Opposing Functions of Calcineurin and CaMKII Regulate G-protein Signaling in Egg-laying Behavior of C. elegans

Ca²⁺/calmodulin-dependent calcineurin has been shown to have important roles in various Ca²⁺ signaling pathways. We have previously reported that cnb-1(jh103) mutants, null mutants of a regulatory B subunit, displayed pleiotropic defects including uncoordinated movement and delayed egg laying in Caenorhabditis elegans. Interestingly, gain-of-function mutants of a catalytic A subunit showed exactly opposite phenotypes to those of cnb-1(null) mutants providing an excellent genetic model to define calcium-mediated signaling pathway at the organism level. Furthermore, calcineurin is also important for normal cuticle formation, which is required for maintenance of normal body size in C. elegans. Genetic interactions between tax-6 and several mutants including egl-30 and egl-10, which are known to be involved in G-protein signaling pathways suggest that calcineurin indeed regulates locomotion and serotonin-mediated egg laying through goa-1(Goα) and egl-30(Gqα). Our results indicate that, along with CaMKII, calcineurin regulates G-protein-coupled phosphorylation signaling pathways in C. elegans.

Introduction

Calcineurin is a calcium/calmodulin-dependent serine/threonine protein phosphatase and is well known to have diverse cellular functions in different cell types and organisms.1, 2, 3, 4 Although it expresses abundantly in neural tissues, calcineurin is well known to express in other tissues as well.⁵ A heterodimer of the catalytic calcineurin A (CnA) and the regulatory calcineurin B (CnB), the structure of the enzyme is highly conserved from yeast to man.⁵ In higher animals, calcineurin translocates NF-AT to the nucleus, thereby activating several cytokine genes, including interleukin-2, which is required for T cell growth. The enzyme is tightly regulated by Ca²⁺/CaM, and plays critical functional roles in several calcium-mediated signal transduction pathways.

The catalytic CnA contains a catalytic and a regulatory domain. The latter contains the CnB-binding domain, and a CaM-binding domain downstream of it. The auto-inhibitory domain (AI) is situated at the C terminus of the enzyme and downstream of the CaM-binding site.6, 7 In the absence of CaM, the AI domain is in close proximity to the catalytic site thereby inhibiting the enzyme activity. Calcium/CaM binding to the enzyme causes conformational changes displacing the AI domain, and leading to a dramatic enhancement of A subunit phosphatase activity. The CaM-binding domain is highly sensitive to proteolytic digestion. When the functional CaM-binding site and the auto-inhibitory domain are truncated, the enzyme transforms into a CaM-independent constitutively active form.6, 8

Calcineurin genes have also been identified in the nematode, Caenorhabditis elegans. tax-6(p675) has been genetically mapped to a locus that encodes the calcineurin A gene.⁹ The enzyme is mainly expressed in neuronal cells and also expressed in the other tissues including hypodermal seam cells, body wall muscle, vulva muscle and spermatheca.¹⁰ Calcineurin regulates fertility, growth, movement, egg laying and multiple aspects of sensory signaling in C. elegans. The enzymatic activity is specifically inhibited by well-known immunosuppressive drugs, FK506 and cyclosporin A in vitro as well as by RCN-1 which is encoded by a member of the calcineurin-regulating family in vivo.¹¹

In C. elegans in vivo evidence for calcineurin functions has come from mutant studies. A loss-of-function mutant of tax-6(p675) exhibits pleiotropic abnormalities including thermotactic and chemotactic defects.⁹ A null mutant of calcineurin B, cnb-1(jh103), shows even more severe defects including lethargic movement and delayed egg laying.¹⁰ Calcineurin has long been implicated to play important roles in the induction of long-term potentiation (LTP) and long-term depression (LTD) in an antagonistic relationship with kinases like the Ca²⁺/calmodulin-dependent kinase II (CaMKII).12, 13 In C. elegans, a G-protein signaling pathway including unc-43/CaMKII, goa-1/G_oα and egl-30/G_qα is known to regulate locomotory and egg laying behaviors. EGL-30/G_qα acts to increase the locomotion rate and egg laying, while the GOA-1/G_oα acts to decrease both behaviors. unc-43/CaMKII is also known to regulate those behaviors through a G_oα/G_qα pathway.¹⁴ Previously, we investigated that calcineurin has an opposing function of unc-43/CaMKII to regulate locomotion and egg laying in C. elegans.¹⁰

In the present study we generated for the first time in C. elegans a gain-of-function mutant of calcineurin by trimethylpsoralen (TMP)/UV mutagenesis. These tax-6(gf) animals lack domains for auto-inhibition and calmodulin-binding, and therefore are expected to synthesize the CaM-independent constitutively active form of a protein phosphatase. Interestingly, these gain-of-function mutants of calcineurin showed phenotypes opposite to those of cnb-1 null mutants. Our genetic studies between the calcineurin mutants and mutants of unc-43, egl-30 and egl-10 further confirm that calcineurin is a component of CaMKII-mediated G-protein-coupled phosphorylation signaling pathways, and regulates locomotory and egg laying behavior in C. elegans.