WNK1 Regulates Phosphorylation of Cation-Chloride-coupled Cotransporters via the STE20-related Kinases, SPAK and OSR1

Tetsuo Moriguchi,Seiichi Urushiyama,Naoki Hisamoto,Shun-Ichiro Iemura,Shinichi Uchida,Tohru Natsume,Kunihiro Matsumoto,Hiroshi Shibuya

doi:10.1074/jbc.m510042200

Abstract

The WNK1 and WNK4 genes have been found to be mutated in some patients with hyperkalemia and hypertension caused by pseudohypoaldosteronism type II. The clue to the pathophysiology of pseudohypoaldosteronism type II was its striking therapeutic response to thiazide diuretics, which are known to block the sodium chloride cotransporter (NCC). Although this suggests a role for WNK1 in hypertension, the precise molecular mechanisms are largely unknown. Here we have shown that WNK1 phosphorylates and regulates the STE20-related kinases, Ste20-related proline-alanine-rich kinase (SPAK) and oxidative stress response 1 (OSR1). WNK1 was observed to phosphorylate the evolutionary conserved serine residue located outside the kinase domains of SPAK and OSR1, and mutation of the OSR1 serine residue caused enhanced OSR1 kinase activity. In addition, hypotonic stress was shown to activate SPAK and OSR1 and induce phosphorylation of the conserved OSR1 serine residue, suggesting that WNK1 may be an activator of the SPAK and OSR1 kinases. Moreover, SPAK and OSR1 were found to directly phosphorylate the N-terminal regulatory regions of cation-chloride-coupled cotransporters including NKCC1, NKCC2, and NCC. Phosphorylation of NCC was induced by hypotonic stress in cells. These results suggested that WNK1 and SPAK/OSR1 mediate the hypotonic stress signaling pathway to the transporters and may provide insights into the mechanisms by which WNK1 regulates ion balance.

Highlights

Identification of the STE20-related Kinases Ste20-related proline-alanine-rich kinase (SPAK) and oxidative stress response 1 (OSR1) as WNK1-associated Molecules—To identify a protein(s) that physically associates with WNK1, we employed two strategies: yeast two-hybrid screening and FLAG tag immunoprecipitation assays coupled with LCMS/MS analysis
To investigate whether endogenous SPAK is associated with WNK1 in living cells, we generated antibodies to a peptide of SPAK and a peptide of human WNK1
WNK1 was detected with immunoprecipitates of SPAK and OSR1 from HEK293 cells or MDCK cells, indicating that these form an endogenous complex in living cells (Fig. 1, left and right)

Summary

Introduction

We have shown that SPAK and OSR1 directly phosphorylate the N-terminal regulatory regions of NKCC1, NKCC2, and NCC. To further investigate the role of these motifs in WNK1 binding to STE20-related kinases, we co-expressed various forms of WNK1 with SPAK or OSR1 and performed co-immunoprecipitation experiments. The mutation of the same site to Ala or Gly, which was expected to abolish phosphorylation, resulted in causing constitutively activation of OSR1 (Fig. 5B, GST-OSR1(S325A), and data not shown).

Results

Conclusion