PKCdelta acts upstream of SPAK in the activation of NKCC1 by hyperosmomtic stress in airway epithelial cells

Smith L, Smallwood N, Altman A, Liedtke CM
Source: J Biol Chem
Publication Date: (2008)
Issue: 283(32): 22147-56
Research Area:
Cancer Research/Cell Biology
Cells used in publication:
Species: human
Tissue Origin: lung
Nucleofector® I/II/2b
Airway epithelial Na-K-2Cl (NKCC1) cotransport is activated through hormonal stimulation and hyperosmotic stress via a PKCdelta-mediated intracellular signalling pathway. Downregulation of PKCdeltaprevents activation of NKCC1 expressed in Calu-3 cells. Previous studies of this signalling pathway identified coimmunoprecipitation of PKCdelta with with SPAK (Ste20-related proline alanine-rich kinase). We hypothesize that endogenous PKCdeltaactivates 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 (86)Rb flux. Using recombinant proteins, we demonstrate direct binding of PKCdelta to SPAK, PKCdelta-mediated activation of SPAK, binding of SPAK to the amino terminus of NKCC1 (NT-NKCC1, aa 1-286), and competitive inhibition of SPAK-NKCC1 binding by a peptide encoding a SPAK binding site on NT-NKCC1. The carboxyl terminus of SAPK (aa 316-548) pulls down endogenous NKCC1 from Calu-3 total cell lysates and GST-tagged NT-NKCC1 pulls down endogenous SPAK. In intact cells, hyperosmotic stress increased phosphorylated PKCdelta, indicating activation of PKCdelta, and activity of endogenous SPAK kinase. Inhibition of PKCdelta activity with rottlerin blocked the increase in SPAK kinase activity. The results indicate that PKCdelta acts upstream of SPAK to increase activity of NKCC1 during hyperosmotic stress.