title: Interactions with WNK (with no lysine) family members regulate oxidative stress response 1 and ion co-transporter activity.
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.
authors.affiliation: Department of Pharmacology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75390-9041, USA.
authors.forename: Samarpita
authors.initials: S
authors.lastname: Sengupta
authors.forename: Szu-Wei
authors.initials: SW
authors.lastname: Tu
authors.forename: Kyle
authors.initials: K
authors.lastname: Wedin
authors.forename: Svetlana
authors.initials: S
authors.lastname: Earnest
authors.forename: Steve
authors.initials: S
authors.lastname: Stippec
authors.forename: Katherine
authors.initials: K
authors.lastname: Luby-Phelps
authors.forename: Melanie H
authors.initials: MH
authors.lastname: Cobb
mesh_terms: D020816:Amino Acid Motifs / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D003593:Cytoplasm / Q000378:metabolism; D036681:Fluorescence Recovery After Photobleaching; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D006982:Hypertonic Solutions / Q000494:pharmacology; D007038:Hypotonic Solutions / Q000494:pharmacology; D015151:Immunoblotting; D047468:Immunoprecipitation; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence; D015778:Minor Histocompatibility Antigens; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D021381:Protein Transport / Q000187:drug effects; D034622:RNA Interference; D051381:Rats; D028021:Sodium-Potassium-Chloride Symporters / Q000235:genetics / Q000378:metabolism*; D064466:Solute Carrier Family 12, Member 1; D064506:Solute Carrier Family 12, Member 2; D000076204:WNK Lysine-Deficient Protein Kinase 1
chemical_list: D006982:Hypertonic Solutions; D007038:Hypotonic Solutions; D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes; D015778:Minor Histocompatibility Antigens; C578714:SLC12A1 protein, human; C578877:SLC12A2 protein, human; C578716:Slc12a1 protein, rat; C578879:Slc12a2 protein, rat; D028021:Sodium-Potassium-Chloride Symporters; D064466:Solute Carrier Family 12, Member 1; D064506:Solute Carrier Family 12, Member 2; D049452:Green Fluorescent Proteins; C118135:OXSR1 protein, human; D017346:Protein Serine-Threonine Kinases; D000076204:WNK Lysine-Deficient Protein Kinase 1; C498743:WNK1 protein, human