WNK3 abrogates the NEDD4-2-mediated inhibition of the renal Na+-Cl- cotransporter.
Am J Physiol Renal Physiol
; 307(3): F275-86, 2014 Aug 01.
Article
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| MEDLINE
| ID: mdl-24920754
ABSTRACT
The serine/threonine kinase WNK3 and the ubiquitin-protein ligase NEDD4-2 are key regulators of the thiazide-sensitive Na+-Cl- cotransporter (NCC), WNK3 as an activator and NEDD2-4 as an inhibitor. Nedd4-2 was identified as an interacting partner of WNK3 through a glutathione-S-transferase pull-down assay using the N-terminal domain of WNK3, combined with LC-MS/MS analysis. This was validated by coimmunoprecipitation of WNK3 and NEDD4-2 expressed in HEK293 cells. Our data also revealed that the interaction between Nedd4-2 and WNK3 does not involve the PY-like motif found in WNK3. The level of WNK3 ubiquitylation did not change when NEDD4-2 was expressed in HEK293 cells. Moreover, in contrast to SGK1, WNK3 did not phosphorylate NEDD4-2 on S222 or S328. Coimmunoprecipitation assays showed that WNK3 does not regulate the interaction between NCC and NEDD4-2. Interestingly, in Xenopus laevis oocytes, WNK3 was able to recover the SGK1-resistant NEDD4-2 S222A/S328A-mediated inhibition of NCC and further activate NCC. Furthermore, elimination of the SPAK binding site in the kinase domain of WNK3 (WNK3-F242A, which lacks the capacity to bind the serine/threonine kinase SPAK) prevented the WNK3 NCC-activating effect, but not the Nedd4-2-inhibitory effect. Together, these results suggest that a novel role for WNK3 on NCC expression at the plasma membrane, an effect apparently independent of the SPAK kinase and the aldosterone-SGK1 pathway.
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1
Bases de datos:
MEDLINE
Asunto principal:
Membrana Celular
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Proteínas Serina-Treonina Quinasas
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Ubiquitina-Proteína Ligasas
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Simportadores del Cloruro de Sodio
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Complejos de Clasificación Endosomal Requeridos para el Transporte
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Riñón
Límite:
Animals
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Female
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Humans
Idioma:
En
Revista:
Am J Physiol Renal Physiol
Asunto de la revista:
FISIOLOGIA
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NEFROLOGIA
Año:
2014
Tipo del documento:
Article