2.3.1. KLHL3-Mediated WNK4 Ubiquitylation and NCC

NCC, a target of thiazide diuretics, modulates sodium reabsorption in the DCT, and accumulating evidence has demonstrated its importance in controlling BP [25]. Familial hyperkalemic hypertension, also known as pseudohypoaldosteronism type II (PHAII) or Gordon syndrome, is characterized by salt-sensitive hypertension, hyperkalemia, and metabolic acidosis [26–28]. The phenotypes in these patients can be reversed by thiazide treatment, thus suggesting the involvement of NCC in the pathogenesis of PHAII. Till date, mutations in four genes are known to cause PHAII, which include serine-threonine kinase with-no-lysine (WNK) 1 and WNK4 and Cullin 3 (CUL3) and Kelch-like 3 (KLHL3), the components of the cullin-RING ubiquitin ligase (CRL) complex [3,28,29]. WNKs are substrates for the KLHL3-CUL3 ligase complex. WNKs phosphorylate the downstream kinases STE20/SPS1-related proline-alanine-rich protein kinase (SPAK) and oxidative stress-responsive 1 [30],

which in turn increase the levels of phosphorylated NCC, an active form of NCC. We and others have identified by mass spectrometry and co-immunoprecipitation that KLHL3 normally binds to WNK1 and WNK4 [4,31–33]. KLHL3-WNK4 binding induces ubiquitylation in at least 15 specific sites, leading to reduced WNK4 levels [4]. KLHL3 is phosphorylated at serine 433 (KLHL3S433) in the Kelch domain, which is regulated by angiotensin II–protein kinase C signaling [34]. Of interest, this site is recurrently mutated in independent PHAII families, and phosphorylation or single amino acid substitution of this site impairs the binding of KLHL3 with WNKs, resulting in its accumulation and activation of downstream signaling. It has also been reported that Akt and protein kinase A (PKA), key downstream substrates of insulin and vasopressin signaling, respectively, increase phosphorylated KLHL3S433 [35]. In addition, calcium-sensing receptor signaling can modulate KLHL3-WNK4-SPAK pathway by phosphorylating KLHL3 and WNK4 [36,37]. Conversely, phosphatase calcineurin is capable of dephosphorylating KLHL3 phosphorylation at KLHL3S433 [38]. These mechanisms probably play important roles in several pathological conditions such as low-K+-induced BP elevation and hypertension associated with obese diabetes mellitus [39,40]. CRLs are activated by neddylation of cullin. It has been demonstrated that CUL3 is also neddylated, and that its neddylation status is regulated by multisubunit deneddylase COP9 signalosome [41,42].
