WNK3-SPAK Interaction is Required for the Modulation of NCC and other Members of the SLC12 Family

Diana Pacheco-Alvarez, Norma Vazquez, Maria Castaneda-Bueno, Paola De-Los-Heros, Cesar Cortes-Gonzalez, Erika Moreno, Patricia Meade, Norma A. Bobadilla, Gerardo Gamba

    Research output: Contribution to journalArticle

    26 Citations (Scopus)

    Abstract

    The serine/threonine with no lysine kinase 3 (WNK3) modulates the activity of the electroneutral cation-coupled chloride cotransporters (CCC) to promote Cl- influx and prevent Cl- efflux, thus fitting the profile for a putative "Cl--sensing kinase". The Ste20-type kinases, SPAK/OSR1, become phosphorylated in response to reduction in intracellular chloride concentration and regulate the activity of NKCC1. Several studies have now shown that WNKs function upstream of SPAK/OSR1. This study was designed to analyze the role of WNK3-SPAK interaction in the regulation of CCCs with particular emphasis on NCC. In this study we used the functional expression system of Xenopus laevis oocytes to show that different SPAK binding sites in WNK3 ((241, 872, 1336)RFxV) are required for the kinase to have effects on CCCs. WNK3-F1337A no longer activated NKCC2, but the effects on NCC, NKCC1, and KCC4 were preserved. In contrast, the effects of WNK3 on these cotransporters were prevented in WNK3-F242A. The elimination of F873 had no consequence on WNK3 effects. WNK3 promoted NCC phosphorylation at threonine 58, even in the absence of the unique SPAK binding site of NCC, but this effect was abolished in the mutant WNK3-F242A. Thus, our data support the hypothesis that the effects of WNK3 upon NCC and other CCCs require the interaction and activation of the SPAK kinase. The effect is dependent on one of the three binding sites for SPAK that are present in WNK3, but not on the SPAK binding sites on the CCCs, which suggests that WNK3 is capable of binding both SPAK and CCCs to promote their phosphorylation. Copyright (C) 2012 S. Karger AG, Basel

    Original languageEnglish
    Pages (from-to)291-302
    Number of pages12
    JournalCellular Physiology and Biochemistry
    Volume29
    Issue number1-2
    DOIs
    Publication statusPublished - 2012

    Cite this

    Pacheco-Alvarez, D., Vazquez, N., Castaneda-Bueno, M., De-Los-Heros, P., Cortes-Gonzalez, C., Moreno, E., ... Gamba, G. (2012). WNK3-SPAK Interaction is Required for the Modulation of NCC and other Members of the SLC12 Family. Cellular Physiology and Biochemistry, 29(1-2), 291-302. https://doi.org/10.1159/000337610
    Pacheco-Alvarez, Diana ; Vazquez, Norma ; Castaneda-Bueno, Maria ; De-Los-Heros, Paola ; Cortes-Gonzalez, Cesar ; Moreno, Erika ; Meade, Patricia ; Bobadilla, Norma A. ; Gamba, Gerardo. / WNK3-SPAK Interaction is Required for the Modulation of NCC and other Members of the SLC12 Family. In: Cellular Physiology and Biochemistry. 2012 ; Vol. 29, No. 1-2. pp. 291-302.
    @article{3c8b51d006844a51a84ae990e14edccd,
    title = "WNK3-SPAK Interaction is Required for the Modulation of NCC and other Members of the SLC12 Family",
    abstract = "The serine/threonine with no lysine kinase 3 (WNK3) modulates the activity of the electroneutral cation-coupled chloride cotransporters (CCC) to promote Cl- influx and prevent Cl- efflux, thus fitting the profile for a putative {"}Cl--sensing kinase{"}. The Ste20-type kinases, SPAK/OSR1, become phosphorylated in response to reduction in intracellular chloride concentration and regulate the activity of NKCC1. Several studies have now shown that WNKs function upstream of SPAK/OSR1. This study was designed to analyze the role of WNK3-SPAK interaction in the regulation of CCCs with particular emphasis on NCC. In this study we used the functional expression system of Xenopus laevis oocytes to show that different SPAK binding sites in WNK3 ((241, 872, 1336)RFxV) are required for the kinase to have effects on CCCs. WNK3-F1337A no longer activated NKCC2, but the effects on NCC, NKCC1, and KCC4 were preserved. In contrast, the effects of WNK3 on these cotransporters were prevented in WNK3-F242A. The elimination of F873 had no consequence on WNK3 effects. WNK3 promoted NCC phosphorylation at threonine 58, even in the absence of the unique SPAK binding site of NCC, but this effect was abolished in the mutant WNK3-F242A. Thus, our data support the hypothesis that the effects of WNK3 upon NCC and other CCCs require the interaction and activation of the SPAK kinase. The effect is dependent on one of the three binding sites for SPAK that are present in WNK3, but not on the SPAK binding sites on the CCCs, which suggests that WNK3 is capable of binding both SPAK and CCCs to promote their phosphorylation. Copyright (C) 2012 S. Karger AG, Basel",
    author = "Diana Pacheco-Alvarez and Norma Vazquez and Maria Castaneda-Bueno and Paola De-Los-Heros and Cesar Cortes-Gonzalez and Erika Moreno and Patricia Meade and Bobadilla, {Norma A.} and Gerardo Gamba",
    year = "2012",
    doi = "10.1159/000337610",
    language = "English",
    volume = "29",
    pages = "291--302",
    journal = "Cellular Physiology and Biochemistry",
    issn = "1015-8987",
    publisher = "Karger",
    number = "1-2",

    }

    Pacheco-Alvarez, D, Vazquez, N, Castaneda-Bueno, M, De-Los-Heros, P, Cortes-Gonzalez, C, Moreno, E, Meade, P, Bobadilla, NA & Gamba, G 2012, 'WNK3-SPAK Interaction is Required for the Modulation of NCC and other Members of the SLC12 Family', Cellular Physiology and Biochemistry, vol. 29, no. 1-2, pp. 291-302. https://doi.org/10.1159/000337610

    WNK3-SPAK Interaction is Required for the Modulation of NCC and other Members of the SLC12 Family. / Pacheco-Alvarez, Diana; Vazquez, Norma; Castaneda-Bueno, Maria; De-Los-Heros, Paola; Cortes-Gonzalez, Cesar; Moreno, Erika; Meade, Patricia; Bobadilla, Norma A.; Gamba, Gerardo.

    In: Cellular Physiology and Biochemistry, Vol. 29, No. 1-2, 2012, p. 291-302.

    Research output: Contribution to journalArticle

    TY - JOUR

    T1 - WNK3-SPAK Interaction is Required for the Modulation of NCC and other Members of the SLC12 Family

    AU - Pacheco-Alvarez, Diana

    AU - Vazquez, Norma

    AU - Castaneda-Bueno, Maria

    AU - De-Los-Heros, Paola

    AU - Cortes-Gonzalez, Cesar

    AU - Moreno, Erika

    AU - Meade, Patricia

    AU - Bobadilla, Norma A.

    AU - Gamba, Gerardo

    PY - 2012

    Y1 - 2012

    N2 - The serine/threonine with no lysine kinase 3 (WNK3) modulates the activity of the electroneutral cation-coupled chloride cotransporters (CCC) to promote Cl- influx and prevent Cl- efflux, thus fitting the profile for a putative "Cl--sensing kinase". The Ste20-type kinases, SPAK/OSR1, become phosphorylated in response to reduction in intracellular chloride concentration and regulate the activity of NKCC1. Several studies have now shown that WNKs function upstream of SPAK/OSR1. This study was designed to analyze the role of WNK3-SPAK interaction in the regulation of CCCs with particular emphasis on NCC. In this study we used the functional expression system of Xenopus laevis oocytes to show that different SPAK binding sites in WNK3 ((241, 872, 1336)RFxV) are required for the kinase to have effects on CCCs. WNK3-F1337A no longer activated NKCC2, but the effects on NCC, NKCC1, and KCC4 were preserved. In contrast, the effects of WNK3 on these cotransporters were prevented in WNK3-F242A. The elimination of F873 had no consequence on WNK3 effects. WNK3 promoted NCC phosphorylation at threonine 58, even in the absence of the unique SPAK binding site of NCC, but this effect was abolished in the mutant WNK3-F242A. Thus, our data support the hypothesis that the effects of WNK3 upon NCC and other CCCs require the interaction and activation of the SPAK kinase. The effect is dependent on one of the three binding sites for SPAK that are present in WNK3, but not on the SPAK binding sites on the CCCs, which suggests that WNK3 is capable of binding both SPAK and CCCs to promote their phosphorylation. Copyright (C) 2012 S. Karger AG, Basel

    AB - The serine/threonine with no lysine kinase 3 (WNK3) modulates the activity of the electroneutral cation-coupled chloride cotransporters (CCC) to promote Cl- influx and prevent Cl- efflux, thus fitting the profile for a putative "Cl--sensing kinase". The Ste20-type kinases, SPAK/OSR1, become phosphorylated in response to reduction in intracellular chloride concentration and regulate the activity of NKCC1. Several studies have now shown that WNKs function upstream of SPAK/OSR1. This study was designed to analyze the role of WNK3-SPAK interaction in the regulation of CCCs with particular emphasis on NCC. In this study we used the functional expression system of Xenopus laevis oocytes to show that different SPAK binding sites in WNK3 ((241, 872, 1336)RFxV) are required for the kinase to have effects on CCCs. WNK3-F1337A no longer activated NKCC2, but the effects on NCC, NKCC1, and KCC4 were preserved. In contrast, the effects of WNK3 on these cotransporters were prevented in WNK3-F242A. The elimination of F873 had no consequence on WNK3 effects. WNK3 promoted NCC phosphorylation at threonine 58, even in the absence of the unique SPAK binding site of NCC, but this effect was abolished in the mutant WNK3-F242A. Thus, our data support the hypothesis that the effects of WNK3 upon NCC and other CCCs require the interaction and activation of the SPAK kinase. The effect is dependent on one of the three binding sites for SPAK that are present in WNK3, but not on the SPAK binding sites on the CCCs, which suggests that WNK3 is capable of binding both SPAK and CCCs to promote their phosphorylation. Copyright (C) 2012 S. Karger AG, Basel

    U2 - 10.1159/000337610

    DO - 10.1159/000337610

    M3 - Article

    C2 - 22415098

    VL - 29

    SP - 291

    EP - 302

    JO - Cellular Physiology and Biochemistry

    JF - Cellular Physiology and Biochemistry

    SN - 1015-8987

    IS - 1-2

    ER -

    Pacheco-Alvarez D, Vazquez N, Castaneda-Bueno M, De-Los-Heros P, Cortes-Gonzalez C, Moreno E et al. WNK3-SPAK Interaction is Required for the Modulation of NCC and other Members of the SLC12 Family. Cellular Physiology and Biochemistry. 2012;29(1-2):291-302. https://doi.org/10.1159/000337610