Mol. Cells, Vol. 18, No. 1, pp. 10-16

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Regulation of Calcineurin, a Calcium/Calmodulin-dependent Protein Phosphatase, in C. elegans

Jaya Bandyopadhyay*, Jungsoo Lee1, and Arun Bandyopadhyay2 Department of Biotechnology, West Bengal University of Technology, Kolkata 700-064, India; 1 Department of Life Science, Gwangju Institute of Science and Technology, Gwangju 500-712, Korea; 2 Indian Institute of Chemical Biology, Kolkata 700-032, India. (Received June 18, 2004; Accepted June 20, 2004) Calcineurin is a calcium/calmodulin-dependent serine/ threonine protein phosphatase. It is a heterodimeric protein consisting of a catalytic subunit calcineurin A, and a regulatory calcium-binding subunit, calcineurin B. The primary sequence of both subunits and het-erodimeric structure is highly conserved from yeast to mammals. Calcineurin has long been implicated in various signaling pathways. Calcineurin genes (cna-1/tax-6 and cnb-1) have been identified in the nema-tode Caenorhabditis elegans, which share high homol-ogy with their Drosophila and mammalian counter-parts. C. elegans calcineurin binds calcium and func-tions as a heterodimeric protein phosphatase establish-ing its biochemical conservation. Calcineurin expresses in diverse tissues implicating its important role in various physiological processes. This review will focus in brief on the expression pattern and regulation of calcineurin including its effect on growth and devel-opment, locomotion, egg-laying, and sensory responses. Keywords: C. elegans; Calcineurin. �

Introduction Alterations in intracellular Ca2+ concentration [Ca2+]i sig-nal diverse physiological responses in different cell types (Clapham, 1995). The amplitude and duration of dynamic Ca2+ signals add to the diversity in signaling of this Ca2+. One of the mechanisms by which Ca2+ acts is by binding to and activating calmodulin (CaM). As an intracellular

Ca2+ receptor, CaM activates a number of target enzymes such as calmodulin-dependent protein kinases and phos- * To whom correspondence should be addressed. Tel: 91-33-2334-0417; Fax: 91-33-2321-7578 E-mail: jban1117@sify.com

phatases. One of these targets is calcineurin that acts as an effector of Ca2+ signalling by regulating the phosphoryla-tion state of proteins (Klee et al., 1988). The enzyme functions as a heterodimer of catalytic A subunit (CaNA; 59 to 62 kDa) and a calcium-binding regulatory B subunit (CaNB; 19 kDa). This two-subunit structure, highly con-served through evolution, is unique among protein phos-phatases, and is essential for activity (Klee et al., 1988).

Calcineurin belongs to a superfamily of protein serine-threonine phosphatases, and is regulated by [Ca2+]i. It is the only protein phosphatase dependent on Ca2+ and CaM for its activity thereby making it one of the most common intracellular transducers of Ca2+ signalling pathways. Changes in [Ca2+]i control gene expression in many cell types by calmodulin-dependent activation of calcineurin (Crabtree, 1999; Olson and Williams, 2000). Calcineurin is well known to express in the nervous system (Goto et al., 1985; Kuno et al., 1992). It plays an important role in the induction of long-term potentiation (LTP) and long-term depression (LTD), and in the establishment of learn-ing and memory (Malenka, 1994; Malleret et al., 2001; Mansuy et al., 1998; Winder et al., 1998). In fact “cal-cineurin” was primarily labeled for its calcium-binding properties and localisation to neuronal tissues (Klee et al., 1979). Although it abundantly expresses in nervous sys-tem, calcineurin is known to express in other tissues also (Kincaid, 1993). The molecular mechanism by which cal-cineurin plays an important role in the regulation of gene expression in mammalian cells has been well documented in the immune system particularly in lymphocytes where it has been intensively characterized (Liu et al., 1991). It was shown that binding of antigen to cell surface recep-tors triggers Ca2+ entry as a result activating calcineurin,

which then removes phosphate groups from transcrip-tional regulatory proteins of the NF-AT family that are necessary for T cell proliferation (Clipstone and Crabtree, 1992; O’Keefe et al., 1992). Dephosphorylated NF-AT

M olecules and

Cells KSMCB 2004

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Jaya Bandyopadhyay et al. 11 �

cna-1/ tax-6 is characterized by a multidomain structure containing a catalytic domain at its N-terminus, similar to that of protein phosphatase 1 (Griffith et al., 1995; Kiss-inger et al., 1995), followed by the binding regions for the CNB-1 subunit and calmodulin (CaM) (Bandyopadhyay et al., 2002; Kuhara et al., 2002). An autoinhibitory (AI) domain is located near the C-terminus, which is probably displaced upon CaM binding thereby activating the en-zyme (Hubbard and Klee, 1989) (Fig. 1). Truncations of the catalytic subunit that remove the AI domain result in a constitutively active enzyme that no longer requires Ca2+ (Hubbard and Klee, 1989). Both cna-1/tax-6 and cnb-1 interact with one another in a Ca2+ dependent manner con-ferring maximum phosphatase activity (18 pmol phos-phate/min/µg protein) (Bandyopadhyay et al., 2002; Cohen, 1989). Therefore, C. elegans calcineurin subunits

(nuclear factor - activated T cell) proteins translocate from the cytoplasm to the nucleus, where they bind similar rec-ognition elements within target genes, in association with other transcription factors such as AP-1. Downstream genes critical for T-cell activation (eg, interleukin-2) are induced in this way (Schreiber and Crabtree, 1992). The pharmacological actions of immunosuppressive drugs

such as cyclosporin A and FK-506 are based on inhibition of calcineurin activity in immune effector cells. Cal-cineurin activity is also critical for other Ca2+-regulated processes, such as neutrophil chemotaxis (Hendey et al., 1992; Lawson and Maxfield, 1995), and in the pathogene-sis of hypertrophic cardiomyopathy (Molkentin, 1998). It is also known to regulate programmed cell death and in-duce apoptosis through Bcl2 (Shibasaki and McKeon, 1995; Wang et al., 1999). Evidence also shows that cal-cineurin modulates Ca2+ release by directly interacting with Ins(1,4,5)P3R (IP3 receptor) and ryanodine receptor (Bandyopadhyay et al., 2000a; 2000b; Cameron et al., 1995). In other cell types, calcineurin has been implicated to regulate Ca2+ pumps and exchangers to maintain Ca2+ homeostasis (Stark, 1996). For example, calcineurin regu-lates the Na+/K+ ATPase in renal tubule cells (Aperia et al., 1992) and the NMDA receptor in neurons (Lieberman and Mody, 1994; Tong et al., 1995). In Saccharomyces cere-visiae, calcineurin is required for cation homeostasis and regulates cell wall biosynthesis by Tcn1/Crz1, a transcrip-tion factor distantly related to NF-AT, promoting translo-cation to nucleus and subsequent gene activation (Cun-ningham and Fink, 1994; 1996; Cyert and Thorner, 1992;

Hemenway et al., 1995; Mazur et al., 1995; Moser et al., 1996; Nakamura et al., 1993). It is also known to regulate adaptation to high salt stress (Hirata et al., 1995). Calcineurin in C. elegans Calcineurin genes have been identified and well charac-terized in lower eukaryotes and in higher animals, includ-ing plants as well (reviewed by Rusnak and Mertz, 2000). However, this review for the most part will focus the structures and physiological roles of calcineurin genes in the nematode, Caenorhabditis elegans.

C. elegans is a free-living soil nematode having a rela-tively short life cycle and a simple body structure. Be-cause of its short generation time and simple structures, C. elegans has always been a very useful and ideal model system for extensive genetic and cell biological studies. Furthermore, C. elegans has always been a popular sys-tem for having several advantages for studying gene func-tion at the organism level. Especially, completion of the genome sequencing in conjunction with forward and re-verse genetics has made it reasonable to study gene struc-ture and function of both known and novel proteins in addition to investigating gene function of vertebrate

homologues. Indeed more than 19,000 proteins were pre-dicted from the nearly complete genome sequence, and approximately 40% of these are related to proteins deter-mined in other organisms (The C. elegans Sequencing Consortium, 1998). Structure and functional domains of cal-cineurin in C. elegans Conserved calcineurin genes have also been found in C. elegans (Wheelan et al., 1999). Three catalytic genes for CaNA subunit have been identified in vertebrate species,

of which calcineurin Aα and calcineurin Aβ are ubiqui-tously expressed, while calcineurin Aγ expression is re-stricted to the testis and brain (Crabtree, 1999; Klee et al., 1998; Rusnak and Mertz, 2000). However, in C. elegans,

the catalytic subunit is encoded by a single cna-1/tax-6 gene (Bandyopadhyay et al., 2002; Kuhara et al., 2002) and exhibits approximately 77% overall amino acid iden-tity to one isoform of human calcineurin A (Kincaid et al., 1990; Kuhara et al., 2002). A conserved regulatory sub-unit (CaNB) like in higher animals and yeast is also found in C. elegans exhibiting 80% amino acid identity to hu-man and Drosophila calcineuin B, and 58% identity with yeast calcineurin B (Bandyopadhyay et al., 2002; Kuhara et al., 2002). Calcineurin B, encoded by the cnb-1 gene is also characterized having the four “EF-hands” for Ca2+

binding. Calcineurin B is known to bind 4 molecules of Ca2+ with high affinity (Kd ≤ 10−6M) and has sequence homology with CaM and troponin C, another calcineurin binding protein (Aitken et al., 1982). Ca2+ binding proper-ties of C. elegans CNB-1 have also been demonstrated biochemically (Bandyopadhyay et al., 2002). Both sub-units of calcineurin migrate as single bands around 60 kDa for CNA-1 and 16 kDa for CNB-1, respectively as evidenced from western blotting experiments with total protein extracts from wild-type worms (Bandyopadhyay et al., 2002).

12 C. elegans Calcineurin �

A

B Fig. 1. Structure of calcineurin subunits. A. Calcineurin A, the catalytic subunit. Shaded regions represent different domains of calcineurin A. B. Calcineurin B, the regulatory subunit. It has four Ca2+-binding EF-hand motifs. represent a conserved branch of PP2B family of protein phosphatases having important physiological signifi-cances (Crabtree, 2001). Expression and physiological significance of calcineurin in C. elegans Calcineurin expresses in diverse tissues in C. elegans (Bandyopadhyay et al., 2002; Kuhara et al., 2002). Both cna-1 and cnb-1 green fluorescent protein (gfp) reporter transgene expressions were detected at all developmental stages starting from early comma stage embryos to adult stages. Calcineurin also expresses in vulval muscle (muscles required for egg-laying), body-wall muscle, and in a majority of neuronal cell bodies in the head and tail (Fig. 2). Distinct expressions of calcineurin have also been detected in hypodermal seam cells / hypodermal tissue that is required for cuticle formation. Calcineurin also expresses in the male germline, and therefore may have possible roles in germline development. Kuhara et al. (2002) have shown calcineurin to express specifically in sensory neurons and interneurons including muscle cells.

Like mammalian and Drosophila homologues, C. ele-gans calcineurin also plays important role in various physiological processes ranging from locomotion to egg laying. In C. elegans in vivo evidences of calcineurin functions have came from mutant studies. Kuhara et al. (2002) reported that mutations of calcineurin A (tax-6 mutant) display pleiotropic abnormalities, including sev-eral abnormal sensory behaviours. Loss of functions of calcineurin A results in thermotactic and chemotactic de-fects. In contrast, over-activation of calcineurin A leads to cryophilic phenotypes. These animals further displayed body size and growth defects too. Genetic and physio-logical data from Kuhara et al. (2002) suggests that cal-cineurin is required for controlling sensory neuron re-sponsiveness, which is Ca2+-dependent. Particularly, TAX- 6 calcineurin is activated by the influx of Ca2+ through

hypothetical Ca2+ channels such as TAX-4/TAX-2 chan-nels as a result negatively regulating sensory responses in C. elegans (Kuhara et al., 2002).

On the other hand our studies on calcineurin mutants have further revealed some significant roles of the phos-phatase in C. elegans (Bandyopadhyay et al., 2002). Null mutants of calcineurin B, cnb-1(jh103), generated by re-verse genetics (Park et al., 2001), displayed lethargic movement and delayed egg-laying. Furthermore, our mu-tant studies revealed that cnb-1 is involved in normal cu-ticle formation, sperm morphology, and brood size. In C. elegans G-protein signalling via Ca2+/CaM-dependent protein kinase II (CaMKII) is known to regulate locomo-tory and egg-laying behaviours (Robatzek and Thomas, 2000). Phenotypes of loss of function mutants of cnb-1 interestingly resembled the gain-of function mutants of CaMKII, encoded by the unc-43 gene (Reiner et al., 1999) (Fig. 3). Besides, a transgenic gain-of-function mu-tant of G0-protein α-subunit, goa-1, also displayed lethar-gic movement and egg retention phenotypes similar to those of unc-43(gf) mutants (Mendel et al., 1995). On the contrary, loss-of-function mutants of unc-43 and goa-1 showed hyperactive movement and premature egg laying (Mendel et al., 1995). Thus, mutations in goa-1 exhibit similar phenotypes to those of unc-43 mutants, but oppo-site to that of cnb-1 mutants. In particular, the egg-laying phenotypes ascertained by the experiments with exogenous serotonin, known to stimulate egg-laying in wild-type worms (Trent et al., 1983), suggest that calcineurin may have an antagonistic role in CaMKII-regulated phos-phorylation signalling pathways (Bandyopadhyay et al., 2002) (Table 1).

Additionally, a gain-of-function mutant of calcineurin A, generated by chemical mutagenesis, showed phenotypes opposite to those of cnb-1 null mutants (Lee et al., unpub-lished observation). In fact these cna-1 mutant worms are characterized having the constitutively active form of the enzyme as they lack the C-terminal CaM-binding and AI domains (Hubbard and Klee, 1989). Moreover, epistasis studies with double mutants of Gq-�������� �-subunit (en-coded by egl-30) and gain-of-function mutant of cal-cineurin A, cna-1 (gf) suggest that cna-1 may function up-stream of egl-30 (Gqα) (Lee et al., unpublished observa-tion). These results further confirm that calcineurin is in-deed a component of CaMKII-mediated G-protein-coupled phosphorylation signaling pathways, and regulates locomo-tory and egg-laying behaviors in C. elegans. Regulators of C. elegans calcineurin Calcineurin has long been known to be inhibited by im-munosuppressant drugs cyclosporine A (CsA) and FK506 that are specific potent inhibitors of the enzyme. These drugs inhibit calcineurin phosphatase activity when com-

Jaya Bandyopadhyay et al. 13 �

A B C D Fig. 2. Expression and localization of calcineurin. CNA-1::GFP expression in (A) ventral nerve cord (arrow), vulva muscles (arrow-head), and (B) body-wall muscles of the mid-body region. CNB-1::GFP expression in (C) spermatheca (arrowhead) and intestine (ar-row). Bar indicates 20 µm. (D) Immunostaining of wild-type sperm with anti-CNA-1 antibodies (upper panel) and the respective nu-clei are shown by DAPI staining (lower panel). Bar indicates 2 µm. Table 1. Characterization of serotonin-mediated egg-laying phenotypes.

No. of animals laying the indicated number of eggs after treatment with:

M9 buffer 5-HT Imipramine

Genotype >7 4-7 1-3 0 >7 4-7 1-3 0 >7 4-7 1-3 0

N2 (WT) cnb-1 cnb-1;Ex[cnb-1] unc-43(null) unc-43(gf) unc-43(gf);cnb-1

0 0 0 0 0 0

0 0 0 0 1 0

2 1 3 2

11 10

46 47 45 46 36 38

19 0 6

26 1 0

6 0 4

18 6 0

20 8

21 4

19 12

3 40 17

0 3

36

20 0

22 3 4 0

18 5

19 15 18

0

6 21

7 26 18 14

4 22

0 4 8

34

Worms were treated with either exogenous 5-HT, imipramine or control M9 buffer for 90 min after which laid eggs were counted.

plexed to their respective cytoplasmic receptors, cyclo-phillin and FKBP12 (Liu et al., 1991). A number of other compounds have also been demonstrated to exhibit inhibi-tory activity against calcineurin (reviewed by Rusnak and Mertz, 2000). In addition to synthetic inhibitors, cal-cineurin protein phosphatase activity is also known to be potentially inhibited by certain endogenous regulators like protein kinase A anchoring protein (AKAP79) (Coghlan et al., 1995), cain/cabin 1 (Lai et al., 1998; Sun et al., 1998), and calcineurin homologous protein (CHP) (Lin et al., 1999). In C. elegans, in vitro phosphatase activity of calcineurin was shown to be completely inhibited by CsA implicating a conserved phenomenon of phosphatase in-hibition by these pharmacological inhibitors even in a distantly related organism like the nematode. On the other hand, in several organisms including yeast, mice, and humans, members of a conserved family of calcineurin-regulating proteins have been identified to have signifi-cant roles in a number of disease models such as Alz-heimer’s disease, Down syndrome, and cardiac hypertro-

phy. For example, genes encoding the yeast and mammal-ian homologues of these inhibitors, Rcn1p and Down syndrome critical region 1 (DSCR1), are shown to regu-late calcineurin activity and vice versa in a negative feed back mechanism. (Fuentes et al., 2000; Kingsbury and Cunningham, 2000; Rothermel et al., 2000). In C. elegans, inhibitory actions of synthetic inhibitors as well as en-dogenous regulators have also been illustrated. The C. elegans homologue of DSCR1 (Ce-DSCR1L) was origi-nally identified by Strippoli et al. (2000). We have further characterized the C. elegans Ce-DSCR1L as rcn-1 (Lee et al., 2003). It belongs to this family of calcineurin regula-tors, and shows approximately 40% identity with the hu-man counterpart. rcn-1 has been shown to express in hypodermal cells, nerve cords and various neurons, vulva epithelial and muscle cells, marginal cells of the pharynx, and structures of the male tail. The expression of rcn-1 is upregulated by calcineurin activity. Biochemically RCN-1 has been shown to bind calcineurin A and inhibit phos- phatase activity in vitro (Lee et al., 2003). In addition,

14 C. elegans Calcineurin � A B C

D E F

Fig. 3. Locomotory defects in mutants of cnb-1 and unc-43. Five animals were placed in the center of a bacterial lawn and photographed 5 min later. (A) Tracks of movement by wild-type animals, (B) cnb-1mutants, (C) rescued cnb-1 mutants, (D) unc-43(lf) mutants, (E) unc-43(gf) mutants, and (F) cnb-1; unc-43(gf) double mutants. Bar indicates 1 mm. Fig. 4. A model for regulation of locomotion and egg-laying behavior in C. elegans. GOA-1(Goα) and EGL-30(Gqα) are known to be coupled to receptors in the plasma membrane and both are regulated by RGS proteins (EGL-10 and EAT-16). Lo-comotion and egg-laying behavior are activated through EGL-30(Gqα) pathway. On the other hand, GOA-1(Goα) inhibits locomotion and egg laying. We suggest that calcineurin (CN) regulate locomotion and egg-laying activity via GOA-1(Goα)/ EGL-30(Gqα) by counter-acting unc-43/CaMKII in C. elegans. overexpression of RCN-1 results in calcineurin-deficient phenotypes such as small body size, cuticle defects, fertile-ity defects, slow growth, and serotonin-resistant egg-laying defects in the worms. Also, phenotypes displayed by the gain-of-function calcineurin mutant animals were restored to normal phenotype by RCN-1 overexpression. These results demonstrate an effective and specific inhibition of calcineurin in vitro as well as in vivo by RCN-1.

Conclusions and Perspectives During the last two decades the biological roles of cal-cineurin have progressed from a putative inhibitor of the calmodulin-dependent phosphodiesterase to the revolu-tionized discovery that it is the target of the immuno-suppressant drugs cyclosporine A and FK506. Various studies have utilized these immunosuppressant drugs to demonstrate calcineurin as a major player in Ca2+-de-pendent eukaryotic signal transduction pathways. Subse-quently, modern genetic techniques have been used to explore its biological function in model organism such as yeast, filamentous fungi, plants, vertebrates, and mam-mals. In recent years the structure of calcineurin have been determined using X-ray diffraction methods, bio-chemical, spectroscopic, and physical studies to unravel its catalytic mechanism. Further insights into the role of calcineurin in the signal transduction pathway have been revealed from the genetic studies of calcineurin mutants in C. elegans. Calcineurin may function upstream or downstream of CaMKII, which is known to specifically regulate locomotion via the Go/Gq signaling network (Ro-batzek and Thomas, 2000). Epistatic phenotypes between these two functionally antagonistic proteins, i.e. cal-cineurin and CaMKII would allow us to distinguish where calcineurin may function in relation to CaMKII (UNC-43). Phosphatase and kinase, two functionally differing mole-cules like calcineurin and CaMKII, are well known to work in an opposing manner to induce either LTP or LTD in mammalian neurons. It has also been demonstrated that differential Ca2+ signaling can be regulated by distinct Ca2+ oscillations. CaMKII is known to be regulated by transient Ca2+ spikes, while calcineurin is associated with prolonged increases in basal [Ca2+]i (Dolmetsch et al., 1998). Hence, it may be proposed that Ca2+ levels or Ca2+ oscillations in cells affecting locomotion and egg-laying may be directing the opposing functions of calcineurin and CaMKII towards either activating or deactivating locomotory and egg-laying activities via the Go/Gq -mediated signaling network (Fig. 4). Acknowledgments We gratefully acknowledge Dr. Joohong Ahnn for his continuous support and encouragement. We also acknowledge Dr. Byung Jae Park, Dr. Jeonghoon Cho, Jiyeon Lee, Jin Il Lee, Sunki Jung, and Changhoon Jee for their signifi-cant contributions during their tenure in J. Ahnn’s laboratory. J. Lee was supported by the grant M103KV010019-03K2201-01920 from the Korean Ministry of Science and Technology.

References Aitken, A., Cohen, P., Santikarn, S., Williams, D. H., Calder, A.

G., Smith, A., and Klee, C. B. (1982) Identification of the

Jaya Bandyopadhyay et al. 15 �

NH2-terminal blocking group of calcineurin B as myristic acid. FEBS Lett. 150, 314−318.

Aperia, A., Ibarra, R., Svensson, L.-B., Klee, C., and Greengard, P. (1992) Calcineurin mediates α-adrenergic stimulation of Na1, K1-ATPase activity in renal tubule cells. Proc. Natl. Acad. Sci. USA 89, 7394−7397.

Bandyopadhyay, A., Shin, D. W., Ahn, J. O., and Kim, D. H. (2000a) Calcineurin regulates ryanodine receptor/Ca2+-release channels in rat heart. Biochem. J. 352, 61−70.

Bandyopadhyay, A., Shin, D. W., and Kim, D. H. (2000b) Regu-lation of ATP-induced calcium release in COS-7 cells by cal-cineurin. Biochem. J. 348, 173−181.

Bandyopadhyay, J., Lee, J., Lee, J. I., Yu, J. R., Jee, C., Cho, J. H., Jung, S., Lee, M. H., Zannoni, Z., Singson, A., Kim, D. H., Koo, H. S., and Ahnn, J. (2002) Calcineurin, a cal-cium/calmodulin-dependent protein phosphatase, is involved in movement, fertility, egg-laying, and growth in Caenor-habditis elegans. Mol. Biol. Cell 13, 3281−3293.

Cameron, A. M., Steiner, J. P., Roskams, A. J., Ali, S. M., Ron-nett, G. V., and Snyder, S. H. (1995) Calcineurin associated with the inositol 1,4,5-trisphosphate receptor-FKBP12 com-plex modulates Ca2+ flux. Cell 83, 463−472.

Clapham, D. E. (1995) Calcium signaling. Cell 80, 259−268. Clipstone, N. A. and Crabtree, G. R. (1992) Identification of

calcineurin as a key signalling enzyme in T-lymphocyte acti-vation. Nature 357, 695−697.

Coghlan, V. M., Perrino, B. A., Howard, M., Langeberg, L. K., Hicks, J. B., Gallatin, W. M., and Scott, J. D. (1995) Associa-tion of protein kinase A and protein phosphatase 2B with a common anchoring protein. Science 267, 108−111.

Cohen, P. (1989) The structure and regulation of protein phos-phatases. Annu. Rev. Biochem. 58, 453−508.

Crabtree, G. R. (1999) Generic signals and specific outcomes: signaling through Ca2+, calcineurin, and NF-AT. Cell 96, 611− 614.

Crabtree, G. R. (2001) Calcium, calcineurin and the control of transcription. J. Biol. Chem. 276, 2313−2316.

Cunningham, K. W. and Fink, G. R. (1994) Calcineurin-dependent growth control in Saccharomyces cerevisiae mu-tants lacking PMC1, a homolog of plasma membrane Ca2+ ATPases. J. Cell Biol. 124, 351−363.

Cunningham, K. W. and Fink, G. R. (1996) Calcineurin inhibits VCX1-dependent H+/Ca2+ exchange and induces Ca2+ AT-Pases in Saccharomyces cerevisiae. Mol. Cell. Biol. 16, 2226− 2237.

Cyert, M. S. and Thorner, J. (1992) Regulatory subunit (CNB1 gene product) of yeast Ca2+/calmodulin-dependent phospho-protein phosphatases is required for adaptation to pheromone. Mol. Cell. Biol. 12, 3460−3469.

Dolmetsch, R. E., Xu, K., and Lewis, R. S. (1998) Calcium os-cillations increase the efficiency and specificity of gene ex-pression. Nature 392, 933−936.

Fuentes, J. J., Genesca, L., Kingsbury, T. J., Cunningham, K. W., Perez-Riba, M., Estivill, X., and de la Luna, S. (2000) DSCR1, overexpressed in Down syndrome, is an inhibitor of calcineurin-mediated signaling pathways. Hum. Mol. Genet. 9, 1681−1690.

Goto, S., Yamamoto, H., Fukunaga, K., Iwasa, T., Matsukado, Y., and Miyamoto, E. (1985) Dephosphorylation of micro-tubule-associated protein 2, tau factor, and tubulin by cal-

cineurin. J. Neurochem. 45, 276−283. Griffith, J. P., Kim, J. L., Kim, E. E., Sintchak, M. D., Thomson,

J. A., Fitzgibbon, M. J., Fleming, M. A., Caron, P. R., Hsiao, K., and Navia, M. A. (1995) X-ray structure of calcineurin inhibited by the immunophilin-immunosuppressant FKBP12-FK506 complex. Cell 82, 507−522.

Hemenway, C. S., Dolinski, K., Cardenas, M. E., Hiller, M. A., Jones, E. W., and Heitman, J. (1995) vph6 mutants of Sac-charomyces cerevisiae require calcineurin for growth and are defective in vacuolar H(+)-ATPase assembly. Genetics 141, 833−844.

Hendey, B., Klee, C. B., and Maxfield, F. R. (1992) Inhibition of neutrophil chemokinesis on vitronectin by inhibitors of cal-cineurin. Science 258, 296−299.

Hirata, D., Harada, S. I., Namba, H., and Miyakawa, T. (1995) Adaptation to high salt stress in Saccharomyces cerevisiae is regulated by Ca2+/calmodulin-dependent phosphoprotein phosphatase (calcineurin) and cAMP-dependent protein kinase. Mol. Gen. Genet. 249, 257−264.

Hubbard, M. J. and Klee, C. B. (1989) Functional domain struc-ture of calcineurin A: mapping by limited proteolysis. Bio-chemistry 28, 1868−1874.

Kincaid, R. (1993) Calmodulin-dependent protein phosphatases from microorganisms to man: a study in structural conserva-tism and biological diversity. Adv. Second Messenger Phos-phoprotein Res. 27, 1−23.

Kincaid, R. L., Giri, P. R., Higuchi, S., Tamura, J., Dixon, S. C., Marietta, C. A., Amorese, D. A., and Martin, B. M. (1990) Cloning and characterization of molecular isoforms of the catalytic subunit of calcineurin using nonisotopic methods. J. Biol. Chem. 265, 11312−11319.

Kingsbury, T. J. and Cunningham, K. W. (2000) A conserved family of calcineurin regulators. Genes Dev. 14, 1595−1604.

Kissinger, C. R., Parge, H. E., Knighton, D. R., Lewis, C. T., Pelletier, L. A., Tempczyk, A., Kalish, V. J., Tucker, K. D., Showalter, R. E., Moomaw, E. W., et al. (1995) Crystal struc-tures of human calcineurin and the human FKBP12-FK506-calcineurin complex. Nature 378, 641−644.

Klee, C. B., Crouch, T. H., and Krinks, M. H. (1979) Subunit structure and catalytic properties of bovine brain Ca2+-dependent cyclic nucleotide phosphodiesterase. Biochemistry 18, 722−729.

Klee, C. B., Draetta, G. F., and Hubbard, M. J. (1988) Cal-cineurin. Adv. Enzymol. Relat. Areas Mol. Biol. 61, 149−200.

Klee, C. B., Ren, H., and Wang, X. (1998) Regulation of the calmodulin-stimulated protein phosphatase, calcineurin. J. Biol. Chem. 273, 13367−13370.

Kuhara, A., Inada, H., Katsura, I., and Mori, I. (2002) Negative regulation and gain control of sensory neurons by the C. ele-gans calcineurin TAX-6. Neuron 33, 751−763.

Kuno, T., Mukai, H., Ito, A., Chang, C. D., Kishima, K., Saito, N., and Tanaka, C. (1992) Distinct cellular expression of cal-cineurin A alpha and A beta in rat brain. J. Neurochem. 58, 1643−1651.

Lai, M. M., Burnett, P. E., Wolosker, H., Blackshaw, S., and Snyder, S. H. (1998) Cain, a novel physiological protein in-hibitor of calcineurin. J. Biol. Chem. 273, 18325−18331.

Lawson, M. A. and Maxfield, F. R. (1995) Ca2+- and cal-cineurin-dependent recycling of an integrin to the front of migrating neutrophils. Nature 377, 75−79.

16 C. elegans Calcineurin �

Lee, J. I., Dhakal, B. K., Lee, J., Bandyopadhyay, J., Jeong, S. Y., Eom, S. H., Kim, D. H., and Ahnn, J. (2003) The Caenorhabditis elegans homologue of Down syndrome criti-cal region 1, RCN-1, inhibits multiple functions of the phos-phatase calcineurin. J. Mol. Biol. 328, 147−156.

Lieberman, D. N. and Mody, I. (1994) Regulation of NMDA channel function by endogenous Ca2+-dependent phosphatase. Nature 369, 235−239.

Lin, X., Sikkink, R. A., Rusnak, F., and Barber, D. L. (1999) Inhibition of calcineurin phosphatase activity by a cal-cineurin B homologous protein. J. Biol. Chem. 274, 36125− 36131.

Liu, J., Farmer, J. D. Jr., Lane, W. S., Friedman, J., Weissman, I., and Schreiber, S. L. (1991) Calcineurin is a common target of cyclophilin-cyclosporin A and FKBP-FK506 complexes. Cell 66, 807−815.

Malenka, R. C. (1994) Synaptic plasticity in the hippocampus: LTP and LTD. Cell 78, 535−538.

Malleret, G., Haditsch, U., Genoux, D., Jones, M. W., Bliss, T. V., Vanhoose, A. M., Weitlauf, C., Kandel, E. R., Winder, D. G., and Mansuy, I. M. (2001) Inducible and reversible en-hancement of learning, memory, and long-term potentiation by genetic inhibition of calcineurin. Cell 104, 675−686.

Mansuy, I. M., Mayford, M., Jacob, B., Kandel, E. R., and Bach, M. E. (1998) Restricted and regulated overexpression reveals calcineurin as a key component in the transition from short-term to long-term memory. Cell 92, 39−49.

Mazur, P., Morin, N., Baginsky, W., El-Sherbeini, M., Clemas, J. A., Nielsen, J. B., and Foor, F. (1995) Differential expression and function of two homologous subunits of yeast 1,3-β-D-glucan synthase. Mol. Cell. Biol. 15, 5671−5681.

Mendel, J. E., Korswagen, H. C., Liu, K. S., Hajdu-Cronin, Y. M., Simon, M. I., Plasterk, R. H. A., and Sternberg, P. W. (1995) Participation of the protein Go in multiple aspects of behavior in C. elegans. Science 267, 1652−1655.

Molkentin, J. D. (1998) Calcineurin inhibition and cardiac hy-pertrophy. Science 282, 1007a.

Moser, M. J., Geiser, J. R., and Davis, T. N. (1996) Ca2+-calmodulin promotes survival of pheromone-induced growth arrest by activation of calcineurin and Ca2+-calmodulin-dependent protein kinase. Mol. Cell. Biol. 16, 4824−4831.

Nakamura, T., Liu, Y., Hirata, D., Namba, H., Harada, S.-I., Hirokawa, T., and Miyakawa, T. (1993) Protein phosphatase type 2B (calcineurin)-mediated, FK506-sensitive regulation of intracellular ions in yeast is an important determinant for adaptation to high salt stress conditions. EMBO J. 12, 4063− 4071.

O’Keefe, S. J., Tamura, J., Kincaid, R. L., Tocci, M. J., and O’Neill, E. A. (1992) FK-506- and CsA-sensitive activation of the interleukin-2 promoter by calcineurin. Nature 357, 692−694.

Olson, E. N. and Williams, R. S. (2000) Calcineurin signaling and muscle remodeling. Cell 101, 689−692.

Park, B. J., Lee, J. I., Lee, J., Kim, S., Choi, K. Y., Park, C. S.,

and Ahnn, J. (2001) Isolation of deletion mutants by reverse genetics in Caenorhabditis elegans. Korean J. Biol. Sci. 5, 65−69.

Reiner, D. J., Newton, E. M., Tian, H., and Thomas, J. H. (1999) Diverse behavioral defects caused by mutations in Caenor-habditis elegans unc-43 CaM kinase II. Nature 402, 199−203.

Robatzek, M. and Thomas, J. H. (2000) Calcium/calmodulin-dependent protein kinase II regulates C. elegans in concert with a Go/Gq signaling network. Genetics 156, 1069−1082.

Rothermel, B., Vega, R. B., Yang, J., Wu, H., Bassel-Duby, R., and Williams, R. S. (2000) A protein encoded within the Down syndrome critical region is enriched in striated mus-cles and inhibits calcineurin signaling. J. Biol. Chem. 275, 8719−8725.

Rusnak, F. and Mertz, P. (2000) Calcineurin: form and function. Physiol. Rev. 80, 1483−1521.

Schreiber, S. L. and Crabtree, G. R. (1992) The mechanism of action of cyclosporin A and FK506. Immunol. Today 13, 136−142.

Shibasaki, F. and McKeon, F. (1995) Calcineurin functions in Ca2+-activated cell death in mammalian cells. J. Cell Biol. 131, 735−743.

Stark, M. J. (1996) Yeast protein serine/threonine phosphatases: multiple roles and diverse regulation. Yeast 12, 1647−1675.

Strippoli, P., Lenzi, L., Petrini, M., Carinci, P., and Zannotti, M. (2000) A new gene family including DSCR1 (Down syndrome candidate region 1) and ZAKI-4: characterization from yeast to human and identification of DSCR1-like 2, a novel human member (DSCR1L2). Genomics 64, 252−263.

Sun, L., Youn, H. D., Loh, C., Stolow, M., He, W., and Liu, J. O. (1998) Cabin 1, a negative regulator for calcineurin signal-ling in T lymphocytes. Immunity 8, 703−711.

The C. elegans Sequencing Consortium (1998) Genome se-quence of the nematode C. elegans: a platform for investigat-ing biology. Science 282, 2012−2018.

Tong, G., Shepherd, D., and Jahr, C. E. (1995) Synaptic desensi-tization of NMDA receptors by calcineurin. Science 267, 1510−1512.

Trent, C., Tsung, N., and Horvitz, H. R. (1983) Egg-laying de-fective mutants of the nematode C. elegans. Genetics 104, 619−647.

Wang, H. G., Pathan, N., Ethell, I. M., Krajewski, S., Yamagu-chi, Y., Shibasaki, F., McKeon, F., Bobo, T., Franke, T. F., and Reed, J. C. (1999) Ca2+-induced apoptosis through cal-cineurin dephosphorylation of BAD. Science 284, 339−343.

Wheelan, S. J., Boguski, M. S., Duret, L., and Makalowski, W. (1999) Human and nematode orthologs--lessons from the analysis of 1800 human genes and the proteome of Caenor-habditis elegans. Gene 238, 163−170.

Winder, D. G., Mansuy, I. M., Osman, M., Moallem, T. M., and Kandel, E. R. (1998) Genetic and pharmacological evidence for a novel, intermediate phase of long-term potentiation suppressed by calcineurin. Cell 92, 25−37.