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J. Biol. Chem., Vol. 279, Issue 27, 28257-28265, July 2, 2004
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¶
From the
Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115 and Department of Medical Oncology, Cancer Institute Hospital and Division of Clinical Chemotherapy, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Tokyo 170-8455, Japan
Received for publication, March 22, 2004 , and in revised form, April 26, 2004.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The N termini of NFAT family members share a conserved sequence about 
300 amino acids in length known as the NFAT homologous region (NFAT-h). The NFAT-h is known to regulate nuclear/cytoplasmic trafficking of NFATs in response to changes in intracellular Ca2+ concentrations. In resting T cells, NFATs are retained in the cytoplasm, and the NFAT-h is heavily phosphorylated. Engagement of the T-cell antigen receptor or treatment of cells with Ca2+ ionophores activates calcineurin, a Ca2+/calmodulin-dependent Ser/Thr phosphatase, which dephosphorylates the NFAT-h, resulting in rapid translocation of the proteins to the nucleus (10). The nuclear translocation of NFATs is critically important for regulating NFAT activity. Immunosuppressive drugs FK506 and cyclosporin A block this translocation by inhibiting calcineurin activity in T cells as well as in nonhemopoietic cells, which leads to undesired side effects (11, 12). Considerable efforts are being made to identify additional mechanisms that regulate the nuclear translocation of NFATs with the hope of elucidating isoform-specific or cell type-specific mechanisms that would allow the development of new drugs without unpleasant side effects.
Protein posttranslational modifications by a variety of mechanisms play crucial roles in regulating protein function. Ubiquitination represents a particular case where ubiquitin, itself a small polypeptide, is covalently linked to lysine residues in a protein to target it for proteasomal degradation or other signaling pathways. Recently, several proteins that share similarity with ubiquitin have been identified. One member of this ubiquitin-like protein family is SUMO-1 (also known as sentrin, GMP1, UBL1, and PIC1), a polypeptide of 101 amino acids that can be attached covalently to proteins in a process that is mechanically analogous to ubiquitination (13). The SUMO-1 activating enzyme catalyzes the ATP-dependent activation of SUMO-1, the first step in the conjugation pathway (14–16), and transfers the activated SUMO-1 to Ubc9, the E2-conjugating enzyme involved in this process (17–20). To date, the known substrates of SUMO-1 include RanGAP1 (21, 22), PML (23, 24), Sp100 (25), and I
B
(26, 27). The observation that RanGAP1 and PML are targeted to distinct subcellular structures upon conjugation to SUMO-1 suggests that modification by SUMO-1 might play an important role in regulating the subcellular localization of proteins. Modification by SUMO has been shown to play critical roles in both nuclear and cytoplasmic processes, such as nuclear transport, transcription, and subnuclear targeting, which ultimately contribute to regulation of the cell cycle, cell growth, and apoptosis.
To explore the mechanisms and functions of sumoylation in regulating cellular processes, we developed a screening system for sumoylated proteins. Using this system, we determined that NFAT1 is sumoylated and that sumoylation of NFAT1 regulates the cytoplasmic-nuclear trafficking, subnuclear localization, and transcription of NFAT1. We show that ionomycin and phorbol 12-myristate 13-acetate (PMA), agents commonly used to activate the transcriptional activity of NFATs and T-cell activation, exhibit distinct effects on the sumoylation of NFAT1. Our study identified sumoylation as a novel nuclear anchorage and transcriptional regulatory mechanism for NFAT1 that may provide isoform-specific regulation.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In Vitro Expression Cloning for Sumoylation Substrates—Pools from a mouse spleen cDNA library (28) were in vitro transcribed and translated as [35S]methionine-labeled proteins as described previously. Sumoylation assays were performed in a total volume of 20 µl in reaction buffer (20 mM Hepes-NaOH, pH 7.4, 5 mM MgCl2, 0.05% Nonidet P-40, 2 mM ATP, 3.5 units of creatine phosphokinase, and 5 mM phosphocreatine) containing 200 µg/ml recombinant human SUMO-1 activating enzyme 1/SUMO-1 activating enzyme 2 as E1 and 50 µg/ml Ubc9 as E2 (plasmids expressing human E1 and Ubc9 were kind gifts from Dr. Ronald T. Hay; University of Saint Andrews, Saint Andrews, UK). Reactions were incubated at 30 °C for 1 h and terminated by the addition of sample buffer.
Cell Culture and Transient Transfection—BHK cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. Cells were transfected with Mirus TransIT LT1 (Mirus Corp.) according to the manufacturer's instructions.
Electroporation—Jurkat cells were cultured at 37 °C in RPMI 1640 medium with 10% fetal bovine serum. Fifteen million Jurkat cells were washed once in phosphate-buffered saline and resuspend in 1 ml of Eppendorf Hypoosmolar electroporation buffer (Fisher) and 1.25% Me2SO. Twenty µg of His-tagged sumo-1 construct was aliquoted into the 4-mm electroporation cuvettes, and then the cells were added and mixed. Electroporation was performed at 130 V and 900 microfarads, and cells were kept at room temperature for 5 min. They were plated in 4 ml of complete RPMI 1640 media plus 50 µlofMe2SO. The cells were incubated at 37 °C for 3.5 h and then overlaid on top of 3 ml of Ficoll-Plaque Plus solution (Amersham Biosciences) and spun down at 800 x g for 40 min. The interface band was collected and washed once with complete media. Jurkat cells were incubated with or without ionomycin or ionomycin plus PMA in the presence or absence of cyclosporin A for 1 h, and cells were lysed with 6 M guanidine buffer (Invitrogen). The lysates were applied onto Probond resin (Invitrogen), and His-tagged sumo-1-conjugated proteins were purified under denaturing conditions according to the manufacturer's instructions (Invitrogen).
Immunocytochemistry/Subcellular Localization—BHK cells were transfected with myc-tagged full-length wild-type or mutant NFAT1. Myc-tagged NFAT1 was detected with anti-myc antibody followed by Alexa594-conjugated anti-mouse IgG (Molecular Probes). For endogenous proteins, Jurkat cells were incubated with or without ionomycin or ionomycin plus PMA in the presence or absence of cyclosporin A. After 1 h, cells were fixed and immunostained with monoclonal anti-NFAT1 antibody (G1-D10) and polyclonal anti-sumo-1 antibody (FL101). Alexa594-conjugated anti-mouse IgG antibody and Alexa488-conjugated anti-rabbit IgG antibody were used as secondary antibodies. Immunofluorescence microscopic analysis was performed by laser scanning fluorescence confocal microscopy (Bio-Rad).
In Vivo Sumoylation Assay—Cells were co-transfected with the expression constructs of GFP-SUMO-1 and myc-tagged wild-type NFAT1 or mutants. Twenty-four h after transfection, the cells were lysed with lysis buffer (200 mM NaCl and 0.25% SDS) and then sonicated. Samples were centrifuged at 13,000 rpm for 10 min, and supernatants were collected. For immunoprecipitation, supernatant was diluted 1:10 with Nonidet P-40 buffer (20 mM potassium phosphate, pH 7.5, 50 mM sodium fluoride, 10 mM 
-glycerophosphate, 2 mM EDTA, 0.5% Nonidet P-40, and 20 mM dithiothreitol). After pre-clearing with protein A/G-agarose, NFAT1 was immunoprecipitated with anti-myc antibody (9E10) complexed protein A/G beads.
Luciferase Assay—BHK cells were transfected with NFAT1 expression plasmids, NFAT/AP1 promoter-driven luciferase expression plasmid, Fas ligand (FasL) promoter-driven luciferase expression plasmid (a kind gift from Dr. Shyr-Te Ju, Boston University of School of Medicine), or Renilla luciferase expression plasmid (Promega). FasL mutant promoter-driven luciferase expression plasmid and nuclear factor-B promoter-driven luciferase expression plasmid were used as negative controls. Twenty-four h after transfection, cells were treated for 3 h with control or ionomycin plus PMA in the presence or absence of leptomycin B (LMB) (10 ng/ml). Luciferase units were normalized to Renilla luciferase readings within each transfection and are expressed as relative luciferase units. To determine the protein levels of wild-type NFAT1 and NFAT1 mutants, immunoblotting was performed at the same time.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Like all members of the NFAT family, NFAT1 is normally present in the cytoplasm of resting cells; when T cells are activated, NFAT1 is dephosphorylated by the Ca2+/calmodulin-dependent phosphatase calcineurin, translocates to the nucleus, and becomes transcriptionally active (1, 30, 31). Nuclear localization of NFAT1 can be induced by low, sustained levels of elevated intracellular free Ca2+, which can be achieved by stimulation of self-antigen, low-affinity peptide-major histocompatibility complex complexes, or most conveniently, low concentrations of Ca2+ ionophore ionomycin (32). To examine the sumoylation of NFAT1 in vivo and a possible effect of intracellular free Ca2+ on sumoylation, BHK cells were transfected with the expression constructs of myc-tagged NFAT1 and GFP-SUMO-1; 24 h later, the cells were treated with control, ionomycin, or ionomycin plus PMA. The results were analyzed by Western blots and immunoprecipitation (Fig. 1, C and D). Consistent with previously published reports (30), when myc-NFAT1 was transfected alone without additional treatment, NFAT1 was expressed as a single protein species that was known to be phosphorylated. Treatment with ionomycin, with or without co-treatment with PMA, induced dephosphorylation of NFAT1 as indicated by a downshift on SDS-PAGE (Fig. 1C) (30). Interestingly, co-transfection of myc-NFAT1 with GFP-SUMO-1 significantly elevated the myc-NFAT1 levels (Fig. 1C); furthermore, a portion of myc-NFAT1 now migrated as two slower-migrating species consistent with singly (177 kDa) and doubly (214 kDa) sumoylated forms (Fig. 1C). These two bands were confirmed as NFAT1 by anti-NFAT1 antibody (data not shown). Treatment with ionomycin with or without PMA induced dephosphorylation of myc-NFAT1 and increased the levels of sumoylated NFAT1 (Fig. 1C).
To confirm that the two slower-migrating myc-NFAT1 forms are indeed sumoylated, total myc-tagged NFAT1 protein was immunoprecipitated with anti-myc antibody and then analyzed by Western blotting with anti-GFP antibody to detect GFP-SUMO-1-conjugated proteins (Fig. 1D). This analysis confirmed that both slower-migrating bands are indeed SUMO-1-modified myc-NFAT1. These results suggest that overexpression of SUMO-1 can increase the level of NFAT1 and induce the sumoylation of NFAT1, which can be further stimulated by treatment with ionomycin.
In ionomycin- and ionomycin + PMA-treated but not control or ionomycin + cyclosporin A-treated Jurkat cells, our Western blots using anti-NFAT1 detected two additional species with a larger apparent molecular mass than that of phosphorylated and unphosphorylated NFAT1 (Fig. 1E). As commonly found in sumoylation studies, the presence of possible efficient desumoylation mechanisms in lysed cells prevented us from immunoprecipitating these two larger forms of NFAT1 efficiently. To circumvent this problem and determine whether endogenous NFAT1 can be sumoylated, we electroporated Jurkat cells with His-tagged SUMO-1 expression construct. Twenty-four h after electroporation, the cells were treated with ionomycin, ionomycin + PMA, or ionomycin + cyclosporin A or left untreated for 1 h, and then the cells were lysed directly in 6 M guanidine. The cell lysate was applied to a Ni-column, and the proteins eluted from the Ni-column were Western blotted using anti-NFAT1 (Fig. 1E). Consistent with our above-mentioned data regarding transfected NFAT1, the two slower-migrating species of endogenous NFAT1 can only be detected in cells treated with ionomycin or ionomycin + PMA. Therefore, our data indicated that endogenous NFAT1 can be sumoylated in response to ionomycin and ionomycin + PMA stimulation.
Determination of Sumoylation Sites of NFAT1—Sumoylation is known to occur on the lysine residue with a consensus sequence of 
KXE, with representing a hydrophobic residue (33). To determine the lysine residues of NFAT1 serving as the SUMO-1 attachment sites, we focused on Lys684, Lys897, and Lys532 because they reside in a motif that fits the KXE consensus sumoylation site (Fig. 2A). The amino acids before Lys684 or Lys897 are leucine or valine, respectively, which are hydrophobic amino acid residues and therefore fit the preferred sumoylation sites from previous studies (33). The amino acid residue before Lys532 is arginine, which is not a hydrophobic amino acid residue; however, because this lysine resides in a domain that is highly conserved among all members of NFAT family, we included Lys532 in our analysis as well. Each of the three lysines was mutated individually to arginine, and the resulting NFAT1 point mutant cDNA clones were in vitro transcribed and translated into 35S-labeled proteins and analyzed in the in vitro sumoylation assay (Fig. 2B). The K532R mutation did not have any effect on in vitro sumoylation, indicating that Lys532 is not a sumoylation site. Interestingly, the K897R mutant had only one slower-migrating band corresponding to SUMO-1-modified NFAT1, whereas the K684R mutation abrogated both SUMO-1-modified NFAT1 species completely. These results suggest that NFAT1 has two sumoylation sites, Lys684 and Lys897, and that sumoylation of Lys684 is required for that of Lys897 in vitro. Consistent with this proposal, a C-terminal deletion (amino acids 1–683) mutant (delC) and a double mutant (K684R/K897R) completely eliminated sumoylation of NFAT1 in vitro (Fig. 2B).
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A Functional Role of Sumoylation at Lys897 in the Nuclear Retention of NFAT1—Sumolyation has been shown to promote the nuclear presence and function of proteins (33). Because NFAT1 is known to shuttle between the cytoplasm and the nucleus, we determined whether sumoylation of NFAT1 plays a role in the nuclear translocation of NFAT1. As reported, wild-type NFAT1 expressed in BHK cells is predominantly cytoplasmic without additional treatment; the addition of ionomycin induced NFAT1 nuclear translocation efficiently (Fig. 3A). Treatment with LMB alone increased the nuclear presence of NFAT1, suggesting that NFAT1 shuttles between the cytoplasm and nucleus in the unstimulated cells. The expressed K684R, K897R, and K684R/K897R NFAT1 in unstimulated BHK cells were indistinguishable from those of wild-type NFAT1 (Fig. 3A, top row; Fig. 3B, top left panel). In contrast to wild-type NFAT1, however, a considerable portion of NFAT1 mutants in ionomycin-treated BHK cells remained cytoplasmic (Fig. 3A, second row). A quantitation of staining such as that in Fig. 3A showed that wild-type NFAT1 was nuclear in 75% of cells after ionomycin treatment, whereas the proportion of cells with nuclear NFAT1 in cells expressing K684R, K897R, and K684R/K897R was 30.1%, 28.3%, and 30.3%, respectively (Fig. 3B, top right panel). An additional portion of cells showed mutant NFAT1 in both the cytoplasm and nucleus (Fig. 3B, top right panel). These results suggest that mutations in these three lysine residues had a significant impact on either the nuclear transport or retention of NFAT1.
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80% of cells expressing K684R, K897R, or K684R/K897R mutant as well as wild-type NFAT1 (WT) (Fig. 3A, last row; and Fig. 3B, bottom right panel). These results suggest that sumoylation is required not for nuclear import but for the nuclear retention of NFAT1 in control as well as in ionomycin-treated cells. Because the K897R mutation minimally affects the appearance of 214-kDa sumoylated NFAT1, these results suggest that sumoylation of Lys897 is critically needed for the nuclear retention of NFAT1.
A Functional Role of Sumoylation at Lys684 in the Transcriptional Activity of NFAT1—To assess the role of sumoylation on NFAT1 function, we determined the impact of sumoylation on the transcriptional activity of NFAT1 using two known NFAT1-targeting promoters, a FasL promoter (34) and a NFAT/AP-1 promoter, in promoter-driven luciferase expression assays (Fig. 4A, left panels). FasL mutant promoter- and nuclear factor-B promoter-driven luciferase assays were used as negative controls (Fig. 4A, right panels). As reported previously (30), wild-type NFAT1 (WT) showed a high level of transcriptional activity after ionomycin plus PMA stimulation (Fig. 4A, left panels). The expression of the K684R, K897R, and K684R/K897R NFAT1 mutants showed a low level of transcriptional activity that did not respond to ionomycin plus PMA stimulation, consistent with their defects in nuclear retention. Because, as shown in Fig. 3, treatment with LMB and ionomycin promoted the nuclear presence of the wild-type, K684R, K897R, and K684R/K897R mutants, we examined whether treatment with LMB and ionomycin plus PMA could enhance the transcriptional activity of the wild-type, K684R, K897R, and K684R/K897R mutants (Fig. 4A, left panels). Treatment with LMB enhanced the transcriptional activity of wild-type NFAT1 (WT) slightly, probably in proportion to the slight increase in its nuclear localization(Fig. 4A, left panels). Surprisingly, treatment with LMB in the presence of ionomycin and PMA enhanced the transcription of the K897R mutant, but not that of the K684R and K684/897R mutants (Fig. 4A, left panels). This result suggests that the sumoylation at Lys684 of NFAT1 is important for transcriptional activity of NFAT1; whereas sumoylation at Lys897 is only required for nuclear retention of NFAT1.
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30% of cells expressing NFAT mutants K684R, K897R, and K684R/K897R showed nuclear localization of mutant proteins (Fig. 3B). To determine whether mutations in the sumoylation sites affect the recruitment of NFAT1 mutants into the SUMO-1 bodies, we determined the subnuclear distribution of NFAT1 mutants with nuclear localization by immunostaining and confocal microscopy. K897R (Fig. 5C), but not K684R (Fig. 5B) and K684R/K897R NFAT1 mutants (Fig. 5D; Table I), was recruited into SUMO-1 bodies after treatment with ionomycin plus PMA. From these results, we conclude that Lys684, but not Lys897, of NFAT1 is required for the recruitment into the nuclear SUMO-1 bodies. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Because Lys897 is localized to the C terminus of NFAT1 and distal to the NFAT-h, which is known to regulate nuclear translocation by interacting with calcineurin, our study identified a new mechanism that contributes to the regulation of the nuclear presence of NFAT1. Our study identified a previously unexpected role of Lys897, located in the C terminus of NFAT1, in the nuclear retention of NFAT1 and suggests the possibility that the C terminus of NFAT1 may interact with the nuclear export signal localized in the N terminus of the protein. The C-terminal tails of the NFAT family, where Lys897 is localized in NFAT1, are highly divergent, and a similar KXE sequence cannot be found in the other members of NFAT family. Although it is possible that sumoylation at Lys residues less conforming to the KXE consensus sequence occur in the C termini of other NFAT family members, our study suggests that sumoylation may provide one of the regulatory mechanisms differentiating the nuclear presence of different NFAT family members.
Sumoylation has been shown to have diverse effects on proteins. Sumoylation of RanGAP1 promotes the interaction of RanGAP1 with the Ran-GTP-binding protein RanBP2 at the cytoplasmic face of the nuclear pore complex (21, 35), whereas SUMO-1 modification of the promyelocytic leukemia gene product (PML) targets the protein to PML oncogenic domains (23, 25, 36). Sumoylation of NFAT1 appears to enhance both the nuclear retention of NFAT1 and SUMO-1 body targeting. Although Lys897 is localized in the C terminus of NFAT1, in which no homologous Lys residue is found in other members of NFAT1 family, homologous Lys residues for Lys684 NFAT1 that fit the motif KXE can be found at least in NFAT3 and NFAT4, suggesting that NFAT3 and NFAT4 may be similarly sumoylated at this Lys as well. Because Lys684 is localized immediately after the known DNA binding domain of NFAT1, our results suggest that sumoylation at this Lys and its interaction with the DNA binding domain may be a shared mechanism for regulation of transcriptional activity in the NFAT family.
How might sumoylation of Lys684 regulate the transcriptional activity of NFAT1? Overexpression of GFP-SUMO-1 significantly increased the levels of both sumoylated and unsumoylated NFAT1 (Fig. 2), suggesting that sumoylation of NFAT1 may increase the stability of NFAT1, which may account in part for the transcriptional activation. Furthermore, because K684R NFAT1 is transcriptionally inactive even in the presence of LMB, which prevents its exit from the nucleus, and is unable to be recruited into the nuclear SUMO-1 bodies in the presence of ionomycin and PMA, the sumoylation of Lys684 may allow NFAT1 to interact with a key protein complex critical for effective transcription. We propose that the sumoylation of Lys684 NFAT1 may provide a license for transcriptional activity of NFAT1.
In lymphocytes, integration of Ca2+ and other signaling pathway results in productive activation, whereas unopposed Ca2+ signaling leads to tolerance or anergy. Calcium-regulated transcription factor NFAT has an integral role in both aspects of lymphocyte function. Ca2+/calcineurin signaling induces a limited set of anergy-associated genes, distinct from genes induced in the productive immune response. Thus, NFAT1 induces T-cell anergy in the absence of AP-1, and it induces a program of productive activation mediated by the cooperative NFAT·AP-1 complex in the presence of AP-1. Stimulation by ionomycin alone mimics anergy by inducing a limited set of anergy-associated genes, whereas ionomycin + PMA stimulation induces the productive immunoresponsive genes (37). Given the distinct effects of stimulation with ionomycin alone and combined stimulation with both ionomycin and PMA, we speculate that perhaps the activation of anergy-associated genes can occur without association with the SUMO-1 bodies, whereas the activation of a productive immune response requires recruitment of NFAT1 into the nuclear SUMO-1 bodies, where the cooperative NFAT1·AP-1 complex may form.
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FOOTNOTES |
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¶ To whom correspondence should be addressed: Dept. of Cell Biology, Harvard Medical School, 240 Longwood Ave., Boston, MA 02115. Tel.: 617-432-4170; Fax: 617-432-4177; E-mail: jyuan{at}hms.harvard.edu.
1 The abbreviations used are: NFAT, nuclear factor of activated T cells; PMA, phorbol 12-myristate 13-acetate; NFAT-h, NFAT homologous region; E2, ubiquitin carrier protein; E1, ubiquitin-activating enzyme; FasL, Fas ligand; LMB, leptomycin B; GFP, green fluorescent protein.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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