C-terminally truncated, kidney-specific variants of the WNK4 kinase lack several sites that regulate its activity
| Autor: | Agnieszka Wengi, Junhui Zhang, María Castañeda-Bueno, Johannes Loffing, Richard P. Lifton, Alejandro Rodríguez-Gama, Diana Pacheco-Alvarez, Adrián Rafael Murillo-de-Ozores, David H. Ellison, Gerardo Gamba, Karla Leyva-Ríos, Silvana Bazúa-Valenti, Inti A. De La Rosa-Velázquez, Chao Ling Yang, Kathryn L. Stone, Norma Vázquez |
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| Rok vydání: | 2018 |
| Předmět: |
Male
0301 basic medicine Phosphatase Serine threonine protein kinase Protein Serine-Threonine Kinases Kidney Biochemistry Mice 03 medical and health sciences Protein Domains medicine Animals Phosphorylation Kinase activity Protein kinase A Molecular Biology Sequence Deletion Mice Knockout urogenital system Kinase Chemistry Cell Biology Molecular biology WNK4 030104 developmental biology medicine.anatomical_structure Organ Specificity Protein Binding |
| Zdroj: | Journal of Biological Chemistry. 293:12209-12221 |
| ISSN: | 0021-9258 |
| DOI: | 10.1074/jbc.ra118.003037 |
| Popis: | 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 Zn(2+)-dependent metalloprotease, as recombinant full-length WNK4 was cleaved when incubated with kidney lysate. |
| Databáze: | OpenAIRE |
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