Abstract
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.
Highlights
Stoichiometry of 2:1:1 [1]
We have shown a critical role for activity of protein kinase C (PKC)␦ during regulation of airway NKCC1 by intracellular Cl and hyperosmotic stress and during ␣1-adrenergic stimulation in airway epithelial cells [21, 27] and for stable binding of PKC␦ to actin for activation of NKCC1 [28, 29]
Treatment with non-targeting siCONTROL RNA did not affect the amount of SPAK, NKCC1, PP2A, PKC␦, or actin detected in total cell lysates (Fig. 1B)
Summary
Baculovirus-expressed recombinant PKC␦ was purchased from Pan Vera (Madison, WI). Goat polyclonal antibody to NKCC1 (N-16), rabbit polyclonal antibody to PKC␦, polyclonal anti-GST antibody, anti-GST conjugated to agarose beads, and horseradish peroxidase-coupled secondary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit polyclonal antibody to SPAK was obtained from Abgent (San Diego, CA) and mouse monoclonal antibody to actin from Cytoskeleton (Denver, CO). (San Diego, CA), Talon beads from Clontech (Mountain View, CA), and an enhanced chemiluminescence reagent from Denville Scientific, Inc. Tissue culture supplies were purchased from Invitrogen and pre-cast 4 –15% gradient slab gels from Bio-Rad. All other chemicals were reagent grade
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