine,
adjacent residues are critical mediators of the specificity of this
interaction. Upon treatment of cells with interferon (IFN
), the
IFNaR1 subunit of the IFN
receptor becomes tyrosine phosphorylated
at position 466. The region surrounding phosphorylated tyrosine 466 subsequently acts as a docking site for the SH2 domain of Stat2,
facilitating phosphorylation of the latter and, thus, the transduction
of the IFN
signal. In this report site-specific mutagenesis was
employed to analyze the nature of the interaction between the SH2
domain of Stat2 and the region surrounding tyrosine 466 on IFNaR1.
Mutation of the valine at the +1 position carboxyl-terminal to tyrosine 466 or of the serine at the +5 position inhibits the association of
Stat2 with phosphorylated IFNaR1. Moreover, receptors mutated at either
of these two positions act in a dominant manner to decrease IFN
signaling, as assayed by both Stat2 phosphorylation and expression of
an IFN
-responsive reporter. The demonstration that these two residues are critical in mediating the interaction between Stat2 and
IFNaR1 suggests that STAT proteins might utilize a structurally distinct subset of SH2 domains to mediate signal transduction from the
cell surface to the nucleus.
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INTRODUCTION |
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Interferon (IFN
)1
is a member of the cytokine superfamily of effector molecules. First
identified as an anti-viral agent, IFN
has also been shown to affect
cell growth by inducing the transcription of growth inhibitory genes
(for a review, see Ref. 1), following interaction with widely expressed
cell surface receptors. Two subunits of the IFN
receptor (designated
IFNaR1 and IFNaR2) have been identified (2-5), both encoded by genes localized within a 400-kilobase region on chromosome 21 (6). As with
other cytokine receptors, both subunits are rapidly phosphorylated on
tyrosine following binding of the cognate ligand (7-9).
Ligand binding activates transcription via a signaling pathway mediated
by members of the Janus kinase and signal
transducers and activators of
transcription (JAK and STAT) families of proteins. The JAK
family of tyrosine kinases includes four mammalian members. Genetic
evidence has demonstrated that two of these, Tyk2 and Jak1, are
involved in IFN signal transduction (10, 11). Each of these JAK
kinases has been shown to constitutively bind to a subunit of the
IFN
receptor; Tyk2 associates with IFNaR1 (12-14) and Jak1 with
IFNaR2 (15). In vitro, all of the JAK kinases can
phosphorylate IFNaR1, predominantly on tyrosine 466 (Tyr-466) (13, 16).
Homodimerization of a chimeric protein composed of the CD4
extracellular domain fused to the IFNaR1 intracellular domain resulted
in its tyrosine phosphorylation. This phosphorylation was, again, found
to occur primarily on Tyr-466 and to be dependent on the association of
IFNaR1 with Tyk2 (17). The pattern of phosphorylation of IFNaR1 under
physiologic conditions, though, has not been determined.
The STAT proteins are a family of latent cytoplasmic transcription
factors employed in signaling pathways activated by various cytokines
and growth factors (for a review, see Ref. 18). STAT proteins share a
common domain structure; each has a DNA-binding domain, a
transactivation domain, a Src-homology 3 domain, a Src-homology 2 (SH2)
domain for binding phosphotyrosine, and a single tyrosine phosphorylation site near the C terminus. Complementation of mutant cell lines nonresponsive to IFN revealed that two STAT family members, Stat1 and Stat2, are involved in the IFN
signal
transduction pathway (19, 20). After IFN
stimulation, Stat2 docks to
IFNaR1 via an interaction between the Stat2 SH2 domain and the
phosphorylated Tyr-466 (Tyr(P)-466) of IFNaR1 (16, 17). The critical
role of Tyr(P)-466 and the Stat2 SH2 domain in this docking interaction has been demonstrated by in vitro binding studies (16). In
addition, phosphopeptides corresponding to the region surrounding
Tyr-466 and dominant-negative IFNaR1 constructs in which Tyr-466 has
been mutated to phenylalanine inhibit Stat2 phosphorylation in
vivo (16). Phosphotyrosine dependent recruitment by IFNaR1
positions Stat2 to become tyrosine phosphorylated, presumably by one of the associated JAK kinases. Phosphorylated Stat2, in turn, provides a
docking site for the SH2 domain of Stat1, positioning it for tyrosine
phosphorylation (16, 19). The two STAT molecules heterodimerize via
SH2-phosphotyrosine interactions, translocate to the nucleus, and
effect transcription of genes under control of
interferon-stimulated gene response
elements (ISREs) (21-23).
SH2 domains were first identified as conserved noncatalytic domains in the Src and Fps proteins (24). These domains were subsequently found in many proteins and shown to mediate interactions between cytoplasmic proteins by binding to phosphorylated tyrosine (25). Site-specific mutations in the residues surrounding tyrosines that function as SH2 domain-docking sites (summarized in Ref. 26) and subsequent studies that identified ligands for recombinant SH2 domains from degenerate phosphopeptide libraries indicated that the residue immediately C-terminal and the residue three amino acids C-terminal to the phosphorylated tyrosine (the +1 and +3 positions with respect to the tyrosine), were of critical importance in binding and conference of specificity for many SH2 domains (27, 28). In this report, we examine the role of the residues C-terminal to Tyr-466 of IFNaR1 in the docking of Stat2 and subsequent signaling events and demonstrate that the amino acids at positions +1 and +5 relative to Tyr-466 are particularly critical for these molecular events.
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EXPERIMENTAL PROCEDURES |
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DNA Constructs--
All of the constructs were expressed from
the vector pMT2T (29). Plasmid constructs encoding the wild type
full-length IFNaR1, the Y466F mutant of full-length IFNaR1, and the
wild type CD4-IFNaR1 chimera have been previously described (16, 17). A
series of five mutant constructs, which convert residues 467 through 471 to alanine were generated by a single step polymerase chain reaction approach, using a 5' primer containing the appropriate mutation and a wild type 3' primer. Mutant polymerase chain reaction products spanning positions 1462-1617 of the IFNaR1 cDNA (5) were
first cloned into the pGEM-T vector (Promega) and sequenced to ensure
fidelity. NsiI-MfeI restriction fragments,
containing the various mutations, were transferred into versions of the
receptors cloned in Bluescript (Stratagene). Subsequently,
PstI or EcoRI fragments containing the entire
mutant full-length or chimeric receptor, respectively, were transferred
into the pMT2T expression vector. The -galactosidase expression
plasmid (pCH110) used to control for transfection efficiency, and the
ISRE-luciferase reporter plasmid (pZ-ISRE-luc; from R. Pine, Public
Health Research Institute, New York, NY), which contains four tandem
copies of the ISRE, have been previously described (30, 31).
Cell Culture and Transfection-- The human embryonic kidney 293T cell line and the human osteogenic sarcoma U2OS cell line were maintained in Dulbecco's modified Eagle's medium containing 10% fetal calf serum (Atlanta Biologicals, Atlanta, GA). Transfections were performed using calcium phosphate as described previously (16).
Antibodies, Immunoprecipitation, and Immunoblotting-- The monoclonal anti-CD4 antibody used for cross-linking (Leu3a) was obtained from Becton-Dickinson Immunochemistry. Rabbit polyclonal anti-CD4 antisera used for immunoblotting (32) was from R.W. Sweet (Smith Kline-Beecham Pharmaceuticals). The rabbit polyclonal anti-Stat2 antisera, used for immunoprecipitation and immunoblotting (22), was from C. Schindler (Columbia University, New York, NY). The anti-Stat2 monoclonal antibody used in the co-immunoprecipitation experiments was obtained from Transduction Laboratories (Lexington, KY). The polyclonal anti-Tyk2 antisera used for immunoprecipitation and immunoblotting has been previously described (8). The monoclonal anti-phosphotyrosine antibody, 4G10, was obtained from Upstate Biotechnology Inc. (Lake Placid, NY). Preparation of lysates, immunoprecipitation, electrophoresis, and immunoblotting were performed as described previously (16, 17).
Fluorescence-activated Cell Sorting-- Transfected cells (105) were stained with isotype-matched phycoerythrin-conjugated anti-CD4 and anti-IgG2a murine monoclonal antibodies (Caltag, S. San Francisco, CA) and analyzed as described (14).
Luciferase Assays--
For each individual assay, duplicate
subconfluent 10-cm dishes of 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. 30 h after
transfection, one dish was treated with 3000 units/ml of IFN
2 (from
M. Brunda; Hoffman-La Roche, Nutley, NJ) for 18 h at 37 °C, and
the other dish was left untreated. Cells were then washed twice with
cold phosphate-buffered saline, lysed, and rapidly frozen on dry ice.
Lysates were thawed, and cellular debris was pelleted by centrifugation
at 10,000 × g for 5 min at room temperature.
Luciferase activity was measured as per the manufacturer's protocol
(Promega) and
-galactosidase activity was measured as described
(30).
Statistical Analysis-- Data from the luciferase assays was analyzed using an unpaired t test to compare wild type transfectants with each of the mutants.
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RESULTS |
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To study the contribution of various amino acid residues in the
SH2-mediated docking of Stat2 to the IFNaR1 receptor, we took advantage
of a chimeric receptor system that reconstitutes the initial events in
IFN signaling, including kinase activation, receptor
phosphorylation, and STAT docking (17). More distal events, such as
Stat2 tyrosine phosphorylation, are not observed in this chimeric
system, presumably because additional receptor subunit(s) (33) are
required. Cells transfected with chimeric molecules composed of the CD4
extracellular domain fused to the intracellular domain of IFNaR1
(CD4-IFNaR1) are tyrosine phosphorylated, predominantly at Tyr-466 of
IFNaR1, following cross-linking with an anti-CD4 antibody (17). Stat2
is recruited to the phosphorylated chimera, as evidenced by the
co-immunoprecipitation of the chimeric receptor by anti-Stat2
antibodies (17). Co-immunoprecipitation was demonstrated to be specific
for Stat2 and dependent on the phosphorylation of Tyr-466. In agreement
with the in vitro and in vivo data cited above
(16), this association supports the idea that Stat2 is recruited to
phosphorylated Tyr-466 on IFNaR1. Furthermore, because more distal
signaling events do not occur in the chimeric system, the Stat2-IFNaR1
association is unusually stable and can be much more readily detected
than the equivalent interaction occurring under physiologic
conditions.
We therefore reasoned that any mutation altering a site required for SH2-receptor interaction will inhibit the co-immunoprecipitation of Stat2 and the CD4-IFNaR1 chimera. Five mutant CD4-IFNaR1 chimeric molecules were constructed, substituting alanine for each of the five amino acids immediately C-terminal to Tyr-466 (amino acids 467 through 471, corresponding to the sequence VFFPS). To ensure that the mutations do not effect induced tyrosine phosphorylation, we first performed cross-linking experiments with the mutants and measured chimeric receptor phosphorylation. As the top panel of Fig. 1 shows, anti-CD4 treatment of 293T cells transfected with either the wild type chimera or any of the five mutant constructs results in a strong induced tyrosine phosphorylation of the chimeric receptor. Stripping the anti-phosphotyrosine immunoblot and reprobing with a polyclonal anti-CD4 antibody (Fig. 1, bottom panel) showed that each of the chimeric constructs was expressed, indicating that these single amino acid substitutions do not dramatically alter protein stability. In addition, fluorescence-activated cell sorting analysis with an anti-CD4 antibody showed that the each of the various chimeric receptors was expressed on the cell surface in approximately equivalent levels (data not shown). It is important to note that, although we have observed differences in the level of expression of the various chimeric constructs, the extent of tyrosine phosphorylation is similar in all cases. IFNaR1-Stat2 docking is completely dependent on tyrosine phosphorylation of the chimeric receptor. Thus, this is the most relevant control criteria for comparing the constructs in the recruitment assay described below.
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Having demonstrated that mutation of residues adjacent to Tyr-466 does not effect IFNaR1 phosphorylation, we sought to determine which, if any, of these residues influence the association of the Stat2 SH2 domain with the phosphotyrosine on IFNaR1. To test this, wild type and mutant CD4-IFNaR1 chimeras were transfected into 293T cells and cross-linked. Lysates were immunoprecipitated with an anti-Stat2 monoclonal antibody and immunoblotted with an anti-CD4 antisera. As shown in Fig. 2, co-immunoprecipitation and therefore association of CD4-IFNaR1 with Stat2 was detected at similar levels in cells transfected with the wild type construct (lane 1), as well as constructs expressing the mutants F468A (lane 5), F469A (lane 7), and P470A (lane 9). On the other hand, receptors encoding two mutant constructs, V467A (lane 3) and S471A (lane 11), were co-immunoprecipitated significantly less efficiently than the wild type. Thus, three residues (+2, +3, and +4 with respect to Tyr-466) have little effect on the binding of the Stat2 SH2 domain to IFNaR1, whereas two others (+1 and +5) appear to be important for this association.
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To demonstrate the importance of the +1 and +5 residues in a more
physiologic setting, we employed an IFN-driven reporter gene system.
As noted in the Introduction, docking of Stat2 to the phosphorylated
IFNaR1 is believed to position Stat2 so that it may be tyrosine
phosphorylated by one of the JAK kinases. Phosphorylated STAT molecules
dimerize, translocate to the nucleus and stimulate transcription of
genes located downstream of an ISRE. Overexpression of a mutant
receptor proficient in IFN
binding but deficient in Stat2 docking,
we reasoned, would have a dominant inhibitory effect on IFN
signaling.
Adenovirus-transformed 293T cells cannot be employed in reporter gene
assays because the E1A gene product binds p300/cAMP response
element-binding protein and prevents Stat2-mediated transactivation (34). Therefore, we transfected U2OS cells with a plasmid containing the firefly luciferase gene downstream of a minimal promoter; upstream
of this gene are four tandem-arranged ISRE enhancer elements. Constructs overexpressing either wild type or mutant forms of the
full-length IFNaR1 receptor protein, as well as a lacZ
expressing construct to control for transfection efficiency, were
co-transfected with this reporter gene construct. The extent of
dominant negative inhibition is quantified by measuring activity of the
reporter gene in the presence and absence of IFN. In cells
transfected with either wild type IFNaR1 (Fig.
3, first bar from the
left) or an empty vector (data not shown), IFN
treatment
produces an approximately 10-fold increase in luciferase activity.
Overexpression of IFNaR1-Y466F, which cannot be phosphorylated at the
Stat2 docking site, inhibits IFN
-induced luciferase activity 70%
(Fig. 3, seventh bar). In cells transfected with the
construct containing IFNaR1 mutated at the +3 site with respect to the
tyrosine (F469A), an increase in luciferase activity similar to that
seen in cells transfected with the wild type is observed following
treatment (Fig. 3, fourth bar). In contrast, constructs
mutated at either the +1 (V467A, second bar) or +5 (S471A,
sixth bar) positions produced an unequivocal dominant
negative effect on induced luciferase activity, though less pronounced
than the Y466F mutant construct. Finally, mutations at the +2 and +4
positions (F468A and P470A, respectively), which did not appreciably
effect the ability of Stat2 to co-immunoprecipitate the receptor (Fig.
2), produced intermediate effects on reporter gene activity when
overexpressed in U2OS cells.
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To determine if the dominant inhibitory effect of either the +2 or +4 mutant is significant, an unpaired t test was used to compare the induction of reporter gene activity observed in cells transfected with wild type IFNaR1 and the induction observed in cells transfected with each of the mutants. As expected from inspection of Fig. 3, a significant difference in induced luciferase activity was observed between the wild type and cells overexpressing either the +1 or +5 mutants (p < 0.0005), whereas the induction of reporter gene activity in cells overexpressing the +3 mutant was not different from the wild type (p > 0.05). Interestingly, the induction of luciferase activity in cells overexpressing the +4 mutant was found to be significantly different from that in wild type IFNaR1 transfected cells (p < 0.005), indicating some inhibitory effect of this mutation. Statistical analysis of the data for the +2 mutant indicates no difference from wild type (p > 0.05). Thus, the reporter gene assay and the co-immunoprecipitation data both suggest that residues at the +1 and +5 positions are critically involved in the interaction between the Tyr-466 docking site and the Stat2 SH2 domain. Mutation at the +4 position may have a smaller effect on signaling that is not revealed in the co-immunoprecipitation experiment.
Finally, to elucidate the molecular nature of the inhibition observed
in cells overexpressing IFNaR1 mutants, we examined the phosphorylation
state of signaling cascade components following IFN treatment. Wild
type and mutant IFNaR1 constructs were overexpressed in 293T cells.
These cells were used because they are IFN
responsive and can be
transfected at the very high levels required to permit the proteins
produced by the transfected genes to displace endogenous molecules
involved in signaling interactions (17). Tyrosine phosphorylation of
signaling components was assayed by immunoblotting anti-Tyk2 and
anti-Stat2 immunoprecipitates with an anti-phosphotyrosine monoclonal
antibody (4G10). In cells transfected with either wild type IFNaR1
(Fig. 4A, lane 1)
or any of the five IFNaR1 constructs mutated near Tyr-466 (Fig.
4A, lanes 3, 5, 7,
9, and 11), IFN
treatment induces the tyrosine
phosphorylation of Tyk2. On the other hand, when Stat2 phosphorylation
was assayed, differences were observed between the various mutants.
Stat2 phosphorylation in cells transfected with the mutants F468A,
F469A, and P470A (Fig. 4B, lanes 5, 7,
and 9, respectively) occurs at levels similar to that of
wild type IFNaR1 (Fig. 4B, lane 1). On the other
hand, cells transfected with mutants V467A and S471A (Fig.
4B, lanes 3 and 11, respectively)
showed a decreased induction of Stat2 tyrosine phosphorylation,
relative to the wild type. Tyrosine phosphorylation of the Stat1
protein, which co-immunoprecipitates with Stat2, is also
diminished.
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As shown previously (16), overexpression of the Y466F mutant of IFNaR1
has a stronger effect on IFN induced Stat2 and Stat1 tyrosine
phosphorylation (Fig. 4C). Thus, consistent with the idea
that SH2-dependent docking on IFNaR1 positions Stat2 for tyrosine phosphorylation, mutations at the +1 and +5 positions show a
dominant inhibitory effect on Stat2 activation, independent of Tyk2
activation. Furthermore, as in Fig. 3, IFNaR1 constructs containing
mutations in the carboxyl-terminal flanking amino acids produce a more
modest dominant negative effect compared with that observed with a
construct in which the critical tyrosine has been mutated to
phenylalanine.
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DISCUSSION |
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We have previously demonstrated that following IFN treatment,
Stat2 is specifically recruited to Tyr(P)-466 on the IFNaR1 subunit of
the receptor (16, 17). SH2-mediated recruitment of STATs to
phosphorylated cytokine receptors is believed to be the mechanism by
which STAT specificity is determined (35). To confirm that the region
surrounding Tyr-466 is required for Stat2 recruitment, and to begin to
characterize the residues that confer specificity for Stat2 binding, we
systematically mutated the five amino acids C-terminal to Tyr-466 on
the IFNaR1 receptor subunit. We employed two experimental approaches
previously used to characterize the IFN
-signaling pathway: (i)
inducible activation of a chimeric receptor by anti-CD4 antibodies
(17), and (ii) overexpression of dominant inhibitory versions of IFNaR1
(16). Our results indicate that residues at the +1 and +5 positions C-terminal to Tyr-466 are critically important in mediating the binding
of Stat2 to Tyr(P)-466. Specifically, mutation of either of these
residues to alanine did not effect the anti-CD4 induced tyrosine
phosphorylation of the chimeric receptor (Fig. 1) but did drastically
disrupt the ability of the receptor to co-immunoprecipitate Stat2 (Fig.
2). Second, full-length receptors with alanine at either position
function in a dominant inhibitory fashion to suppress both
ISRE-dependent reporter gene activity (Fig. 3) and Stat2 tyrosine phosphorylation (Fig. 4B) in response to IFN
.
Significantly, overexpression of constructs with these same mutations
did not suppress Tyk2 tyrosine phosphorylation (Fig. 4A),
consistent with the idea that the effect we have observed is occurring
at the level of Stat2 docking to the IFNaR1 subunit and not in earlier steps in the signaling cascade, such as kinase activation.
Stark, Kerr, and colleagues (36) have produced mutant cell lines that
are deficient in various components of the IFN signaling pathway.
Complementation of these mutant lines proved invaluable in establishing
the role of these components under physiologic conditions.
Unfortunately, no IFNaR1-deficient mutants have been identified,
preventing us from performing similar experiments. In addition, the
murine IFNaR1 receptor does not contain an obvious region of homology
to the Tyr-466 docking site on the human version, nor has a
functionally equivalent site been identified (37) (it is unclear how
Stat2 is recruited to the murine receptor). Thus, complementation
experiments, which might confirm our results, await the development of
human cell lines that are null at the IFNaR1 locus.
Although residues carboxyl-terminal to the tyrosine contribute to the specificity of an SH2-phosphotyrosine interaction, the strongest binding occurs between the SH2 domain and the phosphotyrosine itself, which fits into a deep pocket containing a positively charged arginine residue (38). Thus, Tyr(P)-466 is the main determinant of affinity for Stat2 when it binds the docking site on IFNaR1. As such, mutation of residues carboxyl-terminal to the phosphorylated tyrosine will likely weaken, but not entirely abrogate Stat2 binding. This is consistent with our observation of some residual co-immunoprecipitation for the +1 and +5 mutant constructs in Fig. 2. Other explanations, such as low level binding of Stat2 to the region surrounding Tyr-481 (16) or an incomplete dominant negative effect because of technical limitations might also account, at least in part, for the residual co-immunoprecipitation. The secondary role of the carboxyl-terminal residues is also well demonstrated in the reporter gene assay, where the Y466F mutant is clearly a stronger inhibitor than either the +1 or +5 mutants (Fig. 3), as well as the dominant inhibition of Stat2 tyrosine phosphorylation (Fig. 4).
The proline residue at the +4 position may also contribute to the Stat2 SH2 domain binding site on IFNaR1. This is evidenced by the modest, but statistically significant dominant inhibitory effect of an alanine mutation at this position on the ISRE-driven reporter gene assay (Fig. 3). In contrast, two biochemical measures of the interaction, Stat2-IFNaR1 co-immunoprecipitation (Fig. 2) and Stat2 tyrosine phosphorylation (Fig. 4), showed little or no difference for the +4 alanine mutation when compared with the wild type controls. One possible explanation for the difference between the reporter assay and the biochemical assays may lie in the increased sensitivity of reporter gene activation to small alterations in phenotype. Another possibility is that the biochemical measurements are made at a single point in time and do not necessarily reflect the kinetics of signaling, whereas reporter gene activation measures the accumulated activation of the signaling pathway.
It should be noted that our results are based entirely on the use of alanine substitution to identify residues that are critical for the Stat2-IFNaR1 interaction. Although alanine is widely employed as a substitute amino acid because its methyl group side chain is unlikely to participate in most hydrophobic or hydrophilic interactions, we cannot rule out the possibility that mutation of the residues in question to amino acids other than alanine might affect the interaction. It is of interest to note that experiments aimed at identifying a consensus sequence for Stat2 SH2 domain binding partners, using a partially degenerate library of phosphopeptides, were not successful because of technical limitations (28). Thus, additional data that might guide the choice of other substitutions is lacking at present.
Co-crystallization of the Src SH2 domain with a phosphotyrosine
containing peptide ligand corresponding to a known high affinity binding site revealed not only a conserved binding pocket for phosphotyrosine but also a set of interactions between the SH2 domain
and residues of the peptide carboxyl-terminal to the tyrosine (39). The
amino acid immediately carboxyl-terminal to the tyrosine (the +1
residue) was found to make direct contact with the side chains of two
amino acids (D3 and
D5) within the
D-strand of the central
-sheet conserved in SH2 domains. SH2 domains with glutamine at the
D3 position preferentially bind peptides with valine, leucine, or
isoleucine at the +1 position (27). Stat2 contains a glutamine at the
position corresponding to
D3 (40) and binds to a phosphotyrosine
motif containing a valine at the +1 position. As shown in the Fig. 2,
mutation of this valine to alanine substantially reduces the
association between Stat2 and phosphorylated IFNaR1. The Stat2 SH2
domain also interacts with a phosphorylated tyrosine on the Stat1
protein (Tyr-701) when the Stat1-Stat2 heterodimer is formed (21, 41).
Interestingly, the +1 position with respect to Tyr-701 of Stat1 is an
isoleucine, one of the amino acids predicted to interact with a
D3
glutamine (27). Thus, our finding that Val-467 on IFNaR1 is important for Stat2 SH2 binding fits with one of the predicted structural features of the Stat2 SH2 domain.
X-ray crystallographic data also revealed that the SH2 domains of both Src (38, 39) and Lck (42) fold to create a hydrophobic pocket into which the side chain of the +3 amino acid fits. Other SH2 domains were subsequently found to make direct contacts with the +3 position. However, mutation of the +3 site in IFNaR1 (Phe-469) to alanine does not effect association of the receptor with Stat2 (Fig. 2), nor does it effect downstream signaling (Figs. 3 and 4). In contrast, our data suggest that the serine in the +5 position is a likely site of contact between the Stat2 SH2 domain and IFNaR1. The involvement of a residue at the +5 position in SH2-phosphotyrosine interaction is not without precedent. For example, x-ray crystallographic analysis of the N-terminal SH2 domain of Syp complexed with high affinity peptides revealed strong interaction between the SH2 domain and the +5 residue of the phosphopeptide (43). This SH2 domain, however, also forms a hydrophobic channel that contacts the +1 and +3 residues, in a manner similar to Src. In fact, the side chain of the +3 residue projects deep within the SH2 domain of Syp, providing extensive contacts and stability. Thus, the structure of the SH2 domain of Stat2 is unlikely to be analogous to that of the N-terminal SH2 domain of Syp.
The SH2 domain of Stat1 was also recently shown to display a
distinctive binding specificity. In a manner similar to IFN, IFN
induces tyrosine phosphorylation of its receptor at a specific site,
tyrosine 440, which is subsequently used as a docking site for the SH2
domain of Stat1 (44). Using changes in surface plasmon resonance as a
measure of binding affinities, Greenlund et al. (45) showed
that mutation of the +1 and +4 positions to alanine reduced the
affinity of the Stat1 SH2 domain for the IFN
receptor. The effect of
mutation of the +5 position, however, was not tested in this system,
and thus at this time it is premature to draw conclusions about the
similarity of the binding specificities of these SH2 domains. Marengere
et al. (46) classify SH2 domains into two groups - those
that select hydrophobic residues at the +1, +2, and +3 positions; and
those that select hydrophilic residues at the +1 and +2 positions, and
a hydrophobic amino acid at the +3 position. The data for selectivity
of both Stat1 and Stat2, though, indicate that they may be structurally
distinct and define a new class or classes of SH2 domains.
In most cases, the role of the SH2 domain is to stably localize a protein to the inner surface of the cell membrane via an interaction with phosphotyrosine on either catalytic receptors or adaptor molecules. The Stat2 SH2 domain, on the other hand, must interact sequentially with at least two separate phosphotyrosines. First, it transiently binds Tyr(P)-466 on IFNaR1. Subsequently, it uncouples from the receptor and dimerizes with Stat1, prior to translocating to the nucleus. Thus, functionally, the STAT SH2 domains are distinct from previously characterized SH2 domains. Although additional studies, including crystallographic analyses, will be required to confirm our hypothesis, the data presented in this report suggest that the SH2 domain of Stat2 may be structurally distinct from previously characterized SH2 domains.
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ACKNOWLEDGEMENTS |
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We thank C. Schindler, R. Pine, R.W. Sweet, and M. Brunda for anti-Stat2 antibody, pZ-ISRE-luc plasmid, anti-CD4 antibody, and interferon, respectively.
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FOOTNOTES |
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* This work was supported by National Institutes of Health 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.
Medical Scientist Training Program trainee.
¶ To whom correspondence should be addressed. Dept. of Pathology, University of California Irvine, Medical Sciences D-440, Irvine, CA 92697. Tel.: 949-824-4089; Fax: 949-824-2160; E-mail: jkrolews{at}uci.edu.
§ Current address: Dept. of Pathology, Georgetown University Medical Center, Lombardi Cancer Center, Washington, DC 20007.
1
The abbreviations used are: IFN, interferon
; SH2, Src-homology domain 2; STAT, signal transducer and activator
of transcription; JAK, Janus family tyrosine kinase; ISRE,
interferon-stimulated gene response element; WT, wild type; IFN
,
interferon gamma.
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REFERENCES |
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