Serum and Glucocorticoid-induced Kinase (SGK) 1 and the Epithelial Sodium Channel Are Regulated by Multiple with No Lysine (WNK) Family Members

Abstract

The four WNK (with no lysine (K)) protein kinases affect ion balance and contain an unusual protein kinase domain due to the unique placement of the active site lysine. Mutations in two WNKs cause a heritable form of ion imbalance culminating in hypertension. WNK1 activates the serum-and glucocorticoidinduced protein kinase SGK1; the mechanism is noncatalytic. SGK1 increases membrane expression of the epithelial sodium channel (ENaC) and sodium reabsorption via phosphorylation and sequestering of the E3 ubiquitin ligase neural precursor cell expressed, developmentally down-regulated 4-2 (Nedd4-2), which otherwise promotes ENaC endocytosis. Questions remain about the intrinsic abilities of WNK family members to regulate this pathway. We find that expression of the N termini of all four WNKs results in modest to strong activation of SGK1. In reconstitution experiments in the same cell line all four WNKs also increase sodium current blocked by the ENaC inhibitor amiloride. The N termini of the WNKs also have the capacity to interact with SGK1. More detailed analysis of activation by WNK4 suggests mechanisms in common with WNK1. Further evidence for the importance of WNK1 in this process comes from the ability of Nedd4-2 to bind to WNK1 and the finding that endogenous SGK1 has reduced activity if WNK1 is knocked down by small interfering RNA. WNKs5 (with no lysine (K)) are large protein-serine/threonine kinases found in all multicellular and a few unicellular eukaryotes (1). WNK1, the first member of the family identified in mammals, was found in searches for novel components of protein kinase cascades (2). WNK1 is expressed ubiquitously, consistent with effects on many cell types (2-4). Numerous splice forms, containing from just under 2000 to almost 2400 residues, are known, including one lacking most of the kinase domain (KS-WNK1), which is enriched in kidney (5, 6).The four WNK family members are distinct from all other protein kinases in that their catalytic lysine is shifted from its usual position buried in the N-terminal part of the kinase core to a more exposed position in the glycine-rich loop (2, 7). The strict conservation of the unique catalytic core structure of the WNK family in organisms such as Chlamydomonas, Phycomyces, Arabidopsis, and mammals suggests conserved properties for these kinases.Our initial characterization of WNK1 revealed that the kinase activity is sensitive to hypertonic stress (2). The subsequent discovery that WNK1 and WNK4 are genetically linked to a rare type of hypertension, pseudohypoaldosteronism type 2 (PHA2) (4), demonstrated the importance of WNK function in man and an implicit significance of the sensitivity of WNK1 kinase activity to osmotic stress. Further characterization showed that activity is increased in response to increased and decreased ionic strength (8). The consequences of WNK mutations in PHA2 are hyperkalemia, renal tubular acidosis, and eventually hypertension (1, 9 -11). WNK1 knock-out mice do not survive, but heterozygotes have low blood pressure (12), ...

Af17 Deficiency Increases Sodium Excretion and Decreases Blood Pressure

“…WNK1 and WNK4 can upregulate ENaC activity through SGK1 and Nedd4 -2. 8,9 Although the BP phenotype of Period Ϫ/Ϫ mice has not been reported, these mice fed the standard diet also had higher Na ϩ excretion and lower ␣ENaC expression than WT controls. 20 Another feature not limited to Af17 Ϫ/Ϫ mice is that the changes in Na ϩ reabsorption are not accompanied by an inverse effect on K ϩ , indicating that the impaired ENaC function can not account for the complete panoply of the renal dysfunction.…”

“…WNK1 and WNK4 have been shown to regulate ENaC. 8,9 A number of inherited disorders characterized by salt wasting and hypotension are caused by loss-of-function mutations impairing salt homeostasis. Examples include Bartter's and Gitelman's syndromes, with defects in six genes encoding renal tubular transporters and ion channels (reviewed in ref.…”

Regulation of OSR1 and the sodium, potassium, two chloride cotransporter by convergent signals

“…Phosphorylation of this site has little effect on WNK1 kinase activity. However, mutation of T60 inhibits SGK activation by WNKs, suggesting that phosphorylation of T60 is a permissive event that exerts positive feedback on some WNK1 functions (24,36). Phosphorylation of T60 is required for maximal activation of ENaC by WNK1, consistent with the effect on SGK1 activation (36); regulation of the renal outer medullary potassium channel by WNK1 also depends on phosphorylation of T60 (56).…”

“…WNKs are interconnected with PI3K-dependent pathways. Akt phosphorylates WNK1 on a threonine residue, T60 (T58 in rat), preceding the kinase domain and the comparable residue in WNK4 (24,36,54,55). Phosphorylation of this site has little effect on WNK1 kinase activity.…”

“…WNKs also regulate serum-and glucocorticoid-inducible protein kinases (SGKs) through a noncatalytic mechanism leading to increased sodium influx through the epithelial sodium channel (ENaC) (23,24). SGKs and the related Akt enzymes are activated by phosphorylation on multiple sites, most prominently a residue in the activation loop by the phosphoinositide-dependent protein kinase and on a second site in a C-terminal hydrophobic motif (25).…”