From Tularik Inc., South San Francisco, California 94080
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ABSTRACT |
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The SH2 domain of the STAT family of transcription factors is essential for STAT binding to phosphorylated cytoplasmic domains of activated cytokine receptors. Furthermore, the same domain mediates dimerization of activated STAT monomers, a prerequisite for DNA binding by this family of proteins. To identify amino acid residues within the STAT protein that mediate these various interactions, we have carried out an extensive mutational analysis of the Stat6 SH2 domain. Recombinant proteins carrying C-terminal deletions or double alanine substitutions were expressed in mammalian and insect cells and assayed for DNA binding, transcription activation, tyrosine phosphorylation, and the ability to interact with a tyrosine-phosphorylated peptide derived from the interleukin-4 receptor signaling chain. From these studies, we have identified amino acids that are required for both DNA binding and interleukin-4 receptor interaction, as well as residues that when mutated impair only one of the two functions. Our results suggest that the structural homology between the SH2 domain of Stat6 and that of the distantly related Src protein may be higher than predicted on the basis of primary amino acid sequence comparisons. However, the two types of SH2 domains may differ at their C-terminal ends.
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INTRODUCTION |
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Tyrosine phosphorylation and specific recognition of these
phospho-residues by SH21
domain-containing proteins are critical features of many cellular signaling pathways. Hence, this protein-protein interaction domain has
been the focus of many studies (1, 2). The best characterized class of
SH2 containing proteins is the Src family. Structural analysis of the
Src SH2 domain bound to a high affinity phosphopeptide revealed three
key features necessary for selective protein recognition (3, 4). First,
phosphotyrosine binding is mediated through a mostly polar pocket that
contains the conserved GTFLLR motif found in most SH2 domains. Second,
a -sheet structure interacts with the first two amino acids
(i + 1 and i + 2) immediately C-terminal to the
phosphotyrosine residue. Third, the i + 3 residue is
recognized specifically by a second, more hydrophobic pocket (4). Thus, binding selectivity is determined largely by the three amino acids following the phosphotyrosine and specific residues in the interacting SH2 domain (5, 6). In general these features are shared by all SH2
domain-containing proteins for which structural information is
available (6-9).
The STAT proteins are the only transcription factors known to contain SH2 domains (10). Thus far, seven STAT proteins have been characterized. Some are activated by multiple cytokines or growth factors, whereas others are only activated in response to a specific stimulus (11-14). Activation of STAT proteins involves cytokine binding to its receptor, which triggers tyrosine phosphorylation of the intracellular receptor domain by an associated Jak kinase (10, 15, 16). The phosphorylated receptor chain provides a docking site for the latent STAT protein, which resides in the cytoplasm. Once recruited to the receptor, the STAT protein is phosphorylated at a single tyrosine residue by Jak kinase (12). Phosphorylated STAT monomers dimerize, translocate to the nucleus, and modulate transcription through STAT-specific DNA sequence elements (11, 17).
Receptor-associated Jak kinases are relatively nonselective in their
ability to phosphorylate individual cytokine receptors and/or STAT
proteins (18, 19). Furthermore, many STAT proteins, once activated,
bind to similar DNA motifs, although not all STAT proteins can activate
transcription from the same motif (11, 17, 20-23). Selectivity in gene
activation upon stimulation with different cytokines appears to be
achieved at the STAT-receptor interaction (11, 18, 24). This
interaction is mediated by the STAT-SH2 domain, which also dictates the
specificity in STAT:STAT dimer formation (25-27). The selectivity of
Stat1 for the IFN- receptor can be transferred to Stat2 via the SH2
domain, suggesting that the STAT-SH2 domain is a modular structure as
are other SH2 domains (2, 18, 28). Strikingly, STAT-SH2 domains share little sequence similarity with other SH2 domains (Fig. 1). Most of the
similarity is restricted to the N-terminal half which contains the
conserved GTFLLR motif (4). As with other SH2 domains, mutation of the
invariant arginine leads to loss of phosphotyrosine recognition in
STATs (5, 21, 29, 30).
Specific peptide recognition is mediated by residues in the C-terminal half of the SH2 domain (4, 6) where little homology exists between the STAT-SH2 and other SH2 domains (Fig. 1). In addition, no structural information is available for any of the STAT-SH2 domains. Given the involvement of this domain in selective protein-protein interactions, we sought to define aspects of this domain that participate in achieving the fidelity of the Jak/STAT signaling pathway.
We are interested primarily in the IL-4-inducible protein, Stat6. To determine the C terminus of the Stat6 SH2 domain, we generated a series of C-terminal deletion mutants. To identify residues critical for Stat6 function, we carried out a systematic mutational analysis of the SH2 domain by changing two amino acids at a time in the context of the full-length protein. Recombinant mutant proteins were tested for DNA binding, tyrosine phosphorylation, and transcription activation. Proteins were also tested for their ability to interact with a tyrosine-phosphorylated peptide derived from the IL-4 receptor. Mutants that were unable to bind DNA but did interact with the receptor-derived peptide could partially inhibit IL-4-induced gene expression when overexpressed in cells that contain endogenous Stat6. Our analysis provides insight into the structure and function of the Stat6 SH2 domain.
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EXPERIMENTAL PROCEDURES |
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Cell Culture and Transfections--
BJAB cells were grown in
RPMI 1640(1×) (Hyclone) supplemented with 10% fetal calf serum (PAA
Laboratories), 2 mM L-glutamine (Life
Technologies, Inc.), and 5 × 105 M
-mercaptoethanol. Stably transfected BJAB cell lines were grown in
the same media supplemented with 1 mg/ml G418 (Sigma). BJAB cells were
stably transfected with the use of electroporation as described by
Tewari and Dixit (31). Human embryonic kidney 293 cells and HepG2 cells
were grown in Dulbecco's modified Eagle's medium (Mediatech)
containing 10% fetal calf serum. Transfections in embryonic kidney 293 cells and HepG2 cells were carried out as described (21). Luciferase
and
-galactosidase activity was determined 48 h following
transfection using the Promega assay systems. The cells were stimulated
with 5 ng/ml IL-4 6 h before harvesting.
Expression and Purification of Recombinant STAT Proteins with the
Use of the Baculovirus Expression System--
Expression constructs
for full-length Stat6 (TPU272) and Stat6C (TPU285) carrying a stop
codon at position 662 have been described previously (21, 22). All
C-terminal deletion mutants (aa 602, TPU601; aa 620, TPU600; aa 633, TPU599; aa 636, TPU598; aa 641, TPU597; aa 645, TPU596; aa 650, TPU595)
were generated by replacing the XmaI/SacI
fragment of TPU285 with DNA fragments that carried stop codons at the
appropriate residues. The fragments were generated with the polymerase
chain reaction (PCR). Double amino acid substitutions in the Stat6-SH2
domain were generated by substituting the
XmaI/SacI fragment of TPU595 with mutated DNA
fragments that were prepared by PCR. The integrity of the resulting
clones was determined by DNA sequence analysis. All proteins carried
nine histidine residues and a pentapeptide substrate (RRASV) for
protein kinase A at their C terminus. With the exception of three
mutants (WS, FS, and LY), all proteins were expressed in Hi-5 cells and
purified using Ni2+ affinity chromatography (22).
Expression in and Purification of Recombinant STAT Proteins from Mammalian Cells-- The expression construct for full-length Stat6 (TPU389) differs from the previously described construct TPU388 (20) in that a flag epitope tag was introduced at the C terminus. All C-terminal deletion constructs (aa 602, TPU608; aa 620, TPU607; aa 636, TPU605; aa 641, TPU604; aa 645, TPU603; aa 650, TPU602) were generated by replacing the BglII/SpeI fragment of TPU389 with a DNA fragment carrying the indicated 3' deletion. Double alanine mutants were prepared by replacing the BglII/SacI fragment of TPU389 with a DNA fragment that carried the indicated mutation. In each case, the fragments were generated by PCR, and the integrity of the clones was determined by DNA sequence analysis. Mutants WS, FS, and LY failed to express in insect cells and were consequently purified from stably transfected BJAB cells. Proteins expressed in mammalian cells contained C-terminal flag epitope tags, which allowed purification on anti-flag M2 affinity resin (Kodak). BJAB (108) cells were resuspended in lysis buffer (30 ml; 0.2 M NaCl, 30 mM Hepes, pH 7.6, 10% glycerol, 0.1% Nonidet P-40, 1 mM EDTA), incubated on ice for 10 min, and sonicated and clarified by centrifugation at 10,000 × g for 10 min. The supernatant was incubated with anti-flag M2 (1 ml) resin for 1 h at 4 °C. Unbound material was washed from the resin with lysis buffer (50 ml). Bound protein was eluted with flag peptide (400 ng/ml).
Peptide Binding Assays--
Biotinylated peptides (2.5 mmol:
IL-4R peptide, ASSGEEGPYKPFQDLI, or IFN- peptide, GGGGGFGYPDKPHVL)
were coupled to 25 µl of packed streptavidin-agarose beads (Sigma) in
0.5 ml of binding buffer (0.1 M NaCl, 30 mM
Hepes, pH 7.6, 10% glycerol, 0.1% Nonidet P-40, 1 mM
EDTA) for 30 min at 4 °C. Unbound peptide was removed by washing the
resin 4 times with binding buffer (1 ml). Purified protein (20 µg)
was incubated with peptide-coupled streptavidin beads (25 µl in a
final volume of 500 µl binding buffer) for 90 min at 4 °C. Unbound
protein was removed by washing the resin 4 times with binding buffer (1 ml) for 10 min each. Bound protein was eluted with 50 µl of SDS
sample buffer, and 5 µl were subjected to Western analysis with
antibodies directed against Stat6. An aliquot of the starting material
(25 µl) was separated on an SDS-polyacrylamide gel, and proteins were
visualized by Coomassie R-250 staining to ensure that the input was
equivalent for each binding reaction.
In Vitro Phosphorylation of Purified Stat6 Mutants and DNA Binding Assays-- Purified Stat6 was activated in vitro with Jak1 kinase (both Stat6 and Jak1 were expressed and purified from insect cells (13)). Phosphorylation conditions were 10 mM Hepes, pH 7.4, 50 mM NaCl, 50 mM MgCl2, 50 µM ATP, 0.1 mM Na3VO4, 0.5 µg of Jak1, and 1 µg of Stat6 in a 50-µl reaction volume. Reactions were incubated at room temperature for 30 min. Typically, 1 µl of the reaction was used for mobility shift assays. Nuclear extract preparations and mobility shift assays have been described previously (21).
Immune Precipitations and Phosphotyrosine Blots-- BJAB cells (1.5 × 10 6) stably expressing wild-type Stat6 or mutant derivatives or 293 cells (2 × 106) transiently transfected with Stat6 C-terminal deletion mutants were either treated or not treated with IL-4 for 15 min. Cells were lysed in immune precipitation buffer (50 mM Hepes, pH 7.9, 200 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, 10% glycerol, and 0.5% Nonidet P-40), and recombinant STAT proteins were immune-precipitated with anti-flag M2-coupled beads. The beads were washed 5 times with immune precipitation buffer, and bound proteins were eluted in SDS sample buffer (50 µl). Proteins were subjected to Western blots with anti-phosphotyrosine antibody (4G10, Upstate Biotechnology) and anti-flag M2 antibody (Kodak).
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RESULTS |
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Amino Acids C-terminal to Tyr-641 Are Required for Tyrosine Phosphorylation in Vivo-- A sequence alignment of the Stat6 and Src SH2 domains is shown in Fig. 1. The conserved tryptophan residue constitutes the N-terminal border of the Src SH2 domain. The GTFLLR motif, which is involved in phosphotyrosine recognition, represents the most conserved region (28). Some sequence identity exists N-terminal to the GTFLLR motif, whereas very little similarity is observed in the C-terminal half. Thus it is impossible to determine the C-terminal border of the Stat6 SH2 domain on the basis of sequence similarity. We showed previously that a truncated version of Stat6 (amino acids (aa) 1-661) that lacks the C-terminal 186 aa binds with wild-type affinity to a tyrosine-phosphorylated peptide derived from the IL-4 receptor (21). This mutant retains the tyrosine residue (aa 641) critical for phosphorylation and dimerization; hence, the protein is able to bind DNA when activated in cells. In order to define more precisely the C-terminal border of the Stat6 SH2 domain, we generated a series of C-terminal deletion mutants by inserting a flag epitope tag and a stop codon at various positions between aa 602 and 650 (Fig. 1). The proteins were expressed in embryonic kidney 293 cells and assayed for DNA binding upon IL-4 stimulation. The 293 cells lack endogenous Stat6 protein but express all other components of the IL-4 signaling pathway. Therefore, any IL-4-inducible DNA binding activity is due to the recombinant Stat6 protein (21). Fig. 2A shows the DNA binding activity of nuclear extracts prepared from IL-4-treated 293 cells transiently expressing the truncated Stat6 proteins. Only the wild-type protein and the mutant ending with aa 650 bound DNA. Mutants ending with aa 645, 641, and 636 did not bind DNA, although the proteins were expressed at the same level as the wild-type (Fig. 2B). Proteins ending with aa 620 and 602 were not expressed in mammalian cells, suggesting that these truncated proteins are unstable (data not shown). These results indicate that only the mutant ending at aa 650 is, upon phosphorylation, able to dimerize, translocate to the nucleus, and bind DNA. Mutant proteins (ending with aa 645 and 641) retain the critical tyrosine residue but were unable to bind DNA. Hence, we investigated whether these truncated proteins became tyrosine-phosphorylated upon cytokine stimulation. Proteins were immune-precipitated from IL-4-treated 293 cells and probed with anti-phosphotyrosine antibodies (Fig. 2C). Only the DNA-binding positive derivative ending with aa 650 was tyrosine-phosphorylated. The absence of phosphorylation with mutants 645, 641, and 635 showed that no nonspecific tyrosine phosphorylation occurs in response to IL-4. Furthermore, the data suggest that the 9 aa following the critical tyrosine (aa 641) are required for proper cytokine-induced Stat6 activation.
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Delineation of the C-terminal Border of the Stat6 SH2
Domain--
We next assessed the C-terminal boundary of the SH2 domain
with respect to peptide binding. We previously identified two
tyrosine-containing peptides derived from the IL-4 receptor signaling
chain that, when phosphorylated (YPKAFS and
YPKPFQ), associate specifically with Stat6 (25). Similarly,
a tyrosine-phosphorylated peptide derived from the interferon- receptor bound specifically to Stat1 but not Stat6 (22, 32). Thus, the
specificity of the STAT-receptor interaction can be accurately mimicked
with the use of peptides derived from individual receptor chains. We
investigated whether truncated Stat6 proteins purified from insect
cells could bind specifically to the IL-4 receptor-derived peptide
YPKPFQ (S, Fig. 2E). A phosphorylated
peptide derived from the interferon-
receptor served as a negative
control (N, Fig. 2E). Biotinylated peptides were
attached to streptavidin beads and incubated with purified Stat6
proteins. After binding the beads were washed, and the eluted proteins
were analyzed by Western blot. All C-terminal deletion mutants (with
the exception of mutant 602 which did not express) bound selectively to
peptides with affinities equal to that of wild-type Stat6 (Fig.
2E). Consistent with the peptide binding results is the
observation that the mutants exhibited a dominant negative phenotype
when overexpressed in an IL-4-responsive cell line (data not shown).
Similar results were obtained for Stat6 mutants that retained
receptor/peptide binding activity but lacked the ability to bind DNA
(21). From the above results we tentatively designated aa 620 as the
most C-terminal aa of the Stat6-SH2 domain.
Alanine Substitutions: Effect on DNA Binding and Transcription Activation in Vivo-- To identify more precisely amino acid residues critical for function of the Stat6 SH2 domain, we carried out an extensive mutational analysis. We substituted 2 aa at a time with alanines in the context of the full-length protein. Mutagenesis began at the conserved Trp-533 and ended at Pro-620. The mutants were expressed in lymphoid (BJAB) and nonlymphoid cells (293 cells), and their DNA binding properties and tyrosine phosphorylation status were determined.
Fig. 3 shows the DNA binding activity of these mutants in nuclear extracts prepared from transiently transfected 293 cells following IL-4 treatment. Identical results were obtained with stably transfected BJAB cell extracts (data not shown). Many of the double alanine substitutions did not impair DNA binding. Because dimerization is a prerequisite for DNA binding, a positive DNA binding signal indicates that the SH2 domain of these mutants is capable of mediating both receptor binding and dimerization. However, certain substitutions reduced or completely abolished Stat6 DNA binding. Some of these loss-of-function substitutions target residues that are conserved between the SH2 domains of Stat6 and Src. For example, all changes in the conserved GTFLLR motif completely abolished DNA binding, consistent with the observation that this region is required for phosphotyrosine binding. Other double amino acid changes in conserved residues (WS, LI, and LY) also abolished DNA binding, whereas changes in the conserved LLLN sequence had no effect. Substitutions in certain regions without homology to Src (SK, DS, IT, IA, EN, IQ, PF, IR, RI, and RD) interfered with DNA binding.
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Alanine Substitutions: Effect on Tyrosine Phosphorylation in Vivo-- Various reasons could account for the lack of DNA binding seen with some of our mutants. One possibility is that these mutants are not phosphorylated in the cell following IL-4 treatment, because they either failed to bind the receptor or bound the receptor-Jak complex in an unproductive manner. In contrast, mutants that do get phosphorylated completed the receptor binding and activation steps but failed subsequently, most likely because they are impaired in dimerization or nuclear translocation.
Stably transfected BJAB cells expressing flag-tagged versions of Stat6 proteins were treated with IL-4, and extracts were prepared. Recombinant Stat6 proteins were immune-precipitated with anti-flag antibodies, and immunoblotted with anti-phosphotyrosine antibodies. The extracts were also probed with anti-flag antibodies to determine the amount of STAT protein present. Wild-type Stat6 and two mutants (GG and DG) that were active in DNA binding were included as controls. As expected, strong IL-4-dependent tyrosine phosphorylation was observed for these three proteins (Fig. 4). The DNA binding inactive mutants SK, FL, LR, FS, EN, IQ, PF, IR, and RD were not tyrosine-phosphorylated (data for mutant FL is not shown). Hereafter we refer to this class of mutants as group 1. Group 1 mutants either fail to bind the IL-4 receptor or, if bound, fail to become phosphorylated. In any event, their lack of tyrosine phosphorylation explains their inability to bind DNA. However, some of the DNA binding negative mutants WS, LI, GT, DS, IT, IA, RI, and LY showed very weak to moderate tyrosine phosphorylation (group 2). It is conceivable that the gel mobility shift assay is not sensitive enough to detect the small amount of tyrosine-phosphorylated active protein, which could explain the lack of DNA binding in group 2 mutants. Alternatively, these mutations may affect dimerization or nuclear translocation. Therefore, we investigated further the nature of the defects in groups 1 and 2.
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Alanine Substitutions: Effect on Peptide Binding--
Mutations
that affect DNA binding may also affect receptor docking, which would
explain the decrease in tyrosine phosphorylation of group 1 and 2 mutants. In order to determine whether these DNA-binding inactive
proteins can interact with the IL-4 receptor, we purified them from
insect cells and assayed their ability to bind a
tyrosine-phosphorylated peptide derived from the IL-4 receptor signaling chain. The proteins were expressed as C-terminal deletions (ending at aa 650); this modification does not alter the peptide binding properties of Stat6 (see above). Three mutants (WS, FS, and LY)
could not be expressed in insect cells and were purified from mammalian
cells. Proteins were incubated with immobilized peptides representing
either the specific IL-4 receptor sequence (S) or the
nonspecific IFN- sequence (N) (Fig.
5). Specifically bound Stat6 proteins
were detected by Western blot.
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Alanine Substitutions: Effect on DNA Binding upon Activation in Vitro-- On the basis of our data, the lack of DNA binding for both group 1 and 2 mutants can be explained either by the lack of tyrosine phosphorylation and/or the inability of the proteins to interact with the receptor. However, group 2b mutants are tyrosine-phosphorylated and interact with receptor peptide but are still unable to bind DNA. Hence, these mutations may affect the DNA binding and/or dimerization properties of the protein.
We asked next whether mutant Stat6 proteins could be activated in vitro to bind DNA. All proteins belonging to groups 1 and 2 were purified from insect cells, tyrosine-phosphorylated with purified Jak1, and assayed for DNA binding (Fig. 6). Wild-type Stat6 and the DNA-binding positive mutants DR and LA served as positive controls for activation and DNA binding. The three mutants WS, FS, and LY were purified from mammalian cells and tested in this assay (data not shown). With the exception of the mutant EN, all members of group 1 were negative for DNA binding when activated in vitro. The fact that EN can be activated in vitro to bind DNA suggests that alanine substitution at these residues selectively incapacitates the receptor binding function of the SH2 domain, while leaving its dimerization function largely intact.
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Alanine Substitutions: Dominant Negative Effects in
Vivo--
Previously we showed that a Stat6 derivative lacking the
transcription activation domain is able to completely inhibit the function of the endogenous Stat6 protein in vivo. In
contrast, Stat6 mutants that bound the receptor peptide but failed to
bind DNA functioned as partial dominant negative as they could only interfere at the receptor binding but not at the DNA binding level. Mutants that abolished peptide binding and DNA binding had no effect
(21). Five of our Stat6 derivatives (group 1b: FL, IR, and RD; group
2b: LI and GT) carry mutations that allow receptor peptide binding but
completely inhibit DNA binding. Group 1b proteins bind the IL-4
receptor derived peptide very strongly, whereas group 2b proteins were
less effective. The question remains whether the in vitro
peptide binding assay completely mimics the in vivo interaction between the IL-4 receptor -chain and Stat6. To address this issue we determined whether these mutants could function as
dominant negative proteins in vivo. We overexpressed the
mutant proteins in HepG2 cells in the presence of the IL-4 inducible luciferase reporter (21). Fig. 7 shows that all five proteins were able
to inhibit the activity of endogenous Stat6 in a
dose-dependent manner. Mutant FL was less effective than
the other four proteins. Neither one of the five mutants was as active
as Stat6
C which lacks the transcription activation domain. One other
mutant (LR) belonging to group 1a was used as negative control. Hence,
the five mutants that bind the receptor-derived peptide but fail to interact with DNA can interfere with the IL-4 signaling pathway in vivo suggesting that the proteins bind the receptor and
consequently interfere with the activation of the endogenous Stat6
protein.
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DISCUSSION |
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Amino Acids C-terminal to Tyrosine 641 Are Crucial for Stat6 Activation-- STAT proteins are unique in that they are the only known class of transcription factors that contain SH2 domains. By employing this domain to mediate selective binding to the intracellular chains of activated cytokine receptors, as well as for STAT dimerization, an elegant economy of structure and function is employed to achieve both signal transduction and transcriptional activation from a single protein (11). Analysis of truncated Stat6 proteins allowed us to define the C-terminal border of the SH2 domain with respect to DNA and peptide binding functions.
As expected, the formation of active dimers requires the integrity of Tyr-641, which becomes phosphorylated upon IL-4 stimulation. Our data reveal that the 9-aa stretch following Tyr-641 is essential for Stat6 activation in vivo. These aa were not simply deleted in our truncated proteins but were replaced by aa residues that constitute the flag epitope tag. Hence, the nature of these 9 aa must be preserved for Stat6 activation in vivo. In contrast, only 4 aa following Tyr-641 were required for Stat6 activation in a cell-free system using purified Jak1. One possible explanation for this result is that the phosphorylation step in the cell depends on the formation of a ternary complex consisting of IL-4 receptor, Jak, and Stat6. The in vivo phosphorylation event may be characterized by greater constraints than those present in the in vitro reaction where Stat6 and Jak are part of a binary association. The data also highlight similarities and differences between Stat6 and Stat3. Sasse et al. (33) made the observation that all STATs have a valine 8 or 9 aa C-terminal to the phosphorylated tyrosine. Deletion or mutation of this residue in Stat3 interferes with dimer formation but does not inhibit Stat3 phosphorylation in cells. In contrast, deletion of the corresponding valine (Val-650) in Stat6 impaired tyrosine phosphorylation in vivo without affecting dimer formation in vitro. Another study involving Stat3 showed that the 5 aa immediately C-terminal to Tyr-705 could not be deleted, or replaced by residues from Stat1, without blocking the IL-6-dependent activation of the protein, even though it could still bind the IL-6 receptor (34). Thus, in both Stat3 and Stat6, there are residues immediately C-terminal to the essential tyrosine that influence Jak-mediated phosphorylation but not receptor binding.Mutations in Specific Amino Acids of the SH2 Domain Have Different Effects on Stat6 Function-- The use of truncated proteins in functional analyses allowed us to determine the C-terminal boundary of the Stat6 SH2 domain. Taken together with data from our previous experiments, we can now argue that about 100 aa (aa 523-620) of Stat6 are required for specific binding to tyrosine-phosphorylated peptides (22). We have identified residues in the SH2 domain that have differential effects on individual steps of the Stat6 activation pathway. All mutant proteins that bind DNA are also transcriptionally active, whereas no activation was seen with any of the DNA binding defective mutants. Mutants that are unable to bind DNA fell into different groups which are summarized in Fig. 8.
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Structural Implications--
Structural studies on Src and other
SH2 domain-containing proteins have led to a detailed understanding of
how SH2 domains selectively recognize different tyrosine-phosphorylated
proteins. On the basis of the Src SH2 domain structure, it has been
proposed that tyrosine-phosphorylated peptides bind to this domain in a two-pronged manner (4, 6). First, phosphotyrosine binds to one protein
pocket that is universal to all SH2 domains and formed by a cluster of
residues located in the N-terminal half of the SH2 domain including the
GTFLLR motif. Interactions between this pocket and the tyrosine
phosphate residue contribute to high affinity binding. Second, the
i + 3 residue is bound by a second, more hydrophobic pocket.
Formation of this pocket involves residues clustered in the C-terminal
half of the SH2 domain, and interactions mediated by this pocket
contribute greatly to binding specificity. Additional interactions are
mediated by a -sheet structure, between the two pockets, which makes
contacts to the i + 1 and i + 2 positions of the
phosphopeptide.
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ACKNOWLEDGEMENTS |
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We thank Keith Williamson for DNA sequence analysis; Linda Huang for peptide synthesis; Paige Nittler for the help in the purification of proteins; Tim Hoey, Judi McKinney, Todd Dubnicoff, and Greg Peterson for critical comments and helpful discussions.
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FOOTNOTES |
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* 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.
Both authors contributed equally to this manuscript.
§ To whom correspondence should be addressed: Tularik Inc., Two Corporate Drive, South San Francisco, CA 94080. Fax: 650-829-4400; E-mail: uli{at}tularik.com.
1 The abbreviations used are: SH2, Src homology domain; aa, amino acids; PCR, polymerase chain reaction; STAT, signal transducer and activator of transcription; IFN, interferon.
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REFERENCES |
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