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PKCδ Acts Upstream of SPAK in the Activation of NKCC1 by Hyperosmotic Stress in Human Airway Epithelial Cells*

By Laura Smith, Nicole Smallwood, Amnon Altman and Carole M. Liedtke

Abstract

Airway epithelial Na-K-2Cl (NKCC1) cotransport is activated through hormonal stimulation and hyperosmotic stress via a protein kinase C (PKC) δ-mediated intracellular signaling pathway. Down-regulation of PKCδ prevents activation of NKCC1 expressed in Calu-3 cells. Previous studies of this signaling pathway identified coimmunoprecipitation of PKCδ with SPAK (Ste20-related proline alanine-rich kinase). We hypothesize that endogenous PKCδ activates SPAK, which subsequently activates NKCC1 through phosphorylation. Double-stranded silencing RNA directed against SPAK reduced SPAK protein expression by 65.8% and prevented increased phosphorylation of NKCC1 and functional activation of NKCC1 during hyperosmotic stress, measured as bumetanide-sensitive basolateral to apical 86Rb flux. Using recombinant proteins, we demonstrate direct binding of PKCδ to SPAK, PKCδ-mediated activation of SPAK, binding of SPAK to the amino terminus of NKCC1 (NT-NKCC1, amino acids 1–286), and competitive inhibition of SPAK-NKCC1 binding by a peptide encoding a SPAK binding site on NT-NKCC1. The carboxyl terminus of SPAK (amino acids 316–548) pulls down endogenous NKCC1 from Calu-3 total cell lysates and glutathione S-transferase-tagged NT-NKCC1 pulls down endogenous SPAK. In intact cells, hyperosmotic stress increased phosphorylated PKCδ, indicating activation of PKCδ, and activity of endogenous SPAK kinase. Inhibition of PKCδ activity with rottlerin blocked the increase in SPAK kinase activity. The results indicate that PKCδ acts upstream of SPAK to increase activity of NKCC1 during hyperosmotic stress

Topics: Mechanisms of Signal Transduction
Publisher: American Society for Biochemistry and Molecular Biology
OAI identifier: oai:pubmedcentral.nih.gov:2494923
Provided by: PubMed Central
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