Interactions with WNK (with no lysine) family members regulate oxidative stress response 1 and ion co-transporter activity

J Biol Chem. 2012 Nov 2;287(45):37868-79. doi: 10.1074/jbc.M112.398750. Epub 2012 Sep 18.

Abstract

Two of the four WNK (with no lysine (K)) protein kinases are associated with a heritable form of ion imbalance culminating in hypertension. WNK1 affects ion transport in part through activation of the closely related Ste20 family protein kinases oxidative stress-responsive 1 (OSR1) and STE20/SPS1-related proline-, alanine-rich kinase (SPAK). Once activated by WNK1, OSR1 and SPAK phosphorylate and stimulate the sodium, potassium, two chloride co-transporters, NKCC1 and NKCC2, and also affect other related ion co-transporters. We find that WNK1 and OSR1 co-localize on cytoplasmic puncta in HeLa and other cell types. We show that the C-terminal region of WNK1 including a coiled coil is sufficient to localize the fragment in a manner similar to the full-length protein, but some other fragments lacking this region are mislocalized. Photobleaching experiments indicate that both hypertonic and hypotonic conditions reduce the mobility of GFP-WNK1 in cells. The four WNK family members can phosphorylate the activation loop of OSR1 to increase its activity with similar kinetic constants. C-terminal fragments of WNK1 that contain three RFXV interaction motifs can bind OSR1, block activation of OSR1 by sorbitol, and prevent the OSR1-induced enhancement of ion co-transporter activity in cells, further supporting the conclusion that association with WNK1 is required for OSR1 activation and function at least in some contexts. C-terminal WNK1 fragments can be phosphorylated by OSR1, suggesting that OSR1 catalyzes feedback phosphorylation of WNK1.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Motifs / genetics
  • Amino Acid Sequence
  • Animals
  • Cytoplasm / metabolism
  • Fluorescence Recovery After Photobleaching
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism
  • HeLa Cells
  • Humans
  • Hypertonic Solutions / pharmacology
  • Hypotonic Solutions / pharmacology
  • Immunoblotting
  • Immunoprecipitation
  • Intracellular Signaling Peptides and Proteins / genetics
  • Intracellular Signaling Peptides and Proteins / metabolism*
  • Isoenzymes / genetics
  • Isoenzymes / metabolism
  • Microscopy, Fluorescence
  • Minor Histocompatibility Antigens
  • Protein Binding
  • Protein Serine-Threonine Kinases / genetics
  • Protein Serine-Threonine Kinases / metabolism*
  • Protein Transport / drug effects
  • RNA Interference
  • Rats
  • Sodium-Potassium-Chloride Symporters / genetics
  • Sodium-Potassium-Chloride Symporters / metabolism*
  • Solute Carrier Family 12, Member 1
  • Solute Carrier Family 12, Member 2
  • WNK Lysine-Deficient Protein Kinase 1

Substances

  • Hypertonic Solutions
  • Hypotonic Solutions
  • Intracellular Signaling Peptides and Proteins
  • Isoenzymes
  • Minor Histocompatibility Antigens
  • SLC12A1 protein, human
  • SLC12A2 protein, human
  • Slc12a1 protein, rat
  • Slc12a2 protein, rat
  • Sodium-Potassium-Chloride Symporters
  • Solute Carrier Family 12, Member 1
  • Solute Carrier Family 12, Member 2
  • Green Fluorescent Proteins
  • OXSR1 protein, human
  • Protein Serine-Threonine Kinases
  • WNK Lysine-Deficient Protein Kinase 1
  • WNK1 protein, human