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Methylation of STAT6 Modulates STAT6 Phosphorylation, Nuclear Translocation, and DNA-Binding Activity¹

Division of Allergy and Immunology, Department of Pediatrics, Cincinnati Children’s Hospital, Medical Center, Cincinnati, OH 45229

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Signal transducer and activator of transcription 6 is a transcription factor important for the development of Th2 cells and regulation of gene expression by IL-4 and IL-13. It has been reported that STAT1 activity is regulated by methylation of a conserved arginine residue in the N-terminal domain. Methylation of STAT6 has not yet been explored. We observed methylation of STAT6 in cells transfected with wild-type STAT6, but not in cells transfected with Arg²⁷Ala mutant, confirming that STAT6 is methylated on Arg²⁷. Transfectants expressing mutant Arg²⁷Ala STAT6 displayed markedly diminished IL-4-dependent STAT6 phosphorylation and nuclear translocation, and no STAT6 DNA-binding activity compared with wild-type STAT6 transfectants. To confirm this, the experiments were repeated using inhibitors of methylation. In the presence of methylation inhibitors, STAT6 methylation was diminished, as was phosphorylation of STAT6 and STAT6 DNA-binding activity. Thus, methylation is a critical regulator of STAT6 activity, necessary for optimal STAT6 phosphorylation, nuclear translocation, and DNA-binding activity. Furthermore, methylation of STAT6 has distinct effects from those reported with STAT1.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Signal transducers and activators of transcription proteins are mediators of transcription of several cytokine-inducible genes. These proteins exist as latent cytoplasmic monomers until cytokine stimulation, whereupon they are phosphorylated by the Janus family of kinases (JAKs)³, dimerize, and translocate to the nucleus. Once in the nucleus, activated STATs bind specific canonical DNA elements and initiate cytokine-specific gene transcription in what is commonly referred to as the JAK-STAT pathway of signal transduction (1). To date, six STAT proteins have been described (2), and, of these, only STAT6 has been shown to be specifically activated by IL-4 (3, 4).

It is clear from the characterization of STAT6-deficient mice that STAT6 is required for IL-4-dependent gene induction. STAT6^–/– mice are deficient in their ability to mount a Th2 response following nematode infection, fail to produce IgE, and do not develop airway hypersensitivity following Ag challenge (3, 4, 5, 6, 7, 8, 9). STAT6 is also activated by IL-13 via the IL-13R complex, and STAT6-deficient mice display impaired IL-13 responses (10).

Activation of STAT6 following receptor engagement involves multiple steps. First, association of STAT6 with tyrosine-phosphorylated regions of cytokine receptors occurs via its SH2 domain. Subsequently, STAT6 is phosphorylated on Y641, a residue critical for STAT6 function (11), and phosphorylated STAT6 monomers dimerize via their respective amino-terminal domains (12). Activated STAT6 dimers then translocate to the nucleus, bind specific consensus sequences, and promote transcription of downstream genes. The crystal structure of STAT1-DNA complexes revealed that dimeric interactions between the two respective SH2 domains were critical for the formation of the DNA binding region (13). Nuclear translocation of STAT6 depends on its phosphorylation and dimerization (11, 14); however, the elements responsible for nuclear localization of STAT6 have not been identified.

Although STAT6 activation in response to IL-4 and IL-13 has been well documented, the molecular mechanisms responsible for the regulation of STAT signaling are less well understood. A number of negative regulators of the JAK-STAT signaling pathway have been described, including silencers of cytokine signaling (SOCS) and protein inhibitors of activated STAT (PIAS). SOCS proteins form a negative feedback loop whereby SOCS genes are induced following cytokine stimulation and inhibit cytokine signaling (15, 16). SOCS-1 has been shown to bind and inhibit JAK kinases, but this may not be true of all SOCS family members. A number of PIAS proteins have recently been described with specificity for some STAT family members (17, 18). These proteins bind specifically to phosphorylated STAT dimers and prevent them from binding DNA (19).

Recent studies have demonstrated that STAT1 function is modulated by methylation of Arg³¹ (20). For STAT1, the mechanism of this methylation-dependent modulation appears to be regulation at the level of STAT1-PIAS1 association. PIAS1 binds to STAT1 dimers and prevents STAT1 DNA binding. Methylation of STAT1 does not alter STAT1 phosphorylation, but reduces the ability of STAT1 to associate with PIAS, and thus, increases STAT1 DNA-binding activity (20). No PIAS has yet been identified for STAT6. Several studies have implicated tyrosine phosphatases in the regulation of STAT signaling (21, 22, 23, 24). Consistent with this possibility, studies examining STAT1 have shown that activated STAT1 disappears from the nucleus within 60 min and that removal of the activated STAT1 is dependent on a protein tyrosine phosphatase (21). Recently, a nuclear phosphatase, TC45 (also referred to as TcPTP), a nuclear isoform of T cell protein phosphatase, was identified as the tyrosine phosphatase responsible for STAT1 dephosphorylation in the nucleus (25). Interestingly, another recent study demonstrated that dephosphorylation of STAT1 by TC45 (or TcPTP) is regulated by arginine methylation of STAT1, and that this regulation is dependent on PIAS (26). Their data are consistent with a model whereby inhibition of methylation increases association of STAT1 with PIAS with a concomitant decrease in binding of STAT1 to TcPTP, and thus delayed dephosphorylation of STAT1. Arg³¹ is conserved among the STAT molecules, and methylation is most likely generally important for STAT function. In STAT6, the conserved arginine is at position 27. In this study, we investigated whether STAT6 was methylated on Arg²⁷ and the mechanism by which STAT6 methylation affects its function. Our results demonstrate that STAT6 is methylated on Arg²⁷ and that methylation of STAT6 has a distinct regulatory effect from that reported with STAT1.

	Materials and Methods

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Constructs

Murine STAT6 cDNA was amplified with forward primer 5'-GGGGTACCACCATGTCTCTGTGGGGCCTAATTTCC-3' and reverse primer 5'-CCCTCGAGTCATTTATCATCATCATCTTTATAATCCCAGCTGGGGTTGGTCCTTAGGTC-3' to add a Flag tag at the C terminus, digested with KpnI and XhoI, and cloned into pBKS. Primers 5'-CTTTCCACAACGCCTAGCGCATCTCCTGGCTGAC-3' and 5'-GTCAGCCAGGAGATGCGCTAGGCGTTGTGGAAAG-3' were used in Quickchange mutagenesis (Invitrogen, Carlsbad, CA) to mutate Arg²⁷ to Ala²⁷ according to the manufacturer’s instruction. The Flag-tagged STAT6 and the Flag-tagged STAT6-R27A were then cloned into KpnI and XhoI sites of pREP9 (Invitrogen). Murine STAT6 cDNA was amplified with primers 5'-GGGGTACCACCATGTCTCTGTGGGGCCTAATTTCC-3' and 5'-CCCTCGAGTCAATGGTGATGGTGATGATGCCAGCTGGGGTTGGTCCTT-3' to add a His tag at the C terminus, digested with KpnI and XhoI, and cloned into pCEP4 (Invitrogen).

Transfection

HEK293 cells were transfected with STAT6-Flag-pREP9 or STAT6-R27A-Flag-pREP9 using Effectene (Qiagen, Valencia, CA). Stable transfected cell lines of STAT6-Flag-pREP9 and STAT6-R27A-Flag-pREP9 were selected in medium containing 400 µg/ml G418. STAT6-Flag-pREP9 cells or STAT6-R27A-Flag-pREP9 stable-transfected cells were further transfected with STAT6-His-pCEP4, and the double transfectants were selected in medium containing 400 µg/ml G418 and 200 µg/ml hygromycin. Expression of the transfected Flag- or His-tagged protein was determined by Western blot using anti-Flag (Sigma-Aldrich, St. Louis, MO) or anti-His (Roche Molecular Biochemicals, Mannheim, Germany) Abs.

Immunoprecipitation and immunoblotting

Immunoprecipitation and immunoblotting were performed, as previously described (27). Briefly, cells were stimulated with 10 ng/ml human IL-4 (PeproTech, Rocky Hill, NJ) or 200 U/ml human IFN- (R&D Systems, Minneapolis, MN), or medium for the desired time. For methylation inhibition, cells were pretreated with adenosine, D,L-homocysteine, and N-methyl-2-deoxyadenosine (MDA) (Sigma-Aldrich) (26). Cells were lysed in immunoprecipitation lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% Nonidet P-40, 1 mM PMSF, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 5 mM iodoacetamide, 1 mM sodium orthovanadate, 20 mM NaF, and 1 mM EDTA) and then centrifuged at 20,000 x g for 20 min. The supernatant was precleared with protein A/G plus agarose (Santa Cruz Biotechnology, Santa Cruz, CA), and immunoprecipitated with anti-Flag, anti-His, anti-mono/di-methylarginine (Abcam, Cambridge, U.K.), anti-STAT6 (Santa Cruz Biotechnology), anti-STAT1 (Cell Signaling Technology, Beverly, MA), or isotype control Abs (BD PharMingen, San Diego, CA) and protein A/G plus agarose. The samples from immunoprecipitation were resolved on 10% SDS-PAGE and analyzed by Western blot using anti-Flag, anti-His, anti-STAT1, anti-phospho-STAT1 (Tyr⁷⁰¹) (Cell Signaling Technology), anti-STAT6, or anti-phospho-STAT6 (Tyr⁶⁴¹) (Cell Signaling Technology) Abs. The blots were developed with HRP-conjugated secondary Abs (Santa Cruz Biotechnology) and ECL reagents (Amersham Biosciences, Piscataway, NJ).

EMSA

EMSA was performed, as previously described (27). Briefly, 5 x 10⁶ cells were lysed with 20 µl of EMSA lysis buffer (10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 1.5 mM MgCl₂, 0.2% Nonidet P-40, 1.0 mM DTT, and 0.5 mM PMSF), incubated on ice for 5 min, and centrifuged 10,000 x g for 5 min, and the supernatant was collected as cytoplasmic extract. The pellet was resuspended in 20 µl of EMSA extraction buffer (20 mM HEPES, pH 7.9, 420 mM NaCl, 0.1 mM EDTA, 1.5 mM MgCl₂, 25% glycerol, 1.0 mM DTT, and 0.5 mM PMSF), incubated on ice for 30 min, and centrifuged 20,000 x g for 20 min. The supernatant was collected as nuclear extract. The nuclear extract was used for EMSA using ³²P-labeled STAT6 (or STAT1) probe (Santa Cruz Biotechnology), as previously described (27). For pervanadate treatment, pervanadate was produced by the equimolar combination of sodium orthovanadate and hydrogen peroxide, and the cells were treated with 1 mM pervanadate for 30 min (28, 29).

Pulse chase experiments

Cells were starved in methionine/cysteine/pyruvate/FBS-free DMEM medium (Invitrogen) for 1 h and then incubated in fresh methionine/cysteine/pyruvate/FBS-free DMEM medium containing 10 µCi/ml ³⁵S-methionine (PerkinElmer, Shelton, CT) for 3 h. Complete medium (containing methionine, cysteine, pyruvate, and FBS) with or without IL-4 (10 ng/ml) was added and incubated for desired chase time. Total cell lysate was immunoprecipitated, resolved by 10% SDS-PAGE, and visualized by autoradiography or analyzed by Western blot.

Immunostaining and confocal microscopy

Cells were growing on cell culture coverslips (Fisher Scientific, Pittsburgh, PA) in six-well plates. After treatment or stimulation, the cells were washed with cold PBS for three times and fixed with 3% paraformaldehyde (Electron Microscopy Sciences, Fort Washington, PA) for 10 min and incubated with 15 mM glycine in PBS for 10 min to quench endogenous fluorescence. The cells were incubated with blocking solution (PBS with 3% BSA, 3% cold fish gelatin, 3% FBS, and 0.2% saponin) for 30 min at room temperature. Cells were then incubated with 50 µl of anti-Flag Ab (5 µg/ml) or isotype control mouse IgG1 (5 µg/ml) for 30 min. The cells on the coverslips were washed three times with PBS with 1% FBS and 0.2% saponin. The cells were incubated with 50 µl of 10 µg/ml FITC-conjugated anti-mouse IgG1 (BD PharMingen) for 30 min at room temperature. The coverslips were washed three times, rinsed with H₂O once, and inverted onto slides with Vectorshield (Vector Labarotories, Burlingame, CA), and the edges were sealed with nail polish. The slides were observed under a Leica DM IRBC confocal microscope (Leica Microsystems, Heidelberg, Germany) (27, 30).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
STAT6 is methylated at Arg²⁷

Arg²⁷ at the N terminus of STAT6 is conserved in all STAT family members. Studies on STAT1 have demonstrated that this conserved arginine is methylated and that methylation modulates STAT1 function (20). We first examined whether STAT6 was methylated. HEK293 cells were chosen for stable expression of epitope-tagged STAT6 because this cell line has no endogenous STAT6 activity. These cells were stably transfected with either wild-type STAT6-Flag or Arg²⁷Ala mutant STAT6-Flag. As shown in Fig. 1, wild-type STAT6 was methylated. Notably, STAT6 was methylated at baseline, and IL-4 treatment did not alter STAT6 methylation. However, when Arg²⁷ was mutated to alanine, no methylated STAT6 was detected (Fig. 1) in the presence or absence of IL-4, demonstrating that STAT6 is methylated at Arg²⁷. The expression of transfected wild-type STAT6 was 10-fold higher than the transfected R27A STAT6 mutant. Even when we corrected for this difference (Fig. 1), we did not observe any methylation of the R27A STAT6.

View larger version (31K): [in this window] [in a new window]   FIGURE 1. STAT6 is methylated at Arg27. Flag-tagged wild-type STAT6 or R27A STAT6 mutant was stably expressed in HEK293 cells. A, Western blot of total cell lysate from untransfected HEK293 cells (10 µg), wild-type STAT6-Flag transfected (1 µg), or STAT6-R27A-Flag transfected (10 µg). B, Immunoprecipitation of total cell lysate from untransfected HEK293 cells, wild-type STAT6-Flag transfected, or STAT6-R27A-Flag transfected using anti-mono/di-methylarginine Ab (anti-M/DMA) or isotype control IgG1 and Western blot using anti-Flag Ab. The cells were stimulated with human IL-4 (10 ng/ml) for 15 min, as indicated.

Next, we examined whether Arg²⁷ is important for the STAT6 function. We first investigated whether mutation of Arg²⁷ affected IL-4-induced tyrosine phosphorylation of STAT6. As shown in Fig. 2, tyrosine phosphorylation of the R27A STAT6 mutant was nearly abolished compared with wild-type STAT6. We next examined IL-4-dependent STAT6 DNA-binding activity in the transfectants (Fig. 2). STAT6 DNA-binding activity was absent in transfectants expressing the R27A STAT6 mutant compared with wild-type STAT6.

View larger version (49K): [in this window] [in a new window]   FIGURE 2. R27A STAT6 mutation results in decreased tyrosine phosphorylation and DNA-binding activity of STAT6. Total cell lysate from untransfected HEK293 cells, wild-type STAT6-Flag transfected, or STAT6-R27A-Flag transfected was immunoprecipitated using anti-Flag Ab and blotted using anti-Flag or anti-phospho-STAT6 (Tyr641) Abs. Nuclear extracts from untransfected HEK293 cells (10 µg), wild-type STAT6-Flag transfected (1 µg), or STAT6-R27A-Flag transfected (10 µg) were used to assess STAT6 DNA-binding activity by EMSA. The cells were stimulated with human IL-4 (10 ng/ml) for 15 min, as indicated.

View larger version (22K): [in this window] [in a new window]   FIGURE 3. IL-4-induced nuclear translocation of STAT6 is inhibited by the R27A mutation. Cells was stimulated (or unstimulated) with human IL-4 (10 ng/ml) for 5 or 60 min, and the subcellular localization of Flag-tagged STAT6 or STAT6 R27A mutant was analyzed by confocal microscopy.

Our data to date revealed that Arg²⁷, which we have shown to be methylated, was critical for STAT6 phosphorylation, translocation, and DNA-binding activity. In order to confirm that our observations were reflecting the absence of STAT6 methylation rather than a structural problem with the transfected R27A STAT6 protein, we used an inhibitor of protein methylation. It has been shown that treatment of cells with adenosine, D,L-homocysteine, and MDA resulted in complete inhibition of STAT1 arginine methylation (26). Incubation of HEK293 cells expressing Flag-tagged STAT6 with MDA inhibited the methylation of STAT6, as shown in Fig. 4. Under these conditions, both tyrosine phosphorylation and STAT6 DNA-binding activity of STAT6 were nearly abolished, as shown in Fig. 5. This together with our data on the R27A STAT6 transfectants confirm that methylation of STAT6 on Arg²⁷ is important for tyrosine phosphorylation of STAT6 and subsequent nuclear translocation and DNA-binding activity. We compared the effect of MDA treatment on STAT6 and STAT1 in Hela cells for the tyrosine phosphorylation and DNA-binding activity. The MDA treatment resulted in the inhibition of both IL-4-induced tyrosine phosphorylation and DNA-binding activity of STAT6 (Fig. 6A), as seen in STAT6-transfected HEK293 cells. With the MDA treatment, the IFN--induced DNA-binding activity of STAT1 was inhibited, but the tyrosine phosphorylation of STAT1 was not affected (Fig. 6B), which is consistent with the results reported by others (26), suggesting the different role of methylation in activation of STAT6 and STAT1.

View larger version (58K): [in this window] [in a new window]   FIGURE 4. MDA treatment inhibits methylation of STAT6. Cells were treated with or without MDA (600 µM) for 6 h and then stimulated or not with human IL-4 (10 ng/ml) for 15 min. Total cell lysate was subject to immunoprecipitation using anti-mono/di-methylarginine Ab (anti-M/DMA) or isotype control IgG1 and Western blot using anti-Flag Ab.

View larger version (67K): [in this window] [in a new window]   FIGURE 5. MDA treatment inhibits IL-4-dependent STAT6 tyrosine phosphorylation and DNA-binding activity in STAT6-transfected HEK293 cells. Cells were treated with MDA (600 µM) for 6 h and then stimulated (or unstimulated) with human IL-4 (10 ng/ml) for 15 min. Total cell lysate from untransfected HEK293 cells, wild-type STAT6-Flag transfected, or STAT6-R27A-Flag transfected was immunoprecipitated using anti-Flag Ab and then immunoblotted using anti-Flag or anti-phospho-STAT6 (Tyr641) Abs. The nuclear extract (5 µg) was used for STAT6 DNA-binding activity by EMSA.

View larger version (36K): [in this window] [in a new window]   FIGURE 6. MDA treatment inhibits IL-4-dependent STAT6 tyrosine phosphorylation and DNA-binding activity, and IFN--dependent STAT1 DNA-binding activity in Hela cells. Hela cells were treated with MDA (300 µM) for 3 h and then stimulated (or unstimulated) with human IL-4 (10 ng/ml) for 15 min (A) or human IFN- (200 U/ml) for 30 min (B). Total cell lysate was immunoprecipitated using anti-STAT6 or anti-STAT1 Abs and then immunoblotted using anti-STAT6, anti-phospho-STAT6 (Tyr641), anti-STAT1, or anti-phospho-STAT1 (Tyr701) Abs. The nuclear extract (10 µg) was used for STAT6 or STAT1 DNA-binding activity by EMSA.

To explore possible mechanisms underlying the inhibition of tyrosine phosphorylation and loss of DNA-binding activity of R27A STAT6, we treated R27A STAT6-transfected cells with pervanadate, an inhibitor of protein tyrosine phosphatases, and then examined the tyrosine phosphorylation and DNA-binding activity. As shown in Fig. 7, the tyrosine phosphorylation of R27A STAT6 was not affected by pervanadate treatment in the presence or absence of IL-4 stimulation, suggesting that the R27A STAT6 undergoes tyrosine phosphorylation at a very low level upon IL-4 stimulation, and R27A mutation mainly affects the phosphorylation rather than dephosphorylation. No STAT6 DNA-binding activity was observed in R27A STAT6-transfected cells after pervanadate treatment and/or IL-4 stimulation. In comparison, the treatment with pervanadate and/or IL-4 stimulation induced significant tyrosine phosphorylation and STAT6 DNA-binding activity in cells transfected with wild-type STAT6. The DNA-binding activity detected in R27A STAT6-transfected HEK293 cells was not STAT6 specific, as it was not supershifted by anti-STAT6 Ab and it was also present in untransfected cells.

View larger version (71K): [in this window] [in a new window]   FIGURE 7. Pervanadate treatment has no effect on the tyrosine phosphorylation and DNA-binding activity of R27A STAT6. Untransfected (UT), STAT6-R27A-Flag-transfected, or wild-type STAT6-Flag-transfected HEK293 cells were treated with pervanadate (PV) (1 mM) for 30 min and then stimulated (or unstimulated) with human IL-4 (10 ng/ml) for 15 min. A, Total cell lysate was immunoprecipitated using anti-Flag Ab and blotted using anti-Flag or anti-phospho-STAT6 (Tyr641) Abs. B, Nuclear extracts from untransfected (10 µg), STAT6-R27A-Flag-transfected (10 µg), or wild-type STAT6-Flag-transfected (1 µg) HEK293 cells were used to assess STAT6 DNA-binding activity by EMSA, and anti-STAT6 Ab was used for the supershift assay.

Because R27A STAT6 had lower expression level compared with wild-type STAT6, we determined their stability by pulse chase experiments with labeling of ³⁵S-methionine and immunoprecipitation. The R27A STAT6 had a shorter t_1/2 than wild-type STAT6 (Fig. 8). The R27A STAT6 was gradually degraded, while the wild-type STAT6 was stable over time. IL-4 stimulation had no effect on the stability of R27A STAT6 (Fig. 8). When IL-4 was added to the chase medium, the stability of R27A STAT6 was not altered. This suggests that arginine methylation not only contributes to the IL-4-induced tyrosine phosphorylation, but also stability of STAT6.

View larger version (41K): [in this window] [in a new window]   FIGURE 8. R27A STAT6 is less stable compared with the wild-type STAT6. Untransfected (UT), STAT6-R27A-Flag-transfected, or wild-type STAT6-Flag-transfected HEK293 cells were labeled with 35S-methionine and then chased for 1–48 h in the absence or presence of IL-4. Total cell lysate was immunoprecipitated using anti-Flag Ab, resolved by SDS-PAGE, and visualized by autoradiography or analyzed by Western blot using anti-Flag Ab.

We next wanted to examine whether the presence of R27A STAT6, incapable of methylation, affected the function of wild-type STAT6. To examine this, cells were cotransfected with Flag-tagged R27A, and His-tagged wild-type STAT6 were compared with transfectants expressing Flag-tagged wild-type and His-tagged wild-type STAT6. STAT6 DNA-binding activity was not affected in wild-type STAT6 and R27A STAT6 mutant double-transfected cells compared with that in wild-type/wild-type double-transfected cells (Fig. 9). However, the expression of the R27A STAT6 was very low compared with wild-type STAT6, and the effect of unmethylated STAT6 may require a larger presence of unmethylated STAT6. Alternatively, a small percentage of the total STAT6 pool may be activated upon cytokine stimulation, and this may be unaffected by the presence of low levels of R27A STAT6.

View larger version (80K): [in this window] [in a new window]   FIGURE 9. Coexpression of R27A STAT6 and wild-type STAT6 in HEK293 cells. Cells were stimulated with human IL-4 (10 ng/ml) for 15 min. Total cell lysate (10 µg) from untransfected HEK293 cells, wild-type STAT6-Flag/wild-type STAT6-His double transfected, or STAT6-R27A-Flag/wild-type STAT6-His double transfected was analyzed by Western blot using anti-Flag or anti-His Abs. The nuclear extract (5 µg) was analyzed for DNA-binding activity by EMSA.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Arginine methylation of STAT1 has been reported previously (20). Arginine methylation has been shown to modulate STAT1 function by affecting its ability to associate with PIAS1 (20). PIAS1 interacts with activated STAT1 dimers and prevents their ability to bind DNA. STAT1 arginine methylation decreases the ability of STAT1 to interact with PIAS, and thus, effectively increases STAT1 activity. A specific PIAS has not been found for STAT6, suggesting that methylation may have a different role in regulation of STAT6 function. It is likely that all STATs are methylated because the arginine residue is highly conserved in the N-terminal domain. However, our data support that methylation may play distinct roles in the activities of STAT proteins. Our results with the Arg²⁷Ala STAT6 mutant establish that STAT6 methylation occurs at this conserved arginine residue. This mutation dramatically decreased the level of tyrosine phosphorylation of STAT6, supporting that this residue contributes to the step of tyrosine phosphorylation in the STAT6 activation cascade. This contrasts with the results from the STAT1 studies, which showed that the methylation of STAT1 is independent of STAT1 tyrosine phosphorylation (20). Fig. 10 schematically summarizes the effect of arginine methylation on STAT6 and STAT1 function. The finding of STAT N-terminal amino acid residues contributing to the tyrosine phosphorylation is not unique to STAT6. In the case of STAT4, selective mutations at the N-terminal domain of STAT4 interfere with the IFN--induced tyrosine phosphorylation of STAT4 (31). It is most likely that the N terminus of each STAT family member has a unique function. This is further supported by recent studies examining STAT1-STAT4 chimeric proteins; the N-terminal domains of STAT1 and STAT4 were not functionally interchangeable (31).

View larger version (19K): [in this window] [in a new window]   FIGURE 10. Comparison of effect of methylation on the activation of STAT6 and STAT1. A, Unmethylated STAT6 has a decreased level of tyrosine phosphorylation upon IL-4 stimulation, resulting in decreased nuclear translocation and DNA-binding activity. B, In contrast, unmethylated STAT1 can undergo normal tyrosinine phosphorylation upon IFN- stimulation, but unmethylated phosphorylated STAT1 dimers have enhanced association with PIAS1, resulting in decreased nuclear translocation and DNA-binding activity.

It is not surprising that the nuclear translocation and DNA-binding activity of STAT6 induced by IL-4 stimulation were affected by the R27A mutation or methylation inhibition because the tyrosine phosphorylation is a requirement for the formation of active dimers, which are subsequently translocated into nucleus and bind specific DNA sequence to activate transcription. To test the effect of STAT6 R27A mutant on the activation of wild-type STAT6, we coexpressed R27A STAT6 mutant and wild-type STAT6. We observed no significant difference in the total STAT6 DNA-binding activity between the wild-type/wild-type double transfectant and wild-type/R27A double transfectant after IL-4 stimulation. However, the R27A mutant was expressed at a substantially lower level than the wild-type STAT6. Thus, direct competition or other regulation between the R27A mutant and wild-type STAT6 cannot be ruled out. Furthermore, because we did observe some minimal phosphorylation of R27A STAT6 following IL-4 treatment, it remains possible that heterodimers can form between unmethylated and methylated STAT6 forms. Because much less STAT6 R27A mutant was tyrosine phosphorylated compared with the wild type, it is likely that the R27A STAT6 mutant affects the early step of STAT6 activation by competitively binding proteins required for the tyrosine phosphorylation of STAT6.

As discussed above, arginine methylation of STAT1 regulates its association with PAIS1; likewise, arginine methylation of STAT6 might regulate its association with other proteins, such as proteins involved in STAT6 tyrosine phosphorylation. The amino acid sequence containing Arg²⁷ is LRHLL, which matches the LXXLL motif found within the trans activation domain of STAT6 mediating the interaction with NcoA-1 (33). It is possible that LXXLL motif within the N-terminal domain mediates the interaction of STAT6 with unidentified cytoplasmic proteins, and the methylated arginine in this motif is the key residue. There are several protein arginine methyl transferases (PRMTs) that have been identified, and STAT1 has been shown to be a substrate for PRMT1 and PRMT3 in vitro (20). It remains unclear how these enzymes are regulated. How arginine methylation is regulated and how methylation of arginine affects the interaction of STAT6 with other proteins remain to be determined.

	Acknowledgments

 
We are grateful to Drs. Jeffrey Whitsett and Fred Finkelman for helpful discussions, and to Dr. Timothy Weaver for help with the confocal microscopy. We thank Connie Petitt for excellent secretarial support.

	Footnotes

 
¹ This work was supported by American Heart Association Grant 0060337B (to G.K.K.H.) and National Institutes of Health Grant R01AI46652-01A1 (to G.K.K.H.).

² Address correspondence and reprint requests to Dr. Gurjit K. Khurana Hershey, Division of Allergy and Immunology, Children’s Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229. E-mail address: Gurjit.Hershey{at}cchmc.org

³ Abbreviations used in this paper: JAK, Janus kinase; MDA, N-methyl-2-deoxyadenosine; PIAS, protein inhibitors of activated STAT; PRMT, protein arginine methyl transferase; SOCS, silencers of cytokine signaling; TcPTP, T cell protein phosphatase.

Received for publication November 25, 2003. Accepted for publication April 1, 2004.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

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