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PKCdelta acts upstream of SPAK in the activation of NKCC1 by hyperosmotic stress in human airway epithelial cells.

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.

SUBMITTER: Smith L

PROVIDER: S-EPMC2494923 | biostudies-literature | 2008 Aug

REPOSITORIES: biostudies-literature

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PKCdelta acts upstream of SPAK in the activation of NKCC1 by hyperosmotic stress in human airway epithelial cells.

Smith Laura L Smallwood Nicole N Altman Amnon A Liedtke Carole M CM

The Journal of biological chemistry 20080611 32

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 activate ...[more]

PMID: 18550547

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Project description:The SPAK (STE20/SPS1-related proline/alanine-rich kinase) and OSR1 (oxidative stress-responsive kinase-1) kinases interact and phosphorylate NKCC1 (Na+-K+-2Cl- co-transporter-1), leading to its activation. Recent studies indicated that SPAK and OSR1 are phosphorylated and activated by the WNK1 [with no K (lysine) protein kinase-1] and WNK4, genes mutated in humans affected by Gordon's hypertension syndrome. In the present study, we have identified three residues in NKCC1 (Thr175/Thr179/Thr184 in shark or Thr203/Thr207/Thr212 in human) that are phosphorylated by SPAK and OSR1, and have developed a peptide substrate, CATCHtide (cation chloride co-transporter peptide substrate), to assess SPAK and OSR1 activity. Exposure of HEK-293 (human embryonic kidney) cells to osmotic stress, which leads to phosphorylation and activation of NKCC1, increased phosphorylation of NKCC1 at the sites targeted by SPAK/OSR1. The residues on NKCC1, phosphorylated by SPAK/OSR1, are conserved in other cation co-transporters, such as the Na+-Cl- co-transporter, the target of thiazide drugs that lower blood pressure in humans with Gordon's syndrome. Furthermore, we characterize the properties of a 92-residue CCT (conserved C-terminal) domain on SPAK and OSR1 that interacts with an RFXV (Arg-Phe-Xaa-Val) motif present in the substrate NKCC1 and its activators WNK1/WNK4. A peptide containing the RFXV motif interacts with nanomolar affinity with the CCT domains of SPAK/OSR1 and can be utilized to affinity-purify SPAK and OSR1 from cell extracts. Mutation of the arginine, phenylalanine or valine residue within this peptide abolishes binding to SPAK/OSR1. We have identified specific residues within the CCT domain that are required for interaction with the RFXV motif and have demonstrated that mutation of these in OSR1 inhibited phosphorylation of NKCC1, but not of CATCHtide which does not possess an RFXV motif. We establish that an intact CCT domain is required for WNK1 to efficiently phosphorylate and activate OSR1. These data establish that the CCT domain functions as a multipurpose docking site, enabling SPAK/OSR1 to interact with substrates (NKCC1) and activators (WNK1/WNK4).

Project description:BackgroundWorsened stroke outcomes with hypertension comorbidity are insensitive to blood pressure-lowering therapies. In an experimental stroke model with comorbid hypertension, we investigated causal roles of ang II (angiotensin II)-mediated stimulation of the brain WNK (with no lysine [K] kinases)-SPAK (STE20/SPS1-related proline/alanine-rich kinase)-NKCC1 (Na-K-Cl cotransporter) complex in worsened outcomes.MethodsSaline- or ang II-infused C57BL/6J male mice underwent stroke induced by permanent occlusion of the distal branches of the middle cerebral artery. Mice were randomly assigned to receive either vehicle dimethyl sulfoxide/PBS (2 mL/kg body weight/day, IP), a novel SPAK inhibitor, 5-chloro-N-(5-chloro-4-((4-chlorophenyl)(cyano)methyl)-2-methylphenyl)-2-hydroxybenzamide (ZT-1a' 5 mg/kg per day, IP) or a NF-κB (nuclear factor-κB) inhibitor TAT-NBD (transactivator of transcription-NEMO-binding domain' 20 mg/kg per day, IP). Activation of brain NF-κB and WNK-SPAK-NKCC1 cascade as well as ischemic stroke outcomes were examined.ResultsStroke triggered a 2- to 5-fold increase of WNK (isoforms 1, 2, 4), SPAK/OSR1 (oxidative stress-responsive kinase 1), and NKCC1 protein in the ang II-infused hypertensive mouse brains at 24 hours after stroke, which was associated with increased nuclear translocation of phospho-NF-κB protein in the cortical neurons (a Pearson correlation r of 0.77, P<0.005). The upregulation of WNK-SPAK-NKCC1 cascade proteins resulted from increased NF-κB recruitment on Wnk1, Wnk2, Wnk4, Spak, and Nkcc1 gene promoters and was attenuated by NF-κB inhibitor TAT-NBD. Poststroke administration of SPAK inhibitor ZT-1a significantly reduced WNK-SPAK-NKCC1 complex activation, brain lesion size, and neurological function deficits in the ang II-hypertensive mice without affecting blood pressure and cerebral blood flow.ConclusionsThe ang II-induced stimulation of NF-κB transcriptional activity upregulates brain WNK-SPAK-NKCC1 cascade and contributes to worsened ischemic stroke outcomes, illustrating the brain WNK-SPAK-NKCC1 complex as a therapeutic target for stroke with comorbid hypertension.

Dataset Information

PKCdelta acts upstream of SPAK in the activation of NKCC1 by hyperosmotic stress in human airway epithelial cells.

Publications

PKCdelta acts upstream of SPAK in the activation of NKCC1 by hyperosmotic stress in human airway epithelial cells.

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