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Originally published In Press as doi:10.1074/jbc.C100368200 on July 20, 2001

J. Biol. Chem., Vol. 276, Issue 37, 34359-34362, September 14, 2001
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ACCELERATED PUBLICATION
Modulation of Ion Transport by Direct Targeting of Protein Phosphatase Type 1 to the Na-K-Cl Cotransporter*

Rachel B. DarmanDagger , Andreas Flemmer§, and Biff Forbush

From the Department of Cellular and Molecular Physiology, Yale University, New Haven, Connecticut 06510 and The Mount Desert Island Biological Laboratory, Salsbury Cove, Maine 04672

The specificity of major protein phosphatases is conferred via targeting subunits, each of which binds specifically to the phosphatase and targets it to the vicinity of substrate proteins. In the case of protein phosphatase 1 (PP1), an RVXFXD motif on a targeting subunit binds to a cleft in PP1c, the catalytic subunit. Here we report that a substrate of PP1, the Na-K-Cl cotransporter (NKCC1), bears this motif in its N terminus near sites of regulatory phosphorylation and that direct binding of PP1 to NKCC1 is functionally important in determining the set point for intracellular chloride regulation. NKCC1 mutants in which the motif is destroyed or improved exhibit dramatically shifted activation curves because of a change in the rate of cotransporter dephosphorylation. Furthermore, direct interaction of NKCC1 and PP1c observed by coprecipitation of the two proteins is not seen in a mutant lacking the site. This establishes a new paradigm of phosphatase specificity, one in which a substrate protein containing an RVXFXD motif binds directly to PP1c; we propose that this may be a quite general mechanism.

* This work was supported by National Institutes of Health Grant DK47661.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Present address: Dept. of Medicine, Molecular Medicine and Renal Unit, Beth Israel Deaconess Medical Center, Harvard Medical School, 330 Brookline Ave., RW763, Boston, MA 02215. Tel.: 617-667-2923; E-mail: rdarman@caregroup.harvard.edu.

§ Dept. of Neonatology, University Children's Hospital of Munich, Lindwurmstr. 4, Munich, Bavaria D-80337, Germany. Tel.: 49-89-5160-2811; E-mail: andreas.flemmer@kk-i.med.uni-muenchen.de.

To whom correspondence should be addressed: Dept. of Cellular and Molecular Physiology, Yale University School of Medicine, 333 Cedar St., New Haven, CT 06520-8026. Tel.: 203-785-4068; Fax: 203-785-6834; E-mail: biff.forbush@yale.edu.

Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.


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