Proc. Natl. Acad. Sci. USA Vol. 93, pp. 9253-9258, August 1996 Neurobiology Direct modulation of calmodulin targets by the neuronal calcium sensor NCS-1 (calmodulin-binding proteins/calcineurin/neuronal calcium-binding protein/nitric oxide synthase/phosphodiesterase) NICOLAS C. SCHAAD*, EDOUARD DE CASTROt, SERGE NEFt, SARAH HEGIt, ROBERT HINRICHSENt, MARYANN E. MARTONE§, MARK H. ELLISMAN§, ROBERT SIKKINK$, FRANK RUSNAK$, JURGEN SYGUSHII, AND PATRICK NEFt** tDepartment of Biochemistry, Science II, University of Geneva, CH-1211 Geneva, Switzerland; *Division of Clinical Psychopharmacology, Institutions Universitaires de Psychiatrie, CH-1225 Geneva, Switzerland; I1Department of Biochemistry, University of Montreal, CP 6128, Station Centreville, Montreal, PQ, Canada H3C 3J7; tDivision of Basic Sciences, Fred Hutchinson Cancer Research Center, 1124 Columbia Street, Seattle, WA 98104-2092; §Microscopy and Imaging Resource, University of California at San Diego, La Jolla, CA 92093-0608; and ISection of Hematology Research and Department of Biochemistry and Molecular Biology, Mayo Clinic and Foundation, Rochester, MN 55905 Communicated by Stephen R. Heinemann, The Salk Institute for Biological Studies, La Jolla, CA, May 14, 1996 (received for review January 15, 1996) ABSTRACT Ca2+ and its ubiquitous intracellular recep- calcium sensor Recoverin (9). NCS-1 inhibits rhodopsin phos- tor calmodulin (CaM) are required in the nervous system, phorylation in a calcium-dependent manner (8). In this assay, among a host of cellular responses, for the modulation of CaM is ineffective. Our previous work (10) indicated that several important enzymes and ion channels involved in NCS-1 binds only two Ca2+ ions with a high cooperativity and synaptic efficacy and neuronal plasticity. Here, we report that with a higher affinity (-3 x 10-7 M) than CaM (-4-20 x 10-6 CaM can be replaced by the neuronal calcium sensor NCS-1 M), which binds four Ca2+ ions. We have also shown that both in vitro and in vivo. NCS-1 is a calcium binding protein NCS-1 changes its conformation, and most likely its activity, in with two Ca2+-binding domains that shares only 21% of a calcium- and magnesium-dependent manner (10). We there- homology with CaM. We observe that NCS-1 directly activates fore concluded that NCS-1 is not a Ca2+-buffering protein like two Ca2+/CaM-dependent enzymes (3':5'-cyclic nucleotide calbindin and parvalbumin, since it exhibits all the properties phosphodiesterase and protein phosphatase calcineurin). Co- of a Ca2+ sensor that responds, like CaM, to Ca2+ fluctuations activation of nitric oxide synthase by NCS-1 and CaM results by exposing hydrophobic surfaces in order to interact with its in a higher activity than with CaM alone. Moreover, NCS-1 is target(s). Since NCS-1 is not only present in photoreceptors, coexpressed with calcineurin and nitric oxide synthase in but also in various neurons throughout the entire nervous several neuron populations. Finally, injections of NCS-1 into system, we wondered what function NCS-1 might fulfill in calmodulin-defective cam' Paramecium partially restore wild- nonretinal neurons. type behavioral responses. With this highly purified prepara- Here we report that a highly purified preparation of NCS-1 tion of NCS-1, we have obtained crystals suitable for crystal- can substitute or potentiate calmodulin effects both in vitro and lographic structure studies. NCS-1, despite its very different in vivo. NCS-1 can directly activate CaM-dependent enzymes structure, distribution, and Ca2+-binding affinity as com- such as the PDE or the protein phosphatase calcineurin, and pared with CaM, can substitute for or potentiate CaM func- is also capable of potentiating the activity of NOS. Coexpres- tions. Therefore, NCS-1 represents a novel protein capable of sion of NCS-1 with these enzymes is observed in several mediating multiple Ca2+-signaling pathways in the nervous neuron populations. In vivo, NCS-1 alone partially restores the system. wild-type behavior in calmodulin-defective cam' Paramecium mutants. In the nervous system, calmodulin (CaM) directly modulates cyclic nucleotide-gated channels in vision and olfaction (1-3), MATERIALS AND METHODS and activates the production of nitric oxide (NO) by NO synthases (NOS) (4, 5). Paradoxically, CaM also stimulates Purification and Enzymatic Assays. Recombinant chicken enzymes with antagonist activities-e.g., kinases (CaMKI-II) NCS-1 (7), bovine Recoverin (11), and chicken Vilipl (12) versus phosphatases (calcineurin), or cyclase (adenylate cy- were produced in Escherichia coli and affinity-purified on a clase) versus 3' :5'-cyclic nucleotide phosphodiesterase (PDE). phenyl-Sepharose CL-4B column to apparent homogeneity as These enzymes can act locally at the synapse or mediate more described (10) and used in the different enzymatic assays. general cellular responses involved in the regulation of tran- Crystals of NCS-1, shown in Fig. 3C, were grown using scription factors (gene expression). CaM is a 148-aa protein conditions obtained from a sparse matrix crystallization screen highly conserved between species-e.g., 88% identity between (13), and were authenticated by SDS/PAGE. Silver staining of plant and human calmodulin proteins-that belongs to the the resulting electrophoretic migration pattern, shown in Fig. large superfamily of Ca2+-binding proteins (6). 3D, revealed a protein of 22 kDa (identical to the relative We have recently characterized a neuron-specific calcium molecular weight of NCS-1) present in crystals shown in Fig. sensor named NCS-1 (7). This 22-kDa protein has 46% identity 3C. The NCS-1 preparation is therefore highly pure since it with Recoverin and 21% with CaM. NCS-1 is also highly forms crystals. Bovine brain PDE was purchased from Sigma conserved in evolution since we characterized NCS-1 or- and was assayed in triplicate as described (14). Recombinant thologs in rat, chicken, mouse (100% identity), and in Caeno- calcineurin A (CNA) and calcineurin B (CNB) subunits were rhabditis elegans (75% identity) (8). One function of NCS-1, tested in vitro, appears to be similar to the retina-specific Abbreviations: CaM, calmodulin, NOS, nitric oxide synthase; PDE, phosphodiesterase; CNA, calcineurin A; CNB, calcineurin B. **To whom reprint requests should be addressed at: Department of The publication costs of this article were defrayed in part by page charge Biochemistry, Sciences II, University of Geneva, 30 quai Ernest payment. This article must therefore be hereby marked "advertisement" in Ansermet, CH-1211 Geneva 4, Switzerland. e-mail: Patrick.Nef@ accordance with 18 U.S.C. §1734 solely to indicate this fact. biochem.unige.ch. 9253 Downloaded by guest on September 30, 2021 9254 Neurobiology: Schaad et al. Proc. Natl. Acad. Sci. USA 93 (1996) purified and reconstituted as described (15). Bovine brain NCS-1 targets could overlap with CaM targets. We first tested NOS was affinity-purified as described (16) and assayed in for a possible effect of NCS-1 on purified PDE which is a triplicate by measuring the conversion of [3H]arginine to well-characterized CaM target that regulates the intracellular [3H]citrulline as described (17) . second messenger levels of cyclic AMP and cyclic GMP (20, Western Blot Analysis. Chicken brain and eye were homog- 21). In this assay, recombinant NCS-1 was able to substitute for enized with a Teflon glass in 5 vol of buffer (20 mM Tris HCl, pH 7.4/0.1 mM phenylmethylsulfonyl fluoride/0.5 ,g of pep- CaM and to function as a direct PDE activator (Fig. 1A). statine per ml/1 mM EGTA/0.1 mM diisopropyl fluorophos- NCS-1 produced a large increase (3.8 ± 0.3 fold, n = 7) in PDE phate/2% SDS) and centrifuged at 30,000 x g for 30 min. The activity which was slightly smaller than with CaM alone (6- to eye supernatant was concentrated 10 times by precipitation 7-fold increase). The NCS-1-dependent activation of PDE was with 70% trichloroacetic acid. Samples were subjected to saturable and concentration-dependent (see below). NCS-1 SDS/PAGE in the presence of 1 mM EDTA, transferred onto itself had no endogenous PDE activity. nitrocellulose, blocked with 0.2% casein/0.033% polyvinylpyr- CaM-Dependent Phosphatase Calcineurin. We next as- rolidone, and incubated with an affinity-purified NCS-1 poly- sayed the phosphatase calcineurin for NCS-1 stimulation since clonal antibody (18) obtained from rabbit. Immunocomplexes its activity is essential for synaptic activity and is subject to dual were detected with anti-rabbit IgG-alkaline phosphatase con- Ca2+ ion control mediated by both CaM and CNB (22, 23). A jugate, then visualized with the substrate system 5-bromo-4- functional calcineurin enzyme consists of two tightly associ- chloro-3-indolyl phosphate/nitro blue tetrazolium (BCIP/ ated subunits (A and B), and is regulated in a reversible and NTB), or with the chemiluminescent substrate 3-(2'- spiroadamantane)-4-methoxy-4-(3"phosphoryloxy) phenyl- Ca2+-dependent fashion by CaM. CNA contains the active site 1,2-dioxetane (AMPPD). and belongs to the family of serine/threonine protein phos- Immunohistochemistry. Vibratome sections (50 ,tm) of phatases. The B subunit (CNB) is a Ca2+-binding protein with adult rat brain were labeled using the avidin/biotin/ four Ca2+-binding sites that possesses 35% identity with CaM peroxidase complex method using affinity-purified polyclonal and 30% with NCS-1. CNA contains distinct binding sites for antibodies against NCS-1 (18), a mouse monoclonal antibody CaM and CNB and neither can substitute for the other to against calcineurin (Transduction Laboratories, Lexington, potentiate CNA phosphatase activity (24). Using recombinant KY), and a rabbit polyclonal antibody against brain NOS forms of CNA and CNB (15), we observed that NCS-1 could (Transduction Laboratories). replace CaM but not CNB to stimulate the phosphatase Paramecium Injection. Using a micropipette, cells were activity (Fig. 1B). Thus, CNA alone had low phosphatase injected with "10 pl of a solution containing either NCS-1, activity that could be stimulated somewhat by either CNB or wild-type Paramecium calmodulin, or a control buffer as CaM The maximum occurred in the described (19).