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J. Biol. Chem., Vol. 281, Issue 28, 19645-19654, July 14, 2006

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Inhibitory Interaction of the Plasma Membrane Na⁺/Ca²⁺ Exchangers with the 14-3-3 Proteins^*

Maria V. Pulina, Rosario Rizzuto, Marisa Brini, and Ernesto Carafoli¹

From the Venetian Institute of Molecular Medicine, Via Orus 2, and Department of Biochemistry, University of Padova, Viale G. Colombo 3, 35100 Padova, Italy and the Department of Experimental and Diagnostic Medicine, Interdisciplinary Center for the Study of Inflammation, University of Ferrara, Via Borsavi 46, 44100 Ferrara, Italy

The three Na⁺/Ca²⁺ exchanger isoforms, NCX1, NCX2, and NCX3, contain a large cytoplasmic loop that is responsible for the regulation of activity. We have used 347 residues of the loop of NCX2 as the bait in a yeast two-hybrid approach to identify proteins that could interact with the exchanger and regulate its activity. Screening of a human brain cDNA library identified the and isoforms of the 14-3-3 protein family as interacting partners of the exchanger. The interaction was confirmed by immunoprecipitation and in vitro binding experiments. The effect of the interaction on the homeostasis of Ca²⁺ was investigated by co-expressing NCX2 and 14-3-3 in HeLa cells together with the recombinant Ca²⁺ probe aequorin; the ability of cells expressing both NCX2 and 14-3-3 to dispose of a Ca²⁺ transient induced by an InsP3-producing agonist was substantially decreased, indicating a reduction of NCX2 activity. The 14-3-3 protein also inhibited the NCX1 and NCX3 isoforms. In vitro binding experiments revealed that all three NCX isoforms interacted with multiple 14-3-3 isoforms. 14-3-3 was bound by both phosphorylated and nonphosphorylated NCX, but the phosphorylated form had much higher binding affinity.

Received for publication, March 3, 2006 , and in revised form, May 4, 2006.

^* This work was supported by the financial contribution of Telethon Foundation of Italy Grants GP0193Y01 (to E. C.) and GGP04169 (to M. B.), Italian Ministry of University and Scientific Research Grants PRIN 2003 and PRIN 2005 (to M. B.) and FIRB 2001 (to E. C.), and the European Community (Network of Excellence NeuroNe (to E. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed. Tel.: 39-049-7923240; Fax: 39-049-7923275; E-mail: ernesto.carafoli{at}unipd.it.

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