HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) All Versions of this Article: 297/3/F685    most recent 00358.2009v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Google Scholar Articles by Ahlstrom, R. Articles by Yu, A. S. L. PubMed PubMed Citation Articles by Ahlstrom, R. Articles by Yu, A. S. L.

Characterization of the kinase activity of a WNK4 protein complex

¹Department of Physiology and Biophysics and ²Division of Nephrology, Department of Medicine, University of Southern California Keck School of Medicine, Los Angeles, California

Submitted 23 June 2009 ; accepted in final form 3 July 2009

Mutations in WNK4 protein kinase cause pseudohypoaldosteronism type II (PHAII), a genetic disorder that is characterized by renal NaCl and K⁺ retention leading to hypertension and hyperkalemia. Consistent with this, WNK4 is known to regulate several renal tubule transporters, including the NaCl cotransporter, NCC, and the K⁺ channel, ROMK, but the mechanisms are incompletely understood, and the role of the kinase activity in its actions is highly controversial. To assay WNK4 kinase activity, we have now succeeded in expressing and purifying full-length, enzymatically active WNK4 protein from HEK293 cells. We show that full-length wild-type WNK4 phosphorylates oxidative stress response kinase 1 (OSR1) and Ste20/SPS1-related proline/alanine-rich kinase (SPAK) in vitro. Introducing the PHAII-associated mutations, E559K, D561A, and Q562E, into our protein had no significant effect on this phosphorylation. We conclude that PHAII is unlikely to be caused by abnormal WNK4 kinase activity. We also made the intriguing observation that inactivating mutations of the WNK4 kinase domain did not completely abolish in vitro phosphorylation of OSR1/SPAK. Led by this, we identified a novel 40-kDa kinase that associates specifically with the COOH-terminal half of WNK4 and is able to phosphorylate both WNK4 and SPAK/OSR1. We suggest that this 40-kDa kinase functions in the WNK4 signal transduction pathway and may mediate some of the physiological actions attributed to WNK4.

protein kinase; pseudohypoaldosteronism type II; in-gel phosphorylation