Premium
PKCδ‐SPAK (Ste20‐related proline alanine‐rich kinase) interaction in the regulation of native human NKCC1
Author(s) -
Liedtke Carole M.,
Smith Laura
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.1_supplement.936.4
Subject(s) - protein kinase c , phosphorylation , kinase , microbiology and biotechnology , chemistry , osmotic concentration , actin cytoskeleton , actin , biochemistry , biology , cell , cytoskeleton
Activation of native NKCC1 by hyperosmotic stress involves a regulatory proteome which includes PKCδ interacting directly with the actin cytoskeleton and with SPAK kinase. NKCC1 activation requires phosphorylation, yet PKCδ does not directly phosphorylate NKCC1. The goal of this study is to determine whether SPAK is part of a kinase cascade linking PKCδ to phosphorylation of native human epithelial NKCC1. NKCC1 activity was measured as bumetanide‐sensitive basolateral to apical 86‐Rb flux in Calu‐3 cells. SPAK was downregulated using siRNA delivered into Calu‐3 cells using an Amaxa nucleofector. siSPAK reduced SPAK protein expression by 73.5% without altering expression of NKCC1, PKCδ, and actin and prevented activation of NKCC1 by hyperosmotic stress. Substitution of a non‐targeting siCONTROL RNA did not affect expression of SPAK, PKCδ, NKCC1, and actin or activation of NKCC1. Using recombinant proteins, we demonstrate direct binding of PKCδ to SPAK and PKCδ‐mediated activation of SPAK. In intact cells, hyperosmotic stress increased native phosphorylated PKCδ (pPKCδ) 2.4‐fold, indicating activation of PKCδ, the amount of PKCδ and pPKCδ coimmunoprecipitating with SPAK 2.9‐fold and 3.6‐fold, respectively, and the amount of SPAK coimmunoprecipitating with PKCδ by 1.9‐fold. The results indicate that PKCδ mediates the stress signal to SPAK. Supported by a grant from the NIH, RO1‐HL‐58598.