SPAK/OSR1 regulate NKCC1 and WNK activity: analysis of WNK isoform interactions and activation by T-loop trans-autophosphorylation

Jacob O. Thastrup, Fatema H. Rafiqi, Alberto C. Vitari, Eulalia Pozo-Guisado, Maria Deak, Youcef Mehellou, Dario R. Alessi

    Research output: Contribution to journalArticle

    56 Citations (Scopus)

    Abstract

    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.

    Original languageEnglish
    Pages (from-to)325-337
    Number of pages13
    JournalBiochemical Journal
    Volume441
    DOIs
    Publication statusPublished - 1 Jan 2012

    Keywords

    • Coiled-coil domain
    • Na+/Cl− co-transporter (NCC)
    • Na+/K+/2Cl− co-transporter 1 (NKCC1)
    • Osmotic stress
    • Protein kinase
    • Signal transduction

    Cite this

    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. 2012 ; Vol. 441. pp. 325-337.
    @article{242b4d1480354ad18729bc7d528d46cb,
    title = "SPAK/OSR1 regulate NKCC1 and WNK activity: analysis of WNK isoform interactions and activation by T-loop trans-autophosphorylation",
    abstract = "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.",
    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",
    month = "1",
    day = "1",
    doi = "10.1042/BJ20111879",
    language = "English",
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    pages = "325--337",
    journal = "Biochemical Journal",
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    publisher = "Portland Press",

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    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

    TY - JOUR

    T1 - SPAK/OSR1 regulate NKCC1 and WNK activity

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

    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 - 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.

    AB - 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.

    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

    M3 - Article

    C2 - 22032326

    VL - 441

    SP - 325

    EP - 337

    JO - Biochemical Journal

    JF - Biochemical Journal

    SN - 0264-6021

    ER -