Role for protein phosphatase 2A in the regulation of Calu-3 epithelial Na+-K+-2Cl-, type 1 co-transport function

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 Rb-86 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 n(M) 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 alpha(1)- adrenergic agonist methoxamine increased NKCC1 activity and transiently increased PP2A activity 3.8- fold but did not alter PP1 activity. OA augmented methoxaminedependent 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-delta ( PKC-delta) and was pulled down by a recombinant N terminus of NKCC1 consisting of amino acids 1 - 286. One novel finding is co- precipitation of STE20related proline- alanine- rich kinase, a regulatory kinase for NKCC1, with PP2A and PKC-delta. The results suggest a model of actin serving as a scaffold for binding and association of PKC-delta, 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.