Mutations in protein kinase with-no-lysine (K) 4 (WNK4) are associated with pseudohypoaldosteronism type II (PHAII), a hereditary form of hypertension. WNK4 is an integrative regulator of renal electrolyte transporters that are involved in blood pressure regulation. Accumulating evidence indicates that WNK4 is a key component of a phosphorylation cascade that links the activation of renin-angiotensin-aldosterone system to electrolyte transport in the kidney. However, the angiotensin II/aldosterone-responsive elements in WNK4 and the aldosterone-responsive regulation of WNK4 stability remain unclear. The long-term goal is to understand renal electrolyte transport physiology via analyzing how mutations in electrolyte transporters and their regulators cause disordered electrolyte homeostasis, so that therapeutic strategies could be developed for relevant disorders of both rare and common causes. The objective in this application is to identify the mechanisms for the activation of WNK4 kinase and for the regulation of WNK4 stability by angiotensin II and/or aldosterone. The preliminary studies indicate that a regulatory domain in WNK4 harbors calmodulin binding and phosphorylation sites. This domain inhibits WNK4 kinase activity and the calmodulin binding site is required for this action. Mimicking phosphorylation in this domain abolishes the inhibitory effect. Furthermore, the stability of WNK4 protein is robustly regulated by Kelch-like 3 (KLHL3), an ubiquitin E3 ligase component mutated in PHAII. The central hypothesis of this proposal is that PHAII mutations in both WNK4 and KLHL3 result in elevated WNK4 activity. Mutations in WNK4 elevate specific kinase activity/protein abundance and those in KLHL3 raise WNK4 protein abundance. This hypothesis will be tested in two specific aims: 1) Determine the regulation of WNK4 kinase activity by the regulatory domain of WNK4;and 2) Determine the regulation of WNK4 protein stability by the ubiquitin E3 ligase containing KLHL3.
In Aim 1, the regulation of WNK4 kinase activity by calmodulin and by phosphorylation will be assessed using in vitro and in vivo assays, and the interaction surfaces of the regulatory domain with calmodulin and the kinase domain will be determined using nuclear magnetic resonance (NMR) spectroscopy.
In Aim 2, the KLHL3-recognition motif at WNK4 C-terminal region will be determined. In addition, the effects of PHAII mutations in KLHL3 will be assessed biochemically and in knock-in mouse model. The responsiveness of KLHL3 to aldosterone will be determined in animals. The mechanisms for WNK4 kinase activation and protein stability regulation are significant, because they are essential for WNK4 to respond to physiological signals. Dysfunction of these mechanisms results in PHAII. Elucidating these mechanisms paves the way to new interventions for hypertension. The interaction surface information of the regulatory domain is crucial for developing small molecule inhibitors of WNK4 as research tools and potentially as new antihypertensive drugs.

Public Health Relevance

High blood pressure is a major risk factor for stroke, heart attack, and chronic kidney disease. The proposed research is relevant to public health because it is expected to elucidate mechanisms controlling WNK4, a protein kinase involved in blood pressure regulation. This project will lay a foundation for developing new ways and new inhibitors to control WNK4 activity and in turn, blood pressure.

National Institute of Health (NIH)
Research Project (R01)
Project #
Application #
Study Section
Program Officer
Ketchum, Christian J
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
University of Alabama Birmingham
Internal Medicine/Medicine
Schools of Medicine
United States
Zip Code
Na, Tao; Peng, Ji-Bin (2014) TRPV5: a Ca(2+) channel for the fine-tuning of Ca(2+) reabsorption. Handb Exp Pharmacol 222:321-57
Wu, Guojin; Peng, Ji-Bin (2013) Disease-causing mutations in KLHL3 impair its effect on WNK4 degradation. FEBS Lett 587:1717-22
Na, Tao; Wu, Guojin; Zhang, Wei et al. (2013) Disease-causing R1185C mutation of WNK4 disrupts a regulatory mechanism involving calmodulin binding and SGK1 phosphorylation sites. Am J Physiol Renal Physiol 304:F8-F18
Na, Tao; Wu, Guojin; Peng, Ji-Bin (2012) Disease-causing mutations in the acidic motif of WNK4 impair the sensitivity of WNK4 kinase to calcium ions. Biochem Biophys Res Commun 419:293-8
Wu, Guojin; Zhang, Wei; Na, Tao et al. (2012) Suppression of intestinal calcium entry channel TRPV6 by OCRL, a lipid phosphatase associated with Lowe syndrome and Dent disease. Am J Physiol Cell Physiol 302:C1479-91
Peng, Ji-Bin (2011) TRPV5 and TRPV6 in transcellular Ca(2+) transport: regulation, gene duplication, and polymorphisms in African populations. Adv Exp Med Biol 704:239-75
Jing, Haiyan; Na, Tao; Zhang, Wei et al. (2011) Concerted actions of NHERF2 and WNK4 in regulating TRPV5. Biochem Biophys Res Commun 404:979-84
Zhang, Wei; Na, Tao; Wu, Guojin et al. (2010) Down-regulation of intestinal apical calcium entry channel TRPV6 by ubiquitin E3 ligase Nedd4-2. J Biol Chem 285:36586-96
Nie, Hong-Guang; Zhang, Wei; Han, Dong-Yun et al. (2010) 8-pCPT-cGMP stimulates alphabetagamma-ENaC activity in oocytes as an external ligand requiring specific nucleotide moieties. Am J Physiol Renal Physiol 298:F323-34
Lieben, L; Benn, B S; Ajibade, D et al. (2010) Trpv6 mediates intestinal calcium absorption during calcium restriction and contributes to bone homeostasis. Bone 47:301-8

Showing the most recent 10 out of 16 publications