PKCdelta acts upstream of SPAK in the activation of NKCC1 by hyperosmotic stress in human airway epithelial cells.
Airway epithelial Na-K-2Cl (NKCC1) cotransport is activated through hormonal stimulation and hyperosmotic stress via a protein kinase C (PKC) delta-mediated intracellular signaling pathway. Down-regulation of PKCdelta prevents activation of NKCC1 expressed in Calu-3 cells. Previous studies of this signaling pathway identified coimmunoprecipitation of PKCdelta with SPAK (Ste20-related proline alanine-rich kinase). We hypothesize that endogenous PKCdelta 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 (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, 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 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.
Smith
Laura
L
Department of Pediatrics, Case Western Reserve University, Cleveland, Ohio 44106, USA.
Smallwood
Nicole
N
Altman
Amnon
A
Liedtke
Carole M
CM
D002460:Cell Line; D015536:Down-Regulation; D004847:Epithelial Cells / Q000201:enzymology / Q000378:metabolism*; D008168:Lung / Q000166:cytology*; D009997:Osmotic Pressure; D010766:Phosphorylation; D011485:Protein Binding; D051745:Protein Kinase C-delta / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D028021:Sodium-Potassium-Chloride Symporters / Q000378:metabolism*; D064506:Solute Carrier Family 12, Member 2
C578877:SLC12A2 protein, human; D028021:Sodium-Potassium-Chloride Symporters; D064506:Solute Carrier Family 12, Member 2; D017346:Protein Serine-Threonine Kinases; C486409:STK39 protein, human; D051745:Protein Kinase C-delta