NRBP1 pseudokinase binds to and activates the WNK pathway in response to osmotic stress

Ramchandra Amnekar; Toby Dite; Paweł Lis; Sebastian Bell; Fiona Brown; Clare Johnson; Stuart G Wilkinson; Samantha L. Raggett; Mark Dorward; Melanie Wightman; Thomas Macartney; Renata Filipe Soares; Fred Lamoliatte; Dario Alessi

doi:10.1126/sciadv.adv4636

NRBP1 pseudokinase binds to and activates the WNK pathway in response to osmotic stress

Ramchandra Amnekar (Lead / Corresponding author)
, Toby Dite
, Paweł Lis
, Sebastian Bell
, Fiona Brown
, Clare Johnson
, Stuart G Wilkinson
, Samantha L. Raggett
, Mark Dorward
, Melanie Wightman
, Thomas Macartney
, Renata Filipe Soares
, Fred Lamoliatte
, Dario Alessi (Lead / Corresponding author)

MRC PPU

Research output: Contribution to journal › Article › peer-review

Abstract

WNK family kinases are regulated by osmotic stress and control ion homeostasis by activating SPAK and OXSR1 kinases. Using a proximity labeling approach, we found that osmotic stress promotes the association of WNK1 with the NRBP1 pseudokinase and TSC22D2/4 adaptor proteins, results that are confirmed by immunoprecipitation, mass spectrometry, and immunoblotting studies. NRBP1 pseudokinase is closely related to WNK isoforms and contains a RΦ-motif–binding conserved C-terminal (CCT) domain, like the CCT domains in WNKs, SPAK, and OXSR1. Knockdown or knockout of NRBP1 markedly inhibited basal as well as sorbitol-induced activation of WNK1 and downstream components. We demonstrate that recombinant NRBP1 can directly induce the activation of WNK4 in vitro. AlphaFold-3 modeling predicts that WNK1, SPAK, NRBP1, and TSC22D4 form a complex, in which two TSC22D4 RΦ-motifs interact with the CCTL1 domain of WNK1 and the CCT domain of NRBP1. Our data indicate that NRBP1 and likely its close homolog NRBP2 function as an upstream activator of the WNK pathway.

Original language	English
Article number	eadv4636
Journal	Science Advances
Volume	11
Issue number	29
Early online date	16 Jul 2025
DOIs	https://doi.org/10.1126/sciadv.adv4636
Publication status	E-pub ahead of print - 16 Jul 2025

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Amnekar, R., Dite, T., Lis, P., Bell, S., Brown, F., Johnson, C., Wilkinson, S. G., Raggett, S. L., Dorward, M., Wightman, M., Macartney, T., Filipe Soares, R., Lamoliatte, F., & Alessi, D. (2025). NRBP1 pseudokinase binds to and activates the WNK pathway in response to osmotic stress. Science Advances, 11(29), Article eadv4636. Advance online publication. https://doi.org/10.1126/sciadv.adv4636

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title = "NRBP1 pseudokinase binds to and activates the WNK pathway in response to osmotic stress",

abstract = "WNK family kinases are regulated by osmotic stress and control ion homeostasis by activating SPAK and OXSR1 kinases. Using a proximity labeling approach, we found that osmotic stress promotes the association of WNK1 with the NRBP1 pseudokinase and TSC22D2/4 adaptor proteins, results that are confirmed by immunoprecipitation, mass spectrometry, and immunoblotting studies. NRBP1 pseudokinase is closely related to WNK isoforms and contains a RΦ-motif–binding conserved C-terminal (CCT) domain, like the CCT domains in WNKs, SPAK, and OXSR1. Knockdown or knockout of NRBP1 markedly inhibited basal as well as sorbitol-induced activation of WNK1 and downstream components. We demonstrate that recombinant NRBP1 can directly induce the activation of WNK4 in vitro. AlphaFold-3 modeling predicts that WNK1, SPAK, NRBP1, and TSC22D4 form a complex, in which two TSC22D4 RΦ-motifs interact with the CCTL1 domain of WNK1 and the CCT domain of NRBP1. Our data indicate that NRBP1 and likely its close homolog NRBP2 function as an upstream activator of the WNK pathway.",

author = "Ramchandra Amnekar and Toby Dite and Pawe{\l} Lis and Sebastian Bell and Fiona Brown and Clare Johnson and Wilkinson, \{Stuart G\} and Raggett, \{Samantha L.\} and Mark Dorward and Melanie Wightman and Thomas Macartney and \{Filipe Soares\}, Renata and Fred Lamoliatte and Dario Alessi",

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doi = "10.1126/sciadv.adv4636",

language = "English",

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T1 - NRBP1 pseudokinase binds to and activates the WNK pathway in response to osmotic stress

AU - Amnekar, Ramchandra

AU - Dite, Toby

AU - Lis, Paweł

AU - Bell, Sebastian

AU - Brown, Fiona

AU - Johnson, Clare

AU - Wilkinson, Stuart G

AU - Raggett, Samantha L.

AU - Dorward, Mark

AU - Wightman, Melanie

AU - Macartney, Thomas

AU - Filipe Soares, Renata

AU - Lamoliatte, Fred

AU - Alessi, Dario

PY - 2025/7/16

Y1 - 2025/7/16

N2 - WNK family kinases are regulated by osmotic stress and control ion homeostasis by activating SPAK and OXSR1 kinases. Using a proximity labeling approach, we found that osmotic stress promotes the association of WNK1 with the NRBP1 pseudokinase and TSC22D2/4 adaptor proteins, results that are confirmed by immunoprecipitation, mass spectrometry, and immunoblotting studies. NRBP1 pseudokinase is closely related to WNK isoforms and contains a RΦ-motif–binding conserved C-terminal (CCT) domain, like the CCT domains in WNKs, SPAK, and OXSR1. Knockdown or knockout of NRBP1 markedly inhibited basal as well as sorbitol-induced activation of WNK1 and downstream components. We demonstrate that recombinant NRBP1 can directly induce the activation of WNK4 in vitro. AlphaFold-3 modeling predicts that WNK1, SPAK, NRBP1, and TSC22D4 form a complex, in which two TSC22D4 RΦ-motifs interact with the CCTL1 domain of WNK1 and the CCT domain of NRBP1. Our data indicate that NRBP1 and likely its close homolog NRBP2 function as an upstream activator of the WNK pathway.

AB - WNK family kinases are regulated by osmotic stress and control ion homeostasis by activating SPAK and OXSR1 kinases. Using a proximity labeling approach, we found that osmotic stress promotes the association of WNK1 with the NRBP1 pseudokinase and TSC22D2/4 adaptor proteins, results that are confirmed by immunoprecipitation, mass spectrometry, and immunoblotting studies. NRBP1 pseudokinase is closely related to WNK isoforms and contains a RΦ-motif–binding conserved C-terminal (CCT) domain, like the CCT domains in WNKs, SPAK, and OXSR1. Knockdown or knockout of NRBP1 markedly inhibited basal as well as sorbitol-induced activation of WNK1 and downstream components. We demonstrate that recombinant NRBP1 can directly induce the activation of WNK4 in vitro. AlphaFold-3 modeling predicts that WNK1, SPAK, NRBP1, and TSC22D4 form a complex, in which two TSC22D4 RΦ-motifs interact with the CCTL1 domain of WNK1 and the CCT domain of NRBP1. Our data indicate that NRBP1 and likely its close homolog NRBP2 function as an upstream activator of the WNK pathway.

UR - https://www.scopus.com/pages/publications/105011840352

U2 - 10.1126/sciadv.adv4636

DO - 10.1126/sciadv.adv4636

M3 - Article

C2 - 40668933

SN - 2375-2548

VL - 11

JO - Science Advances

JF - Science Advances

IS - 29

M1 - eadv4636

ER -

NRBP1 pseudokinase binds to and activates the WNK pathway in response to osmotic stress

Abstract

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