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Phosphoproteomic Strategy for Profiling Osmotic Stress Signaling in Arabidopsis
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Phosphoproteomic Strategy for Profiling Osmotic Stress Signaling in Arabidopsis

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Osmotic stress induces the phosphorylation of WNK4 Ser575 via the p38MAPK-MK pathway

Junichi Maruyama1,2, Yumie Kobayashi1, Tsuyoshi Umeda1

  • 1Laboratory of Cell Signaling, Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.

Scientific Reports
|January 7, 2016
PubMed

Insights

The WNK-SPAK/OSR1 pathway regulates blood pressure. This study reveals apoptosis signal-regulating kinase 3 (ASK3) phosphorylates WNK4, uncovering a new mechanism in osmotic stress response.

Area of Science:

  • Molecular biology
  • Cell signaling
  • Physiology

Background:

  • The With No lysine [K] (WNK)-Ste20-related proline/alanine-rich kinase (SPAK)/oxidative stress-responsive kinase 1 (OSR1) pathway is critical for pseudohypoaldosteronism type II (PHAII), a hypertension-causing disease.
  • The precise regulatory mechanisms of the WNK-SPAK/OSR1 pathway remain incompletely understood.

Purpose of the Study:

  • To elucidate the molecular mechanisms regulating the WNK-SPAK/OSR1 pathway.
  • To identify novel interacting partners and phosphorylation sites of WNK4.

Main Methods:

  • Co-immunoprecipitation to identify WNK4 interacting partners.
  • Kinase assays to assess WNK4 phosphorylation.
  • Liquid chromatography-tandem mass spectrometry (LC-MS/MS) for phosphorylation site identification.
  • Western blotting to analyze pathway activation under various stimuli.

Main Results:

  • Apoptosis signal-regulating kinase 3 (ASK3) was identified as a WNK4 interacting partner.
  • WNK4 is phosphorylated by ASK3 at a novel site, Serine 575 (Ser575).
  • ASK3-dependent WNK4 Ser575 phosphorylation is mediated by the p38MAPK-MAPK-activated protein kinase (MK) pathway.
  • Osmotic stress and hypotonic low-chloride stimulation enhance WNK4 Ser575 phosphorylation via the p38MAPK-MK pathway.
  • ASK3 is essential for hypotonic but not hypertonic or hypotonic low-chloride-induced p38MAPK activation.

Conclusions:

  • The p38MAPK-MK pathway regulates WNK4 phosphorylation in an osmotic stress-dependent manner.
  • Upstream regulators of the p38MAPK-MK pathway may differ based on the type of osmotic stimulus.
  • This study reveals a novel regulatory mechanism for the WNK-SPAK/OSR1 pathway, potentially impacting hypertension research.

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