SPAK/OSR1 kinases directly phosphorylate the K+-Cl- co-transporters (1109.7)

Precise homeostasis of the intracellular chloride (Cl-) concentration is critical for cell volume regulation, epithelial transport, and neuronal excitability, and is achieved via the coordinated activities of the Cl- influx N(K)CCs and efflux KCCs members of the cation- Cl- cotransporter family. Here we present biochemical, genetic, and pharmacological evidence that the WNK kinase substrates SPAK/OSR1 directly phosphorylate the KCCs at one of the two recently-described cell swelling-regulated C-terminal threonines, Thr 1048 (Site 2) but not Thr 991 (Site 1) in KCC3a. First, we demonstrate that SPAK and OSR1, in the presence of the MO25 regulatory subunit, robustly phosphorylates all KCC isoforms at Site-2 in vitro. Second, STOCK 1S-50699, a selective WNK pathway inhibitor, suppresses SPAK/OSR1 activation and Site 2 phosphorylation. Third, the endogenous phosphorylation of KCC2, 3 and 4 at Site-2 is abolished in double SPAK/OSR1 knock in embryonic stem cells, where T-loop phosphorylation by WNK isoforms is...