title: N-terminal serine dephosphorylation is required for KCC3 cotransporter full activation by cell swelling.
abstract: The K(+):Cl(-) cotransporter (KCC) activity is modulated by phosphorylation/dephosphorylation processes. In isotonic conditions, KCCs are inactive and phosphorylated, whereas hypotonicity promotes their dephosphorylation and activation. Two phosphorylation sites (Thr-991 and Thr-1048) in KCC3 have been found to be critical for its regulation. However, here we show that the double mutant KCC3-T991A/T1048A could be further activated by hypotonicity, suggesting that additional phosphorylation site(s) are involved. We observed that in vitro activated STE20/SPS1-related proline/alanine-rich kinase (SPAK) complexed to its regulatory MO25 subunit phosphorylated KCC3 at Ser-96 and that in Xenopus laevis oocytes Ser-96 of human KCC3 is phosphorylated in isotonic conditions and becomes dephosphorylated during incubation in hypotonicity, leading to a dramatic increase in KCC3 function. Additionally, WNK3, which inhibits the activity of KCC3, promoted phosphorylation of Ser-96 as well as Thr-991 and Thr-1048. These observations were corroborated in HEK293 cells stably transfected with WNK3. Mutation of Ser-96 alone (KCC3-S96A) had no effect on the activity of the cotransporter when compared with wild type KCC3. However, when compared with the double mutant KCC3-T991A/T1048A, the triple mutant KCC3-S96A/T991A/T1048A activity in isotonic conditions was significantly higher, and it was not further increased by hypotonicity or inhibited by WNK3. We conclude that serine residue 96 of human KCC3 is a third site that has to be dephosphorylated for full activation of the cotransporter during hypotonicity.
authors.affiliation: From the Molecular Physiology Unit, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México and Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Tlalpan, 14000 Mexico City, Mexico.
authors.forename: Zesergio
authors.initials: Z
authors.lastname: Melo
authors.forename: Paola
authors.initials: P
authors.lastname: de los Heros
authors.forename: Silvia
authors.initials: S
authors.lastname: Cruz-Rangel
authors.forename: Norma
authors.initials: N
authors.lastname: Vázquez
authors.forename: Norma A
authors.initials: NA
authors.lastname: Bobadilla
authors.forename: Herminia
authors.initials: H
authors.lastname: Pasantes-Morales
authors.forename: Dario R
authors.initials: DR
authors.lastname: Alessi
authors.forename: Adriana
authors.initials: A
authors.lastname: Mercado
authors.forename: Gerardo
authors.initials: G
authors.lastname: Gamba
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D002460:Cell Line; D057809:HEK293 Cells; D006801:Humans; D020125:Mutation, Missense; D009865:Oocytes / Q000166:cytology / Q000378:metabolism; D009997:Osmotic Pressure / Q000502:physiology*; D010766:Phosphorylation / Q000502:physiology; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D012694:Serine / Q000235:genetics / Q000378:metabolism; D027981:Symporters / Q000235:genetics / Q000378:metabolism*; D014982:Xenopus laevis; D000096922:K Cl- Cotransporters
chemical_list: D017346:Protein Serine-Threonine Kinases; D012694:Serine; D027981:Symporters; C119650:SLC12A6 protein, human; D000096922:K Cl- Cotransporters; C486409:STK39 protein, human; C488586:WNK3 protein, human
keywords: Hypertension; Phosphorylation; Physiology; Potassium Transport; Signal Transduction
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