Alternatively spliced proline-rich cassettes link WNK1 to aldosterone action

Ankita Roy, Lama Al-Qusairi, Bridget F. Donnelly, Caroline Ronzaud, Allison L. Marciszyn, Fan Gong, Y. P. Christy Chang, Michael B. Butterworth, Núria M. Pastor-Soler, Kenneth R. Hallows, Olivier Staub, Arohan R. Subramanya

Research output: Contribution to journalArticle

Abstract

The thiazide-sensitive NaCl cotransporter (NCC) is important for renal salt handling and blood-pressure homeostasis. The canonical NCC-activating pathway consists of With-No-Lysine (WNK) kinases and their downstream effector kinases SPAK and OSR1, which phosphorylate NCC directly. The upstream mechanisms that connect physiological stimuli to this system remain obscure. Here, we have shown that aldosterone activates SPAK/OSR1 via WNK1. We identified 2 alternatively spliced exons embedded within a proline-rich region of WNK1 that contain PY motifs, which bind the E3 ubiquitin ligase NEDD4-2. PY motif-containing WNK1 isoforms were expressed in human kidney, and these isoforms were efficiently degraded by the ubiquitin proteasome system, an effect reversed by the aldosterone-induced kinase SGK1. In gene-edited cells, WNK1 deficiency negated regulatory effects of NEDD4-2 and SGK1 on NCC, suggesting that WNK1 mediates aldosterone-dependent activity of the WNK/SPAK/OSR1 pathway. Aldosterone infusion increased proline-rich WNK1 isoform abundance in WT mice but did not alter WNK1 abundance in hypertensive Nedd4-2 KO mice, which exhibit high baseline WNK1 and SPAK/ OSR1 activity toward NCC. Conversely, hypotensive Sgk1 KO mice exhibited low WNK1 expression and activity. Together, our findings indicate that the proline-rich exons are modular cassettes that convert WNK1 into a NEDD4-2 substrate, thereby linking aldosterone and other NEDD4-2-suppressing antinatriuretic hormones to NCC phosphorylation status.

Original languageEnglish (US)
Pages (from-to)3433-3448
Number of pages16
JournalJournal of Clinical Investigation
Volume125
Issue number9
DOIs
StatePublished - Sep 1 2015
Externally publishedYes

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Aldosterone
Proline
Protein Isoforms
Phosphotransferases
Exons
Kidney
Ubiquitin-Protein Ligases
Proteasome Endopeptidase Complex
Ubiquitin
Lysine
Homeostasis
Salts
Phosphorylation
Hormones
Blood Pressure
Genes

ASJC Scopus subject areas

  • Medicine(all)

Cite this

Roy, A., Al-Qusairi, L., Donnelly, B. F., Ronzaud, C., Marciszyn, A. L., Gong, F., ... Subramanya, A. R. (2015). Alternatively spliced proline-rich cassettes link WNK1 to aldosterone action. Journal of Clinical Investigation, 125(9), 3433-3448. https://doi.org/10.1172/JCI75245

Alternatively spliced proline-rich cassettes link WNK1 to aldosterone action. / Roy, Ankita; Al-Qusairi, Lama; Donnelly, Bridget F.; Ronzaud, Caroline; Marciszyn, Allison L.; Gong, Fan; Christy Chang, Y. P.; Butterworth, Michael B.; Pastor-Soler, Núria M.; Hallows, Kenneth R.; Staub, Olivier; Subramanya, Arohan R.

In: Journal of Clinical Investigation, Vol. 125, No. 9, 01.09.2015, p. 3433-3448.

Research output: Contribution to journalArticle

Roy, A, Al-Qusairi, L, Donnelly, BF, Ronzaud, C, Marciszyn, AL, Gong, F, Christy Chang, YP, Butterworth, MB, Pastor-Soler, NM, Hallows, KR, Staub, O & Subramanya, AR 2015, 'Alternatively spliced proline-rich cassettes link WNK1 to aldosterone action', Journal of Clinical Investigation, vol. 125, no. 9, pp. 3433-3448. https://doi.org/10.1172/JCI75245
Roy, Ankita ; Al-Qusairi, Lama ; Donnelly, Bridget F. ; Ronzaud, Caroline ; Marciszyn, Allison L. ; Gong, Fan ; Christy Chang, Y. P. ; Butterworth, Michael B. ; Pastor-Soler, Núria M. ; Hallows, Kenneth R. ; Staub, Olivier ; Subramanya, Arohan R. / Alternatively spliced proline-rich cassettes link WNK1 to aldosterone action. In: Journal of Clinical Investigation. 2015 ; Vol. 125, No. 9. pp. 3433-3448.
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AU - Al-Qusairi, Lama

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AU - Marciszyn, Allison L.

AU - Gong, Fan

AU - Christy Chang, Y. P.

AU - Butterworth, Michael B.

AU - Pastor-Soler, Núria M.

AU - Hallows, Kenneth R.

AU - Staub, Olivier

AU - Subramanya, Arohan R.

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N2 - The thiazide-sensitive NaCl cotransporter (NCC) is important for renal salt handling and blood-pressure homeostasis. The canonical NCC-activating pathway consists of With-No-Lysine (WNK) kinases and their downstream effector kinases SPAK and OSR1, which phosphorylate NCC directly. The upstream mechanisms that connect physiological stimuli to this system remain obscure. Here, we have shown that aldosterone activates SPAK/OSR1 via WNK1. We identified 2 alternatively spliced exons embedded within a proline-rich region of WNK1 that contain PY motifs, which bind the E3 ubiquitin ligase NEDD4-2. PY motif-containing WNK1 isoforms were expressed in human kidney, and these isoforms were efficiently degraded by the ubiquitin proteasome system, an effect reversed by the aldosterone-induced kinase SGK1. In gene-edited cells, WNK1 deficiency negated regulatory effects of NEDD4-2 and SGK1 on NCC, suggesting that WNK1 mediates aldosterone-dependent activity of the WNK/SPAK/OSR1 pathway. Aldosterone infusion increased proline-rich WNK1 isoform abundance in WT mice but did not alter WNK1 abundance in hypertensive Nedd4-2 KO mice, which exhibit high baseline WNK1 and SPAK/ OSR1 activity toward NCC. Conversely, hypotensive Sgk1 KO mice exhibited low WNK1 expression and activity. Together, our findings indicate that the proline-rich exons are modular cassettes that convert WNK1 into a NEDD4-2 substrate, thereby linking aldosterone and other NEDD4-2-suppressing antinatriuretic hormones to NCC phosphorylation status.

AB - The thiazide-sensitive NaCl cotransporter (NCC) is important for renal salt handling and blood-pressure homeostasis. The canonical NCC-activating pathway consists of With-No-Lysine (WNK) kinases and their downstream effector kinases SPAK and OSR1, which phosphorylate NCC directly. The upstream mechanisms that connect physiological stimuli to this system remain obscure. Here, we have shown that aldosterone activates SPAK/OSR1 via WNK1. We identified 2 alternatively spliced exons embedded within a proline-rich region of WNK1 that contain PY motifs, which bind the E3 ubiquitin ligase NEDD4-2. PY motif-containing WNK1 isoforms were expressed in human kidney, and these isoforms were efficiently degraded by the ubiquitin proteasome system, an effect reversed by the aldosterone-induced kinase SGK1. In gene-edited cells, WNK1 deficiency negated regulatory effects of NEDD4-2 and SGK1 on NCC, suggesting that WNK1 mediates aldosterone-dependent activity of the WNK/SPAK/OSR1 pathway. Aldosterone infusion increased proline-rich WNK1 isoform abundance in WT mice but did not alter WNK1 abundance in hypertensive Nedd4-2 KO mice, which exhibit high baseline WNK1 and SPAK/ OSR1 activity toward NCC. Conversely, hypotensive Sgk1 KO mice exhibited low WNK1 expression and activity. Together, our findings indicate that the proline-rich exons are modular cassettes that convert WNK1 into a NEDD4-2 substrate, thereby linking aldosterone and other NEDD4-2-suppressing antinatriuretic hormones to NCC phosphorylation status.

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