Protein Kinase Cζ Phosphorylates Nuclear Factor of Activated T Cells and Regulates Its Transactivating Activity
2002; Elsevier BV; Volume: 277; Issue: 30 Linguagem: Inglês
10.1074/jbc.m106983200
ISSN1083-351X
AutoresBelén San-Antonio, Miguel A. Íñiguez, Manuel Fresno,
Tópico(s)T-cell and B-cell Immunology
ResumoAlthough several isoforms of protein kinase C (PKC) have been implicated in T lymphocyte activation events, little is known about their mode of action. To address the role of PKCζ in T cell activation, we have generated Jurkat T cell transfectants expressing either the wild type (J-PKCζ) or “kinase-dead” mutant (J-PKCζmut) versions of this protein. Expression of PKCζ but not PKCζmut increased transcriptional activation mediated by the NF-κB or nuclear factor of activated T cells (NFAT). PKCζ cooperates with calcium ionophore and with NFAT1 or NFAT2 proteins to enhance transcriptional activation of a NFAT reporter construct. However, neither NFAT nuclear translocation nor DNA binding were in J-PKCζ cells. Our results show that PKCζ enhanced transcriptional activity mediated by Gal4-NFAT1 fusion proteins containing the N-terminal transactivation domain of human NFAT1. Interestingly, PKCζ synergizes with calcineurin to induce transcriptional activation driven by the NFAT1 transactivation domain. Co-precipitation experiments showed physical interaction between PKCζ and NFAT1 or NFAT2 isoforms. Even more, PKCζ was able to phosphorylate recombinant glutathione S-transferase-NFAT1 (1–385) protein. These data reveal a new role of PKCζ in T cells through the control of NFAT function by modulating the activity of its transactivation domain. Although several isoforms of protein kinase C (PKC) have been implicated in T lymphocyte activation events, little is known about their mode of action. To address the role of PKCζ in T cell activation, we have generated Jurkat T cell transfectants expressing either the wild type (J-PKCζ) or “kinase-dead” mutant (J-PKCζmut) versions of this protein. Expression of PKCζ but not PKCζmut increased transcriptional activation mediated by the NF-κB or nuclear factor of activated T cells (NFAT). PKCζ cooperates with calcium ionophore and with NFAT1 or NFAT2 proteins to enhance transcriptional activation of a NFAT reporter construct. However, neither NFAT nuclear translocation nor DNA binding were in J-PKCζ cells. Our results show that PKCζ enhanced transcriptional activity mediated by Gal4-NFAT1 fusion proteins containing the N-terminal transactivation domain of human NFAT1. Interestingly, PKCζ synergizes with calcineurin to induce transcriptional activation driven by the NFAT1 transactivation domain. Co-precipitation experiments showed physical interaction between PKCζ and NFAT1 or NFAT2 isoforms. Even more, PKCζ was able to phosphorylate recombinant glutathione S-transferase-NFAT1 (1–385) protein. These data reveal a new role of PKCζ in T cells through the control of NFAT function by modulating the activity of its transactivation domain. protein kinase C nuclear factor of activated T cells nuclear factor κB activating protein-1 calcineurin transactivation domain cyclosporin A phorbol 12-myristate 13-acetate A23187 calcium ionophore glutathioneS-transferase fetal bovine serum hemagglutinin relative luciferase unit interleukin thymidine kinase calmodulin phosphate-buffered saline c-Jun N-terminal kinase The serine/threonine protein kinase C (PKC)1 family is constituted by several isoenzymes that have been grouped into three subfamilies according to their activation profile and their regulatory properties (1Newton A.C. J. Biol. Chem. 1995; 270: 28495-28498Abstract Full Text Full Text PDF PubMed Scopus (1466) Google Scholar, 2Newton A.C. Curr Opin Cell Biol. 1997; 9: 161-167Crossref PubMed Scopus (845) Google Scholar). The named atypical subclass of the PKC family comprises two members, PKCζ and PKCλ/ι, which are unresponsive to calcium or phorbol esters. Both PKCζ and PKCλ/ι are more than 70% homologous in primary structure and so far have been implicated in similar activities (3Dı́az-Meco M.T. Municio M.M. Frutos S. Sánchez P. Lozano J. Sanz L. Moscat J. Cell. 1996; 86: 777-786Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar). PKCζ is activated by important lipid co-factors like phosphatidylserine, phosphatidic acid (4Limatola C. Schaap D. Moolenaar W.H. van Blitterswijk W.J. Biochem. J. 1994; 304: 1001-1008Crossref PubMed Scopus (284) Google Scholar), phosphatidylinositol 3,4,5-triphosphate (5Nakanishi H. Brewer K.A. Exton J.H. J. Biol. Chem. 1993; 268: 13-16Abstract Full Text PDF PubMed Google Scholar), and ceramide (6Lozano J. Berra E. Municio M.M. Diaz-Meco M.T. Dominguez I. Sanz L. Moscat J. J. Biol. 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Bandyopadhyay G. Quon M.J. Burke T.R. Farese R.V. J. Biol. Chem. 1999; 274: 30495-30500Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar) and nerve growth factor-dependent signaling (20Wooten M.W. Seibenhener M.L. Neidigh K.B. Vandenplas M.L. Mol. Cell. Biol. 2000; 20: 4494-4504Crossref PubMed Scopus (69) Google Scholar). Diverse studies have implicated PKCζ in the control of NF-κB activation in response to inflammatory cytokines, by mechanisms implying nuclear translocation (21Sanz L. Diaz-Meco M.T. Nakano H. Moscat J. EMBO J. 2000; 19: 1576-1586Crossref PubMed Google Scholar, 22Sanz L. Sanchez P. Lallena M.J. Diaz-Meco M.T. Moscat J. EMBO J. 1999; 18: 3044-3053Crossref PubMed Scopus (323) Google Scholar) and regulation of their intrinsic transactivating activity (23Anrather J. Csizmadia V. Soares M.P. Winkler H. J. Biol. Chem. 1999; 274: 13594-13603Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar, 24Martin A.G. San-Antonio B. Fresno M. J. Biol. Chem. 2001; 276: 15840-15849Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). Several PKC isoforms have been shown to play an important role in T cell activation through the regulation of the activity of transcription factors such as AP-1 (25Werlen G. Jacinto E. Xia Y. Karin M. EMBO J. 1998; 17: 3101-3111Crossref PubMed Scopus (252) Google Scholar) and NF-κB (26Coudronniere N. Villalba M. Englund N. Altman A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3394-3399PubMed Google Scholar, 27Khoshnan A. Bae D. Tindell C.A. Nel A.E. J. Immunol. 2000; 165: 6933-6940Crossref PubMed Scopus (123) Google Scholar, 28Trushin S.A. Pennington K.N. Algeciras-Schimnich A. Paya C.V. J. Biol. Chem. 1999; 274: 22923-22931Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). However, the importance of PKCζ in T cell activation-mediated events is unclear. PKCζ is expressed in T lymphocytes that are involved in IL-2-mediated proliferation (29Gomez J. Pitton C. Garcia A. Martinez de Aragon A. Silva A. Rebollo A. Exp. Cell Res. 1995; 218: 105-113Crossref PubMed Scopus (36) Google Scholar) and in the actin cytoskeleton organization (30Gomez J. Martinez de Aragon A. Bonay P. Pitton C. Garcia A. Silva A. Fresno M. Alvarez F. Rebollo A. Eur. J. Immunol. 1995; 25: 2673-2678Crossref PubMed Scopus (60) Google Scholar). We have recently described the involvement of PKCζ in the regulation of NF-κB transactivation in T cells (24Martin A.G. San-Antonio B. Fresno M. J. Biol. Chem. 2001; 276: 15840-15849Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar), but little is known about the effect of this atypical PKC on the function of other transcription factors known to be essential for T cell activation as the nuclear factor of activated T cells (NFAT). This transcription factor plays a prominent role in T cell activation by regulating the expression of a large number of inducible genes encoding cytokines and cell surface receptors that are essential for a productive immune response (31Crabtree G.R. Cell. 1999; 96: 611-614Abstract Full Text Full Text PDF PubMed Scopus (661) Google Scholar, 32Rao A. Luo C. Hogan P.G. Ann Rev Immunol. 1997; 15: 707-747Crossref PubMed Scopus (2200) Google Scholar). The NFAT family of transcription factors consists of four “classical” members: NFAT1, NFAT2, NFAT3, and NFAT4 (33Hoey T. Sun Y.L. Williamson K. Xu X. Immunity. 1995; 2: 461-472Abstract Full Text PDF PubMed Scopus (352) Google Scholar, 34Masuda E.S. Naito Y. Tokumitsu H. Campbell D. Saito F. Hannum C. Arai K. Arai N. Mol. Cell. Biol. 1995; 15: 2697-2706Crossref PubMed Scopus (198) Google Scholar, 35McCaffrey P.G. Luo C. Kerppola T.K. Jain J. Badalian T.M., Ho, A.M. Burgeon E. Lane W.S. Lambert J.N. 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Ann Rev Immunol. 1997; 15: 707-747Crossref PubMed Scopus (2200) Google Scholar). The activity of Cn (and therefore NFAT activation) can be inhibited by the immunosuppressive drug cyclosporin A (CsA) (39O'Keefe S.J. Tamura J. Kincaid R.L. Tocci M.J. O'Neill E.A. Nature. 1992; 357: 692-694Crossref PubMed Scopus (781) Google Scholar, 40Schreiber S.L. Crabtree G.R. Immunol Today. 1992; 13: 136-142Abstract Full Text PDF PubMed Scopus (1947) Google Scholar). Phorbol esters indirectly stimulate NFAT activity by promoting the synthesis and activation of Fos and Jun, components of the AP-1 family of transcription factors (41Jain J. Loh C. Rao A. Curr Opin Immunol. 1995; 7: 333-342Crossref PubMed Scopus (499) Google Scholar), which cooperatively bind with NFAT to composite DNA elements found in the promoter region of many NFAT target genes (42Chen L. Glover J.N. Hogan P.G. Rao A. Harrison S.C. Nature. 1998; 392: 42-48Crossref PubMed Scopus (410) Google Scholar, 43Macian F. Garcı́a-Rodrı́guez C. Rao A. EMBO J. 2000; 19: 4783-4795Crossref PubMed Scopus (258) Google Scholar). At the structural level, NFAT proteins contain highly conserved DNA-binding domains responsible for cooperation with Fos/Jun (32Rao A. Luo C. Hogan P.G. Ann Rev Immunol. 1997; 15: 707-747Crossref PubMed Scopus (2200) Google Scholar, 33Hoey T. Sun Y.L. Williamson K. Xu X. Immunity. 1995; 2: 461-472Abstract Full Text PDF PubMed Scopus (352) Google Scholar, 44Beals C.R. Clipstone N.A., Ho, S.N. Crabtree G.R. Genes Dev. 1997; 11: 824-834Crossref PubMed Scopus (341) Google Scholar), as well as regulatory domains that are the targets for dephosphorylation by Cn (44Beals C.R. Clipstone N.A., Ho, S.N. Crabtree G.R. Genes Dev. 1997; 11: 824-834Crossref PubMed Scopus (341) Google Scholar, 45Aramburu J. Garcia-Cózar 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, 46Shibasaki F. Price E.R. Milan D. McKeon F. Nature. 1996; 382: 370-373Crossref PubMed Scopus (430) Google Scholar). NFAT1-dependent transactivation has been shown to be controlled by two different transactivation domains (TAD), at the N- and C-terminal ends of the protein (32Rao A. Luo C. Hogan P.G. Ann Rev Immunol. 1997; 15: 707-747Crossref PubMed Scopus (2200) Google Scholar, 47Luo C. Burgeon E. Rao A. J. Exp. Med. 1996; 184: 141-147Crossref PubMed Scopus (86) Google Scholar). The N-terminal TAD (amino acids 1–100) of NFAT1, the stronger of both domains, is rich in acidic residues and prolines, characteristic of acidic transactivation domains. This region is able to recruit the p300/CBP co-activator and is regulated by the adjacent regulatory domain, a serine- and proline-rich region with sequence motifs conserved within the NFAT family (48Avots A. Buttmann M. Chuvpilo S. Escher C. Smola U. Bannister A.J. Rapp U.R. Kouzarides T. Serfling E. Immunity. 1999; 10: 515-524Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 49Garcı́a-Rodrı́guez C. Rao A. J. Exp. Med. 1998; 187: 2031-2036Crossref PubMed Scopus (164) Google Scholar). Recent findings suggest that signals mediated by PMA plus ionomycin induced phosphorylation of the N-terminal TAD of NFAT1, strongly enhancing the transcriptional activity of this factor (50Garcı́a-Rodriguez C. Rao A. Eur. J. Immunol. 2000; 30: 2432-2436Crossref PubMed Scopus (18) Google Scholar).Here, we show that PKCζ modulates NFAT-mediated signaling through potentiation of its transactivating function in T cells. Even more, physical interaction between PKCζ and NFAT suggest direct regulation of this factor by PKCζ. We propose that phosphorylation of the N-terminal transactivation domain of NFAT by PKCζ might mediate modulation of gene expression.DISCUSSIONIt is well established that atypical PKCs play an important role in transcription factor regulation such as NF-κB and AP-1 activation (13Dı́az-Meco M.T. Dominguez I. Sanz L. Dent P. Lozano J. Municio M.M. Berra E. Hay R.T. Sturgill T.W. Moscat J. EMBO J. 1994; 13: 2842-2848Crossref PubMed Scopus (219) Google Scholar, 14Dominguez I. Sanz L. Arenzana-Seisdedos F. Diaz-Meco M.T. Virelizier J.L. Moscat J. Mol. Cell. Biol. 1993; 13: 1290-1295Crossref PubMed Google Scholar, 64Bonizzi G. Piette J. Schoonbroodt S. Merville M.P. Bours V. Biochem. Pharmacol. 1999; 57: 713-720Crossref PubMed Scopus (36) Google Scholar, 65Huang C., Li, J. Chen N., Ma, W. Bowden G.T. Dong Z. Mol Carcinog. 2000; 27: 65-75Crossref PubMed Scopus (43) Google Scholar). We and others have shown that overexpression of PKCζ is sufficient to activate κB-dependent transcription in synergism with tumor necrosis factor-α (24Martin A.G. San-Antonio B. Fresno M. J. Biol. Chem. 2001; 276: 15840-15849Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 66Lallena M.J. Diaz-Meco M.T. Bren G. Paya C.V. Moscat J. Mol. Cell. Biol. 1999; 19: 2180-2188Crossref PubMed Google Scholar). However, contradictory data about the involvement of PKCζ in NFAT and NF-κB activation have been documented. Whereas some groups have described different roles of PKCζ in NF-κB signaling (6Lozano J. Berra E. Municio M.M. Diaz-Meco M.T. Dominguez I. Sanz L. Moscat J. J. Biol. Chem. 1994; 269: 19200-19202Abstract Full Text PDF PubMed Google Scholar, 13Dı́az-Meco M.T. Dominguez I. Sanz L. Dent P. Lozano J. Municio M.M. Berra E. Hay R.T. Sturgill T.W. Moscat J. EMBO J. 1994; 13: 2842-2848Crossref PubMed Scopus (219) Google Scholar, 14Dominguez I. Sanz L. Arenzana-Seisdedos F. Diaz-Meco M.T. Virelizier J.L. Moscat J. Mol. Cell. Biol. 1993; 13: 1290-1295Crossref PubMed Google Scholar), other authors discard a role of this kinase in NF-κB and NFAT transactivation (67Genot E.M. Parker P.J. Cantrell D.A. J. Biol. Chem. 1995; 270: 9833-9839Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar). Here we describe for the first time the regulation of NFAT activity by PKCζ in T lymphocytes through a novel mechanism. The mechanisms described so far by which some kinases and phosphatases regulate NFAT activation imply modulation of nuclear translocation of this factor or its binding to DNA. Thus, it is well established that nuclear import of NFAT factors requires dephosphorylation by the phosphatase Cn (62Clipstone N.A. Crabtree G.R. Nature. 1992; 357: 695-697Crossref PubMed Scopus (1462) Google Scholar, 68Jain J. McCaffrey P.G. Miner Z. Kerppola T.K. Lambert J.N. Verdine G.L. Curran T. Rao A. Nature. 1993; 365: 352-355Crossref PubMed Scopus (673) Google Scholar, 69Loh C. Shaw K.T. Carew J. Viola J.P. Luo C. Perrino B.A. Rao A. J. Biol. Chem. 1996; 271: 10884-10891Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar). On the other hand, increasing evidence supports the contribution of several kinases to nuclear shuttling of this factor, thus promoting opposed effects to those driven by Cn signaling. Thus, casein kinase Iα and JNK have been shown to phosphorylate NFAT4, resulting in inhibition of its nuclear translocation (70Zhu 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, 71Chow C.W. Rincon M. Cavanagh J. Dickens M. Davis R.J. Science. 1997; 278: 1638-1641Crossref PubMed Scopus (300) Google Scholar). JNK is also able to phosphorylate NAFT1 or NFAT2 isoforms likely favoring nuclear shuttling of these factors (55Martı́nez-Martı́nez S. Gómez del Arco P. Armesilla A.L. Aramburu J. Luo C. Rao A. Redondo J.M. Mol. Cell. Biol. 1997; 17: 6437-6447Crossref PubMed Scopus (59) Google Scholar, 63Chow C.W. Dong C. Flavell R.A. Davis R.J. Mol. Cell. Biol. 2000; 20: 5227-5234Crossref PubMed Scopus (119) Google Scholar). In a similar way it has been shown that glycogen synthase kinase-3 enhanced nuclear export of NFAT2, thus inhibiting NFAT activation (72Beals C.R. Sheridan C.M. Turck C.W. Gardner P. Crabtree G.R. Science. 1997; 275: 1930-1934Crossref PubMed Scopus (633) Google Scholar). In contrast, induction of NFAT signaling by kinases as extracellular signal-regulated kinase 1 seems to be mediated by enhancing its DNA binding activity (73Park J.H. Levitt L. Blood. 1993; 82: 2470-2477Crossref PubMed Google Scholar). Nevertheless, our results show that modulation of NFAT activity by PKCζ does not involve either the shuttling to the nucleus or DNA binding of this factor to its recognition sequences. In contrast, our data show that PKCζ modulates NFAT-mediated transcription by increasing the activity of its N-terminal transactivation domain.NFAT1-dependent transactivation seems to map in two different domains of this protein, located at the N and the C termini (47Luo C. Burgeon E. Rao A. J. Exp. Med. 1996; 184: 141-147Crossref PubMed Scopus (86) Google Scholar). We have established that PKCζ strongly potentiates transactivation mediated by amino acids 1–415 of the N terminus of NFAT1. This region contains the N-terminal TAD-A of NFAT1 that has been defined as the stronger transactivation domain (47Luo C. Burgeon E. Rao A. J. Exp. Med. 1996; 184: 141-147Crossref PubMed Scopus (86) Google Scholar, 61Okamura 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 (381) Google Scholar). In agreement with previous reports showing that this region still requires the inputs from Ca2+ and phorbol ester-mediated signaling pathways for optimal function, we have shown that treatment with PMA + Io increase its activity (50Garcı́a-Rodriguez C. Rao A. Eur. J. Immunol. 2000; 30: 2432-2436Crossref PubMed Scopus (18) Google Scholar). It is important to emphasize that PKCζ cooperates with Ca2+-mediated signaling in the induction of NFAT-dependent transactivation. Thus, transfection of a constitutively active Cn mimicking Ca2+ requirement cooperated with PKCζ to further augment transactivation mediated by the N-terminal TAD of NFAT1. Cn is required not only for the nuclear import of NFAT proteins but also to regulate the intrinsic transcriptional activity of the TAD-A of NFAT1 (50Garcı́a-Rodriguez C. Rao A. Eur. J. Immunol. 2000; 30: 2432-2436Crossref PubMed Scopus (18) Google Scholar). Cooperation between Cn and PKCs has been well established for other PKC isoforms in the regulation of NFAT signaling (25Werlen G. Jacinto E. Xia Y. Karin M. EMBO J. 1998; 17: 3101-3111Crossref PubMed Scopus (252) Google Scholar, 32Rao A. Luo C. Hogan P.G. Ann Rev Immunol. 1997; 15: 707-747Crossref PubMed Scopus (2200) Google Scholar, 62Clipstone N.A. Crabtree G.R. Nature. 1992; 357: 695-697Crossref PubMed Scopus (1462) Google Scholar). The fact that PKCζ cooperates with Ca2+ signaling and did not affect nuclear translocation together with the lack of effect of CsA on the induction of NFAT by PKCζ support the hypothesis that this kinase does not seem to be required for the initiation of NFAT signaling but rather for the potentiation of their transactivation function in conjunction with signals converging on NFAT activation. Moreover, the increase in the transactivating function of NFAT induced by PKCζ seems to be unrelated to that induced by PMA + Io/Cn signaling because PKCζ and PMA + Io/Cn-mediated signals were always additive. Moreover, in Jurkat cells stably expressing the dominant-negative mutant of PKCζ (J-PKCζmut), there was no inhibition of PMA + Io or Cn-induced NFAT activation. More importantly, PKCζ but not PMA + Io was able to activate the GAL4 NFAT1 (1–104) construct. Taken together, it is likely that PKCζ activates NFAT in a Ca2+/Cn-independent pathway.Acidic transactivation domains are usually regulated by phosphorylation (74Hahn S. Cell. 1993; 72: 481-483Abstract Full Text PDF PubMed Scopus (120) Google Scholar, 75Martı́n A.G. Fresno M. J. Biol. Chem. 2000; 275: 24383-24391Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). A recent report describes that phosphorylation of a novel site in the TAD-A of NFAT1 upon PMA + Io stimulation is important for transcriptional activity (61Okamura 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 (381) Google Scholar). We report here that PKCζ is able to physically interact with both NFAT1 and NFAT2. This interaction was independent of the kinase activity of PKCζ, because it also took place with a kinase-dead mutant. Interestingly, we have been able to identify PKCζ as a kinase able to phosphorylate the N-terminal region (amino acids 1–385) of NFAT1. Although our experiments suggest that phosphorylation by PKCζ might contribute to the activation of the transactivating activity of NFAT1, with residues 1–104 in the TAD-A responsible for this activation, additional experiments need to be performed to address the importance of NFAT-1 phosphorylation by PKCζ and to determine the exact residues involved. Transactivation activity of transcription factors such as NFAT or NF-κB is modulated by interaction with basal transcription machinery or with coactivators such as CBP/p300 (65Huang C., Li, J. Chen N., Ma, W. Bowden G.T. Dong Z. Mol Carcinog. 2000; 27: 65-75Crossref PubMed Scopus (43) Google Scholar, 49Garcı́a-Rodrı́guez C. Rao A. J. Exp. Med. 1998; 187: 2031-2036Crossref PubMed Scopus (164) Google Scholar, 48Avots A. Buttmann M. Chuvpilo S. Escher C. Smola U. Bannister A.J. Rapp U.R. Kouzarides T. Serfling E. Immunity. 1999; 10: 515-524Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). Thus, PKCζ actions regulating NFAT-dependent transactivation could be achieved by modulation interaction of NFAT and coactivator factors such as CBP/p300. However, preliminary results trying to address this hypothesis have failed to establish any effect of CBP/p300 on PKCζ actions (data not shown).This effect of PKCζ on NFAT signaling is similar to that we have described on c-Rel, a transcription factor of the NF-κB family (24Martin A.G. San-Antonio B. Fresno M. J. Biol. Chem. 2001; 276: 15840-15849Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). There is increasing evidence that PKCζ can play a dual role in the activation of NF-κB through the activation of the IκB kinases (66Lallena M.J. Diaz-Meco M.T. Bren G. Paya C.V. Moscat J. Mol. Cell. Biol. 1999; 19: 2180-2188Crossref PubMed Google Scholar) and also by regulating the transactivating function of several members of the Rel family of proteins, through the phosphorylation of the DNA-binding subunits (RHD) or the transactivation domains (reviewed in Ref. 76Schmitz M.L. Bacher S. Kracht M. Trends Biochem. Sci. 2001; 26: 186-190Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar). Thus, in endothelial cells, phosphorylation of p65 RHD by PKCζ- and Ras-derived signals contributes to p65 transcriptional activity (23Anrather J. Csizmadia V. Soares M.P. Winkler H. J. Biol. Chem. 1999; 274: 13594-13603Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar). In addition, we have shown in a previous work that PKCζ is a critical downstream signaling molecule in tumor necrosis factor-α-dependent activation of c-Rel transactivating activity, and we have identified the target sites for PKCζ in the TAD of c-Rel (24Martin A.G. San-Antonio B. Fresno M. J. Biol. Chem. 2001; 276: 15840-15849Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar).In the past, much work has focused on the identification of pathways regulating dephosphorylation/nuclear translocation and rephosphorylation/nuclear export of NFAT proteins, as well as the identification of kinases and phosphatases involved in these processes. However, there is increasing evidence supporting a role for serine/threonine kinases in the regulation of the intrinsic NFAT transactivation function in T cells. Thus, we have described that Cot/Tpl2 kinase, a mitogen-activated protein kinase kinase kinase, increases transcription mediated by NFAT through enhancement of the transactivation function of the NFAT1 N-terminal TAD (54de Gregorio R. Iniguez M.A. Fresno M. Alemany S. J. Biol. Chem. 2001; 276: 27003-27009Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). The kinase activity of the CaM-dependent kinase IV has been also demonstrated to be required for potentiation of transactivation mediated by this domain (50Garcı́a-Rodriguez C. Rao A. Eur. J. Immunol. 2000; 30: 2432-2436Crossref PubMed Scopus (18) Google Scholar). In a very recent report, it has been shown that Pim-1 is able to interact with NFAT and phosphorylate it in several serine residues enhancing NFAT-dependent transactivation in Jurkat cells (77Rainio E.M. Sandholm J. Koskinen P.J. J. Immunol. 2002; 168: 1524-1527Crossref PubMed Scopus (107) Google Scholar). Together with the results presented here, these data support a role of serine/threonine kinases in the regulation of NFAT-dependent gene expression in T cells through the potentiation of the activity of its transactivation domain. However, future work is needed to address the exact mechanism by which these kinases are achieving these effects on NFAT. The serine/threonine protein kinase C (PKC)1 family is constituted by several isoenzymes that have been grouped into three subfamilies according to their activation profile and their regulatory properties (1Newton A.C. J. Biol. Chem. 1995; 270: 28495-28498Abstract Full Text Full Text PDF PubMed Scopus (1466) Google Scholar, 2Newton A.C. Curr Opin Cell Biol. 1997; 9: 161-167Crossref PubMed Scopus (845) Google Scholar). The named atypical subclass of the PKC family comprises two members, PKCζ and PKCλ/ι, which are unresponsive to calcium or phorbol esters. Both PKCζ and PKCλ/ι are more than 70% homologous in primary structure and so far have been implicated in similar activities (3Dı́az-Meco M.T. Municio M.M. Frutos S. Sánchez P. Lozano J. Sanz L
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