C-terminally truncated, kidney-specific variants of the WNK4 kinase lack several sites that regulate its activity
Journal
Journal of Biological Chemistry
ISSN
0021-9258
Date Issued
2018
Author(s)
Murillo-de-Ozores, Adrián Rafael
Rodríguez Moya, Alejandro
Bazúa-Valenti, Silvana
Vázquez, Norma
Rosa-Velázquez, Inti A. de la
Wengi, Agnieszka
Stone, Kathryn L.
Junhui, Zhang
Loffing, Johannes
Lifton, Richard P.
Chao-Ling , Yang
Ellison, David H.
Gamba, Gerardo
Castañeda-Bueno, María
Type
Resource Types::text::journal::journal article
Abstract
WNK lysine-deficient protein kinase 4 (WNK4) is an important regulator of renal salt handling. Mutations in its gene cause pseudohypoaldosteronism type II, mainly arising from overac-tivation of the renal Na/Cl cotransporter (NCC). In addition to full-length WNK4, we have observed faster migrating bands (between 95 and 130 kDa) in Western blots of kidney lysates. Therefore, we hypothesized that these could correspond to uncharacterized WNK4 variants. Here, using several WNK4 antibodies and WNK4/ mice as controls, we showed that these bands indeed correspond to short WNK4 variants that are not observed in other tissue lysates. LC-MS/MS confirmed these bands as WNK4 variants that lack C-terminal segments. In HEK293 cells, truncation of WNK4’s C terminus at several positions increased its kinase activity toward Ste20-related proline/ alanine-rich kinase (SPAK), unless the truncated segment included the SPAK-binding site. Of note, this gain-of-function effect was due to the loss of a protein phosphatase 1 (PP1)-bind-ing site in WNK4. Cotransfection with PP1 resulted in WNK4 dephosphorylation, an activity that was abrogated in the PP1-binding site WNK4 mutant. The electrophoretic mobility of the in vivo short variants of renal WNK4 suggested that they lack the SPAK-binding site and thus may not behave as constitutively active kinases toward SPAK. Finally, we show that at least one of the WNK4 short variants may be produced by proteolysis involving a Zn2-dependent metalloprotease, as recombinant full-length WNK4 was cleaved when incubated with kidney lysate. © 2018 American Society for Biochemistry and Molecular Biology Inc. All rights reserved.