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
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
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Research Article Nephrology

Alternatively spliced proline-rich cassettes link WNK1 to aldosterone action

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.

Authors

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

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Figure 10

Model of aldosterone-mediated activation of SPAK/OSR1 and NCC via WNK1.

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Model of aldosterone-mediated activation of SPAK/OSR1 and NCC via WNK1. ...
In the baseline euvolemic state, active NEDD4-2 binds to the PY motifs in exons 11 and 12 of WNK1, ubiquitylating the WNK1 C-terminus and targeting it for proteasomal degradation. This limits total WNK1 protein expression, acting as a closed gate that shuts off downstream SPAK/OSR1 and NCC activity. During states where aldosterone levels are high, such as extracellular fluid volume depletion, SPAK and OSR1 are phosphorylated and active. Aldosterone contributes to this process by driving the transcription of SGK1 in the distal nephron. Active SGK1 phosphorylates and inactivates NEDD4-2, reducing its interaction with exons 11 and 12 of WNK1. This opens the gate, increasing total WNK1 protein expression. Once WNK1 protein is present, SGK1 can directly phosphorylate the N-terminus of L-WNK1, resulting in selective L-WNK1 activation (43). Active L-WNK1 then stimulates SPAK and OSR1 to directly phosphorylate NCC. Previous data suggest that the SGK1 pathway may be particularly active during states that require the conservation of sodium (59, 68). Other stimuli for aldosterone secretion, such as hyperkalemia, may selectively upregulate KS-WNK1 protein expression via additional undefined mechanisms to attenuate L-WNK1 and NCC activity (55, 59, 60). Such effects may act in concert with changes in intracellular [Cl–] to suppress WNK activity (61). Additionally, other SGK1-dependent effects on NCC, including suppression of WNK4-mediated NCC degradation (46) or ubiquitylation of NCC by NEDD4-2 (35), are not shown here to highlight the mechanism of SPAK/OSR1 activation described in this study.