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.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, Non-P.H.S.
MeSH terms
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Aldosterone / pharmacology*
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Alternative Splicing / drug effects*
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Alternative Splicing / physiology
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Animals
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Endosomal Sorting Complexes Required for Transport / genetics
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Endosomal Sorting Complexes Required for Transport / metabolism
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Enzyme Activation / drug effects
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Enzyme Activation / physiology
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Gene Expression Regulation, Enzymologic / drug effects*
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Gene Expression Regulation, Enzymologic / physiology
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HEK293 Cells
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Humans
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Intracellular Signaling Peptides and Proteins / biosynthesis*
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Intracellular Signaling Peptides and Proteins / genetics
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Mice
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Mice, Knockout
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Minor Histocompatibility Antigens
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Nedd4 Ubiquitin Protein Ligases
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Protein Serine-Threonine Kinases / biosynthesis*
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Protein Serine-Threonine Kinases / genetics
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Protein Serine-Threonine Kinases / metabolism
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Signal Transduction / drug effects*
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Signal Transduction / physiology
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Ubiquitin-Protein Ligases / genetics
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Ubiquitin-Protein Ligases / metabolism
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WNK Lysine-Deficient Protein Kinase 1
Substances
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Endosomal Sorting Complexes Required for Transport
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Intracellular Signaling Peptides and Proteins
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Minor Histocompatibility Antigens
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Aldosterone
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Nedd4 Ubiquitin Protein Ligases
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Nedd4 protein, human
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Nedd4L protein, human
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Nedd4l protein, mouse
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Ubiquitin-Protein Ligases
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OXSR1 protein, human
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Stk39 protein, mouse
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OXSR1 protein, mouse
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Protein Serine-Threonine Kinases
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STK39 protein, human
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WNK Lysine-Deficient Protein Kinase 1
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WNK1 protein, human
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Wnk1 protein, mouse