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• W2012564453 abstract "The nuclear factor of activated T cells (NFAT) family of transcription factors regulates the transcription of cytokine genes and other genes involved in the regulation and function of the immune system. NFAT activity is regulated by the phosphatase calcineurin, which binds and dephosphorylates the NFAT N-terminal regulatory domain, a critical step required for nuclear translocation and transcriptional activity. Here we show that the mitogen-activated protein kinase (MAPK) JNK activates NFATc2-dependent transcription. Mass spectrometry revealed that JNK phosphorylates at least six residues within the NFATc2 regulatory domain in vitro. Transfection of cells with a chimeric construct encoding the GAL-4 DNA binding domain linked to wild-type NFATc2 showed that JNK stimulates the NFATc2 transactivation domain in activated Jurkat T lymphocytes, an effect that is inhibited by dominant-negative versions of JNK. Likewise, the mutation of the phosphorylation sites identified revealed that Thr116 and Ser170 are critical for the transactivation of NFATc2 by JNK. In addition, clustered mutation of the SP-conserved motifs of NFATc2 showed that SP1 and SP2, but not SP3, are also important for the inducible transactivation of NFATc2. Furthermore, mass spectrometry analysis of NFATc2-transfected cells indicated that the activation of the JNK pathway results in the in vivo phosphorylation of Thr116. Our results indicate that, unlike other NFAT members, the transcriptional activity of NFATc2 is up-regulated by JNK. JNK-mediated phosphorylation of NFATs thus appears to play a differential physiological role among NFAT family members. The nuclear factor of activated T cells (NFAT) family of transcription factors regulates the transcription of cytokine genes and other genes involved in the regulation and function of the immune system. NFAT activity is regulated by the phosphatase calcineurin, which binds and dephosphorylates the NFAT N-terminal regulatory domain, a critical step required for nuclear translocation and transcriptional activity. Here we show that the mitogen-activated protein kinase (MAPK) JNK activates NFATc2-dependent transcription. Mass spectrometry revealed that JNK phosphorylates at least six residues within the NFATc2 regulatory domain in vitro. Transfection of cells with a chimeric construct encoding the GAL-4 DNA binding domain linked to wild-type NFATc2 showed that JNK stimulates the NFATc2 transactivation domain in activated Jurkat T lymphocytes, an effect that is inhibited by dominant-negative versions of JNK. Likewise, the mutation of the phosphorylation sites identified revealed that Thr116 and Ser170 are critical for the transactivation of NFATc2 by JNK. In addition, clustered mutation of the SP-conserved motifs of NFATc2 showed that SP1 and SP2, but not SP3, are also important for the inducible transactivation of NFATc2. Furthermore, mass spectrometry analysis of NFATc2-transfected cells indicated that the activation of the JNK pathway results in the in vivo phosphorylation of Thr116. Our results indicate that, unlike other NFAT members, the transcriptional activity of NFATc2 is up-regulated by JNK. JNK-mediated phosphorylation of NFATs thus appears to play a differential physiological role among NFAT family members. Reversible phosphorylation of proteins is a common process in the regulation of most cellular functions. The mitogen-activated protein kinase (MAPK) 1The abbreviations used are: MAPK, mitogen-activated protein kinase; PMA, phorbol 12-myristate 13-acetate; wt, wild type; HA, hemagglutinin; JNK, c-Jun N-terminal kinase; Io, calcium ionophore A23187; GST, glutathione S-transferase; RIPA, radioimmune precipitation assay buffer; IL, interleukin; ERK, extracellular signal-regulated kinase; NFAT, nuclear factor of activated T cells; HEK, human embryonic kidney cells; RP-HPLC, reverse phase high pressure liquid chromatography; NLS, nuclear localization signal. 1The abbreviations used are: MAPK, mitogen-activated protein kinase; PMA, phorbol 12-myristate 13-acetate; wt, wild type; HA, hemagglutinin; JNK, c-Jun N-terminal kinase; Io, calcium ionophore A23187; GST, glutathione S-transferase; RIPA, radioimmune precipitation assay buffer; IL, interleukin; ERK, extracellular signal-regulated kinase; NFAT, nuclear factor of activated T cells; HEK, human embryonic kidney cells; RP-HPLC, reverse phase high pressure liquid chromatography; NLS, nuclear localization signal. family is a set of protein kinases that are activated by extracellular stimuli, whose function and regulation has been conserved during evolution from yeast to mammals (1Widmann C. Gibson S. Jarpe M.B. Johnson G.L. Physiol. Rev. 1999; 79: 143-180Crossref PubMed Scopus (2255) Google Scholar). The major role of these enzymes is to amplify and integrate signals from a wide variety of extracellular stimuli, thereby allowing cells to respond and adapt to changes in their environment. MAPK signal transduction pathways control many processes, including gene expression, protein synthesis, the cell cycle, differentiation, transformation, and programmed cell death. The MAPK family comprises the extracellular signal-regulated kinases (ERK), the p38 MAPKs, and the stress-activated protein kinases (SAPK/JNK). All MAPKs are activated through the dual phosphorylation of conserved TXY motifs by upstream kinases termed MAPK kinases (MAPKK), which are themselves activated by Ser/Thr phosphorylation by the third component of the MAPK system, the MAPKK kinases (MAPKKK). Members of the SAPK/JNK family phosphorylate the N-terminal transactivation domain of c-Jun and other transcription partners of AP-1 (2Hibi M. Lin A. Smeal T. Minden A. Karin M. Genes Dev. 1993; 7: 2135-2148Crossref PubMed Scopus (1708) Google Scholar, 3Derijard B. Raingeaud J. Barrett T. Wu I.H. Han J. Ulevitch R.J. Davis R.J. Science. 1995; 267: 682-685Crossref PubMed Scopus (1407) Google Scholar). There are at least three JNK MAPKs, called JNK-1, JNK-2, and JNK-3, and each is encoded by a different gene. Although the MAPKKs MKK4 and MKK7 have been established as specific activators of JNK, more than a dozen additional MAPKKKs are also known to activate this pathway (reviewed in Ref. 4Manning A.M. Davis R.J. Nat. Rev. Drug. Discov. 2003; 2: 554-565Crossref PubMed Scopus (533) Google Scholar). This huge diversity of activators allows a wide range of stimuli to activate JNK, although JNKs are more commonly activated in response to environmental stress or signaling through GTPases of the rho family, which can trigger the MAPKKK cascade pathway. Once activated, JNKs phosphorylate an array of different substrates ranging from transcription factors to apoptosis regulatory molecules, involving these kinases in regulation of cell viability, cellular stress, induction of apoptosis, and cell proliferation (reviewed in Ref. 4Manning A.M. Davis R.J. Nat. Rev. Drug. Discov. 2003; 2: 554-565Crossref PubMed Scopus (533) Google Scholar). Unlike JNK-3, whose expression is restricted to brain, heart, and testis, isoforms 1 and 2 are ubiquitously expressed (reviewed in Ref. 5Davis R.J. Cell. 2000; 103: 239-252Abstract Full Text Full Text PDF PubMed Scopus (3609) Google Scholar). Much attention has been paid, however, to their roles in T cell development and function (6Crabtree G.R. Science. 1989; 243: 355-361Crossref PubMed Scopus (914) Google Scholar). In these cells, complete activation of JNK requires signals from both the T cell receptor (TCR)-CD3 complex and the costimulatory factor CD28. These signals, which can be replaced by combined treatment with the pharmacological agents phorbol 12-myristate 13-acetate (PMA) and calcium ionophore A23187 (Io), lead to T cell activation (7Su B. Jacinto E. Hibi M. Kallunki T. Karin M. Ben-Neriah Y. Cell. 1994; 77: 727-736Abstract Full Text PDF PubMed Scopus (849) Google Scholar, 8Rincon M. Flavell R.A. EMBO J. 1994; 13: 4370-4381Crossref PubMed Scopus (246) Google Scholar). The transcription factors c-Jun and NFAT are JNK substrates, implicated in T cell function as effector molecules of JNK signaling in these cells. NFAT was initially identified in T cells as a transcription factor responsible for mediating Ca2+/calcineurin-dependent transcription of genes including IL-2 and other important cytokines involved in T cell activation (9Rao A. Luo C. Hogan P.G. Annu. Rev. Immunol. 1997; 15: 707-747Crossref PubMed Scopus (2209) Google Scholar, 10Jain J. Loh C. Rao A. Curr. Opin. Immunol. 1995; 7: 333-342Crossref PubMed Scopus (500) Google Scholar, 11Crabtree G.R. Clipstone N.A. Annu. Rev. Biochem. 1994; 63: 1045-1083Crossref PubMed Scopus (626) Google Scholar). Further studies identified new members of the family that participate in many physiological processes in different cell types inside and outside of the immune system. The NFAT family of transcription factors contains five members, which are related to the Rel/NFκB family. Four of them NFATc1 (NFAT2/c), NFATc2 (NFAT1/p), NFATc3 (NFAT4/x), and NFATc4 (NFAT3) are regulated by calcineurin and share a highly homologous region located at their regulatory N-terminal domain (reviewed in Ref. 12Hogan P.G. Chen L. Nardone J. Rao A. Genes Dev. 2003; 17: 2205-2232Crossref PubMed Scopus (1535) Google Scholar). Activation of these members is primarily regulated through the control of their subcellular localization. In the absence of Ca2+ signals, NFAT proteins are highly phosphorylated and remain in the cytoplasm of resting cells. When intracellular calcium levels rise, the phosphatase calcineurin is activated and dephosphorylates NFATs. This favors the exposure of a nuclear localization signal (NLS) and the masking of a nuclear export signal (NES), thus promoting the translocation of NFATs from the cytosol to the nucleus, their binding to target sequences, and therefore the transcription of different NFAT-dependent genes (12Hogan P.G. Chen L. Nardone J. Rao A. Genes Dev. 2003; 17: 2205-2232Crossref PubMed Scopus (1535) Google Scholar, 13Okamura H. Aramburu J. Garcia-Rodriguez C. Viola J.P. Raghavan A. Tahiliani M. Zhang X. Qin J. Hogan P.G. Rao A. Mol. Cell. 2000; 6: 539-550Abstract Full Text Full Text PDF PubMed Scopus (385) Google Scholar). Calcineurin-dependent nuclear accumulation of NFAT is subject to further regulation by the activity of a number of serine/threonine kinases (14Shibasaki F. Price E.R. Milan D. McKeon F. Nature. 1996; 382: 370-373Crossref PubMed Scopus (432) Google Scholar). These either act in the nucleus to promote the export of nuclear NFAT, or, alternatively, in the cytosol to inhibit NFAT import. Many protein kinases, including the MAPK group (p38, JNK, and ERK), GSK3β, PKA, MEKK1, and CK1α, have been reported to phosphorylate different members of NFAT at different serine residues (15Chow C.W. Rincon M. Cavanagh J. Dickens M. Davis R.J. Science. 1997; 278: 1638-1641Crossref PubMed Scopus (300) Google Scholar, 16Beals C.R. Sheridan C.M. Turck C.W. Gardner P. Crabtree G.R. Science. 1997; 275: 1930-1934Crossref PubMed Scopus (634) Google Scholar, 17Zhu J. Shibasaki F. Price R. Guillemot J.C. Yano T. Dotsch V. Wagner G. Ferrara P. McKeon F. Cell. 1998; 93: 851-861Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar, 18Graef I.A. Mermelstein P.G. Stankunas K. Neilson J.R. Deisseroth K. Tsien R.W. Crabtree G.R. Nature. 1999; 401: 703-708Crossref PubMed Scopus (451) Google Scholar, 19Yang T.T. Xiong Q. Graef I.A. Crabtree G.R. Chow C.W. Mol. Cell. Biol. 2005; 25: 907-920Crossref PubMed Scopus (43) Google Scholar, 20Chow C.W. Dong C. Flavell R.A. Davis R.J. Mol. Cell. Biol. 2000; 20: 5227-5234Crossref PubMed Scopus (119) Google Scholar, 21Porter C.M. Havens M.A. Clipstone N.A. J. Biol. Chem. 2000; 275: 3543-3551Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 22Neal J.W. Clipstone N.A. J. Biol. Chem. 2001; 276: 3666-3673Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 23Sheridan C.M. Heist E.K. Beals C.R. Crabtree G.R. Gardner P. J. Biol. Chem. 2002; 277: 48664-48676Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 24Okamura H. Garcia-Rodriguez C. Martinson H. Qin J. Virshup D.M. Rao A. Mol. Cell. Biol. 2004; 24: 4184-4195Crossref PubMed Scopus (150) Google Scholar). In most cases rephosphorylation is believed to facilitate NFAT inactivation and export to cytoplasm, there are data indicating that phosphorylation can also positively regulate NFAT transactivation. For example, Okamura et al. reported inducible phosphorylation within a functional site of the NFATc2 transactivation domain upon stimulation of T cells with PMA and Io (13Okamura H. Aramburu J. Garcia-Rodriguez C. Viola J.P. Raghavan A. Tahiliani M. Zhang X. Qin J. Hogan P.G. Rao A. Mol. Cell. 2000; 6: 539-550Abstract Full Text Full Text PDF PubMed Scopus (385) Google Scholar). In addition, Pim kinase 1 (25Rainio E.M. Sandholm J. Koskinen P.J. J. Immunol. 2002; 168: 1524-1527Crossref PubMed Scopus (109) Google Scholar) and Cot kinase (26de Gregorio R. Iniguez M.A. Fresno M. Alemany S. J. Biol. Chem. 2001; 276: 27003-27009Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar) have been shown to phosphorylate NFATc1 and NFATc2, respectively, and to enhance their transactivation properties. Despite the importance of phosphorylation in the regulation of NFAT activity, the detailed molecular mechanisms underlying this process are little known. Particularly, it is still not clear which kinases phosphorylate NFATs under basal conditions and which ones mediate rephosphorylation and nuclear exclusion of the transcription factor after activation. Although proteomic analysis has allowed the identification of NFAT sites phosphorylated under basal conditions and after stimulation with PMA and Io (13Okamura H. Aramburu J. Garcia-Rodriguez C. Viola J.P. Raghavan A. Tahiliani M. Zhang X. Qin J. Hogan P.G. Rao A. Mol. Cell. 2000; 6: 539-550Abstract Full Text Full Text PDF PubMed Scopus (385) Google Scholar), the exact identification of sites dynamically phosphorylated by particular kinases upon cell activation using these techniques, are currently a technical challenge because of the implication of different kinases and phosphatases and the low stoichiometry of transient phosphorylation. We have previously shown that p38 and JNK MAPKs phosphorylate NFATc2 in vitro and interact with it in vivo. However, activation of the p38 pathway (but not JNK), results in the inhibition of NFATc2-driven transcription and nuclear accumulation (27Gomez del Arco P. Martinez-Martinez S. Maldonado J.L. Ortega-Perez I. Redondo J.M. J. Biol. Chem. 2000; 275: 13872-13878Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). In this work we analyze the specific role of JNK pathway in the dynamic regulation of NFATc2 by phosphorylation, identifying by mass spectrometry amino acid residues of NFATc2 phosphorylated both in vitro and in vivo by JNK, and systematically addressing the physiological relevance of these sites. Our results show that activation of this pathway enhances the transcriptional activity of this factor, and that mutation of identified residues, located in conserved regions (Fig. 1) results in the inhibition of JNK-mediated NFAT transcriptional activity in Jurkat T cells. Cell Culture and Reagents—HeLa and Jurkat T cells were cultured in Dulbecco's modified Eagle's medium or RPMI medium respectively, supplemented with 10% fetal calf serum, 2 mm glutamine, and a penicillin-streptomycin mixture. The JNK inhibitor SP600125 (46Bennett B.L. Sasaki D.T. Murray B.W. O'Leary E.C. Sakata S.T. Xu W. Leisten J.C. Motiwala A. Pierce S. Satoh Y. Bhagwat S.S. Manning A.M. Anderson D.W. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13681-13686Crossref PubMed Scopus (2210) Google Scholar) was purchased from Biomol, and PMA, Io, calcineurin, and calmodulin were purchased from Sigma. Expression Constructs and Transfections—Expression constructs for wild-type (wt) HA-tagged mouse NFATc2 (full-length), NFATLuc, MEKK1, JNK1, MKK6b(E), and p38α were as previously described (27Gomez del Arco P. Martinez-Martinez S. Maldonado J.L. Ortega-Perez I. Redondo J.M. J. Biol. Chem. 2000; 275: 13872-13878Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). GAL-DBD Luc was kindly provided by Rosario Perona, and JIP1, JBD, and JNK KR were kind gifts from Pura Muñoz. The empty vector pcDNA 3.0 was from Invitrogen. pAB GAL4-mouse NFATc2-(3–385) constructs were created by PCR from wt full-length HA-NFAT and subcloned into the pAB empty vector (47Baniahmad A. Kohne A.C. Renkawitz R. EMBO J. 1992; 11: 1015-1023Crossref PubMed Scopus (238) Google Scholar) between the SalI and PvuII sites. Mutations were conducted with the QuikChange site-directed mutagenesis kit from Stratagene, in accordance with the manufacturer's indications. Western blot experiments using extracts of HeLa cells transfected with 1 μg of each construct confirmed comparable expression levels (data not shown). Transfections in HeLa cells were performed with Lipofectamine Plus reagent (Invitrogen). Cells were cotransfected at 60–70% confluence in OPTIMEM medium (Invitrogen). The transfection mixture consisted of 0.2 μg of reporter plasmid (one among NFAT Luc, IL-13 Luc, Ref. 32Macian F. Garcia-Rodriguez C. Rao A. EMBO J. 2000; 19: 4783-4795Crossref PubMed Scopus (260) Google Scholar, or IL-4 Luc, Ref. 33Szabo S.J. Gold J.S. Murphy T.L. Murphy K.M. Mol. Cell. Biol. 1993; 13: 4793-4805Crossref PubMed Scopus (235) Google Scholar), the HA-mouse NFATc2 full-length expression plasmid (0.3 μg), and a kinase combination (0.3 μg each of constitutively active MKK6b (E) plus p38α, or constitutively active MEKK1 plus JNK1). In control experiments the empty vector pcDNA 3.0 (0.6 μg), from Invitrogen, was substituted for the kinase combination. Transfection took place over 3 h, and afterward cells were maintained in Dulbecco's modified Eagle's medium, 10% fetal calf serum. 24 h later, cells were split, and after one night of recovery were stimulated with PMA and Io. Luciferase was assayed with the luciferase kit from Promega. Jurkat T cells were also transfected with Lipofectamine reagent. In these experiments, 6 × 106 cells were cotransfected with 3 μg of total DNA distributed as follows: 0.1 μg of expression vector pAB GAL4 NFATc2 (wt or mutant); 1.2 μg of GAL-DBD Luc reporter plasmid; and either 1.8 μg of JIP1, JBD, or JNK KR, or 1.8 μg of pcDNA 3.0 empty vector, as indicated. For the JIP1 dose dependence experiment, the amounts of plasmid transfected were 1.8, 0.9, 0.45, and 0.2 μg, made up to 1.8 μg with pcDNA 3.0 empty vector as required. Transfections were performed for 4 h, and after that, cells were recultured in RPMI, 10% fetal calf serum at a concentration of 106 cells/ml. Stimuli were added 16 h post-transfection. Luciferase determination was as described for HeLa cells. When required, cells were stimulated with PMA and calcium ionophore at a concentration of 20 ng/ml and 1 μm, respectively, for 3–5 h. In the case of HeLa cells, we supplemented stimulation medium with 3 mm Ca2+ to obtain a strong NFAT dephosphorylation and activation. The JNK inhibitor SP600125 was added 1 h before stimulation, at a concentration of 20 μm. For in vivo phosphorylation experiments, HEK cells were transfected by the calcium phosphate method (48Rodriguez A. Flemington E.K. Anal. Biochem. 1999; 272: 171-181Crossref PubMed Scopus (28) Google Scholar), with the constructs encoding the full-length FLAG-tagged NFATc2 (10 μg), MEKK1 (10 μg), and JNK1 (5 μg). In control experiments pcDNA 3.0 substituted the kinase combination. The total amount of DNA was completed up to 30 μg with pGL3 basic vector. Activation of JNK was determined by Western blot analysis using an antiphospho-JNK antibody purchased from Cell Signaling Technology (cat. 9251). In Vitro Calcineurin Binding Assay and Western Blot Experiments— The mouse NFAT N-terminal domain (amino acids 4–385) was HA-tagged and fused to GST. Mutants were based on this construct and were generated by using the Stratagene mutation kit. 1 μg of GST-NFAT (either wt or the indicated mutant), adsorbed to glutathione-Sepharose beads (from Amersham Biosciences), was incubated with 20 nm calcineurin and 600 nm calmodulin in a binding buffer containing 20 mm Tris, pH 8.0, 100 mm NaCl, 6 mm MgCl2, 1.5 mm CaCl2, 0.2% Triton X-100, and a protease and phosphatase inhibitors mixture, in a final reaction volume of 60 μl. The reaction was incubated on a rocking platform at 4 °C for 30 min. Beads were then washed five times with binding buffer and prepared for SDS-PAGE and Western blot analysis. Immunodetection was done with purified mouse monoclonal anti-calcineurin antibody, acquired from Pharmingen (Pharmingen Europe), diluted 1:1000 in phosphate-buffered saline-0.1% Tween 20. Western blot assays to check MAPK activation were performed on total extracts from 106 JK cells, lysed in a buffer containing 20 mm HEPES pH 8, 5 mm EDTA, 10 mm EGTA, 5 mm NaF, 10% glycerol, 1 mm dithiothreitol, 400 mm KCl, 0.4% Triton X-100, 20 mm β-glycerophosphate, and inhibitors (0.2 μm okadaic acid, 0.1 mm sodium orthovanadate, 2 μg/ml aprotinin, 5 μg/ml leupeptin, and 1 mm phenylmethylsulfonyl fluoride). Lysates were rotated at 4 °C for 30 min and then centrifuged at maximum speed to sediment cellular debris. Supernatants were subjected to SDS-PAGE and immunodetection. JNK activation was verified by c-Jun phosphorylation, using the monoclonal antiphospho-c-Jun antibody SC-822 purchased from Santa Cruz Biotechnology diluted 1:1000 in TBS-0.1% Tween. Phospho-Erk and phospho-p38 were detected with anti-ACTIVE™ MAPK from Promega (V6671), and antiphospho-p38 from Cell Signaling. Antibody binding was visualized in all cases using the enhanced chemiluminiscence (ECL) system from Amersham Biosciences. In Vitro Kinase Assay and Proteolytic Digestions—1 μg of His-JNK2, kindly provided by Dr. J. Han (The Scripps Research Institute), and 2 μg of GST-HA-mouse NFATc2-(4–385) were incubated at 37 °C for 30 min in a kinase buffer containing 20 mm HEPES pH 7.5, 20 mm β-glycerophosphate, 20 mm MgCl2, 0.1 mm sodium orthovanadate, and 2 mm dithiothreitol. The reaction took place in the presence of 250 μm ATP and 10 μCi of [γ32-P]ATP. Assays were stopped with Laemmli sample buffer and loaded onto a 7% SDS-PAGE gel. Protein Analysis by Mass Spectrometry and Phosphopeptide Mapping—The bands corresponding to phosphorylated proteins, were excised and digested with trypsin (Promega) or V8 (Sigma) proteases, as described (49Shevchenko A. Jensen O.N. Podtelejnikov A.V. Sagliocco F. Wilm M. Vorm O. Mortensen P. Boucherie H. Mann M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14440-14445Crossref PubMed Scopus (1293) Google Scholar). Peptides extracted from radiolabeled bands were separated by reverse phase HPLC as described (50Ogueta S. Rogado R. Marina A. Moreno F. Redondo J.M. Vazquez J. J. Mass Spectr. 2000; 35: 556-565Crossref PubMed Scopus (29) Google Scholar), and analyzed by nanospray off-line with ion trap mass spectrometry as described (50Ogueta S. Rogado R. Marina A. Moreno F. Redondo J.M. Vazquez J. J. Mass Spectr. 2000; 35: 556-565Crossref PubMed Scopus (29) Google Scholar, 51Marina A. Garcia M.A. Albar J.P. Yague J. Lopez de Castro J.A. Vazquez J. J. Mass Spectr. 1999; 34: 17-27Crossref PubMed Scopus (58) Google Scholar), using an LCQ Classic model (Finnigan, ThermoQuest, San Jose, CA). Peptides derived from the digestion of unlabeled NFAT bands, were alternatively analyzed by RP-HPLC-MS/MS using a 0.18 mm × 150 mm BioBasic 18 RP column (ThermoHypersil-Keystone), operating at ∼1.5 μl/min, connected to a Surveyor HPLC system on-line with a LCQ-DECA XP ion trap mass spectrometer (Thermo Finnigan, San Jose, CA). Peptides were eluted using a 90-min gradient from 5 to 60% solvent B (solvent A: 0.5% acetic acid; solvent B: 0.5% acetic acid, 80% acetonitrile). Peptides were detected in survey scans from 400 to 1600 amu (8 μscans), followed by a ZoomScan and a MS/MS analysis, using an isolation width of 3 amu, a normalized collision energy of 35%. Phosphopeptides were mapped by two-dimensional separation on 20 × 20 thin layer cellulose plates purchased from Sigma. Electrophoresis was carried out in a Hunter HTLE-7200 thin layer peptide mapping electrophoresis system (CBS Scientific Company, Del Mar, California). Gels were run for 45 min in pH 8.9 buffer containing 10% w/v ammonium carbonate. The chromatography dimension was performed overnight in standard phosphochromatography buffer composed of 3.7% n-butyl alcohol (v/v), 2.5% pyridine (v/v), and 0.75% (v/v) glacial acetic acid (52Boyle W.J. van der Geer P. Hunter T. Methods Enzymol. 1991; 201: 110-149Crossref PubMed Scopus (1273) Google Scholar). Phosphopeptide signals were visualized by autoradiography. In Vivo Analysis of Phosphorylation—A minimum of 300 × 106 HEK cells were transfected as described above. Forty-eight hours after transfection, every 5 × 106 cells were lysed in 100 μl of RIPA buffer (50 mm Tris-HCl pH 7.4, 150 mm NaCl, 1 mm EDTA, 1% Nonidet P-40, 0.25% sodium deoxycholate, and a combination of proteases and phosphatases inhibitors: leupeptin, aprotinin, pepstatin, phenylmethylsulfonyl fluoride, Na3VO4, and NaF). The lysates were incubated at 4 °C on a rocking platform for 20 min. Lysates were cleared by centrifugation for 15 min at 14,000 × g in a precooled centrifuge. Supernatants were incubated for 1 h with 10 μl of RIPA-equilibrated anti-FLAG M2 agarose beads to immunoprecipitate the FLAG-NFATc2 protein. Inmunoprecipitates were washed three times with RIPA and loaded onto a 6% gel for SDS-PAGE. The gel was stained with Sypro Ruby, and the band corresponding to NFATc2 was excised and trypsin digested. The mixture was then analyzed by RP-HPLC on-line with an LTQ linear ion trap mass spectrometer, using the same HPLC setup and conditions as described above. Single ion reaction monitoring was programmed over selected ions along the entire gradient. JNK Activation Enhances NFAT-dependent Transcription— We have previously shown that activation of the p38, but not the JNK/MAPK pathway reduces NFATc2 nuclear accumulation upon cell stimulation and results in inhibition of NFAT-driven transcription. However, p38 and JNK both interact with NFATc2 in vivo (27Gomez del Arco P. Martinez-Martinez S. Maldonado J.L. Ortega-Perez I. Redondo J.M. J. Biol. Chem. 2000; 275: 13872-13878Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). For this reason, we decided to analyze the potential role of the JNK/MAPK pathway in the regulation of NFATc2. Fig. 1 shows the main regulatory sites in the regulatory domain of NFATc2 that are conserved among other NFAT family members, and indicates the positions of potential target sites for JNK-mediated phosphorylation analyzed in the experiments described here. We initially carried out transient transfection experiments in HeLa cells, in which NFATc2 has been shown to translocate to the nucleus in response to Ca2+/calcineurin stimulation (28Aramburu J. Garcia-Cozar F. Raghavan A. Okamura H. Rao A. Hogan P.G. Mol. Cell. 1998; 1: 627-637Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar, 29Aramburu J. Yaffe M.B. Lopez-Rodriguez C. Cantley L.C. Hogan P.G. Rao A. Science. 1999; 285: 2129-2133Crossref PubMed Scopus (516) Google Scholar). We cotransfected cells with JNK and constitutively active MEKK1 to achieve complete activation of the JNK pathway, together with full-length NFATc2 and a luciferase reporter plasmid directed by a triple repeat of the NFAT/AP1 composite site from the interleukin-2 promoter (NFATLuc) (30Rooney J.W. Sun Y.L. Glimcher L.H. Hoey T. Mol. Cell. Biol. 1995; 15: 6299-6310Crossref PubMed Scopus (219) Google Scholar). Activation of the JNK pathway led to an increase in NFAT-dependent transcription and further potentiated the activation provoked by pharmacological stimulation of cells with PMA and Io (Fig. 2A). In contrast, parallel cotransfections using constitutively active MKK6 and p38, to activate the p38 pathway, resulted in the predicted inhibition of NFAT-driven transcription (27Gomez del Arco P. Martinez-Martinez S. Maldonado J.L. Ortega-Perez I. Redondo J.M. J. Biol. Chem. 2000; 275: 13872-13878Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). Because the reporter construct used in these first experiments contains an NFAT/AP-1 composite site, the possibility exists that AP-1, not NFAT, could be the target of the JNK MAPK pathway (31Derijard B. Hibi M. Wu I.H. Barrett T. Su B. Deng T. Karin M. Davis R.J. Cell. 1994; 76: 1025-1037Abstract Full Text PDF PubMed Scopus (2953) Google Scholar). Therefore, in a new set of experiments we examined the ability of JNK to activate NFAT-driven luciferase reporter plasmids, lacking NFAT:AP1 composite sites, such as those directed by the IL13 or the IL4 promoters (32Macian F. Garcia-Rodriguez C. Rao A. EMBO J. 2000; 19: 4783-4795Crossref PubMed Scopus (260) Google Scholar, 33Szabo S.J. Gold J.S. Murphy T.L. Murphy K.M. Mol. Cell. Biol. 1993; 13: 4793-4805Crossref PubMed Scopus (235) Google Scholar). In both cases, the luciferase reporter activity was activated by JNK, which also potentiated the action of PMA and Io, and this activation was similar to that displayed by the composite NFAT/AP-1 (NFATLuc) reporter construct (Fig. 2B). These results suggested that the JNK/MAPK pathway plays a role on the regulation of NFATc2. Mapping of the NFATc2 Residues Phosphorylated by JNK in Vitro.—To determine which of the NFATc2 residues phosphorylated by JNK could be responsible for the increased NFAT transcriptional activity we observed, we first performed in vitro phosphorylation experiments. We generated a recombinant construct containing the N-terminal part of NFATc2 protein (amino acids 4–385) fused to GST, and this construct was subjected to in vitro phosphorylation by recombinant His-JNK in the p" @default.
• W2012564453 created "2016-06-24" @default.
• W2012564453 creator A5015102352 @default.
• W2012564453 creator A5038752178 @default.
• W2012564453 creator A5048692628 @default.
• W2012564453 creator A5059973854 @default.
• W2012564453 creator A5081217694 @default.
• W2012564453 creator A5088049796 @default.
• W2012564453 date "2005-05-01" @default.
• W2012564453 modified "2023-10-12" @default.
• W2012564453 title "c-Jun N-terminal Kinase (JNK) Positively Regulates NFATc2 Transactivation through Phosphorylation within the N-terminal Regulatory Domain" @default.
• W2012564453 cites W135983215 @default.
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