The with no lysine (WNK) protein kinase family is conserved among many species. Some mutations in human WNK gene are associated with pseudohypoaldosteronism type II, a form of hypertension, and hereditary sensory and autonomic neuropathy type 2A. In kidney, WNK regulates the activity of STE20/SPS1-related, proline alanine-rich kinase and/or oxidative-stress responsive 1, which in turn regulate ion co-transporters. The misregulation of this pathway is involved in the pathogenesis of pseudohypoaldosteronism type II. In the neural system, WNK is involved in the specification of the cholinergic neuron, but the pathogenesis of hereditary sensory and autonomic neuropathy type 2A is still unknown. To better understand the WNK pathway, we isolated WNK-associated genes using Drosophila. We identified Glycogen synthase kinase 3ß (GSK3ß)/Shaggy (Sgg) as a candidate gene that was shown to interact with the WNK signaling pathway in both Drosophila and mammalian cells. Furthermore, GSK3ß was involved in neural specification downstream of WNK. These results suggest that GSK3ß/Sgg functions as a positive effector in the WNK signaling pathway.

WNK kinase family is conserved among many species and regulates SPAK/OSR1 and ion co-transporters. Some mutations in human WNK1 or WNK4 are associated with Pseudohypoaldosteronism type II, a form of hypertension. WNK is also involved in developmental and cellular processes, but the molecular mechanisms underlying its regulation in these processes remain unknown. Here, we identify a new target gene in WNK signaling, Arrowhead and Lhx8, which is a mammalian homologue of Drosophila Arrowhead. In Drosophila, WNK was shown to genetically interact with Arrowhead. In Wnk1 knockout mice, levels of Lhx8 expression were reduced. Ectopic expression of WNK1, WNK4 or Osr1 in mammalian cells induced the expression of the Lhx8. Moreover, neural specification was inhibited by the knockdown of both Wnk1 and Wnk4 or Lhx8. Drosophila WNK mutant caused defects in axon guidance during embryogenesis. These results suggest that WNK signaling is involved in the morphological and neural development via Lhx8/Arrowhead.

With no lysine kinase 1 (WNK1) phosphorylates and activates STE20/SPS1-related proline-alanine-rich protein kinase (SPAK) and oxidative stress responsive kinase 1 (OSR1) to regulate ion homeostasis in the kidney. Mutations in WNK1 result in dysregulation of the WNK1-SPAK/OSR1 pathway and cause pseudohypoaldosteronism type II (PHAII), a form of hypertension. WNK1 is also involved in the autosomal recessive neuropathy, hereditary sensory and autonomic neuropathy type II (HSANII). Mutations in a neural-specific splice variant of WNK1 (HSN2) cause HSANII. However, the mechanisms underlying HSN2 regulation in neurons and effects of HSN2 mutants remain unclear. Here, we found that HSN2 regulated neurite outgrowth through OSR1 activation and glycogen synthase kinase 3β (GSK3β). Moreover, HSN2-OSR1 and HSN2-GSK3β signalling induced expression of LIM homeobox 8 (Lhx8), which is a key regulator of cholinergic neural function. The HSN2-OSR1/GSK3β-LHX8 pathway is therefore important for neurite outgrowth. Consistently, HSN2 mutants reported in HSANII patients suppressed SPAK and OSR1 activation and LHX8 induction. Interestingly, HSN2 mutants also suppressed neurite outgrowth by preventing interaction of between wild-type HSN2 and GSK3β. These results indicate that HSN2 mutants cause dysregulation of neurite outgrowth via GSK3β in the HSN2 and/or WNK1 pathways.