Date Published: March 1, 2018
Publisher: Public Library of Science
Author(s): Atsushi Sato, Hiroshi Shibuya, Renping Zhou.
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.
The with no lysine (WNK) protein kinases are atypical members of the serine/threonine kinase family, and are conserved among many species [1–3]. The mammalian WNK family has four members: WNK1–4. WNK1 and WNK4 have been identified as causative genes of pseudohypoaldosteronism type II (PHAII) , and WNK1 is also a causative gene of hereditary sensory and autonomic neuropathy type 2A (HSAN2A) . Several groups including ours have attempted to identify the functions of the WNK family. In the kidney, WNK1 and WNK4 phosphorylate and activate STE20/SPS1-related, proline alanine-rich kinase (SPAK) and oxidative-stress responsive 1 (OSR1) kinases, which in turn regulate various ion co-transporters [6–9]. Because knock-in mice of Wnk4D561A (the mutation found in PHAII patients) display similar phenotypes to PHAII, dysregulation of this WNK signaling pathway was thought to cause hypertension in PHAII patients . In the neural system, a neural-specific alternatively spliced isoform of WNK1 is expressed, which includes the neural-specific exon HSN2. In HSAN2A patients, mutations were found in this HSN2 exon [5, 11], but HSN2 knock-out mice have no discernable morphological phenotype [11, 12]. Furthermore, in other familial HSAN2A patients, mutations were found outside the HSN2 exon in WNK1 . Thus, the pathogenesis of HSAN2A remains unclear.
The WNK signaling pathway is involved in many biological processes, but the details of its components are unclear, except for in the kidney. Here, we screened candidate genes that genetically interact with the WNK signaling pathway in Drosophila. Among these, we identified shaggy, which encodes the Drosophila homolog of mammalian GSK3ß (Fig 1). We showed that GSK3ß activated Lhx8 expression and that GSK3ß functions downstream of the WNK–OSR1 pathway by epistasis analysis (Fig 2). We also showed that GSK3ß might form a tertiary complex with WNK1 and OSR1 (Fig 3). Furthermore, GSK3ß was found to be involved in neural specification and neurite elongation (Fig 4), and GSK3ß rescued the neural phenotypes induced by the knockdown of both Wnk1 and Wnk4 (Fig 4). However, we did not observe direct phosphorylation of GSK3ß by WNK1 or OSR1 (Fig 3). This suggests that GSK3ß functions as a positive downstream effector in the WNK signaling pathway, although the regulation of GSK3ß activity by the signaling pathway remains unclear and requires further study to elucidate how WNK–OSR1 transduces the signal to GSK3ß.