Calcium, Calcineurin, and the Control of Transcription

interleukin capacitance-regulated activation channels protein kinase C c-Jun NH2-terminal kinase mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase Calcineurin (PP2B), a serine/threonine phosphatase controlled by cellular calcium, was originally identified in extracts of mammalian brain. Within the past few years this phosphatase has been implicated in a wide variety of biological responses including lymphocyte activation, neuronal and muscle development, neurite outgrowth, and morphogenesis of vertebrate heart valves. Progress in this area has been greatly accelerated by the drugs cyclosporin A and FK506, which use a unique mechanism of action to achieve near genome-specific nanomolar inhibition of calcineurin. This minireview will focus on the role of calcineurin in regulating transcription during development, primarily through its dephosphorylation of downstream targets such as the calcineurin-dependent, cytoplasmic subunits of the NF-AT transcription complex.Calcineurin and Regulation of Nuclear Import of NF-ATc Family MembersThe Ca2+, calcineurin/NF-AT signaling pathway was defined about 10 years ago (1Shaw J.-P. Utz P.J. Durand D.B. Toole J.J. Emmel E.A. Crabtree G.R. Science. 1988; 241: 202-205Crossref PubMed Scopus (10) Google Scholar, 2Flanagan W.M. Corthesy B. Bram R.J. Crabtree G.R. Nature. 1991; 352: 803-807Crossref PubMed Scopus (946) Google Scholar, 3Liu J. Farmer Jr., J.D. Lane W.S. Friedman J. Weissman I. Schreiber S.L. Cell. 1991; 66: 807-815Abstract Full Text PDF PubMed Scopus (3567) Google Scholar, 4Clipstone N.A. Crabtree G.R. Nature. 1992; 357: 695-697Crossref PubMed Scopus (1463) Google Scholar) and was one of the first signaling pathways to bridge the cell membrane with the nucleus. Although the pathway is simple (Fig. 1), multiple levels of regulation impinge upon it, making it adaptable for many functions in a wide variety of cell types. The pathway was defined by a strategy of working backward from the nucleus to the cell membrane Ca2+ channels in T lymphocytes. The regulatory regions of the IL-21 gene were identified and found to bind a cyclosporin-sensitive transcription complex called NF-AT1 (1Shaw J.-P. Utz P.J. Durand D.B. Toole J.J. Emmel E.A. Crabtree G.R. Science. 1988; 241: 202-205Crossref PubMed Scopus (10) Google Scholar, 5Siebenlist U. Durand D.B. Bressler P. Holbrook N.J. Norris C.A. Kamoun M. Kant J.A. Crabtree G.R. Mol. Cell. Biol. 1986; 6: 3042-3049Crossref PubMed Scopus (121) Google Scholar, 6Durand D.B. Bush M.R. Morgan J.G. Weiss A. Crabtree G.R. J. Exp. 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Nature. 1992; 357: 695-697Crossref PubMed Scopus (1463) Google Scholar) established that calcineurin was required for the nuclear import of NF-ATc (8Schreiber S.L. Crabtree G.R. Immunol. Today. 1992; 13: 136-142Abstract Full Text PDF PubMed Scopus (1947) Google Scholar). Finally, a screen for somatic cell mutations that prevented NF-AT transcriptional activation yielded many mutations that abolished the activity of the capacitance-regulated activation channels (CRAC) and hence identified it as the source of Ca2+ that regulated NF-AT import (9Serafini A.T. Lewis R.S. Clipstone N.A. Bram R.J. Fanger C. Fiering S. Herzenberg L.A. Crabtree G.R. Immunity. 1995; 3: 239-250Abstract Full Text PDF PubMed Scopus (58) Google Scholar, 10Fanger C.M. Hoth M. Crabtree G.R. Lewis R.S. J. Cell Biol. 1995; 131: 655-667Crossref PubMed Scopus (164) Google Scholar, 11Timmerman L.A. Clipstone N.A. Ho S.N. Northrop J.P. Crabtree G.R. Nature. 1996; 383: 837-840Crossref PubMed Scopus (472) Google Scholar).Calcineurin functions in this pathway by directly dephosphorylating the cytoplasmic subunits of the NF-AT1 transcription complex (12Jain 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, 13Beals C.R. Clipstone N.A. Ho S.N. Crabtree G.R. Genes Dev. 1997; 11: 824-834Crossref PubMed Scopus (341) Google Scholar, 14Loh 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), which are encoded by four genes (NF-ATc1–4) (15Crabtree G.R. Cell. 1999; 96: 611-614Abstract Full Text Full Text PDF PubMed Scopus (661) Google Scholar) and which undergo cytoplasmic to nuclear translocation as originally described (2Flanagan W.M. Corthesy B. Bram R.J. Crabtree G.R. Nature. 1991; 352: 803-807Crossref PubMed Scopus (946) Google Scholar). Calcineurin binds directly to NF-ATc family members through a conserved motif in the N terminus of the protein, which functions as an effective dominant negative (16Wesselborg S. Fruman D.A. Sagoo J.K. Bierer B.E. Burakoff S.J. J. Biol. Chem. 1996; 271: 1274-1277Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 17Northrop J.P. Ho S.N. Chen L. Thomas D.J. Timmerman L.A. Nolan G.P. Admon A. Crabtree G.R. Nature. 1994; 369: 497-502Crossref PubMed Scopus (522) Google Scholar, 18Aramburu 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). This unconventional direct interaction between the phosphatase and its substrate outside of the enzymatic site results in a surprisingly specific relationship between the phosphatase and its substrate. Calcineurin dephosphorylates serines within the SP repeats (SP1 to SP3) and the serine-rich region of NF-ATc family members (13Beals C.R. Clipstone N.A. Ho S.N. Crabtree G.R. Genes Dev. 1997; 11: 824-834Crossref PubMed Scopus (341) Google Scholar, 19Beals C.R. Sheridan C.M. Turck C.W. Gardner P. Crabtree G.R. Science. 1997; 275: 1930-1934Crossref PubMed Scopus (633) Google Scholar). When phosphorylated, these residues appear to obscure the two nuclear localization sequences required for nuclear import, perhaps by forming salt bridges between the basic nuclear localization sequence and the acidic phosphoserines of NF-ATc family members (13Beals C.R. Clipstone N.A. Ho S.N. Crabtree G.R. Genes Dev. 1997; 11: 824-834Crossref PubMed Scopus (341) Google Scholar, 20Zhu 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).Once dephosphorylated by calcineurin, NF-ATc family members move into the nucleus where they are maintained by persistent elevation of intracellular Ca2+ and the continuous activity of calcineurin (11Timmerman L.A. Clipstone N.A. Ho S.N. Northrop J.P. Crabtree G.R. Nature. 1996; 383: 837-840Crossref PubMed Scopus (472) Google Scholar). Persistent activation of calcineurin is required because NFATc proteins are rapidly exported from the nucleus. Export of NF-ATc1 and -c4 was found to be caused by the phosphorylation of the same residues that are dephosphorylated by calcineurin to bring about nuclear import. Biochemical purification of the nuclear kinase that phosphorylated these residues identified GSK3 as a potential export kinase (19Beals C.R. Sheridan C.M. Turck C.W. Gardner P. Crabtree G.R. Science. 1997; 275: 1930-1934Crossref PubMed Scopus (633) Google Scholar). The persistent activation of calcineurin necessary to maintain NF-ATc family members in the nucleus requires the CRAC channel, which is known to provide a sustained high level of Ca2+ (21Cahalan M.D. Lewis R.S. Semin. Immunol. 1990; 2: 107-117PubMed Google Scholar). Indeed overexpression of constitutively active calcineurin or a NF-ATc1 mutant that is constitutively nuclear suppresses mutations in the CRAC channel allowing the activation of immune response genes such as IL-2 with only a PKC signal (11Timmerman L.A. Clipstone N.A. Ho S.N. Northrop J.P. Crabtree G.R. Nature. 1996; 383: 837-840Crossref PubMed Scopus (472) Google Scholar).NFAT Complexes as Coincidence Detectors and Integrators of Ras and Ca2+ SignalingEarly studies indicated that NF-AT integrated Ca2+signals with signals transduced by mitogen-activated protein kinase and/or protein kinase C (1Shaw J.-P. Utz P.J. Durand D.B. Toole J.J. Emmel E.A. Crabtree G.R. Science. 1988; 241: 202-205Crossref PubMed Scopus (10) Google Scholar, 22Crabtree G.R. Science. 1989; 243: 355-361Crossref PubMed Scopus (907) Google Scholar, 23Woodrow M. Clipstone N.A. Cantrell D. J. Exp. Med. 1993; 178: 1517-1522Crossref PubMed Scopus (122) Google Scholar). Thus, the NF-AT1 transcription complex acts as a coincidence detector in that it requires that both Ras/PKC and Ca2+/calcineurin signaling be coincident to achieve activation (Fig. 2). In fact, DNA binding by NF-ATc proteins is quite weak because of the unusual structural features of its Rel-like DNA binding domain (24Wolfe S.A. Zhou P. Dotsch V. Chen L. You A. Ho S.N. Crabtree G.R. Wagner G. Verdine G.L. Nature. 1997; 385: 172-176Crossref PubMed Scopus (90) Google Scholar), and as a consequence the protein requires a partner for tight association with DNA. Thus, Ca2+ signaling becomes dependent on coincident Ras/PKC signaling and vice versa. In nearly all cell types studied, activation of Rac, Ras, or PKC must accompany a Ca2+ signal for activation of NF-AT-dependent transcription (Fig.2).Figure 2Coincidence detection and signal integration by the NF-AT1 transcriptional complex. Ras or Rac or PKC signaling must be coincident with Ca2+/calcineurin signaling to assemble the NF-AT1 transcription complex and to activate downstream genes. Two receptors are shown that independently activate Ras/Rac/PKC signaling and Ca2+ signaling. However, the T cell receptor as well as a number of other receptors activate both pathways. In some situations, NF-ATn may be a tissue-specific component of the NF-AT complex.View Large Image Figure ViewerDownload Hi-res image Download (PPT)In addition to the effects of GSK3 on NF-ATc1, the movement of NF-ATc3 into the nucleus appears to be opposed by the actions of either JNK kinase (25Chow C.W. Rincon M. Cavanagh J. Dickens M. Davis R.J. Science. 1997; 278: 1638-1641Crossref PubMed Scopus (300) Google Scholar) or perhaps the combination of MEKK1 and CK1 (20Zhu 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). Although the evidence is conflicting, these two kinases have both been reported to provide opposition to the cytoplasmic dephosphorylation by calcineurin in cell lines. However, because the transcriptional activity of NF-AT complexes is enhanced rather than blocked by agents that activate MEKK1 and JNK (1Shaw J.-P. Utz P.J. Durand D.B. Toole J.J. Emmel E.A. Crabtree G.R. Science. 1988; 241: 202-205Crossref PubMed Scopus (10) Google Scholar, 23Woodrow M. Clipstone N.A. Cantrell D. J. Exp. Med. 1993; 178: 1517-1522Crossref PubMed Scopus (122) Google Scholar, 26Holsinger L.J. Graef I.A. Swat W. Chi T. Bautista D.M. Davidson L. Lewis R.S. Alt F.W. Crabtree G.R. Curr. Biol. 1998; 8: 563-572Abstract Full Text Full Text PDF PubMed Google Scholar, 27Rooney J.W. Hoey T. Glimcher L.H. Immunity. 1995; 2: 473-483Abstract Full Text PDF PubMed Scopus (237) Google Scholar, 28Jacinto E. Werlen G. Karin M. Immunity. 1998; 8: 31-41Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar) the physiological roles of these kinases remain to be understood.Signaling through Ca2+, Calcineurin, and NF-ATc Family Members in NeuronsCalcineurin has been shown to have important roles in axonal guidance (29Chang H.Y. Takei K. Sydor A.M. Born T. Rusnak F. Jay D.G. Nature. 1995; 376: 686-690Crossref PubMed Scopus (148) Google Scholar) as well as memory and learning (30Winder D.G. Mansuy I.M. Osman M. Moallem T.M. Kandel E.R. Cell. 1998; 92: 25-37Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar, 31Mansuy I.M. Mayford M. Jacob B. Kandel E.R. Bach M.E. Cell. 1998; 92: 39-49Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar). In general, these functions in neurons have been thought to be independent of transcription. However, recent evidence indicates that the NF-ATc family members may have critical roles in the development of synaptic connections. NF-ATc4 is expressed in hippocampal neurons and undergoes translocation to the nucleus with brief depolarization or even with physiological 5-Hz stimulation (32Graef 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). The latter is significant in that it implies that normal synaptic activity is sufficient to send NF-ATc4 into the nucleus, i.e. when you think about it NF-ATc4 enters the nucleus. A potential downstream gene appears to beIP3R1, which encodes a Ca2+ channel and is activated shortly after birth in response to the neural activity of the newborn animal (33Genazzani A.A. Carafoli E. Guerini D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5797-5801Crossref PubMed Scopus (154) Google Scholar). The promoter region of this gene has a cluster of NF-ATc sites and appears to be dependent on the activity of the NF-ATc4 protein, which is expressed in neurons (32Graef 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). In addition, the activation of this gene after birth or in cultured hippocampal neurons is blocked by FK506 or cyclosporin A (33Genazzani A.A. Carafoli E. Guerini D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5797-5801Crossref PubMed Scopus (154) Google Scholar). These studies suggest that signaling by Ca2+, calcineurin, and NF-ATc4 regulates a positive feedback pathway that could reinforce synaptic connections (Fig. 1). However, additional studies with null mutations of NF-ATc4 and possibly other redundant family members will be necessary to confirm this hypothesis.The Morphogenesis of Heart Valves and the Calcium, Calcineurin, NFAT PathwayNull mutations in the NF-ATc1 gene result in a failure of heart valve development and abnormalities of the cardiac septum that recall the common cardiac abnormalities that occur in nearly 1% of all live births (34Ranger A.M. Grusby M.J. Hodge M.R. Gravallese E.M. de la Brousse F.C. Hoey T. Mickanin C. Baldwin H.S. Glimcher L.H. Nature. 1998; 392: 186-190Crossref PubMed Scopus (508) Google Scholar, 35de la Pompa J.L. Timmerman L.A. Takimoto H. Yoshida H. Elia A.J. Samper E. Potter J. Wakeham A. Marengere L. Langille B.L. Crabtree G.R. Mak T.W. Nature. 1998; 392: 182-186Crossref PubMed Scopus (541) Google Scholar). Although calcineurin is expressed ubiquitously, at embryonic days 8.5–13, NF-ATc1 is expressed only in the cells that are destined to contribute to heart valves. Remarkably, the protein is nuclear in the endocardial cells that are adjacent to the cardiac jelly, suggesting that a local signal results in its nuclear localization (Fig. 1). Either cyclosporin or FK506 blocks this translocation and results in an arrest in valve development, indicating that an as yet uncharacterized signal (presumably a cytokine or growth factor) activates calcineurin leading to nuclear localization of NF-ATc1 and activation of genes that orchestrate the formation of a cardiac valve (Fig. 1). Much needs to be learned about this extremely important pathway including the nature of the signal, the downstream genes, and the role of this pathway in the cardiac valve abnormalities that affect nearly 50 million individuals worldwide.The Role of Signaling through Ca2+, Calcineurin, and NF-ATc4 in Myocardial HypertrophyThe heart responds to stress with the hypertrophy of cardiomyocytes. This response is probably normal during development but becomes damaging in hypertension or after multiple myocardial infarctions. The signals for the stress-induced hypertrophy of cardiac muscle have long been known to be dependent on calcium, and recently it was discovered that severe cardiac hypertrophy could be induced by the overexpression of a truncated constitutively active calcineurin A (36Molkentin J.D. Lu J.R. Antos C.L. Markham B. Richardson J. Robbins J. Grant S.R. Olson E.N. Cell. 1998; 93: 215-228Abstract Full Text Full Text PDF PubMed Scopus (2191) Google Scholar). In addition, a similar pathology could be induced in animals in which a truncated NF-ATc4 was overexpressed (36Molkentin J.D. Lu J.R. Antos C.L. Markham B. Richardson J. Robbins J. Grant S.R. Olson E.N. Cell. 1998; 93: 215-228Abstract Full Text Full Text PDF PubMed Scopus (2191) Google Scholar). Furthermore, cyclosporin A prevents the development of cardiac hypertrophy in response to certain (but apparently not all) stimuli (37Meguro T. Hong C. Asai K. Takagi G. McKinsey T.A. Olson E.N. Vatner S.F. Circ. Res. 1999; 84: 735-740Crossref PubMed Scopus (158) Google Scholar, 38Sussman M.A. Lim H.W. Gude N. Taigen T. Olson E.N. Robbins J. Colbert M.C. Gualberto A. Wieczorek D.F. Molkentin J.D. Science. 1998; 281: 1690-1693Crossref PubMed Scopus (400) Google Scholar). Additional studies with mice bearing mutations in calcineurin genes or the NF-ATc4 gene will be necessary to confirm these pharmacologic and overexpression studies, but the present results indicate that calcineurin and NF-ATc4 represent new targets for development of antihypertrophic agents.Calcineurin and Control of Transcription in YeastIn yeast, signals initiated by pheromone or other agents that increase intracellular Ca2+ lead to the activation of transcription of FKS2, PMR2, PMC1,PMR1, and other genes (39Cunningham K.W. Fink G.R. J. Cell Biol. 1994; 124: 351-363Crossref PubMed Scopus (360) Google Scholar, 40Stathopoulos A.M. Cyert M.S. Genes Dev. 1997; 11: 3432-3444Crossref PubMed Scopus (375) Google Scholar, 41Matheos D.P. Kingsbury T.J. Ahsan U.S. Cunningham K.W. Genes Dev. 1997; 11: 3445-3458Crossref PubMed Scopus (274) Google Scholar). This transcriptional response requires the Crz1/Tcn1 gene and plays an important role in controlling ion homeostasis (40Stathopoulos A.M. Cyert M.S. Genes Dev. 1997; 11: 3432-3444Crossref PubMed Scopus (375) Google Scholar). The mechanism of control ofCrz1p/Tcn1p is remarkably similar to the control of NF-ATc proteins in mammalian cells (Fig. 3). Like NF-ATc proteins that translocate from cytoplasm to nucleus upon activation of calcineurin, Crz1p/Tcn1p also translocates and furthermore is dephosphorylated at critical serines similar to NF-ATc family members (42Stathopoulos-Gerontides A. Guo J.J. Cyert M.S. Genes Dev. 1999; 13: 798-803Crossref PubMed Scopus (193) Google Scholar). Despite these mechanistic similarities, there is relatively little sequence similarity in the two proteins other than a conserved group of serines in the N-terminal translocation domain, and whereas Crz1/Tcn1 use a zinc finger for DNA binding the NFATc family uses a Rel domain.Figure 3Calcineurin regulation of transcription in yeast and mammalian cells. CHP, calcineurin homologous protein.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Calcineurin InhibitorsIn recent years, five different classes of calcineurin inhibitors have been discovered (Table I) raising questions regarding their various roles in Ca2+/calcineurin signaling. Each of these inhibits calcineurin by binding to the protein and inhibiting its ability to dephosphorylate substrates, such as NFATc family members, thereby preventing their nuclear localization. Perhaps the most interesting of these is the DSCR1 gene and its relatives, DSCR2 and ZAK14 (43Fuentes J.J. Genesca L. Kingsbury T.J. Cunningham K.W. Perez-Riba M. Estivill X. Luna S. Hum. Mol. Genet. 2000; 9: 1681-1690Crossref PubMed Google Scholar). DSRC1is located on chromosome 21 in the so-called critical region (hence its name, Down's Syndrome critical region 1 gene). One possibility is that overexpression of DSCR1 as a result of trisomy leads to inhibition of calcineurin and subsequent effects on the development of the brain, immune system, heart, and skeleton. However, the Down's Syndrome critical region includes a number of genes, and it is possible that the syndrome could arise from overexpression of several of these. Two pieces of evidence indicate that overexpression of DSCR1might underlie the pathogenesis of Down's and (43Fuentes J.J. Genesca L. Kingsbury T.J. Cunningham K.W. Perez-Riba M. Estivill X. Luna S. Hum. Mol. Genet. 2000; 9: 1681-1690Crossref PubMed Google Scholar) have shown that DSCR1 protein is overexpressed in the of Down's Syndrome some of the of Down's Syndrome appear in mice with mutations of the different subunits of the transcription complex. yeast have been shown to have a protein, that binds and calcineurin T.J. Cunningham K.W. Genes Dev. 2000; Google Scholar, J. J.R. J. 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Genet. 1995; PubMed Scopus Google nuclear translocation of J.J. Genesca L. Kingsbury T.J. Cunningham K.W. Perez-Riba M. Estivill X. Luna S. Hum. Mol. Genet. 2000; 9: 1681-1690Crossref PubMed Google Scholar, T. S. T. K. H. H. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google to are and for was made with a from and for the protein be T.J. Cunningham K.W. Genes Dev. 2000; Google Scholar, J. J.R. J. J. 2000; PubMed Scopus (148) Google NF-ATc L.C. Science. 1998; 281: PubMed Scopus Google not This was made with a from and for the protein be in a new Two additional interesting classes of calcineurin inhibitors are L. C. M. W. Immunity. 1998; 8: Full Text Full Text PDF PubMed Scopus Google Scholar, H. S. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar), which are and the protein that has similarity to calcineurin X. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: PubMed Scopus Google Scholar, X. Rusnak F. J. Biol. 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