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 overactivation 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)-binding 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.
Highlights
WNK lysine-deficient protein kinase 4 (WNK4) is an important regulator of renal salt handling
Using two different antibodies directed against N-terminal epitopes, we observed, in addition to the band corresponding to the full-length protein, at least two smaller bands that were absent in the WNK4Ϫ/Ϫ mouse samples (Fig. 1A and Fig. S1A)
These smaller bands were more abundant in samples from TgWNK4PHAII mice [4] (Fig. S1B) and were not detected when an antibody directed against a C-terminal WNK4 epitope was used (Fig. 1B)
Summary
C-terminally truncated, kidney-specific variants of the WNK4 kinase lack several sites that regulate its activity. Mutations occurring within a specific motif in WNK4, the acidic motif, produce the human genetic disease pseudohypoaldosteronism type II (PHAII) [2] This disease features hypertension, hyperkalemia, metabolic acidosis, and marked sensitivity to thiazide diuretics, alterations that are thought to be mainly due to higher basal activity of the renal thiazide–sensitive Naϩ/ClϪ cotransporter (NCC), which is expressed in the distal convoluted tubule (DCT) of the nephron [3, 4]. The second known regulatory mechanism of WNK4 kinase activity involves phosphorylation of at least two sites, Ser-64 and Ser-1196, located within the regulatory N- and C-terminal domains of WNK4, respectively [16]. The characterization of these short variants of WNK4 has led us to the identification of a bona fide PP1binding site located at the final portion of WNK4’s C terminus, which regulates WNK4 phosphorylation levels and, kinase activity
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