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J. Biol. Chem., Vol. 280, Issue 27, 25491-25498, July 8, 2005

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Role for Protein Phosphatase 2A in the Regulation of Calu-3 Epithelial Na⁺-K⁺-2Cl^–, Type 1 Co-transport Function^*

Carole M. Liedtke, Xiangyun Wang, and Nicole D. Smallwood

From the Willard Alan Bernbaum Center for Cystic Fibrosis Research, Departments of Pediatrics at Rainbow Babies & Childrens Hospital and Physiology & Biophysics, Case Western Reserve University, Cleveland, Ohio 44106

Activity of Na⁺-K⁺-2Cl^– co-transport (NKCC1) in epithelia is thought to be highly regulated through phosphorylation and dephosphorylation of the transporter. Previous functional studies from this laboratory suggested a role for protein phosphatase 2A (PP2A) as a serine/threonine protein phosphatase involved in the regulation of mammalian tracheal epithelial NKCC1. We expand on these studies to characterize serine/threonine protein phosphatase(s) necessary for regulation of NKCC1 function and the interaction of the phosphatase(s) with proteins associated with NKCC1. NKCC1 activity was measured as bumetanide-sensitive ⁸⁶Rb uptake or basolateral to apical ⁸⁶Rb flux in primary cultures of human tracheal epithelial cells or in Calu-3 airway epithelial cells grown on Transwell filter inserts. Preincubation with 0.1 nM okadaic acid, a PP2A >> phosphatase 1 (PP1) inhibitor, increased NKCC1 activity 3.5-fold in human tracheal epithelial cells and 4.1-fold in Calu-3 cells. Calyculin, a PP1 >> PP2A inhibitor, did not alter NKCC1 activity or percent bumetanide-sensitive flux. The effect of OA was dose-dependent with an IC₅₀ of 0.4 nM. The ₁-adrenergic agonist methoxamine increased NKCC1 activity and transiently increased PP2A activity 3.8-fold but did not alter PP1 activity. OA augmented methoxamine-dependent stimulation of NKCC1 activity. PP1, PP2A, and PP2C but not PP2B were detected in lysates from Calu-3 cells by immunoblot analysis. PP1 was not detected in immunoprecipitates of NKCC1 and vice versa. PP2A co-immunoprecipitated with NKCC1 and protein kinase C- (PKC-) and was pulled down by a recombinant N terminus of NKCC1 consisting of amino acids 1–286. One novel finding is co-precipitation of STE20-related proline-alanine-rich kinase, a regulatory kinase for NKCC1, with PP2A and PKC-. The results suggest a model of actin serving as a scaffold for binding and association of PKC-, PP2A, and STE20-related proline-alanine-rich kinase. The role of the complex of serine/threonine protein kinases and a protein phosphatase is probably the maintenance of optimal phosphorylation of NKCC1 coincident with its physiological function in epithelial absorption and secretion.

Received for publication, April 25, 2005 , and in revised form, May 6, 2005.

^* This work was supported by National Institutes of Health Grant HL 58598 and the Cystic Fibrosis Foundation Research Development Program. 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.

To whom correspondence should be addressed: Pediatric Pulmonology, Case Western Reserve University, Biomedical Research Bldg., Rm. 824, 2109 Adelbert Rd., Cleveland, OH 44106-4948. Tel.: 216-368-4629; Fax: 216-368-4223; E-mail: carole.liedtke{at}case.edu.

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