Characterization of the osmoregulatory mechanism involving WNK3 kinase, K-Cl cotransporters (KCCs) and Protein phosphatase 1 (PP1)

Background: K-Cl cotransporters (KCCs) belong to the SLC12A cation/chloride cotransporters (CCCs) family that mediate epithelial transport, maintain cellular volume, and regulate GABA neurotransmission. Regulation of KCCs activity by WNK3 kinase has been shown, where co-expresion with the kinase under hypotonic conditions; where KCCs are normally active, inhibits their transport. Whereas kinase dead WNK3 activates them under isotonic conditions where they are normally inhibited. Likewise, KCCs activation is abolished by inhibition of PP1A and PP2, demonstrating an essential regulatory role for PPs in this process. Substrate specificity for PPs is believed to be achieved by consensus binding motifs, present on their regulatory subunits stablishing a complex mechanism with several sensors, transducers, and effectors. KCCs regulation upon osmotic changes involves dephosphorylation of important regulatory sites in order to become fully activated, and on the other hand phosphorylation by WNK/SPAK/OSR1 kinases to become inhibited. In the present study we analyzed two putative PP1 binding sites present in the WNK3 kinase amino acid sequence to determine the role of WNK3 and protein phosphatase 1 (PP1) interaction upon KCCs phosphorylation/dephosphorylation regulation during cell volume and electrolyte homeostasis. Methods: KCCs activity was evaluated by Western blot analysis from HEK 293 cells transfected with KCC3, WNK3 WT, catalytically inactive WNK3DA, or PP1 binding sites mutants WNK3 PP1a, WNK3DA PP1A, WNK3 PP1B or WNK3DA PP1B. KCC3 dephosphorylation in response to cell swelling, hypotonic conditions, was evaluated in KCCs regulatory sites Thr1039, Thr991 and Ser86. Phosphorylation of SPAK/OSR1 activating regulatory site Ser373 was analyzed in parallel under the same conditions. Results: WNK3 kinase co-expression inhibited KCC3a Thr1039, Thr991 and Ser86 dephosphorylation under hypotonic conditions, eliminating their response to cell swelling. Nevertheless, when co-expressed with PP1 binding site mutant PP1a, KCC3 regulatory sites become dephosphorylated under isotonic and hypotonic conditions eliminating WNK3 inhibitory effect. WNK3 kinase autophosphorylation was evaluated in WT and PP1a mutant. Ser309 was phosphorylated in both osmotic conditions for WT WNK3, whereas PP1A mutant autophosphorylation was lost under isotonic and hypotonic environments. SPAK Thr373 regulation was unaffected in any of the conditions studied. Conclusions: Our results show a direct interaction between WNK3 and PP1 during KCCs regulation. We suggest WNK3 kinase inhibits PP1 activity, preventing KCC3 regulatory sites dephosphorylation. These data support the hypothesis of a phosphatase/kinase osmoregulatory complex controlling KCCs function during cell volume regulatory response. PAPPIT, Clave: IN222320 This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.