A Conserved Calcineurin-binding Motif in Human T Lymphotropic Virus Type 1 p12I Functions to Modulate Nuclear Factor of Activated T Cell Activation*

Seung-jae KimDagger , Wei DingDagger , Björn AlbrechtDagger §, Patrick L. GreenDagger ||, and Michael D. LairmoreDagger ||**

From the Dagger  Center for Retrovirus Research and Department of Veterinary Biosciences,  Comprehensive Cancer Center, The Arthur G. James Cancer Hospital and Solove Research Institute, and the || Department of Molecular Virology, Immunology, and Medical Genetics, Ohio State University, Columbus, Ohio 43210-1093

Received for publication, October 4, 2002, and in revised form, February 12, 2003

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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The PXIXIT calcineurin binding motif or highly related sequences are found in a variety of calcineurin-binding proteins in yeast, mammalian cells, and viruses. The accessory protein p12I encoded in the HTLV-1 pX ORF I promotes T cell activation during the early stages of HTLV-1 infection by activating nuclear factor of activated T cells (NFAT) through calcium release from the endoplasmic reticulum. We identified in p12I, a conserved motif, which is highly homologous with the PXIXIT calcineurin-binding motif of NFAT. Both immunoprecipitation and calmodulin agarose bead pull-down assays indicated that wild type p12I and mutants of p12I that contained the motif-bound calcineurin. In addition, an alanine substitution p12I mutant (p12I AXAXAA) had greatly reduced binding affinity for calcineurin. We then tested whether p12I binding to calcineurin affected NFAT activity. p12I competed with NFAT for calcineurin binding in calmodulin bead pull-down experiments. Furthermore, the p12I AXAXAA mutant enhanced NFAT nuclear translocation compared with wild type p12I and increased NFAT transcriptional activity 2-fold greater than wild type p12I. Similar to NFAT, endogenous calcineurin phosphatase activity was increased in Jurkat T cells expressing p12I independent of its calcineurin binding property. Thus, the reduced binding of p12I to calcineurin allows enhanced nuclear translocation and transcription mediated by NFAT. Herein, we are the first to identify a retroviral protein that binds calcineurin. Our data suggest that HTLV-1 p12I modulates NFAT activation to promote early virus infection of T lymphocytes, providing a novel mechanism for retrovirus-mediated cell activation.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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As a complex retrovirus, human T lymphotropic virus type 1 (HTLV-1)1 encodes common retrovirus structural and enzymatic proteins as well as regulatory (Tax and Rex) and accessory (p12I, p27I, p13II, and p30II) proteins from four open reading frames (ORFs I-IV) in its unique pX region (1-4). The understanding of the molecular pathogenesis of HTLV-1 infection, immortalization, and transformation has been primarily focused on the role of Tax and Rex (5, 6); however, emerging evidence also indicates the important roles of accessory proteins in establishment of HTLV-1 infection (7).

The ORF I-encoded protein HTLV-1 p12I is highly conserved among different isolates, and its messenger RNA can be detected in infected cell lines and directly from infected patient cells (2-4, 8, 9). Humoral antibody and cytotoxic T lymphocyte responses against ORF I-encoded viral proteins are elicited in infected carriers and diseased patients, indicating the expression of the protein during the natural infection (10, 11). p12I localizes in the endoplasmic reticulum and cis-Golgi compartment (12-14) and associates with the H+ vacuolar ATPase (15), the interleukin-2 receptor beta - and gamma -chain (16), and major histocompatibility complex class I heavy chain (17). A small hydrophobic protein, p12I, contains several potential functional motifs, including two transmembrane domains, leucine zipper motifs, and four Src homology 3 domain-binding motifs (PXXP) (2, 15, 18). However, defined motifs of p12I that may alter key T cell signaling pathways that thereby influence cell activation have not been identified.

We have reported that p12I is required for early establishment of HTLV-1 infection (19, 20). When compared with the wild type infectious molecular clone (ACH), ORF I-mutated ACH-immortalized T cell lines (ACH·p12I) failed to establish infection in rabbits (21, 22). ACH·p12I also exhibited reduced infectivity in quiescent peripheral blood mononuclear cells but not in activated peripheral blood mononuclear cells, suggesting a role for p12I in T cell activation during the early stages of infection (20). Recently, we have demonstrated that p12I localizes to the ER and associates with an ER luminal protein, calreticulin (12), and selectively activates the nuclear factor of activated T cells (NFAT) in a calcium-dependent manner (12, 23). Importantly, p12I expression in lymphocytes directly increases cytoplasmic calcium from ER calcium stores (12, 24), which serves to explain how the protein activates the NFAT signaling pathway.

The transcriptional activation of NFAT is regulated by the calcium/calmodulin-dependent serine/threonine phosphatase, calcineurin (25, 26). Through protein-protein interaction, calcineurin triggers the dephosphorylation and subsequent nuclear translocation of NFAT, which results in transactivation of NFAT-inducible cytokine genes including interleukin-2 (25, 26). A conserved calcineurin-binding motif (PXIXIT) has been identified in the N-terminal regulatory domain of NFAT, which serves as a major binding site with both inactivated and activated calcineurin (27, 28). When this motif is mutated, NFAT is functionally impaired (27, 28). In addition, secondary calcineurin binding sites in NFAT have been identified that contribute to higher affinity binding with calcineurin (29, 30).

The NFAT SPRIEIT calcineurin binding sequence and synthetic peptides containing the critical binding amino acids (PXIXIT) inhibit binding between calcineurin and NFAT without affecting calcineurin phosphatase activity (27, 28). A number of calcineurin-binding proteins inhibit either calcineurin phosphatase activity or its substrate NFAT transcriptional activity. These include the antiapoptotic protein Bcl-2 (31, 32), calcineurin B homologous protein (33), AKAP79 (A kinase anchoring protein) (34), and myocyte-enriched calcineurin-interacting protein 1 (35). Interestingly, the African swine fever virus immunosuppressive protein A238L inhibits NFAT activation and has a calcineurin binding sequence resembling that of NFAT (36, 37).

Herein, we identified a conserved sequence (PSLP(I/L)T) in p12I, which is highly homologous to the PXIXIT calcineurin-binding motif of NFAT. Full-length p12I and the PSLP(I/L)T motif-containing mutants bound calcineurin in both immunoprecipitation and calmodulin bead pull-down assays. In contrast, serial mutations of p12I that lacked PSLP(I/L)T motif or had selective alanine substitutions of the motif (p12I AXAXAA) exhibited abolished or decreased binding affinity with calcineurin. Furthermore, p12I competed with NFAT for calcineurin binding in calmodulin bead pull-down experiments. Interestingly, the p12I AXAXAA mutant induced more NFAT nuclear translocation than wild type and increased NFAT transcriptional activity (~2-fold) in a reporter gene assay when compared with wild type p12I. The existence of a calcineurin binding motif in p12I suggests that there may be at least two regulatory actions for p12I that modulate NFAT activation: 1) to cause calcium release from ER stores and 2) through calcineurin binding. Collectively, our data provide new insight into the role of a retroviral accessory protein in mediating early events in T cell infection and activation.

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ABSTRACT
INTRODUCTION
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Cell Lines-- The 293T HEK (American Type Culture Collection catalog no. 1573) cells and HeLa-Tat cells (AIDS Research and Reference Reagent Program, National Institutes of Health, HLtat, catalog no. 1293) were maintained in Dulbecco's modified Eagle's medium (supplemented with 10% fetal bovine serum, 100 µg of streptomycin plus penicillin/ml, and 2 mM L-glutamine (Invitrogen). Jurkat T cells (clone E6-1; American Type Culture Collection catalog no. TIB-152) were maintained in RPMI medium (Invitrogen) supplemented with 15% fetal bovine serum, 100 µg of streptomycin plus penicillin/ml, 2 mM L-glutamine, and 10 mM HEPES (Invitrogen) (complete RPMI).

Plasmids and Site-directed Mutagenesis-- pME18S and pMEp12I plasmids were kindly provided by G. Franchini (National Institute of Health) (14). pMEp12I expresses an influenza hemagglutinin (HA1)-tagged HTLV-1 p12I fusion protein. HA-NFAT1c-GFP expression vector (28) (from A. Rao, Harvard Medical School) was used for NFAT expression in 293T and HeLa-Tat cell lines. The serial deletion mutants of p12I (1-47, 48-99, 15-69, and 15-86) were previously described (12). Point mutants of p12I were generated by PCR-based site-directed mutagenesis using a QuikChangeTM site-directed mutagenesis kit (Stratagene, La Jolla, CA). Point mutant L74I was made by substituting the 74th leucine residue to isoleucine within the 99-amino acid sequence of p12I. Point mutant p12I AXAXAA was made by multiple alanine substitution of the PXLXLT motif (e.g. 70th proline to alanine, 71st leucine to alanine, 73rd leucine to alanine, and 75th threonine to alanine) motif. To construct the L74I mutant, 5'-CCTCAGCCCGTCGCTGCCGATCACGATGCGTTTCCCC-3' and 5'-GGGGAAACGCATCGTGATCGGCAGCGACGGGCTGAGG-3' primers were used. To construct the p12I AXAXAA mutant, 5'-CCTCTTCTCCTCAGCGCGTCGGCGCCGGCAGCGATGCGTTTCCCC-3' and 5'-GGGGAAACGCATCGCTGCCGGCGCCGACGCGCTGAGGAGAAGAGG-3' primers were used. Sanger sequencing was used to confirm that the sequences of each p12I mutant plasmids were correct and in frame. An NFAT-luciferase construct, pNFAT-luc, which has a trimerized human distal interleukin-2 NFAT site inserted into the minimal interleukin-2 promoter, was used for the reporter gene assay (38).

Transient Transfection and Immunoprecipitation Assay-- 293T cells were seeded at 50% confluence in 10-cm-diameter tissue culture dishes 24 h before transfection. Cells were transfected with 10 µg of pME18S, wild type pMEp12I, and mutants of p12I using LipofectAMINETM reagent (12) (Invitrogen). For Jurkat T cell transfection, 107 cells were electroporated in complete RPMI (350 V, 975 microfarads; Bio-Rad Gene Pulser II) with 30 µg of pME18S, wild type pMEp12I and mutant expression vectors. To test the binding between p12I and calcineurin, 293T cells or Jurkat T cells were transfected with wild type pMEp12I or mutated p12I expression vectors. Transfected 293T cells (1-2 × 107) or Jurkat T cells (1 × 107) were lysed with Triton X-100 buffer (1% deoxycholic acid, 0.1% SDS, 1% Triton X-100, 150 mM NaCl, 50 mM Tris-HCl (pH 7.5), with Complete® Protease Inhibitor (Roche Molecular Biochemicals)). CaCl2 (2 mM), EDTA (5 mM), or EGTA (5 mM) were added to test the calcium chelation effect in p12I and calcineurin binding. Cell lysates were incubated with 1:200 diluted rabbit polyclonal calcineurin antibody (Chemicon International, Inc., Temecular, CA) overnight. The immune complex mixture was then incubated with 50 µl of protein A/G plus agarose beads (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) for 4 h. Beads were washed three times in Triton X-100 buffer and boiled in SDS sample buffer, and supernatants were analyzed by Western immunoblotting. To test pharmacological inhibitors in a p12I and calcineurin binding assay, cyclosporin A (Sigma) (10 µM) or glycine, N,N'-1,2-ethanediylbis(oxy-2,1-phenylene)-bis-N-2-(acetyloxy)methoxy-2-oxoethyl-, bis(acetyloxy)methyl ester (BAPTA-AM; Molecular Probes, Inc., Eugene, OR) (1 µM) were added in culture 30 min before cell lysis.

Calmodulin-Agarose Bead Pull-down Assay-- To test p12I binding with calcineurin and to test for competition between p12I and NFAT for calcineurin binding, a calmodulin bead assay was performed as previously described (39). Briefly, 293T cells or Jurkat T cells were transfected with wild type pMEp12I or mutated p12I expression vectors or an NFAT expression vector. Transfected 293T (2 × 107) or Jurkat T cell (1 × 107) were lysed in Triton X-100 buffer (1% deoxycholic acid, 0.1% SDS, 1% Triton X-100, 150 mM NaCl, 50 mM Tris-HCl (pH 7.5), with Complete® protease inhibitor (Roche Molecular Biochemicals)) with CaCl2 (2 mM). Calmodulin-agarose (phosphodiesterase 3',5'-cyclic nucleotide activator from bovine brain) beads (Sigma) were washed three times in 1 ml of preactivation buffer (10 mM Tris, pH 8.0, 150 mM NaCl, and 2 mM CaCl2) to activate calmodulin. Lysates were then incubated with 60 µl of packed beads for 3 h at 4 °C. To remove unbound proteins, the beads were washed three times with washing buffer (20 mM Hepes, pH 7.4, 150 mM NaCl, 5 mM sodium pyrophosphate, 10% glycerol, 1% Triton X-100, and protease inhibitors supplemented with CaCl2 (2 mM)). The beads were boiled in SDS sample buffer, and supernatants were analyzed by Western immunoblotting.

Immunoblotting-- Western immunoblotting was used to analyze immunoprecipitated and calmodulin bead pull-down products. Protein concentrations in all lysates were determined by BCA assay (micro-BCA Protein Assay; Pierce). Cell lysates were separated by SDS-PAGE followed by transfer to nitrocellulose membranes. Membranes were blocked with 5% nonfat dry milk for 2 h, incubated overnight at 4 °C with monoclonal anti-HA antibody (1:1000) (clone 16B-12; Covance Research Products), with polyclonal calcineurin antibody (1:1000) (Chemicon International) or with monoclonal NFAT1c antibody (1:500) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) for detection of p12I, calcineurin, and NFAT2 (23), respectively. The blots were developed using horseradish peroxidase-labeled secondary antibody and enhanced chemiluminescence (Cell Signaling Technologies, Beverly, MA). The density of each band was measured using a commercial software package (Gel-pro Analyzer software; Media Cybernetic, Inc., Silver Spring, MD).

NFAT Reporter Gene Assay-- For analysis of NFAT transcriptional activity in wild type pMEp12I and alanine-substituted mutant (p12I AXAXAA) transfected Jurkat T cells, NFAT-driven luciferase activity assay was performed as previously described (23). Briefly, Jurkat T cells (107) were electroporated as described above with 30 µg of expression vectors (pME18S, pMEp12I, or p12I AXAXAA mutant), 10 µg of reporter plasmid (NFAT-luc), and 1 µg of pCMV·SPORT-beta -gal to normalize for transfection efficiency. The transfected cells were plated in six-well plates at a density of 5 × 105/ml. Cells were stimulated with 20 ng/ml phorbol 12-myristate 13-acetate (Sigma) at 6 h post-transfection. After 18 h of stimulation, cells were lysed (Cell Culture Lysis Reagent; Promega, Madison, WI) and analyzed for luciferase activity according to the manufacturer's protocol (Promega, Madison, WI). Values were normalized for transfection efficiency based on beta -galactosidase activity. Data expressed in reporter gene assays were the mean of at least two independent experiments conducted in triplicate. Statistical analysis was performed using Student's t test.

NFAT-GFP Localization-- HeLa-Tat cells were seeded into chamber slides (Fisher) and were cotransfected with 2 µg of HA-NFAT1c-GFP expression vector (28) and 4 µg pME18S, pMEp12I, or p12I AXAXAA mutant expression vectors. Two days post-transfection, cells were fixed for 15 min with 2% paraformaldehyde and were visualized by an Olympus BH-2 fluorescence microscope or Zeiss LSM510 confocal microscope. To quantify the HA-NFAT1c-GFP nuclear localization, cells that had sole or predominant nuclear fluorescence were counted as positive cells. Values were obtained from two wells of a single representative experiment (~600 cells). To measure GFP intensity in cytoplasmic and nuclear regions, MetaMorph® Imaging software (Universal Imaging Corp., Downingtown, PA) was used. Values represent nuclear/cytoplasmic ratio of GFP intensity of up to 25 cells.

Calcineurin Phosphatase Activity-- Endogenous calcineurin phosphatase activity of Jurkat cells was measured by using the calcineurin assay kit (Biomol; Plymouth Meeting, PA) according to the manufacturer's instructions. 107 Jurkat cells were transfected with 30 µg of expression vectors (pME18S, pMEp12I, or p12I AXAXAA mutant). At 24 h post-transfection, phosphatase activity was measured using phosphopeptide substrate (RII peptide) in the presence or absence of EGTA. The released free PO4 was measured colorimetrically by using the Biomol Green reagent.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The Putative Calcineurin-binding Motif in p12I Is Highly Conserved-- The peptide alignment of p12I sequences with the calcineurin binding peptide sequence from NFAT (SPRIEIT) was carried out using a commercial software package (Align X software of the Vector NTI suite; InforMax Inc., Bethesda, MD). We compared p12I sequences from pMEp12I plasmid, ACH molecular proviral clone, and five different p12I sequences from the Entrez protein data base (available on the World Wide Web at www.ncbi.nlm.nih.gov). We identified a highly conserved 70PSLP(I/L)T75 sequence in p12I (Fig. 1). p12I sequences from various HTLV-1 strains had nearly identical sequences compared with the consensus calcineurin binding PXIXIT sequence of NFAT except the 72nd leucine residue. The pMEp12I plasmid was the only clone that had a leucine residue at amino acid 74. This putative calcineurin-binding motif is also conserved in the closely related simian T cell leukemia virus type 1 (STLV-1) p12I (40). However, the accessory proteins R3 and G4 of bovine leukemia virus, also a member of the Deltaretrovirus group, did not share these consensus sequences (41).


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Fig. 1.   HTLV-1 p12I contains a highly conserved putative calcineurin-binding motif in p12I. Sequences from HTLV-1 and STLV-1 were aligned with calcineurin binding sequence (SPRIEIT) of NFAT using a commercial software program (Align X, Vector NTI version 7.0). ACH (22), pMEp12I (16), HTLV-1A (Entrez Protein AAA45390), HTLV-1B (AAA85329), HTLV-1C (AAB23360), HTLV-1D (B46181), HTLV-1E (C61547), and STLV-1 isolate number 14 (40) were compared. The conserved SPSLP(I/L)T sequence is indicated in a shaded box, and black boxes indicate amino acid residues that have similar properties. The consensus motif is indicated in the bottom row.

The C-terminal Half (Residues 48-- 99) of p12I Containing PSLP(I/L)T Binds Calcineurin---To test whether p12I binds calcineurin, we transiently transfected p12I into both 293T and Jurkat T cells and performed both immunoprecipitation and calmodulin bead pull-down experiments (Fig. 2). In calcium-containing buffer, calmodulin beads precipitated both calcineurin and its binding protein NFAT (39). p12I was consistently pulled down in calmodulin-agarose bead and immunoprecipitation assays (Fig. 2B). Thus, p12I, as a full-length protein, had the capacity to bind endogenously expressed calcineurin in both 293T and Jurkat T cells. As expected, using the pMEp12I vector, the detected p12I in higher percentage SDS gels (15%) showed typical doublet bands as described (18). The two forms of p12I may be due to post-translational modification of p12I, such as phosphorylation or ubiquitylation (18).


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Fig. 2.   The C-terminal half of p12I containing the PSLP(I/L)T motif is required for calcineurin binding. A, schematic representation of wild type pMEp12I and serial p12I deletion mutants. The shaded boxes indicate PXXP motifs, and the bar represents the location of the PSLP(I/L)T conserved sequence. B, co-precipitation of wild type p12I and serial deletion p12I mutants with calcineurin in immunoprecipitation (IP) and calmodulin agarose bead pull-down (CaM bead) assays. Jurkat T or 293T cells transfected with pME empty vector wild type pMEp12I or mutant p12I plasmids were lysed and analyzed by Western blot. Polyclonal calcineurin antibody was used for immunoprecipitation assay. Monoclonal HA-1 antibody was used to detect wild type and mutant p12I.

To define the regions in p12I that are responsible for binding with calcineurin, we tested serially deleted p12I mutants for calcineurin binding (Fig. 2A). The mutants p12I 48-99, p12I 15-86, and p12I 15-69 bound calcineurin in immunoprecipitation and calmodulin bead pull-down assays (Fig. 2B). However, the mutant p12I 1-47 (the N-terminal half of p12I) failed to bind calcineurin in either assay (Fig. 2B). These data indicated that amino acids 48-99 of p12I, which contains the putative calcineurin-binding (70PSLP(I/L)T75) motif, was probably responsible for calcineurin binding. The mutant p12I 15-69, which did not contain the putative calcineurin binding motif, weakly bound to calcineurin (Fig. 2B). This finding was not unexpected, because secondary calcineurin binding sites are predicted from the p12I amino acid sequence when compared with homologous alignment of secondary binding sequences of NFAT (data not shown).

The PSLP(I/L)T Motif Is Responsible for Interaction between p12I and Calcineurin-- To more specifically test whether the calcineurin-binding region in p12I was due to the PSLP(I/L)T motif, we constructed an alanine substitution mutant of p12I (p12I AXAXAA) and carried out immunoprecipitation and calmodulin bead pull-down assays (Fig. 3). These substitutions were designed to replace critical proline, leucine, isoleucine, and threonine amino residues with alanine residues (Fig. 3A). The p12I AXAXAA mutant had significantly reduced binding affinity for calcineurin (Fig. 3B). This result indicated that the PSLP(I/L)T motif in p12I is largely responsible for calcineurin binding. Our data, however, did not exclude the possibility of secondary binding sites in p12I as implicated by our serially deletion mutant results. In addition, we constructed a mutant L74I that converted a leucine from pMEp12I to an isoleucine more common at this position in p12I sequences from most HTLV-1 strains including the ACH molecular clone (PSLPLT versus PSLPIT) (Fig. 1). There was no significant difference in binding affinity between these two sequences (Fig. 3). The other single substitution mutants in the putative calcineurin-binding (70PSLP(I/L)T75) motif, P70A, S71A, P73A, and T75A, did not result in significant reduction in binding affinity (data not shown).


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Fig. 3.   Alanine substitution mutant of p12I (AXAXAA) decreases binding affinity for calcineurin. A, schematic representation of wild type pMEp12I, alanine substitution mutant p12I AXAXAA, and L74I plasmid. Gray boxes, PXXP motifs; bar, the PSLP(I/L)T conserved sequence. B, 293T cells were transfected with wild type pMEp12I, p12I AXAXAA, and L74I plasmid. Binding was tested by immunoprecipitation (IP) and calmodulin-agarose bead pull-down (CaM bead) assays.

p12I and Calcineurin Binding Is Calcium-dependent but Is Not Affected by Cyclosporin A-- Calcineurin is activated via cooperative interactions between calcium and calmodulin (42). Previous reports indicated that calcium was required for binding of NFAT to calcineurin as a complex with calmodulin-Sepharose beads (39). To test the calcium requirement for the association between p12I and calcineurin, we treated cell lysates with the calcium chelators, EDTA and EGTA, or used BAPTA-AM-treated cells prior to conducting immunoprecipitation experiments. The strong calcium chelator EGTA eliminated p12I binding to calcineurin (Fig. 4, lane 4), whereas the weaker calcium chelator EDTA and BAPTA-AM reduced the binding affinity in immunoprecipitation assays (Fig. 4, lanes 3 and 5). p12I binding was decreased when cell lysates were treated with a decreased concentration of CaCl2 or when treated with increasing concentration of EDTA (data not shown). These data indicated that the binding of p12I to calcineurin required calcium, which is similar to the requirement of calcium for effective NFAT binding to calcineurin (39). In parallel, because the binding of NFAT to calcineurin was not affected by calcineurin inhibitor cyclosoporin A (39), we tested the affect of cyclosporin A in the p12I binding to calcineurin. As expected, p12I binding was not inhibited by cyclosporin A (Fig. 4, lane 6). This result suggested that the cyclosporin A-cyclophilin complex site is distinct from sites required for the calcium/calmodulin activation and NFAT and p12I binding (43). Collectively, our data indicated that the calcineurin binding properties of NFAT and p12I were similar in requiring calcium for calmodulin binding, and each did not compete with binding of the cyclosporin A-cyclophilin complex. The results suggested that both proteins might have similar binding mechanism or share an identical binding site in calcineurin.


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Fig. 4.   p12I and calcineurin binding is inhibited by calcium chelators but not inhibited by cyclosporin A. CaCl2 (2 mM) or calcium chelator EDTA (5 mM) (lane 3) or EGTA (5 mM) (lane 4) was added in Triton X-100 cell lysis buffer, and calcium chelator BAPTA-AM (1 µM) (lane 5) or calcineurin inhibitor cyclosporin A (10 µM) (lane 6) was added in 293T cells 30 min before cell lysis and following immunoprecipitation (IP) assay.

p12I Competes with NFAT for Calcineurin Binding-- Because synthetic peptides or proteins, which contain the calcineurin-binding motif, compete with NFAT for calcineurin binding (27, 28, 37), we tested whether the p12I and calcineurin binding inhibits NFAT and calcineurin binding. We transiently cotransfected 293T cells with a constant amount of NFAT expression plasmid with increasing amounts of the wild type pMEp12I expression plasmid (Fig. 5A) as well as plasmids expressing p12I mutants (p12I 1-47, 48-99, 15-69, or AXAXAA mutants) (Fig. 5B). We then performed a calmodulin bead pull-down assay to detect the amount of bound NFAT and wild type p12I or its mutant proteins in the calcineurin A subunit. As expected, wild type p12I and the calcineurin binding mutants, p12I 48-99, p12I 15-69, and p12I AXAXAA, were bound in a concentration-dependent manner, but the noncalcineurin binding mutant p12I 1-47 expressed in a parallel manner failed to bind calcineurin (Fig. 5B). The amount of calcineurin (A subunit) from pull-down experiments remained constant in all assays (Fig. 5A). Both wild type p12I (Fig. 5A) and p12I 48-99 (Fig. 5B) effectively competed against NFAT for calcineurin binding. In the noncalcineurin binding mutant p12I 1-47 transfection, the amount of pulled-down NFAT was relatively constant regardless of the amount of mutant p12I 1-47 transfection (Fig. 5B). In addition, the weakly calcineurin binding p12I 15-69 mutant, which did not contain calcineurin binding sequence, did not compete with NFAT for calcineurin binding (Fig. 5B). As expected, mutation in calcineurin binding sequence (p12I AXAXAA mutant) resulted in reduction of competition between p12I and NFAT (Fig. 5B). Together, these results indicated that the PSLP(I/L)T calcineurin binding sequence is critical for p12I competition with NFAT for calcineurin binding.


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Fig. 5.   p12I binding to calcineurin decreases the amount of NFAT binding to calcineurin. A, a constant amount of HA-NFAT1c-GFP (7.5 µg) and increasing amounts of pMEp12I (0-15 µg) were cotransfected into 293T cells. The amount of NFAT and wild type p12I proteins bound to calcineurin A subunit was detected by calmodulin pull-down assay. B, 293T cells were cotransfected with increasing amounts (0-15 µg) of wild type pMEp12I and mutant (p12I 1-47, 48-99, or 15-69 or AXAXAA mutants) plasmids and a constant amount of HA-NFAT1c-GFP (7.5 µg). Then wild type p12I or p12I mutants, NFAT, and calcineurin A subunit were precipitated by calmodulin beads. Optical density of NFAT, indicating the amount of calcineurin binding, was measured. Optical density of each NFAT band was normalized by corresponding calcineurin A bands.

The PSLP(I/L)T Calcineurin Binding Sequence in p12I Is an Inhibitory Motif for NFAT Transcriptional Activity-- p12I activates NFAT transcriptional activity in T cells in a calcium-dependent manner (25). In contrast, the NFAT SPRIEIT calcineurin binding sequence and synthetic peptides based on this motif inhibit NFAT transcriptional activation by competing with NFAT for calcineurin binding (28). To further investigate whether p12I binding to calcineurin affected NFAT transcriptional activity, we performed an NFAT-driven reporter gene assay using the p12I alanine substitution mutant, p12I AXAXAA, which exhibited lowered binding affinity for calcineurin in the immunoprecipitation and calmodulin bead pull-down assay. Jurkat T cells were transiently transfected with wild type pMEp12I or p12I AXAXAA expression plasmids and then measured NFAT transcriptional activities. Whereas the wild type pMEp12I showed typical ~20-fold induction in the presence of phorbol 12-myristate 13-acetate stimulation (23) compared with the pME empty vector, the p12I AXAXAA mutant induced an ~40-fold enhancement of luciferase activity compared with the pME control vector (Fig. 6). These data suggested that the lowered binding between calcineurin and the p12I AXAXAA mutant allows more NFAT to be available for the reporter gene assay. Alternatively, the calcineurin-binding motif in p12I may function as a negative modulator motif for NFAT activation. In addition, co-transfection of wild type pMEp12I and p12I AXAXAA mutant resulted in ~25-fold induction over the pME control vector, which indicated that pMEp12I and p12I AXAXAA did not compete for NFAT activation.


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Fig. 6.   The substitution mutation in calcineurin binding sequence in p12I induces increased NFAT transcription activity and does not compete against wild type p12I for NFAT transcription activity. Jurkat T cells were co-transfected with 30 µg of effector plasmid (pME control vector, pMEp12I, p12I AXAXAA mutant, or both pMEp12I and p12I AXAXAA mutant (15 µg each)) and 10 µg of NFAT reporter plasmid and then were left unstimulated or stimulated with phorbol 12-myristate 13-acetate (PMA). Each bar represents the -fold induction of NFAT-luciferase activity in wild type pMEp12I, p12I AXAXAA mutant, or both plasmid-transfected cells over luciferase activity of the pME empty vector-transfected cells. Values were the means of quadruplicate samples in two independent experiments and were statistically significant (*, p < 0.01, pMEp12I compared with p12I AXAXAA alone).

Lowered p12I Binding Affinity to Calcineurin Induces More NFAT Nuclear Translocation-- To test whether p12I binding to calcineurin affected NFAT nuclear translocation, we performed NFAT-GFP fusion protein localization assay when co-expressed with wild type p12I or p12I AXAXAA mutant in HeLa-Tat cells. As expected, both wild type p12I and p12I AXAXAA mutant expression induced NFAT-GFP nuclear translocation, whereas NFAT-GFP was retained in the cytoplasm in HeLa-Tat cells transfected with pME vector control (Fig. 7, A-C). Nuclear translocation of NFAT-GFP was activated by ionomycin and was inhibited by calcineurin inhibitor cyclosporin A (Fig. 7D). When untreated, cells transfected with wild type p12I or p12I AXAXAA mutant had ~80% nuclear NFAT-GFP translocation, which was 5-6-fold greater than in pME control-transfected cells (Fig. 7D). We then measured GFP intensity in nuclear and cytoplasmic regions of NFAT-GFP-expressing cells to quantify NFAT nuclear translocation. As demonstrated in Fig. 7E, The nuclear/cytoplasmic ratio of NFAT-GFP was less than 1 in the control pME-transfected cells, ~2 in pMEp12I, and 2.5 in p12I AXAXAA mutant transfection. Thus, p12I AXAXAA, the lowered binding mutant to calcineurin, induced more NFAT translocation than wild type p12I.


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Fig. 7.   Reduced p12I binding to calcineurin induces increased NFAT nuclear translocation. Confocal microscopic image of HeLa-Tat cell cotransfected with HA-NFAT1c-GFP expression vector and pME empty vector (A), pMEp12I (B), or p12I AXAXAA mutant expression vectors (C). D, the percentage of cells that had nuclear predominant fluorescence. Cells were untreated, stimulated with ionomycin (3 µM, 10 min before fixation), or inhibited with cyclosporin A (1 µM, 16 h before fixation). E, nuclear/cytoplasmic ratio of NFAT-GFP intensity of cells transfected with pME, pMEp12I, or p12I AXAXAA vector (*, p < 0.05, pME vector control compared with pMEp12I. **, p < 0.05, p12I AXAXAA compared with pMEp12I).

p12I Binding to Calcineurin Does Not Affect Phosphatase Activity-- Because many calcineurin binding proteins inhibit calcineurin phosphatase activity (42), we tested whether p12I binding to calcineurin affected calcineurin phosphatase activity. We measured endogenous calcineurin phosphatase activity in Jurkat T cells expressing pME, pMEp12I, and the p12I AXAXAA mutant. As expected, wild type p12I stimulated calcineurin phosphatase activity ~3-fold over pME empty vector (Fig. 8). However, the difference between phosphatase activity induced by wild type p12I and the p12I AXAXAA mutant was not statistically significant (p > 0.5). Wild type p12I, which binds calcineurin with higher affinity compared with the p12I AXAXAA mutant, did not show significant reduction in phosphatase activity (Fig. 8). Similar to NFAT, our data indicated that p12I binding to calcineurin via PSLP(I/L)T calcineurin binding sequence did not inhibit calcineurin phosphatase activity. These data indicate that the increased NFAT transcription and nuclear translocation induced by the p12I AXAXAA mutant was independent of calcineurin binding and was more likely due to the known calcium mobilization properties of the viral protein.


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Fig. 8.   Calcineurin phosphatase activity is not affected by p12I binding to calcineurin. Endogenous calcineurin activity from Jurkat T cells transfected with pME, pMEp12I or p12I AXAXAA mutant was tested by measuring free phosphate (nmol) released from phosphopeptide substrate (RII peptide) (*, p < 0.01 versus pMEp12I and p12I AXAXAA).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Herein, we are the first to demonstrate that the HTLV-1 accessory protein p12I binds to calcineurin and modulates NFAT activation. We identified in p12I a highly conserved calcineurin-binding motif, which is homologous to a PXIXIT calcineurin-binding motif of NFAT.

p12I was consistently co-precipitated with calcineurin in both immunoprecipitation and calmodulin bead pull-down assays. We found that the PSLP(I/L)T motif in p12I was largely responsible for calcineurin binding through the use of serial deletion and alanine substitution mutants. Like NFAT, p12I binding required calcium but was not inhibited by cyclosporin A. Thus, these two proteins appear to bind calcineurin via similar motifs.

The PXIXIT calcineurin binding motif or highly related sequences are found in a variety of calcineurin binding proteins of yeast, mammalian cells, and viruses (44). These proteins are either calcineurin substrates or inhibitors of calcineurin-mediated signaling. For example, the yeast transcription factor Crz1p has a PIISIQ motif that mediates calcineurin interaction (35), and its pattern of regulation is similar to that of NFAT. Therefore, the dephosphorylation, nuclear accumulation, and transcriptional activity of Crz1p depend on this calcineurin-binding motif (44). Like NFAT or Crz1p, p12I itself may be a substrate of calcineurin. We are currently investigating whether p12I is regulated by calcineurin-mediated dephosphorylation. Also, calcineurin binding motifs are found in calcineurin inhibitor proteins. Cellular protein myocyte-enriched calcineurin-interacting protein 1 and cain/cabin1 (calcineurin inhibitor) are endogenous calcineurin inhibitors. Myocyte-enriched calcineurin-interacting protein 1, which contains a PKIIQT calcineurin-binding motif, is expressed abundantly in striated muscle and inhibits calcineurin dependent signaling pathways when overexpressed (35). Cain/cabin1 has a PEITVT motif in a 38-amino acid putative calcineurin-binding domain (45). A viral protein A238L of African swine fever virus, which inhibits NFAT transcriptional activity, interacts with calcineurin via the PKIIIT sequence (37).

Our data indicate that PSLP(I/L)T calcineurin binding sequence of p12I modulates NFAT nuclear translocation and transcription activity. This sequence of p12I is critical for competition with NFAT for calcineurin binding. This result is consistent with our previous study that tested NFAT-driven reporter gene assay using truncation mutants and alanine substitution mutants (AXXA) of putative PXXP motifs (Src homology 3 domain binding motifs).2 There were two positive (aa 33-47 and 87-99) and two negative regions (aa 1-14 and 70-86) for NFAT transcription in p12I, and the mutation in the third PXXP motif (70PXXP73) resulted in increased NFAT activation about 2-fold more than wild type. The 70PSLP(I/L)T75 calcineurin binding site corresponds to the aa 70-86 negative region and third PXXP motif. This NFAT-inhibitory function of the PSLP(I/L)T motif was not from the inhibition of calcineurin phosphatase activity. Calcineurin-binding proteins such as AKAP79, calcineurin B homologous protein, cain/cabin1, and A238L inhibit calcineurin phosphatase activity presumably by affecting calcineurin active sites (42). However, PXIXIT motif-mediated binding itself does not inhibit the catalytic activity of calcineurin, because NFAT activation requires enzymatic activity of calcineurin as well as binding via this motif. Synthetic peptides based on the PXIXIT motif inhibit NFAT activation without affecting calcineurin catalytic activity (27, 28). Likewise, p12I binding to calcineurin via a similar motif does not inhibit calcineurin catalytic activity but instead influences NFAT and calcineurin interaction by competing for binding with NFAT similar to artificial peptides representing this motif (27, 28).

It is unclear why p12I has two regulatory functions for NFAT transcriptional activity: positive modulation by increasing cytosolic calcium concentration from ER stores (12) and negative modulation by calcineurin binding. Interestingly, Bcl-2 has these similar properties with p12I, and the functional relationship between calcium release from the ER and calcineurin binding of Bcl-2 is also still unresolved. Bcl-2 maintains calcium homeostasis and prevents apoptosis by localizing not only at the mitochondrial membrane but also in the ER membranes (46, 47). When Bcl-2 is at the ER membrane, its function appears to increase ER calcium permeability, like p12I (46). Although its function at the ER membrane is still poorly understood, it has been suggested that Bcl-2 functions as an ion channel protein (46). On the other hand, Bcl-2 binds calcineurin via its BH4 domain and inhibits NFAT activity by sequestering calcineurin from NFAT binding without affecting calcineurin catalytic activity (31, 32). By inhibiting NFAT activation, Bcl-2 may prevent Fas ligand (FasL) expression and further apoptosis. Like Bcl-2, p12I may affect apoptosis in HTLV-1-infected T cells. Another ER membrane protein, CAML (calcium modulator and cyclophilin ligand), also has parallel properties to p12I. It activates NFAT by increasing calcium flux when overexpressed in Jurkat T cells (48, 49). Also, CAML binds with calcineurin indirectly, through its association with cyclophilin (50).

In summary, we demonstrate that the HTLV-1 accessory protein p12I interacts with calcineurin, an important regulator of NFAT signaling, via a highly conserved PSLP(I/L)T motif. Thus, HTLV-1, a retrovirus associated with lymphoproliferative disease, expresses a conserved accessory protein to further T cell activation, an important antecedent to effective viral infection, via a calcium/calcineurin/NFAT pathway.

    ACKNOWLEDGEMENTS

We thank A. Phipps for review of the manuscript, T. Vojt for preparation of figures, and G. Franchini and A. Rao for sharing valuable reagents.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants RR-14324, AI-01474, and CA-92009 (to M. D. L.) and NCI, National Institutes of Health, Grant CA-70529 (to the Ohio State University Comprehensive Cancer Center).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.

§ Present address: Howard Hughes Medical Institute, Molecular Pathogenesis Program, The Skirball Institute of Biomolecular Medicine, New York University Medical Center, New York, NY 10016.

** To whom correspondence should be addressed: Center for Retrovirus Research and Department of Veterinary Biosciences, The Ohio State University, 1925 Coffey Rd., Columbus, OH 43210-1093. Tel.: 614-292-4489; Fax: 614-292-6473; E-mail: Lairmore.1@osu.edu.

Published, JBC Papers in Press, February 24, 2003, DOI 10.1074/jbc.M210210200

2 W. Ding, S.-J. Kim, A. M. Nair, B. Michael, K. Boris-Lawrie, A. Tripp, G. Feuer, and M. D. Lairmore, submitted for publication.

    ABBREVIATIONS

The abbreviations used are: HTLV-1, human T lymphotropic virus type 1; ORF, open reading frame; NFAT, nuclear factor of activated T cells; GFP, green fluorescent protein; STLV-1, simian T cell leukemia virus type 1; HA, hemagglutinin; BAPTA-AM, glycine N,N'-1,2-ethanediylbis(oxy-2,1-phenylene)-bis-N-2-(acetyloxy)methoxy-2-oxoethyl-, bis(ace-tyloxy)methyl ester.

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