WNK4

Serine/threonine protein kinase WNK4 also known as WNK lysine deficient protein kinase 4 or WNK4, is an enzyme that in humans is encoded by the WNK4 gene.^[5] Missense mutations cause a genetic form of pseudohypoaldosteronism type 2, also called Gordon syndrome.

WNK4 is a member of a

The WNK4 gene is located on chromosome 17q21-q22. It produces a 1,243-amino acid protein encoded by a 3,732-nucleotide open reading frame within a 4 kb cDNA transcript.^[7] WNK4 protein is highly expressed in the distal convoluted tubule (DCT) and the cortical collecting duct (CDD) of the kidney.^[7] WNK4 is also present in the brain, lungs, liver, heart, and colon of various mammalian species.^[8][9]

Gene mutations in WNK4 has been found in patients with

A chloride ion binding site has been identified in the region ³²⁰DLG³²³ of the kinase domain in WNK4.^[13] The binding of chloride Cl⁻ in this region inhibits the activation of WNK4. The autoinhibitory domain is a homolog of the RFXV-binding PASK/FRAY homology 2 (PF2) domain.^[14] Structural studies have revealed that the autoinhibitory domain consists of three β-strands and two α-helices.^[15] Notably, the RFXV‐binding groove is formed by the β3-αA interface of WNK proteins where RFXV peptide ligand interacts directly with residues Phe524, Asp531, and Glu539 of WNK1.^[15] The interaction between the RFXV motif and the autoinhibitory domain makes it possible for the C-terminal region of WNK4 to be in close proximity of the kinases domain and subsequently regulate its activity.

Function

As a typical

In 2001, four missense mutations in the WNK4 gene were identified in patients with pseudohypoaldosteronism type 2 (PHAII) (Fig. 1).^[7] Three of these mutations (E562K, D564A, and Q565E) are charge-changing substitutions in the acidic motif of WNK4, which are conserved among all members of the WNK family in human and rodent species. The fourth substitution (R1185C) is located in the calmodulin-binding domain near the second coiled-coil domain. Few other PHAII mutations in WNK4 have also been reported. Examples of these mutations include E560G,^[26] P561L,^[27]  and D564H,^[28] all of which are located close to or in the acidic motif; and K1169E ^[29] which is located between the coiled-coil 2 and the calmodulin-binding domain.

The PHAII mutations appear to disrupt the mechanisms underlying Ca²⁺-sensitivity of WNK4 kinase. Two mechanisms are important in this regard. First, the PHAII-causing mutations in the acidic motif make the kinase domain insensitive to Ca²⁺ concentration. The acidic motif of WNK4 potentially acts as a Ca²⁺ sensor, and WNK4 kinase activity rises when Ca²⁺ concentration is elevated. This has been demonstrated using isolated WNK4 kinase domain truncated to contain the acidic motif.^[19] The kinase activity is elevated when a PHAII-causing mutation is present in the acidic motif, similar to what is observed in a Ca²⁺-binding state (Fig. 3). Second, the WNK4 C-terminal region containing the calmodulin-binding domain and multiple SGK1 phosphorylation sites inhibits the WNK4 activity at the resting state.^[30] However, when Ca²⁺ levels are elevated, Ca²⁺/calmodulin complex binds to the C-terminal region, derepressing WNK4 kinase activity. Additionally, the RFXV motif is believed to interact with the autoinhibitory domain and subsequently triggers a conformational change that brings the C-terminal and kinase domain close for the inhibitory effect to take place. Angiotensin II increases the SPAK phosphorylation and activates NCC through a WNK-dependent mechanism.^[31] The activation of SPAK and NCC by angiotensin II is abrogated by WNK4 knockdown.^[32] Activation of angiotensin II receptor AT1 couples to Gq/11 to activate phospholipase C and to increase the intracellular Ca²⁺ concentration. An increase in Ca²⁺ concentration then elevates WNK4 activity through mechanisms described above (Fig. 3, left panel). The PHAII-causing mutations in the acidic motif and the R1185C mutation in the calmodulin-binding domain constitutively activate the WNK4 kinase domain allowing it to function despite the absence of angiotensin II (Fig. 3, right panel).   

Angiotensin II stimulates the secretion of aldosterone, which induces SGK1. SGK1 influences both the WNK-SPAK-NCC ^[33] and SGK1-ENaC signaling cascades.^[34] There are multiple SGK1 phosphorylation sites in the C-terminal region of WNK4 located within or close to the calmodulin-binding domain. SGK1-mediated phosphorylation of these sites is thought to disrupt the effect of the C-terminal inhibitory domain and concomitantly increase WNK4 kinase activity.^[30] The alteration of SGK1 phosphorylation by the R1185C mutation is another indication that the mutation disrupts the C-terminal inhibitory mechanism in WNK4 (Fig. 3, right panel).

Dysregulation of WNK4 abundance

Besides WNK1 and WNK4, mutations in two other genes, CUL3 (encoding Cullin 3) and KLHL3 (encoding Kelch Like Family Member 3) have been found in patients with PHAII.^[35][36] These two proteins are part of the ubiquitin E3 ligase complex involved in the ubiquitin-mediated degradation of WNK1 and WNK4. The PHAII-causing mutations in KLHL3 and cullin 3 prevent the interactions of these proteins with each other and with WNK1/4. The mutations in these proteins impair the degradation of WNK1/4. This in turn increases the protein abundance of WNK1/4 and concomitantly enhances the total kinase activity.^[37] The increased WNK4 kinase activity leads to the hyperactivation of NCC through WNK4-SPAK and/or the OSR1-NCC cascades, ultimately resulting in the retention of sodium and potassium by the kidney.

Elevated WNK4 activity

The primary effect of the elevated WNK4 kinase activity is the increase of NCC-mediated sodium reabsorption in the