Analysis of Protein Phosphatase 1 (PP1) and SPAK/OSR1 as KCC3a Phosphorylation/Dephosphorylation Cycle Regulators

Abstract

Study ObjectiveTo identify the phosphatase/kinase complex that regulates KCC3a activation/deactivation during cell volume regulation and ion transport.BackgroundThe K‐Cl cotransporters (KCCs) are SLC12A cation/chloride cotransporters (CCCs) that mediate epithelial transport, maintain cellular volume, and regulate GABA neurotransmission. These mechanisms are complex, and contain multiple sensors, transducers, and effectors, often operating in parallel pathways. It has been shown that direct phosphorylation by SPAK/OSR1 kinases regulate KCCs activities. In particular for KCC3a phosphorylation of regulatory site Thr1039 by these kinases results in inhibited K‐Cl cotransport, whereas dephosphorylation of this residue greatly stimulates KCC3 activity. Protein phosphatases (PP) inhibitors reduce KCCs function and so, it is generally accepted the necessary presence of phosphatases in the WNKs/SPAK/CCCs/signaling pathway. Several studies have proposed protein phosphatase 1 as the major PP responsible for KCCs dephosphorylation. Specificity of PP1 catalytic domain is determined by its association with one or two regulatory subunits that modulate its activity. In the present study we analyzed how exactly is PP1 regulating KCCs activities and the possible role of SPAK/OSR1 kinases as PP1 regulatory subunits.MethodsHEK 293 cells and Xenopus laevis oocytes were either transfected or microinjected with KCC3 and wild type (WT) or mutant PP1 plasmids (H248K or T320A). KCC3 dephosphorylation in response to cell swelling, hypotonic conditions or presence of “active/inactive” PP1 in dephosphorylation assays, was evaluated by Western blot analysis of KCCs regulatory phosphorylation sites (pThr1039 and pThr991 in KCC3a). Phosphorylation of SPAK/OSR1 activating regulatory site Thr273 was analyzed in parallel under the same conditions. Formation of a phosphatase/kinase/cotransporter complex was evaluated by immunoprecipitation assays. Dephosphorylation assays were performed either with immunoprecipitated PP1 from cell extracts or with recombinant PP1.ResultsWT or constitutive active mutant PP1 T320A induced KCC3a Thr1039 dephosphorylation in HEK cells and X. laevis oocytes even under isotonic conditions, where KCC3a is normally phosphorylated. PP1 inactivating mutation H248K prevented KCC3 dephosphorylation in response to hypotonic conditions and greatly promoted Thr1039 phosphorylation. Dephosphorylation assays in presence of “active” or “inactive” immunoprecipitated WT PP1, showed that “active” PP1 promotes SPAK Thr273 dephosphorylation, while “inactive” PP1 greatly increased SPAK phosphorylation and thereby KCC3 T1039 inmunoprecipitated from hypotonic lysates where both proteins are found dephosphorylated.ConclusionsOur results show a direct role for PP1 in KCC3 function. PP1 is regulating KCC3 activity via SPAK/OSR1 kinases. We show “active” and “inactive” PP1 regulates SPAK/OSR1 activity, that in turn will directly affect KCCs cell volume regulatory response.Support or Funding InformationConacyt 283555Papiit IA207718