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The role of calcineurin in lymphocyte activation
seminars in IMMUNOLOGY, Vol. 12, 2000: pp. 405–415 doi: 10.1006/smim.2000.0221, available online at http://www.idealibrary.com on
The role of calcineurin in lymphocyte activation Shairaz Baksh a and Steven J. Burakoff a,b
vation of calcium responsive proteins. One of the best characterized lymphokine gene transcription pathways is the interleukin (IL)-2 activation pathway in T lymphocytes. The activation of T lymphocytes results in calcium release from the endoplasmic reticulum and the activation of calcium responsive proteins, such as calmodulin. The activation of calmodulin, in turn, results in its binding to and activation of the serine/threonine phosphatase calcineurin (protein phosphatase 2B) and subsequent dephosphorylation of the nuclear transcription factor of activated T cells, NFAT.2 The dephosphorylation of NFAT is required for its translocation to the nucleus (in a complex with calcineurin)3 and heterodimerization with AP-1 family members on NFAT/AP-1 sites in the IL-2 promoter.4 As calcium levels subside, calcineurin loses its phosphatase activity, and NFAT relocalizes to the cytosol.5 Calcineurin/NFAT signaling pathways are utilized to regulate the genes encoding a wide variety of cytokines (IL-2,IL-4, IFN-γ , GMCSF, IL-3, IL-13, TNF-α)2 and cell surface receptors (CD95L, CD40L, CD69).6 NFAT and its role as a key transcriptional activator controlled by calcineurin is well characterized; however, a wide variety of additional biological events that involve calcineurin have recently been discovered, reflecting the diverse role that calcineurin plays in specialized tissues.
Lymphokine gene transcription involves numerous signal transduction molecules and second messengers. The serine/threonine phosphatase calcineurin has been demonstrated to play a central role in the immediate, early activation of numerous lymphokines (such as interleukin [IL]-2) and in the regulation of cell surface receptors such as CD40L, CD95, and recently CD25α (the α chain of the IL-2 receptor). In addition to lymphocyte activation, calcineurin functions include control of neuronal signaling, muscle contraction, muscle hypertrophy and cellular death. Therefore, calcineurin not only plays a vital role in the regulation of T lymphocyte function, but also functions in cellular environments outside the immune system. Key words: calcineurin / T lymphocyte / NFAT / apoptosis / immunophilins c 2000 Academic Press
Lymphokine gene transcription involves a complex array of signal transduction molecules and events that ultimately lead to the activation of nuclear transcription. Central to the activation of lymphokine gene transcription is the elevation of intracellular calcium levels and subsequent activation of calcium responsive proteins.1 Second messengers, such as diacylglycerol (DAG) and inositol-1,4,5-trisphosphate (IP3 ), play important roles in the signaling pathways originating from surface receptor engagement and co-crosslinking. DAG is important in the activation of protein kinase C (PKC) pathways leading to the ras/raf/mitogen-activated kinase (MAPK) cascade. IP3 is an important element controlling the release of calcium from the internal stores of the endoplasmic reticulum into the cytoplasm and the subsequent acti-
Calcineurin: cellular distribution and structure Calcineurin is a heterodimeric phosphatase formed by the association of a 60 kDa phosphatase catalytic subunit (calcineurin A) and a 19 kDa high-affinity calcium binding subunit (calcineurin B). The phosphatase catalytic domain of calcineurin is structurally similar to the catalytic domain of protein phosphatase 1 with the exception of two short insertions towards the end of the catalytic domain that may govern calcineurin substrate specificity (Box 1 and 2, Figure 1). In mammals, calcineurin A exists as three isoforms sharing 75–80% primary sequence similarity. The
From the a Department of Pediatric Oncology, Dana Farber Cancer Institute and b Department of Pediatrics, Harvard Medical School, Boston, MA, 02115 USA. c
2000 Academic Press 1044–5323 / 00 / 000405+ 11 / $35.00/0 / 0
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to calcineurin is obtained by the composite binding of the fungal macrolides, FK506 and cyclosporin A (CsA), to their endogenous proteins, the immunophilins: the FK506 binding proteins (FKBPs) and the cyclosporin A binding proteins (the cyclophilins-CyPs).16 FK506, CsA, or the immunophilins by themselves do not inhibit the phosphatase activity of calcineurin. However, complexes of FK506/FKBP (K i of calcineurin inhibition, 7.9 nM) or CsA/CyP (K i for calcineurin inhibition, 270 nM) can inhibit the activity of calcineurin by 80–90%. In the crystal structure of the calcineurin A/ calcineurin B/FK506/FKBP12 co-complex, FK506/FKBP12 was found to make intimate contacts with both the A and B chains of calcineurin localized no closer than 10 Å from the catalytic active site.17 Inhibition of the phosphatase activity of calcineurin by the complex of FK506/FKBP12 was thus obtained by sterically blocking the access of substrates to the phosphatase catalytic site rather than by directly inhibiting the enzymatic activity of calcineurin.17 Small molecule substrates, such as para-nitrophenyl phosphate (pNPP), were observed to gain access to the phosphatase catalytic site even in the presence of an immunophilin complex. As the potential number of substrates for calcineurin increases, it will be interesting to determine if natural small protein substrates can be found that are allowed to enter into the catalytic site even in the presence of the immunophilin complex.
three isoforms have divergent carboxy terminal residues (Table 1) and distinct tissue distribution and chromosomal localizations: calcineurin Aα (on chromosome 4), calcineurin Aβ (on chromosome 10q21→q22) and calcineurin Aγ (whose localization has yet to be determined).7 High expression of Aβ is found in the thymus, while both Aα and Aβ are found within the peripheral immune system.8 Differences are also observed for the expression patterns of Aα and Aβ within specialized tissues, such as the rat nephron, where Aα expression was observed to be higher in the proximal tubules than in the medullary thick ascending limbs and the cortical collecting ducts.9 Within the central nervous system, calcineurin isoforms (mainly α and β) localize to the Purkinje cells of the cerebellum and the pyramidal cells of the cerebrum.10 Targeted disruption of the calcineurin Aα gene in mice resulted in normal maturation of T and B lymphocytes and normal responses to in vitro mitogenic stimulation, indicating that the β isoform of calcineurin may compensate for most of the functions of the α form in calcineurin Aα −/− T cells.11 However, calcineurin Aα −/− lymph node cells generated defective antigen-specific T cell responses following in vivo stimulation with an OVA peptide, implicating the α form of calcineurin as the predominant form controlling the physiological activation of T lymphocytes in the peripheral immune system. The third isoform of calcineurin, Aγ , is a testis-specific isoform found primarily within the nuclei of spermatids, and functions to control the remodeling of the nuclear chromatin in actively differentiating spermatids.12 Calcineurin B (localized to chromosome 2p16→p15) is the regulatory subunit of calcineurin and exists as two isoforms, CNB1 and CNB2, the latter specifically found in the testis.13 It has 35% sequence identity to calmodulin and contains an amino-terminal myristoylated glycine and four highaffinity EF-hand calcium binding sites. The binding of calcineurin B to calcineurin A allows the enzyme to achieve a two–three fold increase in activity.14 The binding of calcium-bound calmodulin to the calmodulin binding site within the carboxy terminus of calcineurin A results in an additional 10–20 fold increase in the phosphatase activity of the enzyme, which is facilitated by the displacement of a carboxy terminal autoinhibitory domain from the phosphatase active site. These events allow calcineurin substrates to gain access to the catalytic site and undergo dephosphorylation.15 Specific inhibition of the binding of substrates
Calcineurin in immune cells: positive and negative roles Calcineurin: a positive regulator of activation The central role of calcineurin in T cell activation primarily involves the regulation of NFAT. In addition to T cells, NFAT/calcineurin interactions are critical to the activation of other immune cells and to the signaling pathways of numerous other lymphokines. NFAT is a family of transcription factors that include five members mediating distinct biological outcomes in different cellular environments that are regulated by calcineurin.18 The activation of NFAT requires a sustained, low concentration of calcium that functions to maintain calcineurin in an active state and thus initiate events required for the activation of T lymphocytes. Studies have shown that calcium levels above 400 nM for 2 h are required for T lymphocytes to become committed to activation. However, the nuclear 406
Calcineurin in immune cells
Table 1. The mammalian isoforms of calcineurin A. Isoform type is listed in the first column. The next column indicates in which species the isoform has been identified and the name given to it is given in parentheses. Total amino acids are indicated with the isoelectric pH. Genebank accession numbers: human CnA-1: M29550; human CnA-2 (CnAβ): M29551; human CnAα: J05479; human testis: S46622; mouse CnA-1: J05479; mouse CnA-2: M81483; mouse testis: M81475; rat CnAα: M29275; rat Cnβ: M31809 MAMMALIAN CALCINEURIN ISOFORMS Isoform
Species
Divergent features (from CnAα)
α
human, mouse 2, rat α
β1
human (CnA-1)
β2
human (CnA-2), mouse 2, rat β
γ
human, mouse
—Unique amino terminus (MSEPKAIDPKLSTTD) —521 amino acids; pI = 5.6–5.8 —Proline rich amino terminal 25 amino acids MAAPEPAAPPPPPPPPPPPGADR –19 amino acid insertion within the catalytic domain 138 HVLGTEDISINPNNNNINEC —short carboxy terminal sequence after CaM binding domain –514 amino acids; pI = 5.43 —Proline rich amino terminal 25 amino acids MAAPEPARAAPPPPPPPPPPPGADR —524 amino acids; pI = 5.6–5.8 —Unique amino terminus (MSVRRQFSTTE) —Variant carboxy terminal portion of CnB BD and IBD 403 EMNVTDEEGATT –513 amino acids; pI = 7.1
Figure 1. Calcineurin structure. The structure of human calcineurin Aα (accession number: J05480) and human calcineurin B (accession number: M30773) is shown. The sequences of important regulatory regions are indicated. CaM: calmodulin; CnB: calcineurin B; CsA-CyP BD: cyclosporin A/cyclophilin binding domain; FK506/FKBP BD: FK506/FK506 binding protein binding domain; Box 1 and 2 (see text). Sequences important for the carboxyl terminal autoinhibitory function are indicated with an asterisk.15 For calcineurin B, the positions of the EF-hand high-affinity calcium binding sites are indicated; the amino terminus of calcineurin B is myristolyated (C14 -myristate) (see text).
the role of two kinases (casein kinase Iα and mitogenactivated protein/extracellular signal-regulated kinase kinase 1, MEKK1)20 and a nuclear export element (Crml)21 in controlling the intracellular distribution of NFAT. Currently, it is believed the NFAT remains cytoplasmic by virtue of its interaction with casein kinase Iα, a kinase that binds to and possibly phosphorylates the serine-rich amino terminus of NFAT and a putative nuclear localization signal
translocation of NFAT itself occurs within 5 min in response to ionomycin treatment (or T cell receptor activation) and remains nuclear once a sustained level of calcium is achieved.19 When the calcium signal is removed, calcineurin becomes inactive and preexisting NFAT relocalizes to the cytosol. Studies carried out by numerous laboratories have revealed the complexity of calcineurin-mediated regulation of NFAT. Recent findings have implicated 407
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tion of either one reversed the activation of IKK and the phosphorylation levels of IκBα. This synergy was only observed with IKKβ-mediated phosphorylation of IκBα, and was also present in non-T lymphocytes, and thus may be a common mechanism of NF-κB transcriptional activation. Calcineurin and PKC have also been observed to synergize to activate JNK activity in T lymphocytes (Figure 2). In contrast to NF-κB activation which was independent of the PKC isoform, activation of JNK activity was only observed in T lymphocytes and was specific for the PKCθ isoform.27 PKCθ is a novel PKC that binds only to DAG and does not require calcium signals to achieve maximal activation. The synergy between calcineurin and PKCθ was only observed for JNK1 activity towards GST-cJun and not for ERKl activity. Furthermore, it was determined that the synergy was specific for Rac activation of JNK activity, as it was not observed following Ras activation. Since PKCθ activity is calcium independent, the site of interaction of calcineurin presumably is not on PKCθ itself. Furthermore, Werlen et al.27 were not able to observe a similar synergy in HeLa cells, suggesting that calcineurin and/or PKCθ may be acting upon a T-cell-specific factor responsible for the effect on JNK activity. The role of calcineurin in T lymphocyte activation is thus becoming complicated, and may allow the bridging of different pathways converging on a specific biological outcome, IL-2 production. How can calcineurin function to regulate NFAT, NF-κB, and JNK activity mediated by the transient increase in calcium levels that follow T lymphocyte activation? Answers to this question may lie in the ability of calcium levels to be exquisitely controlled spatio-temporally and to rely on the amplitude and frequency changes in the levels of this second messenger in order to control one pathway and leave a parallel pathway unaffected.28 In this manner, elements become activated only when they are needed. Activation pathways within other immune cells, such as mast cells, natural killer (NK) cells, and cytotoxic T cells (CTL) utilize many of the signaling molecules important to T lymphocyte activation. Mast cells and NK cells function as immediate defenses to the presence of foreign agents in our system, and thus play central roles in inflammatory and allergic responses. Calcineurin has been demonstrated to have differential affects on the activation of mousebone-marrow-derived mast cells in response to Fcε receptor (FcεRI) and stem factor receptor (c-kit) ligation.29 Using CsA, Ishizuka et al.29 (1999) demon-
(NLS) masking domain immediately downstream. The latter phosphorylation event allows for the interaction with the NLS of NFAT, masking it and preventing NFAT from localizing to the nucleus. Calcineurin docks at a region upstream from the serine-rich area and functions to dephosphorylate the serine rich area and residues within the putative nuclear localization signal (NLS) masking domain. These events expose the calcium-sensitive NLS of NFAT and allows nuclear translocation of NFAT and activation of target genes. Both calcineurin3 and casein kinase Iα 20 were found in association with NFAT in the nucleus, illustrating that NFAT remains tightly associated with both its regulatory elements. Zhu et al.20 were also able to demonstrate that active MEKK1 stabilized NFAT/casein kinase Iα complexes and contributed to the cytoplasmic localization of NFAT. Additionally, when calcium levels have subsided and calcineurin reverts back to the inactive state, rephosphorylation by casein kinase Iα is thought to occur, followed by the displacement of calcineurin from NFAT by Crml (a nuclear export signal receptor), allowing NFAT to relocalize to the cytosol.21 In addition to casein kinase Iα, glycogen synthase kinase-322 and the c-Jun N-terminal kinase (JNK)23 have been demonstrated to rephosphorylate NFAT and facilitate the return of NFAT to the cytoplasm (Figure 2). The regulation of NFAT is thus spatio-temporally controlled and requires intimate associations with both kinases and phosphatases. In addition to mediating NFAT activation, calcineurin has been demonstrated to synergize with protein kinase C (PKC) to activate IκB kinase (IKK) and NF-κB transcriptional activity in T lymphocytes (Figure 2).24 NF-κB is a ubiquitous heterodimeric transcription factor that activates cis-acting κB enhancer elements present in the promoter region of numerous target genes.25 NF-κB is inactive when in association with IκB proteins, specifically, IκBα, because IκBα masks the nuclear localization signal of NF-κB, thus retaining NF-κB in the cytosol. Following T lymphocyte activation IκBα undergoes serine phosphorylation, mediated by IKK, followed by site-specific ubiquitination and degradation by the 26S proteosome complex.26 This sequence of events releases IκBα from NF-κB and allows NF-κB to translocate to the nucleus and activate target genes. Calcineurin and PKC were observed to increase the phosphorylation level of IκBα (by the regulation of IKK), resulting in maximal degradation of IκBα. In addition, the maximal degradation of IκBα required both second messengers, calcium and DAG; inhibi408
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Figure 2. Calcineurin signal transduction networks in T lymphocytes. T cell receptor engagement results in the activation of receptor tyrosine kinases leading to the activation of PLCγ and the generation of the second messengers IP3 and DAG. IP3 results in the activation of calcium release from the internal stores of the endoplasmic reticulum; DAG activates PKC and thus downstream effectors of PKC. Calcium release from the cytoplasm results in the activation of calcineurin and thus downstream effectors of calcineurin that are dependent on calcineurin (NFAT) or require additional elements (PKC) to synergize and achieve maximal activation (NFκB and JNK). CaM: calmodulin; IKK, IκBα kinase; CD28RE: CD28 responsive element; (→): activation pathway; (- - -|): inhibitory pathway.
strated that calcineurin can transmit signals initiated only from the FcεRI to the JNK MAP kinase pathway without affecting the ERK pathway; furthermore, this was only observed upon FcεRI activation of mast cells and not with c-kit activation. This was in support of an earlier observation of the influence of calcineurin in the degranulation of murine cytotoxic T cells.30 In NK cells, calcineurin was recently demonstrated to
regulate Fas ligand expression resulting from CD16 (Fcγ RIII) cross-linking, and required a reducing environment in order to have maximal Fas ligand expression.31 The latter effect is in agreement with the requirement of a reduced environment in order to protect the catalytic center of calcineurin32 and the consequent activation of NFAT, an activator of Fas ligand expression. Furthermore, Lobo et al.33 409
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infection by Leishmania major. Furthermore, mice lacking NFATl also showed increased accumulation of eosinophils and serum IgE in response to intrathoracically injected ovalbumin, suggesting that NFATl can negatively control the signaling pathways governing eosinophil activation and IgE production. The role of calcineurin may again relate to feedback control mechanisms that control IE gene expression once effectors of the IE genes have carried out their function and their activities are no longer required. The role of calcineurin as a negative regulator of gene transcription has also recently been observed in the regulation of adenylate cyclase activity in AtT20 cells42 and in the control of an element regulating a G2/M checkpoint in yeast. Calcineurin activity in Saccharomyces cerevisiae was found to downmodulate the activity of p34CDC28 , an important G2/M checkpoint cyclin-dependent kinase (cdk) in budding yeast.43 p34CDC28 becomes activated at the onset of mitosis by the coordinated action of kinases and phosphatases. In cooperation with Mpkl (a component of the MAP kinase pathway), the activation of calcineurin was shown to downregulate p34CDC28 by increasing the activity of the inhibitory kinase, Swe1, at the transcriptional and translational levels, thus preventing the entrance into mitosis. We have observed a similar effect of calcineurin on cdk4 activity in Jurkat cells and COS-7 cells, and propose that calcineurin can downregulate the kinase activity of cdk4 by the dephosphorylation of residues necessary for the activation of this mammalian G0/G1 checkpoint kinase (Baksh et al., Oncogene, in press). Thus, the negative control of cell cycle elements by calcineurin illustrates how calcineurin may function in multiple activation pathways that are inhibitory to the control of cellular proliferation.
have extended these observations to two other members of the tumor necrosis family (TNF) of cytokines, CD40L and TNF-α, and demonstrated that calcium/calmodulin kinase IV/Gr (CaMKIV) synergized with calcineurin to activate the transcription of the tumor necrosis family of cytokines. Similarly, in non-immune cells, calcium levels can also control the role played by calcineurin in controlling surface receptor activation, as observed in the control on N -methyl-D-aspartate synaptic transmission34 and Na+ , K+ -ATPase activity in renal tubule cells.35 Calcineurin: a negative regulator of activation In addition to positively regulating elements involved in the activation of immune cells, calcineurin has been demonstrated to negatively regulate immediateearly (IE) gene activation in T lymphocytes,36 erythroleukaemia cells37 and neural PC12 cells.38 Schaefer et al.37 demonstrated that cyclosporin A can increase c-myb, erg-1 and c-fos expression in the erythroleukaemia cell line, ELM-I-l, by inhibiting the activity of calcineurin. Chen et al.36 reported that p2lras may utilize calcineurin in Jurkat cells to negatively regulate erg-1, c-fos, and c-jun expression leading to diminished IL-2 production.36 The negative influence of calcineurin on c-fos expression in Jurkat cells is partly due to the regulation of the phosphorylation state of an important component of c-fos gene expression, Elk-1 (Figure 2).39 By limiting Elk-1 transcriptional activity, calcineurin can negatively regulate c-fos gene expression. In addition, Chen et al.36 observed that the effect of p21ras was specific for calcineurin controlling c-fos, c-jun or erg-1 transcriptional activity, as they were not able to observe changes in PKC isoform distribution nor changes in MAP kinase activity. In contrast, Woodrow et al.40 demonstrated that p21ras can synergize with calcineurin to upregulate the activity of NFAT. It is currently unknown how calcineurin may synergize with p21ras to positively influence NFAT activation and negatively regulate IE gene activation. However, it may influence both elements by initially stimulating the actions of NFAT (which is dependent upon AP-1 transcriptional elements), followed by negative feedback control of AP-1 elements (mediating IE gene expression through p21ras ), thus limiting the activation of T lymphocytes. In support of the role of calcineurin in limiting lymphocyte function, Xanthoudakis et al.41 demonstrated that mice lacking NFATl (NFATp) showed an increased lymphocyte proliferative rate in response to a primary
Calcineurin substrates: key to other cellular functions Numerous substrates for calcineurin (Table 2) have been discovered that have functions ranging from the control of the catalytic activity of calcineurin (Cain/Cabin-1 and superoxide dismutase), modulation of the duration of ER-store-driven calcium release (IP3 receptor, ryanodine receptor), control of amylase secretion in rat pancreatic acinar cells (CRHSP-24),44 control of the interaction with the cytoskeletal matrix (dystrophin),45 synaptic vesicle endocytosis (dynamin),46 control of neuronal signaling elements, such as GAP-4347 and ACtp10,48 and 410
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Table 2. Summary of calcineurin binding proteins. NFAT: nuclear factor of activated T cells; AKAP: A kinase anchor protein; SOD: superoxide dismutase; Cain: calcineurin inhibitor; Cabin-1: calcineurin binding protein; DARPP-22: dopamine- and cAMP-regulated phosphoprotein of molecular mass 32 kDa; MLCK: myosin light chain kinase; IP3 R: inositol-1,4,5-trisphosphate receptor; RyR: ryanodine receptor; CRHSP-24: calcium-regulated heat stable protein with a molecular mass of 24 kDa; ACtp10: adenylyl cyclase type 10 protein; Hsp25: heat shock protein with a molecular mass of 25 kDa; GAP-43: growth-associated protein, also known as B-50 and neuromodulin POTENTIAL CALCINEURIN BINDING PROTEINS Name
Function
Molecular Weight (kDa)
Calmodulin NFAT AKAP-79 SOD Cain Cabin-1 Elk-1 DARPP-32 Inhibitor-1 MLCK Bcl-2 Bad IP3 R RyR Dystrophin Dynamin I CRHSP-24 ACtp10 Hsp25 GAP-43
Calcium responsive regulator Activator of cytokine gene transcription Scafolding protein Free radical scavenger Inhibitor of calcineurin Inhibitor of calcineurin Activator of c-Fos gene Inhibitor of Na+ /K+ ATPase Inhibitor of PP1 Control of muscle contraction Apoptosis Apoptosis Calcium release channel Calcium release channel Cytoskeletal structure Synaptic vesicle endocytosis Amylase secretion Conversion of ATP to cAMP Heat shock response Neuronal signaling
19 120 79 31 240 255 60 32 30 80 27 30 200 565 427 100 24 156 25 30
Localization Cytosol/Nucleus Cytosol/Nucleus Cytosol Cytosol Cytosol Nucleus Cytosol/Nucleus Renal tubule Neurons Muscle Mitochondria Mitochondria ER membrane SR membrane Cytoskeleton Membrane Cytosol Neurons Cytosol Neurons
to affect numerous functions by localizing the kinase to different subcellular compartments.56 AKAP79 is thus thought to localize protein kinase A and calcineurin in neurons to sites that modulate synaptic transmission, such as those regulated by glutamate receptors.54
control of muscle contraction (myosin light chain kinase)49 and skeletal50 and cardiac muscle hypertrophy.51 Superoxide dismutase (SOD) was shown to interact with calcineurin and protect its Fe–Zn catalytic center from the damaging effects of a changed redox environment.32 Cain/Cabin-1 were cloned simultaneously by Sun et al.52 and Lai et al.,53 and Cain was demonstrated to non-competitively inhibit the phosphatase catalytic activity of calcineurin with a K i of 0.44 µM (in comparison, FK506/FKBP12 has a K i of 7.9 nM). Cabin-1 (a nuclear protein) was demonstrated to inhibit NFAT dephosphorylation and NFAT-driven transcription;52 whereas Cain (a cytosolic protein) inhibited calcineurin even in the presence of FK506/FKBP12 and calmodulin indicating that it has a distinct regulatory site on calcineurin. Similarly, A kinase anchor protein-79 (AKAP79) was shown to non-competitively inhibit calcineurin with a K i of 4.2 µM that was obtained in the presence and absence of calmodulin.54 Furthermore, AKAP79 was also found to bind to calcineurin at a site distinct from the immunophilin docking region and to inhibit NFAT dephosphorylation and NFAT-driven transcription.55 A kinase anchor proteins are thought to function as molecular scaffolds that allow kinases
Calcineurin as a modulator of calcium signaling Calcineurin was identified within the complex of two key intracellular sarcoplasmic/endoplasmic reticulum calcium channel proteins, the ryanodine receptor (RyR)57 and the inositol-1,4,5-trisphosphate receptor (IP3 R) (Figure 2).58 Both have been demonstrated to be present in B lymphocytes59 and T lymphocytes60 and function similarly to their muscle counterparts in regulating calcium transients within the cell. In addition, FKBP1261 was identified as an important component of both calcium channel proteins and demonstrated to stably maintain the full conductance state of the channel once the channel has achieved it. Calcineurin is thought to regulate the serine/threonine phosphorylation status of the IP3 receptor calcium channel and thus control the IP3 receptor and possibly the ryanodine receptor by 411
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receptor, Nur77.71 The orphan receptors, Nur77 and Nor1, have been demonstrated to be important mediators of T-cell-receptor-induced apoptosis, and the expression of Nur77 is sensitive to changes in intracellular calcium levels.72 Furthermore, the recently identified endogenous calcineurin inhibitor, Cabin-1, was found to be a protective factor for MEF2-induced apoptosis in Jurkat T cells. MEF2 is believed to associate with Cabin-1 and prevent the activation of Nur77 gene expression. Using a murine T cell hybridoma line sensitive to T-cellreceptor-induced apoptosis, DO11.10, Hong-Duk et al.71 demonstrated that increased levels of calcium following T cell receptor activation were sufficient to allow the formation of activated calmodulin and the displacement of Cabin-1 by calmodulinbound calcineurin from MEF2 complexes. The competitive displacement of Cabin-1 would thus free MEF2 to turn on the expression of Nur77 and thus promote apoptosis. These studies illustrate the complex nature of apoptotic signaling pathways and the role that calcineurin may play in controlling an important component of cellular homeostasis.
influencing the opening potential of these channels. Therefore, both FKBP12 and calcineurin function to prevent aberrant channel activity and thus optimize calcium release from the endoplasmic reticulum. In addition, Genazzani et al.62 demonstrated that calcineurin may also regulate the transcription of the IP3 R by regulation of the stability of its mRNA. Calcineurin and FKBP12 are therefore important regulators of calcium channel function and calcium transients within T and B lymphocytes and thus regulate calcium release from internal stores. Control of cellular apoptosis Programmed cell death, or apoptosis is an important component of cellular homeostasis responsible for the removal of cells by means of an intrinsic programmed signaling cascade. During the development of immune cells, and particularly during T cell ontogeny, apoptosis is an important mechanism for the removal of potentially auto-reactive cells (the process of negative selection), allowing only non-self reactive T cells to reach the peripheral immune system. Calcineurin has been demonstrated to be involved in negative thymic selection by virtue of its role in the removal of self-reactive T cells by apoptosis.63 A direct correlation between the phosphatase activity of calcineurin and programmed cell death was observed in a murine T cell hybridoma,64 in an immature B cell line65 and in a non-immune cell line, BHK-21.66 Recently, the potential targets of calcineurin in the programmed cell death pathway have been characterized, one of which is thought to involve NFAT.67 Calcineurin is thought to control cellular apoptosis by several mechanisms: (1) by the activation of NFAT, allowing NFAT to translocate to the nucleus and upregulate Fas ligand (FasL) expression, an important element mediating Fasdependent apoptosis;68 (2) by the dephosphorylation of the Bcl-XL binding protein, BAD, facilitating the dissociation of BAD from cytosolic 14-3-3 proteins and allowing the reassociation of BAD with Bcl-XL on the outer mitochondrial membrane. The latter event interferes with the protective effects of Bcl-XL and in this manner, calcineurin results in increased apoptosis.69 Furthermore, Shibasaki et al.66 and Srivastava et al.70 have shown that calcineurin can also bind directly to Bcl-2 (specifically to the BH4 domain of Bcl-2) and sequester Bcl-2 away from its protective role in cell survival; (3) by the transcriptional control of myocyte enhancer factor 2 (MEF2), thus directly regulating the expression of the orphan
Summary Calcineurin is involved in many aspects of immune cell regulation and has been demonstrated to utilize similar signaling molecules in order to carry out diverse biological effects. The diversity in the roles played by calcineurin closely parallel the importance of intracellular calcium changes to the function of immune cells and other cell types. The changes in intracellular calcium waves in immune cells vary in both amplitude and duration leading to the differential activation of transcription factors at different thresholds of intracellular calcium. Initially demonstrated to influence the nuclear transcriptional activity of NFAT, the role for calcineurin in immune cell regulation may involve the control of other transcriptional elements important to the activation of immune cells, such as c-fos, Elk-1, and NF-κB, and the involvement in other pathways involving the transient release of intracellular calcium, such as apoptosis and neuronal signaling cascades. These varied effects of calcineurin are thus observed as a result of the differential activation of calcineurin mirrored in the changes in intracellular calcium in different cellular environments. 412
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Acknowledgements 16.
We would like to thank the members of the Burakoff laboratory for their helpful discussions. S. Baksh is a recipient of the Medical Research Foundation of Canada post-doctoral fellowship and is an Abrahams post-doctoral Fellow.
17.
18.
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34. Graef IA, Mermelstein PG, Stankunas K, Neilson JR, Deisseroth K, Tsien RW, Crabtree GR (1999) L-type calcium channels and GSK-3 regulate the activity of NFATc4 in hippocampal neurons. Nature 401:703–708 35. Aperia A, Ibarra F, Svensson LB, Klee C, Greengard P (1992) Calcineurin mediates alphadrenergic stimulation of Na+, K(+)-ATPase activity in renal tubule cells. Proc Natl Acad Sci USA 89:7394–7397 36. Chen CY, Forman LW, Faller DV (1996) Calcium-dependent immediate-early gene induction in lymphocytes is negatively regulated by p21Ha-ras. Mol Cell Biol 16:6582–6592 37. Schaefer A, Magocsi M, Fandrich A, Marquardt H (1998) Stimulation of the Ca2+-mediated egr- l and c-fos expression in murine erythroleukaemia cells by cyclosporin A. Biochem J 335:505–511 38. Enslen H, Soderling TR (1994) Roles of calmodulindependent protein kinases and phosphatase in calciumdependent transcription of immediate-early genes. J Biol Chem 269:20872–20877 39. Tian J, Karin M (1999) Stimulation of Elk1 transcriptional activity by mitogen-activated protein kinases is negatively regulated by protein phosphatase 2B (calcineurin). J Biol Chem 274:15173–15180 40. Woodrow M, Clipstone NA, Cantrell D (1993) p2l ras and calcineurin synergize to regulate the nuclear factor of activated T cells. J Exp Med 178:1517–1522 41. Xanthoudakis S, Viola JP, Shaw KT, Luo C, Wallace JD, Boza PT, Luk DC, Curran T, Rao A (1996) An enhanced immune response in mice lacking the transcription factor NFATl. Science 272:892–895 42. Antoni FA, Barnard RJ, Shipston MJ, Smith SM, Simpson J, Paterson JM (1995) Calcineurin feedback inhibition of agonist-evoked cAMP formation. J Biol Chem 270:28055– 28061 43. Mizunuma M, Hirata D, Miyahara K, Tsuchiya E, Miyakawa T (1998) Role of calcineurin and Mpkl in regulating the onset of mitosis in budding yeast. Nature 392:303–306 44. Grobelwski GE, Yoshida M, Bragado MJ, Ernst SA, Leykam J, Williams JA (1998) Purification and characterization of a novel physiological substrate for calcineurin in mammalian cells. J Biol Chem 273:22738–22744 45. Walsh MP, Busaan JL, Fraser ED, Fu SY, Pato MD, Michalak M (1995) Characterization of the recombinant C-terminal domain of dystrophin: phosphorylation by calmodulindependent protein kinase II and dephosphorylation by type 2B protein phosphatase. Biochemistry 34:5561–5568 46. Lai MM, Hong JJ, Ruggiero AM, Burnett PE, Slepnev VI, De Camilli P, Snyder SH (1999) The calcineurin-dynamin 1 complex as a calcium sensor for synaptic vesicle endocytosis. J Biol Chem 274:25963–25966 47. Lyons WE, George EB, Dawson TM, Steiner JP, Snyder SH (1994) Immunosuppressant FK506 promotes neurite outgrowth in cultures of PC12 cells and sensory ganglia. Proc Natl Acad Sci USA 91:3191–3195 48. Paterson JM, Smith SM, Harmar AJ, Antoni FA (1995) Control of a novel adenylyl cyclase by calcineurin. Biochem Biophys Res Commun 214:1000–1008 49. Hathaway DR, Adelstein RS, Klee CB (1981) Interaction of calmodulin with myosin light chain kinase and cAMPdependent protein kinase in bovine brain. J Biol Chem 256:8183–8189 50. Musaro A, McCullagh KJ, Naya FJ, Olson EN, Rosenthal N (1999) IGF-l induces skeletal myocyte hypertrophy through calcineurin in association with GATA-2 and NF-ATcl. Nature 400:581–585
51. Molkentin JD, Lu JR, Antos CL, Markham B, Richardson J, Robbins J, Grant SR, Olson EN (1998) A calcineurindependent transcriptional pathway for cardiac hypertrophy. Cell 93:215–228 52. Sun L, Youn HD, Loh H, Stolow M, He W, Liu JO (1998) Cabin 1, a negative regulator for calcineurin signaling in T lymphocytes. Immunity 8:703–711 53. Lai MM, Burnett PE, Wolosker H, Blackshaw S, Snyder SH (1998) Cain, a novel physiologic protein inhibitor of calcineurin. J Biol Chem 273:18325–18331 54. Coghlan VM, Perrino BA, Howard M, Langeberg LK, Hicks JB, Gallatin WM, Scott JD (1995) Association of protein kinase A and protein phosphatase 2B with a common anchoring protein. Science 267:108–111 55. Kashishian A, Howard M, Loh C, Gallatin WM, Hoekstra MF, Lai Y (1998) AKAP79 inhibits calcineurin through a site distinct from the immunophilin-binding region. J Biol Chem 273:27412–27419 56. Scott JD (1997) Dissection of protein kinase and phosphatase targeting interactions. Soc Gen Physiol Ser 52:227–239 57. Cameron AM, Nucifora FC Jr, Fung ET, Livingston DJ, Aldape RA, Ross CA, Snyder SH (1997) FKBP12 binds the inositol 1,4,5,-trisphoshate receptor at leucine-proline (1400-1401) and anchors calcineurin to this FK506-like domain. J Biol Chem 272:27582–27588 58. Cameron AM, Steiner JP, Roskams AJ, Ali SM, Ronnett GV, Snyder SH (1995) Calcineurin associated with the inositol 1,4,5-trisphosphate receptor-FKBP12 complex modulates Ca2+ flux. Cell 83:463–472 59. Sei Y, Gallagher KL, Basile AS (1999) Skeletal muscle type ryanodine receptor is involved in calcium signaling in human B lymphocytes. J Biol Chem 274:5995–6002 60. Hakamata Y, Nishimura S, Nakai J, Nakashima Y, Kita T, Imoto K (1994) Involvement of the brain type of ryanodine receptor in T-cell proliferation. FEBS Lett 352:206–210 61. Brillantes AB, Ondrias K, Scott A, Kobrinsky E, Ondriasova E, Moschella MC, Jayaraman T, Landers M, Ehrlich BE, Marks AR (1994) Stabilization of calcium release channel (ryanodine receptor) function by FK506-binding protein. Cell 77:513–523 62. Genazzani AA, Carafoli e, Guerini D (1999) Calcineurin controls inositol 1,4,5-trisphosphate type 1 receptor expression in neurons. Proc Natl Acad Sci USA 96:5797–5801 63. Hollander GA, Fruman DA, Bierer BA, Burakoff SJ (1994) Disruption of T cell development and repertoire selection by calcineurin inhibition in vivo. Transplantation 58:1037–1043 64. Fruman DA, Klee CB, Bierer BE, Burakoff SJ (1992) Calcineurin phosphatase activity in T lymphocytes is inhibited by FK 506 and cyclosporin A. Proc Natl Acad Sci USA 89:3686– 3690 65. Chen W, Wang HG, Srinivasula SM, Alnemri ES, Cooper NR (1999) B cell apoptosis triggered by antigen receptor ligation proceeds via a nove caspase-dependent pathway. J Immunol 163:2483–2491 66. Shibasaki F, Kondo E, Akagi T, McKeon F (1997) Suppression of signaling through transcription factor NF-AT by interactions between calcineurin and Bcl-2. Nature 386:728–731 67. Latinis KM, Norian LA, Eliason SL, Koretzky Z (1997) Two NFAT transcription factor binding sites participate in the regulation of CD95 (Fas) ligand expression in activated human T cells. J Biol Chem 272:1427–1434 68. Toth R, Szegezdi E, Molnar G, Lord JM, Fesus L, Szondy Z (1999) Regulation of cell surface expression of Fas (CD95) ligand and susceptibility to Fas (CD95)-mediated apoptosis in activation-induced T cell death involves cal-
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induced Fas ligand transcription. J Exp Med 190:253–265 71. Youn H-K, Sun L, Prywes R, Liu JO (1999) Apoptosis of T cells mediated by Ca2+-induced release of the transcription factor MEF2. Science 286:790–793 72. Woronicz JD, Lina A, Calnan BJ, Szychowski S, Cheng L, Winoto A (1995) Regulation of the Nur77 orphan steroid receptor in activation-induced apoptosis. Mol Cell Biol 15:6364– 6367
cineurin and protein kinase C, respectively. Eur J Immunol 29:383–393 69. Wang HG, Pathan N, Ethell IM, Krajewski S, Yamaguchi Y, Shibasaki F, McKeon F, Bobo T, Franke TF, Reed JC (1999) Ca2+-induced apoptosis through calcineurin dephosphorylation of BAD. Science 284:339–343 70. Srivastava RK, Sasaki CY, Hardwick JM, Longo DL (1999) Bcl-2-mediated drug resistance: inhibition of apoptosis by blocking nuclear factor of activated T lymphocytes (NFAT)-
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The role of calcineurin in lymphocyte activation Shairaz Baksh a and Steven J. Burakoff a,b
vation of calcium responsive proteins. One of the best characterized lymphokine gene transcription pathways is the interleukin (IL)-2 activation pathway in T lymphocytes. The activation of T lymphocytes results in calcium release from the endoplasmic reticulum and the activation of calcium responsive proteins, such as calmodulin. The activation of calmodulin, in turn, results in its binding to and activation of the serine/threonine phosphatase calcineurin (protein phosphatase 2B) and subsequent dephosphorylation of the nuclear transcription factor of activated T cells, NFAT.2 The dephosphorylation of NFAT is required for its translocation to the nucleus (in a complex with calcineurin)3 and heterodimerization with AP-1 family members on NFAT/AP-1 sites in the IL-2 promoter.4 As calcium levels subside, calcineurin loses its phosphatase activity, and NFAT relocalizes to the cytosol.5 Calcineurin/NFAT signaling pathways are utilized to regulate the genes encoding a wide variety of cytokines (IL-2,IL-4, IFN-γ , GMCSF, IL-3, IL-13, TNF-α)2 and cell surface receptors (CD95L, CD40L, CD69).6 NFAT and its role as a key transcriptional activator controlled by calcineurin is well characterized; however, a wide variety of additional biological events that involve calcineurin have recently been discovered, reflecting the diverse role that calcineurin plays in specialized tissues.
Lymphokine gene transcription involves numerous signal transduction molecules and second messengers. The serine/threonine phosphatase calcineurin has been demonstrated to play a central role in the immediate, early activation of numerous lymphokines (such as interleukin [IL]-2) and in the regulation of cell surface receptors such as CD40L, CD95, and recently CD25α (the α chain of the IL-2 receptor). In addition to lymphocyte activation, calcineurin functions include control of neuronal signaling, muscle contraction, muscle hypertrophy and cellular death. Therefore, calcineurin not only plays a vital role in the regulation of T lymphocyte function, but also functions in cellular environments outside the immune system. Key words: calcineurin / T lymphocyte / NFAT / apoptosis / immunophilins c 2000 Academic Press
Lymphokine gene transcription involves a complex array of signal transduction molecules and events that ultimately lead to the activation of nuclear transcription. Central to the activation of lymphokine gene transcription is the elevation of intracellular calcium levels and subsequent activation of calcium responsive proteins.1 Second messengers, such as diacylglycerol (DAG) and inositol-1,4,5-trisphosphate (IP3 ), play important roles in the signaling pathways originating from surface receptor engagement and co-crosslinking. DAG is important in the activation of protein kinase C (PKC) pathways leading to the ras/raf/mitogen-activated kinase (MAPK) cascade. IP3 is an important element controlling the release of calcium from the internal stores of the endoplasmic reticulum into the cytoplasm and the subsequent acti-
Calcineurin: cellular distribution and structure Calcineurin is a heterodimeric phosphatase formed by the association of a 60 kDa phosphatase catalytic subunit (calcineurin A) and a 19 kDa high-affinity calcium binding subunit (calcineurin B). The phosphatase catalytic domain of calcineurin is structurally similar to the catalytic domain of protein phosphatase 1 with the exception of two short insertions towards the end of the catalytic domain that may govern calcineurin substrate specificity (Box 1 and 2, Figure 1). In mammals, calcineurin A exists as three isoforms sharing 75–80% primary sequence similarity. The
From the a Department of Pediatric Oncology, Dana Farber Cancer Institute and b Department of Pediatrics, Harvard Medical School, Boston, MA, 02115 USA. c
2000 Academic Press 1044–5323 / 00 / 000405+ 11 / $35.00/0 / 0
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to calcineurin is obtained by the composite binding of the fungal macrolides, FK506 and cyclosporin A (CsA), to their endogenous proteins, the immunophilins: the FK506 binding proteins (FKBPs) and the cyclosporin A binding proteins (the cyclophilins-CyPs).16 FK506, CsA, or the immunophilins by themselves do not inhibit the phosphatase activity of calcineurin. However, complexes of FK506/FKBP (K i of calcineurin inhibition, 7.9 nM) or CsA/CyP (K i for calcineurin inhibition, 270 nM) can inhibit the activity of calcineurin by 80–90%. In the crystal structure of the calcineurin A/ calcineurin B/FK506/FKBP12 co-complex, FK506/FKBP12 was found to make intimate contacts with both the A and B chains of calcineurin localized no closer than 10 Å from the catalytic active site.17 Inhibition of the phosphatase activity of calcineurin by the complex of FK506/FKBP12 was thus obtained by sterically blocking the access of substrates to the phosphatase catalytic site rather than by directly inhibiting the enzymatic activity of calcineurin.17 Small molecule substrates, such as para-nitrophenyl phosphate (pNPP), were observed to gain access to the phosphatase catalytic site even in the presence of an immunophilin complex. As the potential number of substrates for calcineurin increases, it will be interesting to determine if natural small protein substrates can be found that are allowed to enter into the catalytic site even in the presence of the immunophilin complex.
three isoforms have divergent carboxy terminal residues (Table 1) and distinct tissue distribution and chromosomal localizations: calcineurin Aα (on chromosome 4), calcineurin Aβ (on chromosome 10q21→q22) and calcineurin Aγ (whose localization has yet to be determined).7 High expression of Aβ is found in the thymus, while both Aα and Aβ are found within the peripheral immune system.8 Differences are also observed for the expression patterns of Aα and Aβ within specialized tissues, such as the rat nephron, where Aα expression was observed to be higher in the proximal tubules than in the medullary thick ascending limbs and the cortical collecting ducts.9 Within the central nervous system, calcineurin isoforms (mainly α and β) localize to the Purkinje cells of the cerebellum and the pyramidal cells of the cerebrum.10 Targeted disruption of the calcineurin Aα gene in mice resulted in normal maturation of T and B lymphocytes and normal responses to in vitro mitogenic stimulation, indicating that the β isoform of calcineurin may compensate for most of the functions of the α form in calcineurin Aα −/− T cells.11 However, calcineurin Aα −/− lymph node cells generated defective antigen-specific T cell responses following in vivo stimulation with an OVA peptide, implicating the α form of calcineurin as the predominant form controlling the physiological activation of T lymphocytes in the peripheral immune system. The third isoform of calcineurin, Aγ , is a testis-specific isoform found primarily within the nuclei of spermatids, and functions to control the remodeling of the nuclear chromatin in actively differentiating spermatids.12 Calcineurin B (localized to chromosome 2p16→p15) is the regulatory subunit of calcineurin and exists as two isoforms, CNB1 and CNB2, the latter specifically found in the testis.13 It has 35% sequence identity to calmodulin and contains an amino-terminal myristoylated glycine and four highaffinity EF-hand calcium binding sites. The binding of calcineurin B to calcineurin A allows the enzyme to achieve a two–three fold increase in activity.14 The binding of calcium-bound calmodulin to the calmodulin binding site within the carboxy terminus of calcineurin A results in an additional 10–20 fold increase in the phosphatase activity of the enzyme, which is facilitated by the displacement of a carboxy terminal autoinhibitory domain from the phosphatase active site. These events allow calcineurin substrates to gain access to the catalytic site and undergo dephosphorylation.15 Specific inhibition of the binding of substrates
Calcineurin in immune cells: positive and negative roles Calcineurin: a positive regulator of activation The central role of calcineurin in T cell activation primarily involves the regulation of NFAT. In addition to T cells, NFAT/calcineurin interactions are critical to the activation of other immune cells and to the signaling pathways of numerous other lymphokines. NFAT is a family of transcription factors that include five members mediating distinct biological outcomes in different cellular environments that are regulated by calcineurin.18 The activation of NFAT requires a sustained, low concentration of calcium that functions to maintain calcineurin in an active state and thus initiate events required for the activation of T lymphocytes. Studies have shown that calcium levels above 400 nM for 2 h are required for T lymphocytes to become committed to activation. However, the nuclear 406
Calcineurin in immune cells
Table 1. The mammalian isoforms of calcineurin A. Isoform type is listed in the first column. The next column indicates in which species the isoform has been identified and the name given to it is given in parentheses. Total amino acids are indicated with the isoelectric pH. Genebank accession numbers: human CnA-1: M29550; human CnA-2 (CnAβ): M29551; human CnAα: J05479; human testis: S46622; mouse CnA-1: J05479; mouse CnA-2: M81483; mouse testis: M81475; rat CnAα: M29275; rat Cnβ: M31809 MAMMALIAN CALCINEURIN ISOFORMS Isoform
Species
Divergent features (from CnAα)
α
human, mouse 2, rat α
β1
human (CnA-1)
β2
human (CnA-2), mouse 2, rat β
γ
human, mouse
—Unique amino terminus (MSEPKAIDPKLSTTD) —521 amino acids; pI = 5.6–5.8 —Proline rich amino terminal 25 amino acids MAAPEPAAPPPPPPPPPPPGADR –19 amino acid insertion within the catalytic domain 138 HVLGTEDISINPNNNNINEC —short carboxy terminal sequence after CaM binding domain –514 amino acids; pI = 5.43 —Proline rich amino terminal 25 amino acids MAAPEPARAAPPPPPPPPPPPGADR —524 amino acids; pI = 5.6–5.8 —Unique amino terminus (MSVRRQFSTTE) —Variant carboxy terminal portion of CnB BD and IBD 403 EMNVTDEEGATT –513 amino acids; pI = 7.1
Figure 1. Calcineurin structure. The structure of human calcineurin Aα (accession number: J05480) and human calcineurin B (accession number: M30773) is shown. The sequences of important regulatory regions are indicated. CaM: calmodulin; CnB: calcineurin B; CsA-CyP BD: cyclosporin A/cyclophilin binding domain; FK506/FKBP BD: FK506/FK506 binding protein binding domain; Box 1 and 2 (see text). Sequences important for the carboxyl terminal autoinhibitory function are indicated with an asterisk.15 For calcineurin B, the positions of the EF-hand high-affinity calcium binding sites are indicated; the amino terminus of calcineurin B is myristolyated (C14 -myristate) (see text).
the role of two kinases (casein kinase Iα and mitogenactivated protein/extracellular signal-regulated kinase kinase 1, MEKK1)20 and a nuclear export element (Crml)21 in controlling the intracellular distribution of NFAT. Currently, it is believed the NFAT remains cytoplasmic by virtue of its interaction with casein kinase Iα, a kinase that binds to and possibly phosphorylates the serine-rich amino terminus of NFAT and a putative nuclear localization signal
translocation of NFAT itself occurs within 5 min in response to ionomycin treatment (or T cell receptor activation) and remains nuclear once a sustained level of calcium is achieved.19 When the calcium signal is removed, calcineurin becomes inactive and preexisting NFAT relocalizes to the cytosol. Studies carried out by numerous laboratories have revealed the complexity of calcineurin-mediated regulation of NFAT. Recent findings have implicated 407
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tion of either one reversed the activation of IKK and the phosphorylation levels of IκBα. This synergy was only observed with IKKβ-mediated phosphorylation of IκBα, and was also present in non-T lymphocytes, and thus may be a common mechanism of NF-κB transcriptional activation. Calcineurin and PKC have also been observed to synergize to activate JNK activity in T lymphocytes (Figure 2). In contrast to NF-κB activation which was independent of the PKC isoform, activation of JNK activity was only observed in T lymphocytes and was specific for the PKCθ isoform.27 PKCθ is a novel PKC that binds only to DAG and does not require calcium signals to achieve maximal activation. The synergy between calcineurin and PKCθ was only observed for JNK1 activity towards GST-cJun and not for ERKl activity. Furthermore, it was determined that the synergy was specific for Rac activation of JNK activity, as it was not observed following Ras activation. Since PKCθ activity is calcium independent, the site of interaction of calcineurin presumably is not on PKCθ itself. Furthermore, Werlen et al.27 were not able to observe a similar synergy in HeLa cells, suggesting that calcineurin and/or PKCθ may be acting upon a T-cell-specific factor responsible for the effect on JNK activity. The role of calcineurin in T lymphocyte activation is thus becoming complicated, and may allow the bridging of different pathways converging on a specific biological outcome, IL-2 production. How can calcineurin function to regulate NFAT, NF-κB, and JNK activity mediated by the transient increase in calcium levels that follow T lymphocyte activation? Answers to this question may lie in the ability of calcium levels to be exquisitely controlled spatio-temporally and to rely on the amplitude and frequency changes in the levels of this second messenger in order to control one pathway and leave a parallel pathway unaffected.28 In this manner, elements become activated only when they are needed. Activation pathways within other immune cells, such as mast cells, natural killer (NK) cells, and cytotoxic T cells (CTL) utilize many of the signaling molecules important to T lymphocyte activation. Mast cells and NK cells function as immediate defenses to the presence of foreign agents in our system, and thus play central roles in inflammatory and allergic responses. Calcineurin has been demonstrated to have differential affects on the activation of mousebone-marrow-derived mast cells in response to Fcε receptor (FcεRI) and stem factor receptor (c-kit) ligation.29 Using CsA, Ishizuka et al.29 (1999) demon-
(NLS) masking domain immediately downstream. The latter phosphorylation event allows for the interaction with the NLS of NFAT, masking it and preventing NFAT from localizing to the nucleus. Calcineurin docks at a region upstream from the serine-rich area and functions to dephosphorylate the serine rich area and residues within the putative nuclear localization signal (NLS) masking domain. These events expose the calcium-sensitive NLS of NFAT and allows nuclear translocation of NFAT and activation of target genes. Both calcineurin3 and casein kinase Iα 20 were found in association with NFAT in the nucleus, illustrating that NFAT remains tightly associated with both its regulatory elements. Zhu et al.20 were also able to demonstrate that active MEKK1 stabilized NFAT/casein kinase Iα complexes and contributed to the cytoplasmic localization of NFAT. Additionally, when calcium levels have subsided and calcineurin reverts back to the inactive state, rephosphorylation by casein kinase Iα is thought to occur, followed by the displacement of calcineurin from NFAT by Crml (a nuclear export signal receptor), allowing NFAT to relocalize to the cytosol.21 In addition to casein kinase Iα, glycogen synthase kinase-322 and the c-Jun N-terminal kinase (JNK)23 have been demonstrated to rephosphorylate NFAT and facilitate the return of NFAT to the cytoplasm (Figure 2). The regulation of NFAT is thus spatio-temporally controlled and requires intimate associations with both kinases and phosphatases. In addition to mediating NFAT activation, calcineurin has been demonstrated to synergize with protein kinase C (PKC) to activate IκB kinase (IKK) and NF-κB transcriptional activity in T lymphocytes (Figure 2).24 NF-κB is a ubiquitous heterodimeric transcription factor that activates cis-acting κB enhancer elements present in the promoter region of numerous target genes.25 NF-κB is inactive when in association with IκB proteins, specifically, IκBα, because IκBα masks the nuclear localization signal of NF-κB, thus retaining NF-κB in the cytosol. Following T lymphocyte activation IκBα undergoes serine phosphorylation, mediated by IKK, followed by site-specific ubiquitination and degradation by the 26S proteosome complex.26 This sequence of events releases IκBα from NF-κB and allows NF-κB to translocate to the nucleus and activate target genes. Calcineurin and PKC were observed to increase the phosphorylation level of IκBα (by the regulation of IKK), resulting in maximal degradation of IκBα. In addition, the maximal degradation of IκBα required both second messengers, calcium and DAG; inhibi408
Calcineurin in immune cells
Figure 2. Calcineurin signal transduction networks in T lymphocytes. T cell receptor engagement results in the activation of receptor tyrosine kinases leading to the activation of PLCγ and the generation of the second messengers IP3 and DAG. IP3 results in the activation of calcium release from the internal stores of the endoplasmic reticulum; DAG activates PKC and thus downstream effectors of PKC. Calcium release from the cytoplasm results in the activation of calcineurin and thus downstream effectors of calcineurin that are dependent on calcineurin (NFAT) or require additional elements (PKC) to synergize and achieve maximal activation (NFκB and JNK). CaM: calmodulin; IKK, IκBα kinase; CD28RE: CD28 responsive element; (→): activation pathway; (- - -|): inhibitory pathway.
strated that calcineurin can transmit signals initiated only from the FcεRI to the JNK MAP kinase pathway without affecting the ERK pathway; furthermore, this was only observed upon FcεRI activation of mast cells and not with c-kit activation. This was in support of an earlier observation of the influence of calcineurin in the degranulation of murine cytotoxic T cells.30 In NK cells, calcineurin was recently demonstrated to
regulate Fas ligand expression resulting from CD16 (Fcγ RIII) cross-linking, and required a reducing environment in order to have maximal Fas ligand expression.31 The latter effect is in agreement with the requirement of a reduced environment in order to protect the catalytic center of calcineurin32 and the consequent activation of NFAT, an activator of Fas ligand expression. Furthermore, Lobo et al.33 409
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infection by Leishmania major. Furthermore, mice lacking NFATl also showed increased accumulation of eosinophils and serum IgE in response to intrathoracically injected ovalbumin, suggesting that NFATl can negatively control the signaling pathways governing eosinophil activation and IgE production. The role of calcineurin may again relate to feedback control mechanisms that control IE gene expression once effectors of the IE genes have carried out their function and their activities are no longer required. The role of calcineurin as a negative regulator of gene transcription has also recently been observed in the regulation of adenylate cyclase activity in AtT20 cells42 and in the control of an element regulating a G2/M checkpoint in yeast. Calcineurin activity in Saccharomyces cerevisiae was found to downmodulate the activity of p34CDC28 , an important G2/M checkpoint cyclin-dependent kinase (cdk) in budding yeast.43 p34CDC28 becomes activated at the onset of mitosis by the coordinated action of kinases and phosphatases. In cooperation with Mpkl (a component of the MAP kinase pathway), the activation of calcineurin was shown to downregulate p34CDC28 by increasing the activity of the inhibitory kinase, Swe1, at the transcriptional and translational levels, thus preventing the entrance into mitosis. We have observed a similar effect of calcineurin on cdk4 activity in Jurkat cells and COS-7 cells, and propose that calcineurin can downregulate the kinase activity of cdk4 by the dephosphorylation of residues necessary for the activation of this mammalian G0/G1 checkpoint kinase (Baksh et al., Oncogene, in press). Thus, the negative control of cell cycle elements by calcineurin illustrates how calcineurin may function in multiple activation pathways that are inhibitory to the control of cellular proliferation.
have extended these observations to two other members of the tumor necrosis family (TNF) of cytokines, CD40L and TNF-α, and demonstrated that calcium/calmodulin kinase IV/Gr (CaMKIV) synergized with calcineurin to activate the transcription of the tumor necrosis family of cytokines. Similarly, in non-immune cells, calcium levels can also control the role played by calcineurin in controlling surface receptor activation, as observed in the control on N -methyl-D-aspartate synaptic transmission34 and Na+ , K+ -ATPase activity in renal tubule cells.35 Calcineurin: a negative regulator of activation In addition to positively regulating elements involved in the activation of immune cells, calcineurin has been demonstrated to negatively regulate immediateearly (IE) gene activation in T lymphocytes,36 erythroleukaemia cells37 and neural PC12 cells.38 Schaefer et al.37 demonstrated that cyclosporin A can increase c-myb, erg-1 and c-fos expression in the erythroleukaemia cell line, ELM-I-l, by inhibiting the activity of calcineurin. Chen et al.36 reported that p2lras may utilize calcineurin in Jurkat cells to negatively regulate erg-1, c-fos, and c-jun expression leading to diminished IL-2 production.36 The negative influence of calcineurin on c-fos expression in Jurkat cells is partly due to the regulation of the phosphorylation state of an important component of c-fos gene expression, Elk-1 (Figure 2).39 By limiting Elk-1 transcriptional activity, calcineurin can negatively regulate c-fos gene expression. In addition, Chen et al.36 observed that the effect of p21ras was specific for calcineurin controlling c-fos, c-jun or erg-1 transcriptional activity, as they were not able to observe changes in PKC isoform distribution nor changes in MAP kinase activity. In contrast, Woodrow et al.40 demonstrated that p21ras can synergize with calcineurin to upregulate the activity of NFAT. It is currently unknown how calcineurin may synergize with p21ras to positively influence NFAT activation and negatively regulate IE gene activation. However, it may influence both elements by initially stimulating the actions of NFAT (which is dependent upon AP-1 transcriptional elements), followed by negative feedback control of AP-1 elements (mediating IE gene expression through p21ras ), thus limiting the activation of T lymphocytes. In support of the role of calcineurin in limiting lymphocyte function, Xanthoudakis et al.41 demonstrated that mice lacking NFATl (NFATp) showed an increased lymphocyte proliferative rate in response to a primary
Calcineurin substrates: key to other cellular functions Numerous substrates for calcineurin (Table 2) have been discovered that have functions ranging from the control of the catalytic activity of calcineurin (Cain/Cabin-1 and superoxide dismutase), modulation of the duration of ER-store-driven calcium release (IP3 receptor, ryanodine receptor), control of amylase secretion in rat pancreatic acinar cells (CRHSP-24),44 control of the interaction with the cytoskeletal matrix (dystrophin),45 synaptic vesicle endocytosis (dynamin),46 control of neuronal signaling elements, such as GAP-4347 and ACtp10,48 and 410
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Table 2. Summary of calcineurin binding proteins. NFAT: nuclear factor of activated T cells; AKAP: A kinase anchor protein; SOD: superoxide dismutase; Cain: calcineurin inhibitor; Cabin-1: calcineurin binding protein; DARPP-22: dopamine- and cAMP-regulated phosphoprotein of molecular mass 32 kDa; MLCK: myosin light chain kinase; IP3 R: inositol-1,4,5-trisphosphate receptor; RyR: ryanodine receptor; CRHSP-24: calcium-regulated heat stable protein with a molecular mass of 24 kDa; ACtp10: adenylyl cyclase type 10 protein; Hsp25: heat shock protein with a molecular mass of 25 kDa; GAP-43: growth-associated protein, also known as B-50 and neuromodulin POTENTIAL CALCINEURIN BINDING PROTEINS Name
Function
Molecular Weight (kDa)
Calmodulin NFAT AKAP-79 SOD Cain Cabin-1 Elk-1 DARPP-32 Inhibitor-1 MLCK Bcl-2 Bad IP3 R RyR Dystrophin Dynamin I CRHSP-24 ACtp10 Hsp25 GAP-43
Calcium responsive regulator Activator of cytokine gene transcription Scafolding protein Free radical scavenger Inhibitor of calcineurin Inhibitor of calcineurin Activator of c-Fos gene Inhibitor of Na+ /K+ ATPase Inhibitor of PP1 Control of muscle contraction Apoptosis Apoptosis Calcium release channel Calcium release channel Cytoskeletal structure Synaptic vesicle endocytosis Amylase secretion Conversion of ATP to cAMP Heat shock response Neuronal signaling
19 120 79 31 240 255 60 32 30 80 27 30 200 565 427 100 24 156 25 30
Localization Cytosol/Nucleus Cytosol/Nucleus Cytosol Cytosol Cytosol Nucleus Cytosol/Nucleus Renal tubule Neurons Muscle Mitochondria Mitochondria ER membrane SR membrane Cytoskeleton Membrane Cytosol Neurons Cytosol Neurons
to affect numerous functions by localizing the kinase to different subcellular compartments.56 AKAP79 is thus thought to localize protein kinase A and calcineurin in neurons to sites that modulate synaptic transmission, such as those regulated by glutamate receptors.54
control of muscle contraction (myosin light chain kinase)49 and skeletal50 and cardiac muscle hypertrophy.51 Superoxide dismutase (SOD) was shown to interact with calcineurin and protect its Fe–Zn catalytic center from the damaging effects of a changed redox environment.32 Cain/Cabin-1 were cloned simultaneously by Sun et al.52 and Lai et al.,53 and Cain was demonstrated to non-competitively inhibit the phosphatase catalytic activity of calcineurin with a K i of 0.44 µM (in comparison, FK506/FKBP12 has a K i of 7.9 nM). Cabin-1 (a nuclear protein) was demonstrated to inhibit NFAT dephosphorylation and NFAT-driven transcription;52 whereas Cain (a cytosolic protein) inhibited calcineurin even in the presence of FK506/FKBP12 and calmodulin indicating that it has a distinct regulatory site on calcineurin. Similarly, A kinase anchor protein-79 (AKAP79) was shown to non-competitively inhibit calcineurin with a K i of 4.2 µM that was obtained in the presence and absence of calmodulin.54 Furthermore, AKAP79 was also found to bind to calcineurin at a site distinct from the immunophilin docking region and to inhibit NFAT dephosphorylation and NFAT-driven transcription.55 A kinase anchor proteins are thought to function as molecular scaffolds that allow kinases
Calcineurin as a modulator of calcium signaling Calcineurin was identified within the complex of two key intracellular sarcoplasmic/endoplasmic reticulum calcium channel proteins, the ryanodine receptor (RyR)57 and the inositol-1,4,5-trisphosphate receptor (IP3 R) (Figure 2).58 Both have been demonstrated to be present in B lymphocytes59 and T lymphocytes60 and function similarly to their muscle counterparts in regulating calcium transients within the cell. In addition, FKBP1261 was identified as an important component of both calcium channel proteins and demonstrated to stably maintain the full conductance state of the channel once the channel has achieved it. Calcineurin is thought to regulate the serine/threonine phosphorylation status of the IP3 receptor calcium channel and thus control the IP3 receptor and possibly the ryanodine receptor by 411
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receptor, Nur77.71 The orphan receptors, Nur77 and Nor1, have been demonstrated to be important mediators of T-cell-receptor-induced apoptosis, and the expression of Nur77 is sensitive to changes in intracellular calcium levels.72 Furthermore, the recently identified endogenous calcineurin inhibitor, Cabin-1, was found to be a protective factor for MEF2-induced apoptosis in Jurkat T cells. MEF2 is believed to associate with Cabin-1 and prevent the activation of Nur77 gene expression. Using a murine T cell hybridoma line sensitive to T-cellreceptor-induced apoptosis, DO11.10, Hong-Duk et al.71 demonstrated that increased levels of calcium following T cell receptor activation were sufficient to allow the formation of activated calmodulin and the displacement of Cabin-1 by calmodulinbound calcineurin from MEF2 complexes. The competitive displacement of Cabin-1 would thus free MEF2 to turn on the expression of Nur77 and thus promote apoptosis. These studies illustrate the complex nature of apoptotic signaling pathways and the role that calcineurin may play in controlling an important component of cellular homeostasis.
influencing the opening potential of these channels. Therefore, both FKBP12 and calcineurin function to prevent aberrant channel activity and thus optimize calcium release from the endoplasmic reticulum. In addition, Genazzani et al.62 demonstrated that calcineurin may also regulate the transcription of the IP3 R by regulation of the stability of its mRNA. Calcineurin and FKBP12 are therefore important regulators of calcium channel function and calcium transients within T and B lymphocytes and thus regulate calcium release from internal stores. Control of cellular apoptosis Programmed cell death, or apoptosis is an important component of cellular homeostasis responsible for the removal of cells by means of an intrinsic programmed signaling cascade. During the development of immune cells, and particularly during T cell ontogeny, apoptosis is an important mechanism for the removal of potentially auto-reactive cells (the process of negative selection), allowing only non-self reactive T cells to reach the peripheral immune system. Calcineurin has been demonstrated to be involved in negative thymic selection by virtue of its role in the removal of self-reactive T cells by apoptosis.63 A direct correlation between the phosphatase activity of calcineurin and programmed cell death was observed in a murine T cell hybridoma,64 in an immature B cell line65 and in a non-immune cell line, BHK-21.66 Recently, the potential targets of calcineurin in the programmed cell death pathway have been characterized, one of which is thought to involve NFAT.67 Calcineurin is thought to control cellular apoptosis by several mechanisms: (1) by the activation of NFAT, allowing NFAT to translocate to the nucleus and upregulate Fas ligand (FasL) expression, an important element mediating Fasdependent apoptosis;68 (2) by the dephosphorylation of the Bcl-XL binding protein, BAD, facilitating the dissociation of BAD from cytosolic 14-3-3 proteins and allowing the reassociation of BAD with Bcl-XL on the outer mitochondrial membrane. The latter event interferes with the protective effects of Bcl-XL and in this manner, calcineurin results in increased apoptosis.69 Furthermore, Shibasaki et al.66 and Srivastava et al.70 have shown that calcineurin can also bind directly to Bcl-2 (specifically to the BH4 domain of Bcl-2) and sequester Bcl-2 away from its protective role in cell survival; (3) by the transcriptional control of myocyte enhancer factor 2 (MEF2), thus directly regulating the expression of the orphan
Summary Calcineurin is involved in many aspects of immune cell regulation and has been demonstrated to utilize similar signaling molecules in order to carry out diverse biological effects. The diversity in the roles played by calcineurin closely parallel the importance of intracellular calcium changes to the function of immune cells and other cell types. The changes in intracellular calcium waves in immune cells vary in both amplitude and duration leading to the differential activation of transcription factors at different thresholds of intracellular calcium. Initially demonstrated to influence the nuclear transcriptional activity of NFAT, the role for calcineurin in immune cell regulation may involve the control of other transcriptional elements important to the activation of immune cells, such as c-fos, Elk-1, and NF-κB, and the involvement in other pathways involving the transient release of intracellular calcium, such as apoptosis and neuronal signaling cascades. These varied effects of calcineurin are thus observed as a result of the differential activation of calcineurin mirrored in the changes in intracellular calcium in different cellular environments. 412
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Acknowledgements 16.
We would like to thank the members of the Burakoff laboratory for their helpful discussions. S. Baksh is a recipient of the Medical Research Foundation of Canada post-doctoral fellowship and is an Abrahams post-doctoral Fellow.
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