Discovery - University of Dundee - Online Publications

Library & Learning Centre

SPAK/OSR1 regulate NKCC1 and WNK activity

Standard

SPAK/OSR1 regulate NKCC1 and WNK activity : analysis of WNK isoform interactions and activation by T-loop trans-autophosphorylation. / Thastrup, Jacob O.; Rafiqi, Fatema H.; Vitari, Alberto C.; Pozo-Guisado, Eulalia; Deak, Maria; Mehellou, Youcef; Alessi, Dario R.

In: Biochemical Journal, Vol. 441, 01.01.2012, p. 325-337.

Research output: Contribution to journalArticle

Harvard

Thastrup, JO, Rafiqi, FH, Vitari, AC, Pozo-Guisado, E, Deak, M, Mehellou, Y & Alessi, DR 2012, 'SPAK/OSR1 regulate NKCC1 and WNK activity: analysis of WNK isoform interactions and activation by T-loop trans-autophosphorylation' Biochemical Journal, vol 441, pp. 325-337., 10.1042/BJ20111879

APA

Thastrup, J. O., Rafiqi, F. H., Vitari, A. C., Pozo-Guisado, E., Deak, M., Mehellou, Y., & Alessi, D. R. (2012). SPAK/OSR1 regulate NKCC1 and WNK activity: analysis of WNK isoform interactions and activation by T-loop trans-autophosphorylation. Biochemical Journal, 441, 325-337. 10.1042/BJ20111879

Vancouver

Thastrup JO, Rafiqi FH, Vitari AC, Pozo-Guisado E, Deak M, Mehellou Y et al. SPAK/OSR1 regulate NKCC1 and WNK activity: analysis of WNK isoform interactions and activation by T-loop trans-autophosphorylation. Biochemical Journal. 2012 Jan 1;441:325-337. Available from: 10.1042/BJ20111879

Author

Thastrup, Jacob O.; Rafiqi, Fatema H.; Vitari, Alberto C.; Pozo-Guisado, Eulalia; Deak, Maria; Mehellou, Youcef; Alessi, Dario R. / SPAK/OSR1 regulate NKCC1 and WNK activity : analysis of WNK isoform interactions and activation by T-loop trans-autophosphorylation.

In: Biochemical Journal, Vol. 441, 01.01.2012, p. 325-337.

Research output: Contribution to journalArticle

Bibtex - Download

@article{242b4d1480354ad18729bc7d528d46cb,
title = "SPAK/OSR1 regulate NKCC1 and WNK activity: analysis of WNK isoform interactions and activation by T-loop trans-autophosphorylation",
keywords = "Coiled-coil domain, Na+/Cl− co-transporter (NCC), Na+/K+/2Cl− co-transporter 1 (NKCC1), Osmotic stress, Protein kinase, Signal transduction",
author = "Thastrup, {Jacob O.} and Rafiqi, {Fatema H.} and Vitari, {Alberto C.} and Eulalia Pozo-Guisado and Maria Deak and Youcef Mehellou and Alessi, {Dario R.}",
year = "2012",
doi = "10.1042/BJ20111879",
volume = "441",
pages = "325--337",
journal = "Biochemical Journal",
issn = "0264-6021",

}

RIS (suitable for import to EndNote) - Download

TY - JOUR

T1 - SPAK/OSR1 regulate NKCC1 and WNK activity

T2 - analysis of WNK isoform interactions and activation by T-loop trans-autophosphorylation

A1 - Thastrup,Jacob O.

A1 - Rafiqi,Fatema H.

A1 - Vitari,Alberto C.

A1 - Pozo-Guisado,Eulalia

A1 - Deak,Maria

A1 - Mehellou,Youcef

A1 - Alessi,Dario R.

AU - Thastrup,Jacob O.

AU - Rafiqi,Fatema H.

AU - Vitari,Alberto C.

AU - Pozo-Guisado,Eulalia

AU - Deak,Maria

AU - Mehellou,Youcef

AU - Alessi,Dario R.

PY - 2012/1/1

Y1 - 2012/1/1

N2 - <p>Mutations in the WNK with no lysine (K) kinase] family instigate hypertension and pain perception disorders. Of the four WNK isoforms, much of the focus has been on WNK1, which is activated in response to osmotic stress by phosphorylation of its T-loop residue (Ser(382)). WNK isoforms phosphorylate and activate the related SPAK (SPS1-related proline/alanine-rich kinase) and OSR1 (oxidative stress-responsive kinase 1) protein kinases. In the present study, we first describe the generation of double-knockin ES (embryonic stem) cells, where SPAK and OSR1 cannot be activated by WNK1. We establish that NKCC1 (Na+/K+/2Cl(-) co-transporter 1), a proposed target of the WNK pathway, is not phosphorylated or activated in a knockin that is deficient in SPAK/OSR1 activity. We also observe that activity of WNK1 and WNK3 are markedly elevated in the knockin cells, demonstrating that SPAK/OSR1 significantly influences WNK activity. Phosphorylation of another regulatory serine residue, Ser(1261), in WNK1 is unaffected in knockin cells, indicating that this is not phosphorylated by SPAK/OSR1. We show that WNK isoforms interact via a C-terminal CCD (coiled-coil domain) and identify point mutations of conserved residues within this domain that ablate the ability of WNK isoforms to interact. Employing these mutants, we demonstrate that interaction of WNK isoforms is not essential for their T-loop phosphorylation and activation, at least for overexpressed WNK isoforms. Moreover, we finally establish that full-length WNK 1, WNK2 and WNK3, but not WNK4, are capable of directly phosphorylating Ser(382) of WNK1 in vitro. This supports the notion that T-loop phosphorylation of WNK isoforms is controlled by trans-autophosphorylation. These results provide novel insights into the WNK signal transduction pathway and provide genetic evidence confirming the essential role that SPAK/OSR1 play in controlling NKCC1 function. They also reveal a role in which the downstream SPAK/OSR1 enzymes markedly influence the activity of the upstream WNK activators. The knockin ES cells lacking SPAK/OSR1 activity will be useful in validating new targets of the WNK signalling pathway.</p>

AB - <p>Mutations in the WNK with no lysine (K) kinase] family instigate hypertension and pain perception disorders. Of the four WNK isoforms, much of the focus has been on WNK1, which is activated in response to osmotic stress by phosphorylation of its T-loop residue (Ser(382)). WNK isoforms phosphorylate and activate the related SPAK (SPS1-related proline/alanine-rich kinase) and OSR1 (oxidative stress-responsive kinase 1) protein kinases. In the present study, we first describe the generation of double-knockin ES (embryonic stem) cells, where SPAK and OSR1 cannot be activated by WNK1. We establish that NKCC1 (Na+/K+/2Cl(-) co-transporter 1), a proposed target of the WNK pathway, is not phosphorylated or activated in a knockin that is deficient in SPAK/OSR1 activity. We also observe that activity of WNK1 and WNK3 are markedly elevated in the knockin cells, demonstrating that SPAK/OSR1 significantly influences WNK activity. Phosphorylation of another regulatory serine residue, Ser(1261), in WNK1 is unaffected in knockin cells, indicating that this is not phosphorylated by SPAK/OSR1. We show that WNK isoforms interact via a C-terminal CCD (coiled-coil domain) and identify point mutations of conserved residues within this domain that ablate the ability of WNK isoforms to interact. Employing these mutants, we demonstrate that interaction of WNK isoforms is not essential for their T-loop phosphorylation and activation, at least for overexpressed WNK isoforms. Moreover, we finally establish that full-length WNK 1, WNK2 and WNK3, but not WNK4, are capable of directly phosphorylating Ser(382) of WNK1 in vitro. This supports the notion that T-loop phosphorylation of WNK isoforms is controlled by trans-autophosphorylation. These results provide novel insights into the WNK signal transduction pathway and provide genetic evidence confirming the essential role that SPAK/OSR1 play in controlling NKCC1 function. They also reveal a role in which the downstream SPAK/OSR1 enzymes markedly influence the activity of the upstream WNK activators. The knockin ES cells lacking SPAK/OSR1 activity will be useful in validating new targets of the WNK signalling pathway.</p>

KW - Coiled-coil domain

KW - Na+/Cl− co-transporter (NCC)

KW - Na+/K+/2Cl− co-transporter 1 (NKCC1)

KW - Osmotic stress

KW - Protein kinase

KW - Signal transduction

U2 - 10.1042/BJ20111879

DO - 10.1042/BJ20111879

M1 - Article

JO - Biochemical Journal

JF - Biochemical Journal

SN - 0264-6021

VL - 441

SP - 325

EP - 337

ER -

Documents

Library & Learning Centre

Contact | Accessibility | Policy