Regulation of K‐Cl cotransporters (KCCs) activity by a PP1 phosphatase/WNK3 kinase complex
Authors: de los Heros Paola , Cabrera Adriana Mercado , Alvarez Diana Pacheco , Florido Ángel Beverido , Gama Alejandro Rodriguez 
Published: June 2021

Study ObjectiveTo identify and characterize the mechanism protein phosphatases use to regulate KCCs activity during ionic homeostasis and the regulation of cell volume via WNK3 kinase.BackgroundSLC12A family are membrane proteins that transport Na+ and/or K+ coupled to Clinside and outside the cell. They regulate cell volume, trans‐epithelial ionic transport, neuron excitability and blood pressure balance. Several members of the family and their regulatory proteins are mutated in human diseases characterized by alteration of ionic homeostasis mainly in the kidney and the brain. The family is formed by KCCs, NCC and NKCCs regulated in a reciprocal manner by phospho‐ and dephosphorylation events. WNKs kinases and their down‐stream substrates, SPAK/OSR1 are their main phospho‐regulatory proteins. Dephosphorylation results in KCCs activation and NCC and NKCCs inhibition, whereas phosphorylation produces the opposite effect. KCCs activation is abolished by inhibition of PP1A and PP2, demonstrating an essential regulatory role for PPs in this process. Substrate specificity for WNK and SPAK/OSR1 kinases is stablished by a SPAK binding motif, present in WNK kinases and the SLC12A. Conversely, some consensus motifs for direct PPs‐dephosphorylation have been stablished. In the present study we analyzed two putative PP1 binding sites present in the WNK3 kinase sequence. Our previous findings showed that WNK3 inhibits all the KCCs and its catalytically inactive form, WNK3 DA activates them. Inhibition of PPs showed regulation of KCCs activity by WNK3 depends on PPs activity. By mutating these putative PP1 binding sites we are aiming to identify the serine (Ser)‐threonine (Thr) kinases/phosphatases complex involved in KCCs response to cell volume changes regulated by WNK3.MethodsKCCs dephosphorylation was evaluated by Western Blot analysis. from HEK 293 cells transfected with either WT WNK3 kinase (WNK3 WT), catalytically inactive WNK3 (WNK3 DA), or PP1 binding sites mutant WNK3 (WNK3 PP1A, WNK3 DA PP1A, WNK3 PP1B or WNK3 DA PP1B) Cells were stimulated with either isotonic or hypotonic buffers in order to stimulate phosphatase activity under KCCs regulatory phosphorylation sites (pT1039 and pT991 in KCC3a). Regulation of KCCs K‐Cl transport by WNK3 WT, DA and mutants was assayed directly in Xenopus laevis oocytes. Formation of a kinase/phosphatase/cotransporter complex was evaluated by immunoprecipitation assays.ResultsData obtained in HEK cells and X. laevis oocytes showed that WNK3 PP1 binding sites mutation modified the effect of WNK3 WT and WNK3 DA previously reported over KCCs in response to cell volume changes.ConclusionsBinding of PP1 to WNK3 and formation of a regulatory complex is necessary for WNK3 to regulate SLC12A family response to osmotic and cell volume changes.Support or Funding InformationPAPIIT IA207718CONACYT, CB‐2016‐01, 283555This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.