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Originally published In Press as doi:10.1074/jbc.M105525200 on February 28, 2002

J. Biol. Chem., Vol. 277, Issue 20, 17556-17563, May 17, 2002
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Interdomain Interaction of Stat3 Regulates Its Src Homology 2 Domain-mediated Receptor Binding Activity*

Tong Zhang, Kah Tong SeowDagger, Chin Thing Ong, and Xinmin Cao§

From the Institute of Molecular and Cell Biology, 30 Medical Drive, Singapore 117609

Received for publication, June 15, 2001, and in revised form, February 26, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Activation of Stat proteins by cytokines is initiated by their Src homology 2 (SH2) domain-mediated association with the cytokine receptors. Previously, we identified an essential role of the coiled-coil domain of Stat3 in binding of the receptor peptides derived from the interleukin-6 receptor subunit, gp130. In this study, we further investigated the molecular basis of this regulation. We found that the C-terminal domain of Stat3 negatively regulates its receptor binding activity only in the absence of the first alpha -helix of the coiled-coil domain, which leads to a hypothesis of intramolecular interaction. Physical interactions between the coiled-coil domain and the C-terminal domain, as well as the SH2 domain, were indeed detected. Furthermore, a sub-region of the C-terminal domain (amino acids 720-740), which is also involved in the interaction with the coiled-coil domain, was demonstrated to be critical for the regulation of the receptor binding. Correspondingly, phosphorylation on Ser-727 within this region inhibits this interaction. In agreement with the peptide binding results, both the coiled-coil domain and the C-terminal sub-region are necessary for the functional recruitment of Stat3 to the cellular gp130 in response to interleukin-6, suggesting that the interdomain interaction is a prerequisite for the SH2-mediated receptor binding in interleukin-6 signaling.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

STAT1 (for signal transducer and activator of transcription) represents a family of latent cytoplasmic transcription factors. Seven mammalian STAT genes have been identified so far, and over 40 different polypeptides, including most cytokines and certain growth factors, are known to activate one or more STATs (reviewed in Refs. 1-3). Stat3 was originally identified as an acute-phase response factor activated by IL-6 in mouse liver, and also by homology to Stat1 (4, 5). IL-6-type cytokines play pleiotropic roles in immune response, hematopoiesis, and neuronal differentiation (6). This family comprises IL-6, IL-11, leukemia inhibitory factor, oncostatin M, ciliary neurotrophic factor, and cardiotrophin, which share receptor gp130 as a common subunit and are able to stimulate Stat3 (7, 8). Binding of IL-6 to its receptor gp80 (subunit alpha ) induces the homodimerization of gp130 (subunit beta ) and trans-phosphorylation of the gp130-associated JAKs (7-9). Activated Jak1 phosphorylates six tyrosine residues on the cytoplasmic domain of gp130. Whereas the second membrane-proximal tyrosine residue (Y2) is required for recruitment of the SH2-containing phosphatase-2, any one of the four tyrosine residues (Y3-Y6) containing the consensus YXXQ motif serves as a docking site for Stat3 binding upon IL-6 stimulation (10-12). The receptor bound Stat3 is subsequently phosphorylated by Jak1 on a single tyrosine residue 705 at the C terminus. Stat3 forms dimers via the reciprocal interactions between its SH2 domain and the phosphorylated tyrosine 705, translocates into the nucleus, binds to DNA, and regulates the expression of their target genes leading to various cellular responses (3, 13-15). Although the recruitment of Stat3 to gp130 is the first and critical step for its subsequent activation, the regulation of Stat3 receptor binding has been remained largely unknown.

STAT proteins share a highly conserved structure with a number of functional domains. The three-dimensional structure of the N-terminal domain (N-domain) of Stat4 contains 130 amino acids was first resolved (16). This domain was originally identified to be involved in tetramer formation, but recently also reported to affect the induction of Stat4 tyrosine phosphorylation stimulated by IFN-alpha (17, 18). Subsequently, the crystal structures of the DNA bound homodimers of truncated Stat1 and Stat3beta , lacking the N-domain and the most of the C-terminal domain, have been reported and exhibit highly similar structures and conserved domains (19, 20). Two new domains have been revealed: an N-terminal coiled-coil domain containing four antiparallel alpha -helices, and a linker domain located between the DNA binding domain and the SH2 domain. The C-terminal domain comprises the transcriptional activation domain (21) and is also involved in the proteasome-dependent turnover in Stat5 (22). However, the very C-terminal region, including 40-50 amino acids, is absent in both crystal structures (19, 20). The linker domain is reported to be important in the transcriptional activity in Stat1 (23), whereas the coiled-coil structure has often been inferred to be involved in protein-protein interaction (24). Indeed, Lys-161 in the helix alpha 1 of Stat1 interacts with p48, a protein from an IFN response factor family, to form interferon-stimulated gene factor 3 complex that regulates IFN-alpha -responsive genes (25). A short region in the first alpha -helix of Stat3 has been demonstrated to associate with another transcription factor, c-Jun, which cooperatively activates transcription of IL-6 inducible alpha 2-macroglobulin gene (26). In addition, the coiled-coil domain of Stat5 associates with Nmi, an N-Myc interactor, which augments Stat5-mediated transcription (27). However, the possible effect of the coiled-coil domain on the activation of Stat protein itself has not been studied. Recently, we have reported that removal of the first alpha -helix, or mutation of the highly conserved Asp-170 or Lys-177 residue, in the first alpha -helix of the coiled-coil domain of Stat3 results in a loss of the SH2 domain-mediated binding of gp130 (28). The data demonstrated a novel role of the coiled-coil domain of Stat3 in the regulation of its receptor binding and the subsequent activation in response to IL-6 and epidermal growth factor. Based on the crystal structure of the Stat3 dimer, the coiled-coil and SH2 domains are separated by the DNA binding and linker domains (19, 20). These results suggest that the coiled-coil domain is able to remotely influence the SH2 function through an undetermined mechanism.

In the present study, experiments were performed to investigate the molecular basis of this novel regulation. We have identified that deletion of the first alpha -helix in the coiled-coil domain abrogates its binding activity. However, further removal of 70 amino acids (700-770) at the C terminus of this mutant restores the impaired binding activity, suggesting a possible interaction of the coiled-coil domain and the C-terminal domain. Such interaction is indeed observed. Furthermore, a sub-region of C-terminal domain was demonstrated to be critical for regulation of the receptor binding and is also essential for the interaction with the coiled-coil domain. Experimental results support the proposed model of the domain-domain interaction and indicate its physiological relevance in the functional recruitment of Stat3 to the gp130 receptor subunit in IL-6 signaling.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Construction of Expression Plasmids-- The expression plasmids of murine Stat3 and the human Stat3beta were obtained from J. E. Darnell, Jr. and R. Jove, respectively (5, 29). Plasmids expressing Jak2, Erk2, and the active mutant of MEK1, MEK1Delta , were described previously (30). A series of N- or C-terminal deletion mutants of Stat3 were generated by PCR using primers containing BamHI site at 5' and XhoI site at 3' and the murine Stat3 expression plasmid as a template. The respective PCR products were cloned either into plasmid pXJ40-FLAG, a FLAG-tagged expression vector provided by Dr. Z. Zhao as described previously (28), or pXJ40-GST provided by E. Manser, which is a plasmid for expression of glutathione S-transferase (GST) fusion protein in mammalian cells (31). The resultant constructs are shown in the diagrams of Fig. 1. The internal deletion mutants of Stat3, Delta C700-720 and Delta C720-740, were constructed with a two-step PCR reaction as described previously (32). In brief, internal primers adjacent to the deletion region were used in the first step to create the templates for the subsequent PCR reaction in which primers containing BamHI site at 5' and XhoI site at 3' end were utilized to generate the final PCR product.

DNA Transfection-- COS-1 and HepG2 cells were grown in Dulbecco's modified Eagle's medium with 10% fetal calf serum purchased from Invitrogen. Transient transfections of the expression plasmids into COS-1 and HepG2 cells were performed with LipofectAMINE (Invitrogen) and FuGENE 6 transfection reagents (Roche Diagnostics Corp.), respectively, following the manufacturers' instructions.

Antibodies, Immunoprecipitation, GST Pull-down, and Immunoblotting-- Anti-Stat3 (C-20 and K15) and anti-GST antibodies were purchased from Santa Cruz Biotechnology and BD PharMingen, respectively. Antibodies against Stat3 N terminus and anti-FLAG M2 were purchased from Transduction Laboratory and Sigma, respectively. Anti-gp130 antibodies were purchased from Upstate Biotechnology and Santa Cruz Biotechnology. Glutathione-Sepharose 4B beads and anti-FLAG M2 affinity gel were purchased from Amersham Biosciences and Sigma, respectively. Transfected cells were washed with cold phosphate-buffered saline (PBS), lysed in radioimmunoprecipitation assay buffer, and subjected to immunoprecipitation as described previously (33). For GST pull-down experiments, the cell lysates containing 500 µg of proteins were incubated with 40 µl of the glutathione-Sepharose 4B beads. The precipitates were washed with radioimmunoprecipitation assay buffer and PBS twice each, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred onto a polyvinylidene difluoride membrane. The membrane was blocked with PBS containing 0.1% Tween 20 and 1% bovine serum albumin before it was incubated with the appropriate primary and secondary antibodies. The bound proteins were visualized by using ECL solution (Amersham Biosciences). For a second blotting, the membrane was incubated in stripping buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, 100 mM beta -mercaptoethanol) for 30 min at 60 °C before re-blotting.

Peptide Binding Assay-- Biotinylated peptides of IL-6 receptor subunit, gp130 (pY3, VVHSG-YPO<UP><SUB>4</SUB><SUP>−</SUP></UP>-RHQVPS; and Y3, VVHSGYRHQVPS) were purchased from Sigma-Genosys. Transfected cells were lysed in lysis buffer, and the peptide binding experiments were performed as described previously (12, 28, 34). Briefly, the peptides (5 µg) were incubated with 40 µl of streptavidin-Sepharose (Pierce) at 4 °C for 2 h. The beads were washed for three times with 20 mM Tris-HCl, pH 7.4, and then incubated with aliquots of lysates (containing 500 µg of proteins). The complexes were washed, boiled, fractionated on SDS-PAGE, and blotted with anti-FLAG or anti-Stat3 antibody.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Regulation of the Receptor Peptide Binding Activity of Stat3 by Its C-terminal Domain-- By systematic deletion and point mutation analysis of the N-terminal region of Stat3, we have previously demonstrated that, in addition to the expected SH2 domain, the coiled-coil domain, particularly the first alpha -helix, is essential for Stat3 binding to the peptide derived from gp130 (28). We proposed a model that the coiled-coil domain may mediate this regulation via an intramolecular interaction with the Stat3 C-terminal domain. In the current studies, we designed a series of experiments to test the proposed hypothesis. The role of the C-terminal domain in receptor binding was first investigated. Constructs lacking the C-terminal domain in combination with the deletion of various N-terminal domains were generated as illustrated in Fig. 1. These constructs are tagged with FLAG epitope at their N termini and transfected into COS-1 cells that express very low endogenous Stat3. The ability of the mutant Stat3 to bind to gp130 was tested using the phosphorylated peptide (pY3) derived from sequences surrounding the third tyrosine residue in the cytoplasmic domain of gp130 and its unphosphorylated counterpart (Y3) as a control as described previously (28).


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  		Fig. 1.   Schematic diagrams of the structural domains and the deletion mutants of Stat3. Name and abbreviation of each domain of Stat3 are indicated on top of the figure. The full-length or the fragments of Stat3 were either tagged with FLAG or fused with GST at their N termini in the expressing plasmids. The names of the constructs are shown on the left, and the numbers indicate the respective amino acids in each domain of Stat3 and the fragments in the constructs.

Stat3 exists in two isoforms: Stat3alpha as the major form and the naturally occurring splice variant, Stat3beta , as the minor form (35). Stat3beta , which lacks 55 amino acid residues with 7 additional amino acids at its C terminus, was used as a C-terminal deletion mutant of Stat3alpha . The results show that Stat3beta can specifically bind to the phosphopeptide pY3, but not the unphosphorylated Y3, with a comparable affinity to Stat3alpha (Fig. 2A). Similarly, the N-domain deletion mutant of Stat3alpha (ST3-Delta N) bound to pY3 strongly, and deletion of the C-terminal 70 amino acids (ST3-Delta N-ct) did not affect its binding affinity. However, removal of the first alpha -helix (ST3-Delta N1H) of the coiled-coil domain resulted in a loss of binding activity as previously observed (28). Surprisingly, this impaired activity was restored when its C terminus was removed (ST3-Delta N1H-ct). The expression levels of each pair of these proteins were comparable (lower panels). These results suggest that the C-terminal domain of Stat3 seems to exert a negative role on the SH2-mediated receptor binding in the absence of the first alpha -helix, and the first alpha -helix may suppress the C-terminal inhibitory effect on the receptor binding via interaction.


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  		Fig. 2.   Role of the C-terminal domain in regulation of the receptor peptide binding. A, the C-terminal 70 amino acids were removed from plasmids encoding deletion mutants of ST3-Delta N and ST3-Delta N1H, and the resultant plasmids were named as ST3-Delta N-ct and ST3-Delta N1H-ct, respectively. COS-1 cells were transfected with various plasmids as labeled on top of the figure, and lysed 48 h later. The biotinylated peptides (pY3 and Y3) derived from gp130 were incubated with the streptavidin-Sepharose. The beads were washed and incubated with lysates containing 500 µg of proteins. The complexes were washed, fractionated on 7.5% SDS-PAGE, and immunoblotted with anti-Stat3 or anti-FLAG antibody as indicated (upper panels). The cell lysates containing 50 µg of proteins per sample (10% input) were subjected to Western blot analysis to monitor the expression of the various Stat3 proteins in transfected cells (lower panels). PB, peptide binding; TC, total cell lysates. B, the C-terminal deletion mutants deleting the amino acids 700-720 (ST3-Delta C700-720), 720-740 (ST3-Delta C720-740), and 740-770 (ST3-Delta C740-770), were transfected into COS-1 cells. Peptide binding experiments were performed as described in A. The peptide binding results are shown in the upper panel, and the expression of the wild-type and the mutants of Stat3 is shown in the lower panel.

Identification of the C-terminal Residues 720-740 As an Essential Region for the Receptor Peptide Binding-- To gain a further insight of the regulatory role of the C terminus on receptor binding, we produced three mutants deleting the amino acids 700-720 (Delta C700-720), 720-740 (Delta C720-740), or 740-770 (Delta C740-770), as shown in Fig. 1, and tested their bindings to the receptor peptides. Delta C700-720, in which the deletion is immediately adjacent to the SH2 domain, binds to pY3 strongly, and the binding affinity of Delta C740-770, which lacks 30 amino acid residues at the end of the C terminus, is slightly decreased. Interestingly, the binding activity of Delta C720-740, containing a deletion in the middle of the C-terminal domain, is abolished completely (Fig. 2B, upper panel). All proteins do not bind to the unphosphorylated peptide, Y3, as expected (upper panel), and the expression level of the wild-type and mutants are comparable (lower panel). These results suggest that the region between 720 and 740 is required for regulation of the receptor peptide binding. Two possibilities are considered. First, this region could be important for maintaining the SH2 in an optimal conformation. However, deletion of the region immediately adjacent to SH2 domain (aa 700-720), or removal of the whole C-terminal region (aa 700-770), has no effect on the receptor binding (Fig. 2), which argues against the hypothesis. Alternatively, this region (720-740) may be involved in the interaction with the N-terminal coiled-coiled domain to restrain the C-terminal inhibitory effect on receptor binding.

Physical Interaction between the N- and C-terminal Domains in Vivo-- To examine the hypothesis of the intramolecular interaction, we sought to obtain direct evidence for a physical interaction between the coiled-coil domain and C-terminal domain in vivo. We first tested the interaction between the N-terminal domains and the C-terminal domains. To this end, a mammalian-expressed fusion construct containing GST N-terminally linked to the SH2 and C-terminal domains of Stat3 (GST-SH2.CT) was generated and cotransfected together with FLAG-tagged plasmids expressing the N-domain and the coiled-coil domain (ST3-ND.4H) of Stat3. As a control, GST-DB.LD containing the DNA binding and linker domains located in the middle portion of Stat3 molecule was also constructed (Fig. 1). Cell lysates were incubated with glutathione-Sepharose beads, and bound proteins were separated on a SDS-PAGE and analyzed with Western blot using anti-FLAG antibody as a probe. As shown in Fig. 3A, ST3-ND.4H coprecipitated strongly with GST-SH2.CT (lane 1), but not with GST-DB.LD (lane 2) or GST alone (lane 3). Expression of the FLAG-tagged ST3-ND.4H is shown in the middle panel, whereas GST-SH2.CT (lane 1), GST-DB.LD (lane 2), and GST alone (lane 3) are indicated in the lower panel.


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  		Fig. 3.   Physical interaction of the coiled-coil domain and C-terminal domains in vivo. A, the fusion constructs containing GST and Stat3 C-terminal domains (GST-SH2.CT), DNA binding and linker domains (GST-DB.LD), or GST alone (GST vector) were cotransfected with the FLAG-tagged plasmid expressing the N-domain and the coiled-coil domain (ST3-ND.4H). The cell lysates containing 500 µg of proteins were incubated with glutathione-Sepharose beads, and the bound proteins were separated on a 12.5% SDS-PAGE and probed with anti-FLAG antibody (upper panel). The middle panel shows the expression of the FLAG-tagged ST3-ND.4H, and the lower panel indicates the expression of the GST-SH2.CT (lane 1), GST-DB.LD (lane 2), and GST alone (lane 3). B, GST fusion constructs containing N-domain (GST-ND) or the coiled-coil domain (GST-4H) were cotransfected with FLAG-tagged C-terminal domain constructs (ST3-SH2 and ST3-CT) and the control plasmids (FLAG-vector and ST3-DB.LD) as labeled on top of the upper panel. The cell lysates (500 µg of total proteins) were incubated with anti-FLAG antibody conjugated with agarose beads, and subjected to the Western blot analysis using anti-GST antibody (upper panel). The expression of the GST-Stat3 fusions and the FLAG-tagged constructs were detected by Western blot analysis with 50 µg of proteins per sample as shown in the middle and lower panels, respectively.

To further delineate the interaction domains, FLAG-tagged SH2 domain (ST3-SH2) and the C-terminal domain (ST3-CT) were cotransfected with either GST-4H containing four alpha -helices of the coiled-coil domain, or GST-ND containing the N-domain. The cell lysates were incubated with anti-FLAG antibody conjugated to agarose beads and subjected to Western blot analysis using GST antibody. The results showed that the coiled-coil domain (GST-4H), interacted with both SH2 domain (ST3-SH2) and C-terminal domain (ST3-CT) (Fig. 3B, lanes 4 and 6), whereas the N-domain (GST-ND) failed to associate with either of them (lanes 3 and 5). As negative controls, both GST-ND and GST-4H did not coprecipitate with either the FLAG epitope (lanes 1 and 2), or FLAG-tagged ST3-DB.LD (lanes 7 and 8). Expressions of the GST fusion proteins and the FLAG-tagged constructs in the transfected cells are shown in the middle and lower panels, respectively. The results suggest that the coiled-coil domain is able to interact with the C-terminal domain and the SH2 domain. In contrast, the N-domain interacts with neither of them. However, because ST3-ND.4H interacts strongly with GST-SH2.CT (as shown in Fig. 3A), the N-domain may stabilize the coiled-coil domain in the absence of the other domains and therefore enhances its binding activity to the SH2 and the C-terminal domains.

Identification of the C-terminal aa 720-740 As Important Interacting Region-- To further investigate whether the aa 720-740 sub-region at the C-terminal domain is involved in the interaction with the coiled-coil domain, internal deletion mutants GST-SH2.CT.Delta C700-720 and GST-SH2.CT.Delta C720-740, lacking indicated amino acids in GST-SH2.CT, were generated, and GST pull-down experiments were performed as described in Fig. 3A. The results showed that deletion of amino acids 700-720 displayed a marginal decrease, whereas deletion of 720-740 exhibits a significant reduction in the interaction with the N-terminal domains (Fig. 4). The expression of the FLAG-tagged ST3-ND.4H is shown in the middle panel, and the GST fusion proteins in the lower panel. These results suggest that amino acids 720-740 are important for the interaction with the N-terminal domains.


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  		Fig. 4.   The effect of the internal deletion mutants of the C terminus on the interaction with the N-terminal domains. GST-SH2.CT.Delta C700-720, GST-SH2.CT.Delta C720-740, GST-SH2.CT, or GST, as indicated on top of the figure, was cotransfected with ST3-ND.4H, and the GST pull-down experiments were performed as described in Fig. 3A. The expressions of FLAG-tagged ST3-ND.4H and GST fusion proteins are shown in the middle and lower panels, respectively.

Phosphorylation on Tyr-705 or Ser-727 Inhibits the Interaction between N- and C-terminal Domains-- Two major phosphorylation sites of Stat3 have been identified so far. Tyr-705 is phosphorylated by receptor-associated JAKs and is absolutely required for its dimer formation and subsequent nuclear translocation (5, 20). On the other hand, Ser-727 is constitutively phosphorylated at basal level in the unstimulated cells and is further induced by various Ser/Thr kinases in response to different extracellular stimuli (30, 36-39). Because both residues are located in the C-terminal domain, we investigated whether their phosphorylation affects the interaction with the N-terminal coiled-coiled domain. Cells were transfected with GST-SH2.CT and FLAG-tagged ST3-ND.4H in the absence or presence of Jak2 or ERK2. Jak2 phosphorylated GST-SH2.CT on Tyr-705 (Fig. 5, second panel, lane 4). Conversely, its interaction with the N-terminal domains decreased in comparison with that in the absence of Jak2 (upper panel, lanes 2 and 4). More dramatically, the interaction was impaired by ERK2 cotransfected with its upstream kinase, MEK1 (lane 6). A basal level of Ser-727 phosphorylation of GST-SH2.CT was observed in the absence of ERK2 (third panel, lanes 2 and 4), which was substantially enhanced in the presence of ERK2 and MEK1 (lane 6). ERK2 alone did not cause obvious increase on Stat3 serine phosphorylation (data not shown). As a control, ST3-ND.4H did not coprecipitate with GST protein in the absence or presence of the kinases (top panel, lanes 1, 3, and 5). Our data suggest that phosphorylation has a negative effect on the domain-domain interaction. It is noteworthy that phosphorylation on Ser-727, which is located within the region of aa 720-740, has a stronger negative effect than that on Tyr-705. This further supports the involvement of this sub-region in the interaction with the coiled-coil domain. Although phosphorylation on other sites cannot be completely excluded, our two-dimensional phosphopeptide mapping has indicated Ser-727 as the major site phosphorylated by ERK2 and MEK1 in vivo (30). Consistently, we observed that increasing Ser-727 phosphorylation by inhibiting Ser/Thr phosphatase also reduces Stat3 binding to the receptor peptide.2


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  		Fig. 5.   Phosphorylation on Tyr-705 or Ser-727 inhibits the association of C-terminal with N-terminal domains. Expression plasmid of GST-SH2.CT and the GST vector were cotransfected with FLAG-tagged ST3-ND.4H in the presence or absence of Jak2, or MEK1Delta and Erk2, as indicated on top of the panels. The GST pull-down experiment was carried out as described in Fig. 3A, and the coprecipitated ST3-ND.4H was detected by anti-FLAG antibody (upper panel). The blot was stripped and re-blotted with antibodies specifically recognizing phosphorylated Stat3 as indicated on the right side of the second and third panels. Expressions of ST3-ND.4H, GST-SH2.CT, and GST, are shown in the fourth and bottom panels as indicated.

The Coiled-coil Domain and the C-terminal aa 720-770 Are Critical for the Recruitment of Stat3 to the Cellular gp130 in Response to IL-6 Stimulation-- The receptor subunit gp130 possesses four consensus sequences in its cytoplasmic region for Stat3 activation, and, more importantly, recruitment of Stat3 to gp130 is initiated by IL-6 stimulation. We wondered whether the results we obtained by peptide binding experiments using one phosphopeptide (pY3) simulate the situation in vivo, and whether the intramolecular interaction is required for the functional recruitment of Stat3 to the gp130 in response to IL-6 stimulation. To address these questions, we examined the association of the endogenous gp130 with the wild-type and the mutant Stat3 lacking the regions that are likely to be involved in the intramolecular interaction. The wild-type Stat3 or the mutant deleting the first alpha -helix of the coiled-coil domain (Delta N1H) was transfected into HepG2 cells, and the interactions with the cellular gp130 were tested. Lysates of the transfected cells were immunoprecipitated by anti-FLAG antibody, and the coprecipitated proteins were detected with anti-gp130 in Western blot analysis. Cellular gp130 did not associate with either the wild-type or the mutant Stat3 in the unstimulated cells. However, it coprecipitated with the full-length Stat3, but not the mutant, upon IL-6 stimulation (Fig. 6A, upper panel). The blot was stripped and re-probed with anti-FLAG antibody to show amounts of immunoprecipitated Stat3 proteins (lower panel). A reciprocal immunoprecipitation/blot analysis was also performed in which lysates were immunoprecipitated with anti-gp130 antibody and probed with anti-FLAG antibody. The results confirmed that only the full-length Stat3, but not the mutant, coimmunoprecipitated with gp130 in response to IL-6 stimulation (Fig. 6B, upper panel). The blot was re-probed with gp130 to indicate equal amounts of gp130 (lower panel).


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  		Fig. 6.   Interaction of the wild-type and the mutant Stat3 with gp130. The FLAG-tagged Stat3 constructs (as labeled on top of the figure) were transfected into HepG2 cells which were either untreated or treated with IL-6 (40 ng/ml) for 15 min (as labeled) before harvesting. A, the lysates containing 500 µg of total proteins were immunoprecipitated with anti-FLAG antibody (IP) and subjected to Western blot analysis with anti-gp130 antibody as a probe. The associated gp130 is indicated by an arrow in the upper panel. The blot was stripped and re-blotted with anti-FLAG antibody to indicate the precipitated Stat3 proteins labeled on the right (lower panel). B, reciprocally, the cell lysates were immunoprecipitated with anti-gp130 antibody and probed with anti-FLAG antibody (upper panel). The blot was stripped and re-probed with anti-gp130 antibody (lower panel). Stat3 and gp130 are indicated with arrows. C, the lysates were immunoprecipitated with anti-FLAG antibody and probed with anti-gp130 to show the associated gp130 as described in A (top panel). The blot was re-probed with anti-Stat3-pY705 antibody to detect the Tyr-phosphorylated Stat3 (second panel) or with anti-FLAG antibody to show amounts of the immunoprecipitated Stat3 (third panel). The bottom panel indicates the expression of the cellular gp130 in each sample by Western blot analysis of the total cell lysates.

Next, we tested the receptor binding activity of the C-terminal internal deletion mutants in a similar assay. The results showed that receptor gp130 was able to associate with the mutant Delta C700-720, but not Delta C720-740, after IL-6 treatment (Fig. 6C, top panel). The tyrosine phosphorylation status of these mutants was examined (second panel). The full-length Stat3 is phosphorylated strongly in response to IL-6 as expected (lane 6). The mutant Delta C700-720 has no Tyr-705 residue and therefore cannot be phosphorylated (lane 7), although it is capable of binding to the gp130. On the other hand, although mutant Delta C720-740 contains the Tyr-705 residue, it cannot be phosphorylated because of the failure of receptor binding (lane 8). Together, these data are consistent with those obtained in the peptide binding experiments, and further indicate that both the coiled-coil domain and the C-terminal region of aa 720-740 are indispensable for the regulation of the SH2-mediated receptor association upon IL-6-stimulation. This suggests that the interaction between the coiled-coil domain and the C-terminal region could be a prerequisite for the activation of Stat3 in IL-6 signaling.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The Coiled-coil Domain and the C-terminal Domain Participate in the Regulation of Stat3 Binding to the Receptor via Intramolecular Interaction-- Stat proteins are recruited to the cytokine receptors via their SH2 domains. Studies have been focused on the identification of the specific phosphotyrosine residues and the amino acid sequences surrounding these residues on the cytoplasmic region of cytokine receptors, whereas other regulations are less known. In our report, we investigated the regulation of Stat3 receptor binding by its structure and demonstrated that both the coiled-coil domain at the N-terminal and the C-terminal domains of Stat3 are involved in the regulation of the SH2 domain-mediated receptor binding. We propose that such regulation is likely to be coordinated via intramolecular interaction. The coiled-coil domain interacts with the flexible C-terminal domain to restrain its possible physical interference to the SH2 domain function. A series of experiments presented here support the hypothesis. We first observed that deletion of the first alpha -helix of the coiled-coil domain, which is structurally distal from the SH2 domain as based on the reported Stat3 dimer structure, abolishes its receptor binding. The impaired binding activity, however, is restored by the removal of the C-terminal domain (Fig. 2A), implying a potential negative effect of the C-terminal region. In the crystal structure of Stat3beta dimer, the majority of the C-terminal domain (aa 722-770) is not included, and some included regions (aa 688-701 and 717-722) are poorly ordered, suggesting the disorder and highly flexibility of this region (20). In fact, a physical interaction between the coiled-coil and the C-terminal domain has been identified (Fig. 3). Additional fine mapping of the C-terminal domain reveals a sub-region (aa 720-740) that is likely to be involved in the interaction with the coiled-coil domain and is essential for the regulation of the receptor binding activity (Figs. 2, 4, and 6C). This interaction is negatively regulated by phosphorylation, especially, on Ser-727, located inside the interacting region (Fig. 5).

Unexpectedly, we also detected an interaction of the coiled-coil domain and the SH2 domain. The role of this interaction is not clear. Perhaps, it is required for the optimal binding conformation of the SH2 domain. In agreement with this, we found that the SH2 domain alone is not sufficient for the receptor binding (data not shown). This is in contrast to the other SH2-containing proteins, such as Src family and phospholipase C-gamma , whereby the SH2 domain alone can bind to their specific phosphotyrosyl peptides (40). Comparison of the SH2 domains reveals less than 25% sequence identity between Stat3 and other SH2-containing proteins. This divergence may explain the dependence of the Stat3 SH2 function on its other domains.

Our results are mostly based on the deletion mutants that may lead to structural disruptions. However, it might not be the case in most situations. For example, deletion of the N-domain or the C-terminal domain, alone or together, has no effect on the SH2 function. In addition, the SH2 domain function remains unchanged by removing immediately adjacent 20 amino acids (700-720) (Fig. 2). Moreover, the impaired binding activity of the mutant lacking the first alpha -helix was rescued by further deletion of the C terminus, which could not be explained by structural perturbations.

A Proposed Model for the Regulation of the SH2 Function Mediated via Intramolecular Interactions-- Based on the experimental data, we suggest a novel model of Stat3 regulation (Fig. 7). In brief, the N-terminal coiled-coil domain interacts with the C-terminal domain and the SH2 domain in the monomeric Stat3alpha before recruitment to the receptor, which "locks" the C-terminal domain into an intramolecular "binding" conformation with an accessible SH2 domain. The first alpha -helix (aa 130-198) and the C-terminal aa 720-740 are likely to be the contact regions indicated by a striped box in step a. This interaction is disrupted by deletion of the first alpha -helix, resulting in a release of the C-terminal domain, which has a potential ability to fold toward the SH2 domain and physically interferes with the SH2 domain function in receptor binding (b). This interference, however, can be eliminated with removal of the whole C-terminal domain, thereby releasing the obstruction on the SH2 domain and restoring its receptor binding activity (c). Similarly, internal deletion of the proposed contacting region of aa 720-740 abolishes its interaction with the coiled-coil domain, and releases the remaining part of the C-terminal region that could interfere with the SH2 domain function (d), whereas further deleting the whole C-terminal domain eliminates its inhibitory effect as seen in the Stat3beta (e). In this model, the intramolecular interaction may serve two purposes. First, it suppresses the inhibitory role of the C-terminal domain toward the SH2 domain. Second, by binding to the SH2 domain, the coiled-coil domain assists the SH2 domain to achieve an optimal conformation required for receptor binding.


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  		Fig. 7.   A proposed model of the intramolecular interaction regulating the SH2 domain-mediated receptor binding. The details of the model are described under "Discussion." (+) and (-) indicate the receptor binding activity.

Regulation of the Latent Stat3 by Intra- and Intermolecular Interaction-- It has been generally accepted that the latent Stat proteins exist as monomeric forms in the cytoplasm of unstimulated cells (14). However, a few reports suggest the existence of the tyrosine phosphorylation-independent preassociated heterodimer of Stat1 and Stat3, or homodimer of Stat3 prior to cytokine stimulation (41-43), giving rise the speculation that the domain-domain association we observed could actually be intermolecular interaction. To address this issue, we found that the minimum region for dimerization is located at the C-terminal portion, including the SH2 domain and the C-terminal domain. On the other hand, the N-terminal domains including the N-domain and the coiled-coil domain are unable to interact each other or form a dimer with the full-length Stat3.3 In the latter situation, the binding site at the C terminus may be "occupied" by the N-terminal domains in a cis manner as reported in c-Abl (44). Therefore, the interaction between the coiled-coil domain and the C-terminal domain is likely to represent an intramolecular rather than an intermolecular interaction, although the latter possibility cannot be completely excluded. Other questions such as whether the intra- and intermolecular interactions exist simultaneously or mutually exclusive, and whether the monomer or the dimer is the major form of Stat proteins in unstimulated situation of a given cell type. Moreover, the Tyr-independent dimer of Stat3 is deficient in the nuclear translocation and DNA binding ability and might play a different physiological role in the cytoplasm (42). In addition, Stat3 protein also associates with other proteins and appears in high molecular mass complexes in the unstimulated cells (45). Thus, the relationship of the intra- and intermolecular interaction and their effects on the regulation of the receptor binding as well as the other functions are a complicated issue, which is the goal for our further investigation.

Physiological Relevance of the Intramolecular Interaction-- To address the physiological relevance of the intramolecular interaction, we demonstrated a functional interaction of Stat3 to the cellular gp130 receptor subunit in response to IL-6 stimulation. In contrast, mutants of Stat3 lacking the putative interaction regions fail to be recruited to the receptor after IL-6 stimulation (Fig. 6C). This confirms their regulatory role in the Stat3-receptor association, and suggests that the intramolecular interaction is a prerequisite for the receptor binding triggered by IL-6 stimulation. Although the current work is based on Stat3, because of high structural conservation among Stat proteins, the findings discussed here may also apply to other members of the Stat family. Moreover, two recent reports demonstrated that the kinase activity of c-Abl and Jak3 is either negatively or positively controlled by intramolecular interaction involving the N-terminal domain and the C-terminal catalytic domains (44, 46). This novel mode of regulation may have profound implications in regulating various proteins with distinct cellular functions.

    ACKNOWLEDGEMENTS

We thank Drs. J. E. Darnell for pRC/CMV-Stat3, R. Jove for Stat3beta , Z. Zhao, E. Manser, and L. Lim for the pXJ40-FLAG and pXJ40-GST vectors. We are grateful to C. P. Lim and V. Novotny for reading the manuscript and helpful discussions.

    FOOTNOTES

* This work was supported by the National Science and Technology Board of Singapore.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.

Dagger Present address: Bio- and Chemoinformatics, Preclinical R&D Medicinal Chemistry, Merck KgaA, 64293 Darmstadt, Germany.

§ Adjunct staff in the Department of Biochemistry, National University of Singapore. To whom correspondence should be addressed: Inst. of Molecular and Cell Biology, 30 Medical Dr., Singapore 117609. Tel.: 65-874-3795, Fax: 65-779-1117; E-mail: mcbcaoxm@imcb.nus.edu.sg.

Published, JBC Papers in Press, February 28, 2002, DOI 10.1074/jbc.M105525200

2 V. Novotny and X. Cao, unpublished observation.

3 T. Zhang and X. Cao, unpublished observation.

    ABBREVIATIONS

The abbreviations used are: STAT, signal transducers and activators of transcription; aa, amino acid(s); JAK, Janus kinase; SH2, Src homology 2; N-domain, N-terminal domain; IFN, interferon; IL, interleukin; PBS, phosphate-buffered saline; ERK, extracellular signal-regulated kinase; GST, glutathione S-transferase.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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