The Caenorhabditis elegans Homologue of Down Syndrome Critical Region 1, RCN-1, Inhibits Multiple Functions of the Phosphatase Calcineurin

doi:10.1016/S0022-2836(03)00237-7

Journal of Molecular Biology

Volume 328, Issue 1, 18 April 2003, Pages 147-156

https://doi.org/10.1016/S0022-2836(03)00237-7 Get rights and content

Abstract

A conserved family of calcineurin-regulating proteins whose members have been implicated in several disease models such as Down syndrome, Alzheimer's disease, and cardiac hypertrophy has been identified in several organisms including yeast, mice, and humans. We have characterized Caenorhabditis elegans rcn-1, which belongs to this family of calcineurin regulators, and shows approximately 40% identity with the human homologue DSCR-1. rcn-1 is expressed in hypodermal cells, nerve cords and various neurons, vulva epithelial and muscle cells, marginal cells of the pharynx, and structures of the male tail. rcn-1 expression is upregulated by calcineurin activity. RCN-1 binds to calcineurin A from C. elegans lysate in a calcium-dependent manner, and inhibits bovine calcineurin phosphatase activity dose-dependently. In addition, overexpression of RCN-1 results in calcineurin-deficient phenotypes such as small body size, cuticle defects, fertility defects, slow growth, and serotonin-resistant egg-laying defects. Moreover, phenotypes observed in gain-of-function calcineurin mutant animals were restored to normal by RCN-1 overexpression. These results demostrate an effective and specific inhibition of calcineurin in vitro as well as in vivo by RCN-1.

Introduction

Calcineurin, a calcium/calmodulin-activated protein phosphatase, is a calcium-sensing cytoplasmic signaling molecule that is a key component of various signal transduction pathways. Calcineurin acts as a heterodimer composed of a catalytic A subunit and a regulatory B subunit.¹ The heterodimeric enzyme can dephosphorylate the transcription factor NFAT (nuclear factor of activated T-cells) in the presence of calcium, which induces its translocation to the nucleus to turn on other transcription factors.² Calcineurin plays important roles in many biological processes such as cardiac function and development,3., 4., 5. skeletal muscle growth and differentiation,6., 7. and T-cell activation and development.8., 9. Two pharmacological inhibitors of calcineurin, FK506 and cyclosporin A, that block calcineurin phosphatase activity10., 11. have been instrumental in suppressing the immune response during transplant therapy. Several cellular inhibitors of calcineurin have been identified as well, including CAIN/CABIN,12., 13. AKAP79,¹⁴ and CHP.¹⁵

Recently, a family of endogenous calcineurin-regulating proteins was identified in fungi, yeast, and mammals.16., 17., 18., 19. All members of this protein family show a characteristic binding to calcineurin causing inhibition of phosphatase activity. The genes encoding the yeast and mammalian members, Rcn1p and Down syndrome critical region 1 (DSCR1), were shown to be regulated by calcineurin phosphatase activity forming a negative feedback inhibition loop.17., 18., 19. The DSCR1 protein (MCIP1) has been implicated in several disease models, including Down syndrome, Alzheimer disease, and cardiac hypertrophy,19., 20., 21. possibly giving insight into the role of calcineurin function in these diseases. In addition, cardiac overexpression of MCIP1 in cardiac hypertrophic mice inhibits both calcineurin-induced and pressure overload-induced cardiac hypertrophy.22., 23. Thus, the future application of MCIP1 as an endogenous genetic treatment of disease is a possibility.

The Caenorhabditis elegans homologue of DSCR1 was identified originally by Strippoli et al.²⁴ as Ce-DSCR1L and consists of four exons encoding a protein with approximately 38% identity and 75% identity in the most conserved region. In this work, we have characterized the Ce-DSCR1L gene, which we have named rcn-1. Our results demonstrate the effective and specific inhibition of multiple functions of calcineurin phosphatase activity by RCN-1.

Section snippets

The rcn-1 gene, expression, and regulation

Strippoli et al.²⁴ identified a gene on C. elegans cosmid F54E7 that encodes a protein that is approximately 38% identical with MCIP1, the protein encoded by the human gene Down syndrome critical region 1 (DSCR1). This protein contains highly conserved SP repeats observed in MCIP1, the yeast calcineurin inhibitor Rcn1p, and the translocation exon of the calcineurin-activated transcription factor NFATc.²⁵ We cloned the cDNA of gene F54E7.7, which we name rcn-1 according to C. elegans standard

Discussion

Calcineurin, as a calcium-signaling molecule, has drawn much attention from scientists particularly because of its involvement in numerous pathways including cardiac and immune system development and function. Thus, the regulation of calcineurin activity to control and adjust these pathways has been of particular interest recently. Among several molecules that can bind directly and inhibit calcineurin phosphatase activity, members of a novel family of calcineurin regulators that includes Down

C. elegans strains and maintenance

Bristol N2, JC0684 tax-6(p675) IV, and DR466 him-5(e1490) V were obtained from the Caenorhabditis Genetics Center (CGC) at the University of Minnesota, USA. KJ300 cnb-1(jh103) V was isolated in our laboratory,²⁶ and KJ306 cna-1(jh107) IV was isolated recently in our laboratory (J.L. & J.A., unpublished results). Worm breeding and handling were conducted as described.³⁰

cDNA cloning and constructs

To obtain a full-length cDNA clone of rcn-1, nested PCR of a mixed-stage C. elegans cDNA library (kindly donated by A. Fire) was

Acknowledgements

The authors thank the following people: A Coulson for cosmids, the CGC for strains, K. Cunningham and A. Fire for expression vectors and a cDNA library. This work was supported by BK21 (to J.L.), Life Phenomena and Function Research Group (to D.H.K.), and Frontier 21 (CFAHG) (to J.A.).

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GST-pull down assay. GST-pull down assay was performed essentially as described previously [16]. Briefly, recombinant GST or GST fused TAX-6 was immobilized on Glutathione-Sepharose beads.
Calcineurin is a Ca²⁺/Calmodulin activated Ser/Thr phosphatase that is well conserved from yeast to human. In Caenorhabditis elegans, tax-6 and cnb-1 encode catalytic and regulatory subunits of calcineurin, respectively. We performed yeast two-hybrid screening using TAX-6 as a bait to identify calcineurin interacting proteins. KIN-29 is one of proteins that specifically interacted with TAX-6. KIN-29 is a Ser/Thr kinase previously shown to be involved in regulating gene expression of a subset of chemoreceptors in specific neurons. Both TAX-6 and KIN-29 are expressed in hypodermis, muscles, and neurons. Moreover, both calcineurin and kin-29 mutants exhibit similar phenotypes, namely small body size, small brood size, and slow growth. Here we describe specific genetic interaction between tax-6 and kin-29 in regulating body size, serotonin mediated egg laying, and chemoreceptor expression.
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Journal of Molecular Biology

The Caenorhabditis elegans Homologue of Down Syndrome Critical Region 1, RCN-1, Inhibits Multiple Functions of the Phosphatase Calcineurin

Abstract

Introduction

Section snippets

The rcn-1 gene, expression, and regulation

Discussion

C. elegans strains and maintenance

cDNA cloning and constructs

Acknowledgements

Cell

Cell

Cell

Cell

Cell

Cell

Cell

J. Biol. Chem.

Immunity

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Sci.

Genomics

Neuron

Biochem. Biophys. Res. Commun.