Ras-ERK MAPK Cascade Regulates GATA3 Stability and Th2 Differentiation through Ubiquitin-Proteasome Pathway*

Differentiation of naive CD4 T cells into Th2 cells requires protein expression of GATA3. Interleukin-4 induces STAT6 activation and subsequent GATA3 transcription. Little is known, however, on how T cell receptor-mediated signaling regulates GATA3 and Th2 cell differentiation. Here we demonstrated that T cell receptor-mediated activation of the Ras-ERK MAPK cascade stabilizes GATA3 protein in developing Th2 cells through the inhibition of the ubiquitin-proteasome pathway. Mdm2 was associated with GATA3 and induced ubiquitination on GATA3, suggesting its role as a ubiquitin-protein isopeptide ligase for GATA3 ubiquitination. Thus, the Ras-ERK MAPK cascade controls GATA3 protein stability by a post-transcriptional mechanism and facilitates GATA3-mediated chromatin remodeling at Th2 cytokine gene loci leading to successful Th2 cell differentiation.

In peripheral lymphoid organs, naive CD4 T cells that have recognized specific antigens differentiate into either one of two distinct helper T cell subsets, Th1 and Th2 cells (1). Upon antigen restimulation, Th1 cells produce large amounts of IFN␥ 1 and direct cell-mediated immunity against intracellular pathogens. Th2 cells produce IL-4, IL-5, and IL-13 and are involved in humoral immunity and allergic reactions. The direction of Th cell differentiation depends on the types of cytokine in the environmental milieu (2,3). Naive CD4 T cells stimulated with antigens in the presence of IL-12 differentiate into Th1 cells, whereas the presence of IL-4 drives differentiation into Th2 cells (4 -6). IL-12-mediated activation of signal transducer and activator of transcription (STAT) 4 is crucial for Th1 cell differentiation, and IL-4-mediated STAT6 activation is important for Th2 cell development (7)(8)(9).
In addition to the cytokines mentioned above, activation of TCR-mediated signaling is also indispensable for both Th1 and Th2 cell differentiation. We reported that Th2 cell differentiation is highly dependent on the extent of TCR-mediated activation of the p56 lck , calcineurin, and Ras-ERK MAPK signaling cascade (10 -12). In particular, inhibition of the activation of the Ras-ERK MAPK cascade caused a shift from Th2 to Th1 cell differentiation, suggesting that the direction of Th1/Th2 cell differentiation could be controlled by TCR-mediated activation of the Ras-ERK MAPK cascade (11,13). On the other hand, Th1 cell development appeared to be regulated by another MAPK, c-Jun N-terminal kinase (14,15).
Recently, several transcription factors that control Th1/Th2 cell differentiation were identified (8,16). Among them, GATA3 appears to be a key factor for Th2 cell differentiation. GATA3 is selectively induced in developing Th2 cells after TCR stimulation in the presence of IL-4, and ectopic expression of GATA3 resulted in the induction of Th2 cell differentiation in the absence of STAT6 (17)(18)(19)(20). GATA3 was found to be important for the maintenance of the Th2 phenotype (21,22).
Th2 cell differentiation is accompanied by chromatin remodeling of the Th2 cytokine (IL-4/IL-5/IL-13) gene loci, e.g. hyperacetylation of histones H3 and H4 (23)(24)(25). Hyperacetylation of the IL-4 and IL-13 gene loci (23) and that of IL-5 gene locus (26) are highly dependent on the expression of GATA3. We described a precise map of the Th2-specific histone hyperacetylation within the Th2 cytokine gene loci, and we identified a 71-bp conserved GATA3-response element at 1.6 kbp upstream of the IL-13 locus (23). The GATA3-response element appears to play a crucial role for GATA3-mediated targeting and downstream spreading of core histone hyperacetylation within the IL-13 and IL-4 gene loci in developing CD4 ϩ Th2 and CD8 ϩ Tc2 cells (23,27).
One of the major pathways of degradation of short lived regulatory proteins, including transcriptional factors, is through ubiquitin-mediated targeting and protein destruction in the 26 S proteasome. Protein ubiquitination is involved in a wide range of cellular processes, including cell cycle progression, signal transduction, transcriptional regulation, DNA re-pair, antigen presentation, and apoptosis (28 -31). Emerging views suggest that various aspects of the immune system are controlled by ubiquitination (32). A well known example of the ubiquitin-dependent regulation in the immune system is the proteasome-dependent processing of peptides in antigen-presenting cells (33). It is also well known that lipopolysaccharide or proinflammatory cytokines such as IL-1 can induce activation of NF-B through ubiquitination and subsequent degradation of the inhibitor of B (34). However, a role for the ubiquitin-mediated regulation of Th1/Th2 cell differentiation has not been reported.
In the present study, we investigated the molecular targets of the Ras-ERK MAPK cascade that control chromatin remodeling of the Th2 cytokine gene loci and subsequent Th2 cell differentiation, and we found that the Ras-ERK MAPK cascade controls the stability of the GATA3 protein through the ubiquitin-proteasome pathway. Moreover, we demonstrated that the ubiquitination of GATA3 by Mdm2 is dependent on a ring finger domain.
Retroviral Vectors and Infection-pMX-IRES-CAR (human coxsackie-adenovirus receptor) plasmid was generated from the pMX-IRES-GFP plasmid by replacing the EGFP gene with the CAR gene. The methods for the generation of virus supernatant and the infection were described previously (37). Infected cells were subjected to intracellular staining with anti-IL-4 and anti-IFN␥ mAb or to cell sorting. To prepare large numbers of infected cells for immunoblotting, the pMX-IRES-CAR or pMXs-IRES-hNGFR vector was used, and the infected cells were enriched by auto-MACS® sorter with anti-CAR mAb (38) or anti-human NGFR (C40 -1457; Pharmingen). cDNA encoding Erk2 sem was described previously (39). pMXs-Mdm2 dR-hNGFR was constructed by inserting Mdm2-dR into a multicloning site of pMXs-IRES-hNGFR. cDNA for human GATA3 or an active form of human Raf-1 (40) was inserted into a multicloning site of pMX-IRES-GFP.
Chromatin Immunoprecipitation (ChIP) Assay-ChIP was performed using the histone H3 assay kit (catalog number 17-245; Upstate Biotechnology, Inc.) as described previously (23). Semi-quantitative PCR was performed with DNA samples from 3 or 1 ϫ 10 4 cells at 28 cycles. PCR products were resolved in an agarose gel and visualized by ethidium bromide. Images were recorded and quantified using ATTO L & S analyzer (ATTO, Tokyo, Japan). The primers used were described previously (23).
For anti-ubiquitin blotting, COS cells were transfected with Myctagged GATA3 vectors (pCMV Tag 3B). Two days later, cells were treated with cycloheximide (100 M) and U0126 (20 M) in the presence or absence of the proteasome inhibitor MG132 (50 M) for 2 h. The cells were then pelleted, resuspended in RIPA buffer, and lysed on ice for 30 min. Insoluble material was removed by centrifugation. The lysates were incubated with 5 g of anti-Myc mAb (MBL, Japan) for 2 h at 4°C. 50 l of protein G-conjugated Sepharose (Amersham Biosciences) was then added and incubated for an additional 1 h. After removal of the supernatant, the beads were washed twice with RIPA buffer. The bound protein was eluted by adding 25 l of SDS sample buffer and was subjected to immunoblot analysis using mAb specific for ubiquitin (FK2; MBL, Tokyo, Japan).
Northern Blotting-Total RNA (20 g) was isolated from cultured cells using TRIzol reagent (Invitrogen), separated on a 1% formaldehyde gel, and transferred to a Nytran Plus membrane (Schleicher & Schuell). Probes for GATA3 and ␤-actin were generated by PCR using the primers described previously (37). The digoxigenin labeling and detection system (Roche Diagnostics) was used for visualization.
Pulse-Chase Experiment-Splenic CD4 T cells were stimulated for 2 days under Th2Ϫ conditions. The cells were washed, preincubated for 30 min in methionine/cysteine-free medium, and pulsed for 30 min with 200 Ci/ml [ 35 S]methionine/cysteine (ICN). Then the cells were washed twice with Dulbecco's modified Eagle's medium containing nonradioactive 5 mM L-methionine, 3 mM L-cysteine, and 0.25% FCS, and chased in the same medium in the presence of PMA (3 ng/ml) or PMA plus U0126 (20 M). U0126 was used in the pulse-chase experiment because it is known to inhibit preactivated MEK as well (41).
In Vitro Ubiquitination Assay-In vitro ubiquitination assay was performed as described previously (42). In brief, 293T cells were transfected with FLAG-tagged GATA3, and 3 days later the cells were treated with MG132 for 2 h. Then the cells were lysed in RIPA buffer (2.5 ϫ 10 6 cells/ml), and the cell lysates (250 l) were subjected to immunoprecipitation with anti-FLAG or anti-GATA3 mAb. The immunoprecipitates were incubated for 2 h at 30°C in 25 l of reaction buffer containing 50 mM Tris-HCl (pH 8.0), 50 ng of recombinant mouse ubiquitin-activating enzyme, 500 ng of ubiquitin carrier protein, 5 g of glutathione S-transferase-Ub, 1 mM dithiothreitol, 2 mM MgCl 2 , and 4 mM ATP. After terminating the reaction by the addition of 2ϫ SDS sample buffer, immunoblotting with anti-GATA3 or anti-FLAG mAb was performed. Recombinant mouse ubiquitin-activating enzyme, ubiquitin carrier protein, and glutathione S-transferase-Ub were kindly provided by Dr. Keiji Tanaka (Tokyo Metropolitan Institute for Medical Science, Tokyo, Japan).
siRNA-Introduction of siRNA into a T cell line TG40 was performed as described (43). In brief, 2 l of TransIT-TKO transfection reagent (Mirus) was diluted in 50 l of serum-free/antibiotic-free RPMI 1640 per well. Ten minutes later, 1 l of 40 M siRNA was added to the diluted transfection reagent and incubated for 30 min with gentle agitation. Then the siRNA solution was added to TG40 cultures containing 5 ϫ 10 5 cells in 500 l of medium per well in a 24-well plate. Three days after transfection, ubiquitination of GATA3 and the expression of Mdm2 protein were assessed by immunoblotting. Pre-designed siRNA for Mdm2 was purchased from Ambion (16704), and control siRNA was from Santa Cruz Biotechnology (sc-37007).

RESULTS
The Ras-ERK MAPK Cascade Controls Histone Hyperacetylation of the Th2Ϫ Cytokine Gene Loci-We reported that Th2 cell differentiation and certain Th2 responses are dependent on the extent of activation of the Ras-ERK MAPK cascade (11,13). Hyperacetylation of the Th2 cytokine gene loci was highly dependent on the expression of GATA3 (23,26). We present here further confirmation of the observations in chromatin remodeling of the Th2 gene loci. The generation of IL-4-produc-ing cells was substantially inhibited in the presence of a specific inhibitor of MEK (an ERK MAPKK), PD98059 (Fig. 1A). Fig. 1B shows that the acetylation levels of histones associated with the Th2 cytokine gene loci (IL-4 promoter, IL-13 promoter, IL-5 promoter, GATA3-response element, CNS1, and IL-4 V A enhancer) were significantly reduced in the presence of PD98059. In the case of RAD50, there was no significant effect with PD98059 treatment, and with the IFN␥ promoter (IFN␥p) there was some enhancement in the acetylation. Under Th1 culture conditions, PD98059 exhibited no detectable inhibition of acetylation at the IFN␥ promoter region (data not shown).
In addition, the effect of ectopic expression of an active form of Raf-1 (active-Raf) on the generation of Th1/Th2 cells was assessed. Significant numbers of IL-4-producing Th2 cells and a suppression of the generation of IFN␥-producing cells were observed in active Raf-infected cells stimulated under Th1Ϫ conditions (Fig. 1C). As a positive control, GATA3 infection was included, and in this case substantial levels of IL-4-producing cells with decreased numbers of IFN␥-producing cells were detected. Assessment of the acetylation status of histones in the active Raf-infected or GATA3-infected T cells defined by GFP expression on day 2 revealed significant increases in acetylation at the IL-4, IL-13, and IL-5 promoters and CNS1 and IL-4 V A enhancer regions; however, the effect on the RAD50 promoter region was marginal (Fig. 1D). A significant decrease in the acetylation of the IFN␥ promoter region was observed in the presence of active Raf. The levels of acetylation induced by active Raf infection were lower than those induced by GATA3. A possible explanation for this could be the limited expression of GATA3 in T cells cultured under Th1Ϫ conditions. Nevertheless, it is clear that the ERK-MAPK cascade controls GATA3-dependent histone hyperacetylation of the Th2 cytokine gene loci in developing Th2 cells.

Activation of the Ras-ERK MAPK Cascade Is Required for Stable Expression of GATA3 Protein in Developing Th2
Cells-GATA3 is a critical transcriptional factor for Th2 cell differentiation (17)(18)(19), and its expression is induced selectively under Th2Ϫ conditions. Here we demonstrate this in freshly prepared splenic CD4 T cells stimulated under Th1 and Th2 conditions ( Fig. 2A, compare lanes 1 and 3 and lanes 2 and 4). Treatment of Th2 condition cultures with PD98059 resulted in decreased protein expression of GATA3 ( Fig. 2A, lanes 5 and 6). Similarly, the induction of GATA3 protein in dominant-negative Ras (dnRas) Tg T cells was significantly lower than that seen in the control, and this is consistent with the observation of impaired Th2 cell differentiation in dnRas Tg mice (11). A specific inhibitor for the p38 MAPK cascade (SB203580) did not affect the GATA3 expression in developing Th2 cells (data not shown). The activation of STAT6 is known to be critical for GATA3 transcription, and as expected, STAT6-deficient (STAT6 KO) CD4 T cells failed to induce GATA3 protein.
Concurrently, the transcriptional levels of GATA3 in T cells cultured under Th2 conditions (12 and 24 h) as in Fig. 2A were assessed (Fig. 2B). The inhibition of activation of the Ras-ERK MAPK cascade by PD98059 or overexpression of dnRas transgene in T cells had no blocking effect on GATA3 mRNA expression. Rather, significant enhancement of GATA3 levels was detected at the 12-h time point. In contrast, GATA3 mRNA was not induced to significant levels in STAT6-deficient CD4 T cells, which is consistent with the lack of induced GATA3 protein.
Consequently, we sought to investigate further the consequence of the inhibition of ERK MAPK activation on the degradation of GATA3 protein. Splenic CD4 T cells were first stimulated under Th2Ϫ conditions for 2 days prior to being cultured without IL-4 or immobilized anti-TCR mAb for various time periods (chase) in either the presence or absence of another specific MEK inhibitor (U0126), which is also effective on activated MEK (Fig. 3, A and B). Under normal culture conditions with 10% FCS, the levels of GATA3 protein remained elevated over 12 h but then they declined significantly at 24 h. In the presence of U0126, significant reduction of GATA3 protein was observed at the 6-and 12-h time points, and the protein was virtually undetectable after a 24-h chase (Fig. 3A). U01126 treatment did not affect the level of another Th2-specific transcription factor, c-Maf. Under culture conditions with low levels of FCS (0.25%), where decreased levels of background stimulation of the MAPK cascades were detected (data not shown), a gradual decrease in GATA3 protein was seen and a modest rescue by the presence of PMA was observed (Fig. 3B). In the presence of U0126, the loss of GATA3 protein was dramatic, and GATA3 was barely detectable at the 12-and 24-h time points.
As an alternative means to assess the effect of ERK MAPK on the stability of the GATA3 protein, we performed pulsechase experiments to follow the degradation of GATA3, and we found that the amount of 35 S-labeled nascent GATA3 protein was degraded very rapidly in the presence of U0126 (Fig. 3C). These results clearly demonstrate that rapid degradation of GATA3 occurs when activation of ERK MAPK is inhibited.
As a more direct test of the requirement for the activation of ERK MAPK to stabilize GATA3 in developing Th2 cells, we introduced an active form of ERK2 (Erk2 sem) (39) or a dominant-negative form of ERK2 (dnErk2) (44) into developing Th2 cells (Fig. 3D). As anticipated, GATA3 protein was significantly retained in cells infected with ERK2 sem, whereas the expres-sion of dnERK2 significantly enhanced its degradation. Thus, the stability of GATA3 protein in developing Th2 cells appears to be highly dependent on the activation of the Ras-ERK MAPK cascade.
GATA3 Is Rapidly Degraded through the 26 S Proteasome Pathway-It would seem very likely that the proteasome pathway would be involved. To determine whether the 26 S proteasome is involved with the rapid degradation of the GATA3 protein, the effect of a proteasome inhibitor MG132 was examined. COS cells were transfected with Myc-tagged GATA3 and treated with cycloheximide (CHX) to inhibit protein synthesis in the presence or absence of MG132 for 1 or 2 h. Myc-tagged GATA3 was predominantly expressed in the nucleus, and after CHX treatment it was degraded rapidly in the absence of MG132 (Fig. 4A, left panel). In contrast, in the presence of MG132, there was a dramatic increase in the amount of Myctagged GATA3 protein in both nuclear and cytoplasmic fractions (Fig. 4A, right panel). Moreover, in developing Th2 cells GATA3 protein was degraded rapidly in the presence of CHX, and the degradation was inhibited by MG132 (Fig. 4B). In addition, another proteasome inhibitor lactacystin was tested in primary T cells for its ability to affect the degradation of GATA3, and a significant blocking of the degradation of GATA3 was observed (Fig. 4C). Collectively, these results point to the involvement of the 26 S proteasome pathway in the degradation of GATA3.
The C-terminal Region of GATA3 Including the Zinc Finger Domain Is Critical for Proteasome-dependent Degradation-In an attempt to map the target region of GATA3 that is critical for proteasome-dependent degradation, we prepared several truncated GATA3 mutants of the N-terminal region, which contains many lysine residues that can be ubiquitinated (Fig.  4D). Myc-tagged wild type (WT), dCT, dCF, and dZF constructs were transfected into COS cells, and the expression levels of GATA3 were assessed after treatment with MG132 (Fig. 4E). In comparison to wild type GATA3, there was essentially no difference in the pattern of disappearance of either the dCT or dCF mutant forms from nuclear fractions following CHX treatment, or in the retention of GATA3 protein by MG132 treatment. In the cytosol fraction of transfectants without drug treatment, slightly increased levels of protein were detected with dCT and dCF mutants, but the levels after treatment with MG132 were indistinguishable (Fig. 4E, lower panels). In sharp contrast, large amounts of dZF mutant protein were detected in the cytosol, and treatment with either CHX or MG132 did not have a significant effect on the levels of the mutant protein.
Small amounts of dZF protein were detected in the nuclear fraction, and a modest increase was detected in the presence of MG132. Thus it would appear the C-terminal region of GATA3, including the zinc finger region (residues 261-315), is critical for proteasome-dependent degradation.
To visualize the dynamics of localization and accumulation, green fluorescence protein (GFP)-fused wild type GATA3 and the dZF mutant were expressed in NIH3T3 cells. As expected, wild type GATA3 showed decreased fluorescence following CHX treatment, and the decrease in fluorescence was prevented to some extent in the presence of MG132 (supplemental Fig. 1). The dZF mutant was expressed in both nuclear and cytosolic fractions, and the fluorescence intensity was not affected by treatment with CHX or MG132. These results are consistent with the results shown in Fig. 4C.
The ERK MAPK Cascade Regulates GATA3 Ubiquitination-In order to assess the involvement of multiubiquitination (Ub) in GATA3 degradation, FLAG-tagged wild type GATA3, dCT, dCF, and dZF mutants were each transfected into 293T cells, and transfectants were treated with MG132. Immuno- blotting with anti-Ub mAb was performed after anti-FLAG immunoprecipitation (Fig. 5A). Significantly increased levels of multiubiquitination (appears as smear) were observed in the wild type GATA3 transfectants compared with control vector, and the levels were significantly increased in the presence of MG132 (Fig. 5A, compare lanes 3 and 4). The levels of multiubiquitination appeared to be equivalent in the case of the dCT mutant (Fig. 5A, lanes 5 and 6), slightly decreased in the dCF mutant (lanes 7 and 8), and greatly reduced in the dZF mutant (lanes 9 and 10). The levels of FLAG-tagged protein in total (nuclear and cytoplasmic) lysates were not reduced in these transfectants (Fig. 5A, right panel). Thus, multiubiquitination of GATA3 protein can be readily demonstrated, and it appears that the dZF mutant is the least modified among the mutant forms tested. The multiubiquitination on truncated GATA3 mutants was further assessed by anti-FLAG immunoblotting (Fig. 5B). The levels of multiubiquitination were slightly decreased in the dCF mutant and greatly reduced in the dZF mutant, indicating again that the C-terminal region of GATA3, including the zinc finger region (residues 261-315), is critical for ubiquitination.
We performed an in vitro ubiquitination assay as a further demonstration of the multiubiquitination on GATA3. 293T cells were transfected with FLAG-tagged GATA3, and 3 days later, the cells were treated with MG132 for 2 h. In vitro ubiquitination was performed after anti-GATA3 immunoprecipitation, and ubiquitinated GATA3 was detected by immunoblotting with anti-GATA3 (Fig. 5C, left panel). Concurrently, anti-FLAG immunoprecipitation and anti-FLAG immunoblot-ting were done (Fig. 5C, right). Although variable levels of multiubiquitinated GATA3 were detected without in vitro ubiquitination (Fig. 5C, lane 4), significantly increased signals with new bands (indicated by *) were detected after in vitro ubiquitination (lane 8). Similarly, increased ubiquitination was readily detected after in vitro ubiquitination in the anti-FLAG immunoblot (Fig. 5C, compare lanes 12 and 14).
Next, in order to examine the involvement of activation of the ERK-MAPK cascade in the GATA3 ubiquitination, we assessed the effect of PMA to activate the MAPK cascades and U0126 to inhibit selectively the ERK-MAPK cascade on the ubiquitination of GATA3. 293T cells transfected with FLAG-tagged GATA3 were treated with PMA in the presence or absence of U0126, and then the levels of ubiquitination on GATA3 were assessed (Fig. 5D). Treatment with PMA resulted in a reduction in the degree of ubiquitination of GATA3, and this effect could be reversed significantly by the addition of U0126, suggesting that the ubiquitination of GATA3 is regulated by the activation of ERK MAPK. Similarly, in developing Th2 cells, the ubiquitination of GATA3 protein was detected when the cells were treated with MG132 (20 M) for 2 h, and the levels in ubiquitination were enhanced in the presence of U0126 (data not shown).
Mdm2 Acts as a Ubiquitin E3 Ligase for GATA3-We wanted to identify possible E3 ligase for GATA3 in developing Th2 cells. Since the association with specific substrates is critical for the function of E3 ligases (32, 45), we first examined the physical association of GATA3 with known E3 ligases that are expressed in lymphocytes (Mdm2, Itch, E6-AP, and Cbl-b). Freshly isolated splenic CD4 T cells were stimulated with immobilized anti-TCR mAb under Th2Ϫ conditions for 3 days in the presence or absence of U0126. Immunoprecipitates with anti-GATA3 mAb were subjected to immunoblotting with anti-Mdm2 mAb and anti-GATA3 mAb (Fig. 6A) and with specific antibodies for several E3 ligases (Fig. 6B). Large amounts of Mdm2 were detected in the GATA3Ϫ precipitates from U0126treated cells, suggesting association of Mdm2 with GATA3, although the amount of GATA3 is significantly reduced (ϳ1/3) in the U0126-treated cells (Fig. 6A). Although there were substantial amounts of E6-AP, Itch, or Cbl-b molecules in developing Th2 cells, no significant quantity of E6-AP, Itch, or Cbl-b was detected in the anti-GATA3 immunoprecipitates under the conditions where substantial amounts of Mdm2 were detected (Fig. 6B). Thus, the association of Mdm2 with GATA3 appeared to be more selective than that of other E3 ligases (Itch, E6-AP, and Cbl-b).
To characterize further the Mdm2 association with GATA3, 293T cells were transfected with FLAG-tagged GATA3 and their mutants (dCT, dCF, and dZF) and were treated with MG132 for 2 h. Immunoprecipitates with anti-FLAG mAb were immunoblotted with anti-Mdm2 mAb. As shown in Fig. 6B, upper panel, association of Mdm2 with GATA3 was readily detected, and the association was apparently decreased in the dCF mutant and almost undetectable in the dZF mutant. The amounts of Mdm2 and FLAG-GATA3 protein in these transfectants were similar (Fig. 6B, middle and bottom panels). Thus Mdm2 appears to be constitutively associated with wild type GATA3 in 293T cells, and the C-terminal region including the zinc finger domain is important for association.
To assess whether Mdm2 has E3 ligase activity for GATA, FLAG-tagged GATA3 and Myc-tagged wild type and a RING finger-deleted Mdm2 were expressed in 293T cells. The RING finger domain of Mdm2 is critical for E3 ligase activity for p53 (46). Immunoprecipitates with an anti-FLAG mAb were subjected to immunoblotting with anti-FLAG mAb. Overexpression of wild type Mdm2 led to increased levels of multiubiquitination of GATA3 (Fig. 6C, 1st two lanes). More interestingly, the ubiquitination of GATA3 was greatly reduced when the RING finger-deleted Mdm2 was expressed. The levels were much lower than those in cells without Mdm2 transfection, suggesting a dominant-negative feature of the RING fingerdeleted Mdm2 to endogenously expressing Mdm2 in 293T cells. The efficiency of expression of the transfected RING fingerdeleted Mdm2 was considerably high (Fig. 6C, right panel), probably because of the inhibition of ubiquitination itself (47,48).
It is known that cyclin-dependent kinase inhibitor 2A, a tumor suppressor molecule (p19 ARF in the mouse and p14 ARF in human cells), binds tightly to Mdm2 and prevents Mdm2mediated p53 ubiquitination (49). Consequently, we tested the effect of expression of ARF in the GATA3 ubiquitination. As shown in Fig. 6D, left panel, the introduction of ARF resulted in nearly complete inhibition of the multiubiquitination of GATA3 in 293T cells. Collectively, these results support the notion that Mdm2 has E3 ligase activity for GATA3.
Mdm2 Is Involved in GATA3 Ubiquitination in T Cells-In order to provide additional evidence to support the role of Mdm2 as a major E3 ligase, we attempted the inhibition of GATA3 ubiquitination in T cells by using Mdm2 RNA interference. Mdm2 siRNA was introduced in a T cell line TG40 at a high level. The expression levels of Mdm2 protein were reduced significantly upon the introduction of the Mdm2 siRNA as compared with the control (Fig. 7A, right top panel). As anticipated, GATA3 ubiquitination was reduced substantially by the Mdm2 siRNA treatment (Fig. 7A, left panel, lane 1.0 versus  0.4). These results help to confirm the involvement of Mdm2 in GATA3 ubiquitination in TG40 T cells.
Finally, we wanted to address the function of Mdm2 in primary developing Th2 cells. The mRNA expression of Mdm2 was similar between developing Th1 and Th2 cells (data not shown). Our attempts to silence Mdm2 by RNA interference were unsuccessful with the primary T cells, probably because of robust proliferation of developing Th2 cells in the in vitro cultures. Thus we took an alternative approach to inhibit GATA3 ubiquitination and to facilitate Th2 cell differentiation by introducing a RING finger-deleted Mdm2 (Mdm2-dR) into developing Th2 cells (Fig. 7, B and C). There was substantial expression of endogenous Mdm2 in primary developing Th2 cells, and furthermore, the level of introduced Mdm2-dR by a retrovirus vector was lower than that of endogenous Mdm2 (Fig. 7B, right top panel). Nevertheless, GATA3 ubiquitination was significantly reduced (Fig. 7B, left panel, lane1.0 versus  0.6). Moreover, there was significant increase in the generation of IL-4-producing Th2 cells (38.4 versus 54.0%) with higher mean fluorescence intensity in IL-4 fluorescence (114.3 versus 163.6) when Mdm2-dR was expressed in developing Th2 cells (Fig. 7C). Thus, we conclude that Mdm2 is involved in GATA3

DISCUSSION
In this paper, we provide evidence indicating that TCRmediated activation of the Ras-ERK MAPK cascade controls GATA3 protein stability through the ubiquitin-proteasome pathway. The induction of GATA3 protein in developing Th2 cells is crucial for the differentiation of Th2 cells (18,19). IL-4-induced STAT6 activation initiates transcription of GATA3 (50). However, among the issues that remain to be clarified is how the expression of GATA3 protein is controlled in developing Th2 cells. Here we demonstrate the following. (i) GATA3 protein is very unstable with a short half-life (ϳ1 h) in transfectants (Fig. 4A) and developing Th2 cells (Fig. 4B). (ii) The degradation of GATA3 is dependent on the 26 S proteasome pathway (Fig. 4, A-C). (iii) GATA3 is ubiquitinated both in vivo and in vitro (Fig. 5). (iv) The deletion of the possible ubiquitination sites of GATA3 led to stable expression of GATA3 and reduced ubiquitination ( Fig. 4E and Fig. 5, A and  B). From these results, we conclude that the fate of GATA3 in developing Th2 is highly dependent on degradation through the ubiquitin-proteasome system. Concurrently, we show that activation of the ERK-MAPK cascade facilitated GATA3-mediated chromatin remodeling at the Th2 cytokine gene loci (Fig.  1) and inhibited the degradation (Figs. 2 and 3) and ubiquitination of the GATA3 molecule (Fig. 5D). Because the Ras-ERK MAPK cascade in naive CD4 T cells is activated by stimulation of TCR and not of IL-4R (11), the activation of the Ras-ERK MAPK cascade detected in the experiments must be a consequence of TCR-mediated signaling. Therefore, stabilization of GATA3 by the activation of the Ras-ERK MAPK cascade could be the mechanism that accounts for an essential role for TCRmediated signaling in Th2 cell differentiation.
Our studies identify Mdm2 as a possible E3 ligase for GATA3. Mdm2 was shown to be associated with GATA3 in developing Th2 cells and 293T cells (Fig. 6, A-C). The overexpression of wild type Mdm2 induced increased ubiquitination on GATA3, whereas that of RING finger-deleted mutant Mdm2 resulted in the inhibition of GATA3 ubiquitination in 293T cells (Fig. 6D). Overexpression of ARF, an inhibitor of Mdm2, resulted in almost complete suppression of multiubiquitination of GATA3 in 293T cells (Fig. 6E). Moreover, the introduction of siRNA for Mdm2 into the T cell line TG40 resulted in the reduction in the ubiquitination of GATA3 protein (Fig. 7A). The generation of IL-4-producing Th2 cell was enhanced by the expression of RING finger-deleted mutant Mdm2, suggesting a physiological role for Mdm2 in Th2 cell differentiation (Fig. 7C).
Mdm2 is known to promote degradation of p53 through a ubiquitin-dependent proteasome pathway (49,51). Mdm2 acts as an E3 ubiquitin ligase specific for p53 in vitro (51). The RING finger domain of Mdm2 is critical for E3 ligase activity for p53 (46). The phosphorylation of p53 at serine 15, threonine 18, and serine 20 led to the reduction of Mdm2 binding and enhancement of p53 stabilization and accumulation (49). Most interestingly, amino acid residues 9 -20 (SVEPPLSQETFS) of human p53, which are reported to be important for the binding for Mdm2, are highly homologous to amino acid residues 131-142 of human GATA3 (SVYPPASSSSLS) and mouse GATA3 (SVYPPASSSSLA). In these regions, serine/threonine phosphorylation sites and surrounding proline residues (indicated in boldface above) occur in similar patterns between p53 and GATA3. Moreover, GATA3 has other structural similarities with p53, e.g. possible lysine ubiquitination sites at the Cterminal region (364 -390 in p53 and 396 -422 in GATA3) and a proline-rich regulatory region (69 -101 in p53 and 146 -178 in GATA3), which are reported to have important roles in posttranslational modification and functions of p53 (52,53). Thus, it is reasonable to expect that a similar set of molecular events operating in ubiquitination of p53 would occur in the case of GATA3 In our experiments with truncation mutants, truncation of the above-mentioned lysine ubiquitination sites in the C-terminal region 396 -422 in GATA3 (dCT mutant) resulted in small effects on degradation (Fig. 4E) and multiubiquitination of GATA3 (Fig. 5, A and B). A small but significant effect was observed in the dCF mutant (Fig. 5, A and B). A more prominent effect was observed by deletion of residues 261-443 (dZF mutant) (Fig. 4E and Fig. 5, A and B). The 261-315 region contains three lysine residues (293, 303, and 305 in human GATA3) and a nuclear localization signal (KPKRR). It is known that the degradation of p53 is controlled also by the localization of p53 and Mdm2 (54,55). Thus, similar to p53, the degradation of GATA3 appears to be controlled by both the ubiquitination process and nuclear/cytosol localization of the protein.
The Ras-ERK MAPK cascade regulates stability of various proteins, including Myc, MKP-1, ATF2, and p53 through a mechanism involving serine phosphorylation (56 -61). In addition, ERK MAPK-dependent phosphorylation and the subsequent enhancement of the transcriptional activities for GATA2 and GATA4 have been suggested (62,63). GATA3 was phosphorylated by activated p38 MAPK in cAMP-treated T cells, suggesting a possible regulatory role for the MAPK cascade in GATA3 function (64). In fact, our preliminary results indicate that an active form of ERK2 directly phosphorylates GATA3 protein in vitro, and PMA-induced GATA3 phosphorylation was significantly inhibited by U0126 in transfected COS7 cells. 2 GATA3 protein contains numerous Ser/Thr residues (93 residues out of 444 residues) and possesses 35 putative phosphorylation sites, and thus the precise location of critical amino acid residues responsible for the MAPK-dependent phosphorylation remains unclear at this time. Thus, it appears to be reasonable to surmise that the activation of the ERK-MAPK cascade induces GATA3 phosphorylation and prevents its ubiquitin-mediated degradation through the 26 S proteasome.
Our studies with primary T cells indicated that the ERK-MAPK cascade plays a major role in the regulation of GATA3 protein expression. Although we observed the activation of the p38 MAPK cascade after PMA treatment in developing Th2 cells, 2 a specific inhibitor for the p38 MAPK cascade (SB203580) did not affect the GATA3 protein expression. However, it is still possible that the activation of the p38 MAPK cascade may have some effect on the expression of GATA3 protein as well as the function of GATA3 (64).
In summary, TCR-mediated activation of the Ras-ERK MAPK cascade controls the stability of GATA3 protein by a ubiquitin-proteasome-dependent mechanism. IL-4-induced STAT6 activation is required for the induction of GATA3 transcription. Thus, efficient activation of both signaling pathways and resulting stable GATA3 expression, therefore, are crucial for chromatin remodeling at the Th2 cytokine gene loci and successful Th2 cell differentiation.