N-terminal Serine Dephosphorylation Is Required for KCC3 Cotransporter Full Activation by Cell Swelling

Abstract

Background: KCC3 lacking the two known phosphorylation sites is still regulated by cell swelling and WNK3.Results: Dephosphorylation of serine 96 is necessary for full activation of the cotransporter.Conclusion: Serine 96 is a third phospho-site involved in KCC3 regulation.Significance: The finding of new phosphorylation sites sheds light on an increasingly complex regulation of K+:Cl− cotransporters.

A Novel Regulatory Locus of Phosphorylation in the C Terminus of the Potassium Chloride Cotransporter KCC2 That Interferes with N-Ethylmaleimide or Staurosporine-mediated Activation*♦

“…Our analyses failed to detect a regulatory role of Ser 25 involved in cell swelling-related activation of the transporter (76). Furthermore, N-terminal truncation disrupted transport activity of KCC1 (77) and KCC2 (9).…”

With no lysine L-WNK1 isoforms are negative regulators of the K+-Cl− cotransporters

“…Ϫ cotransport activity of all four wild-type KCCs (KCC1-KCC4) and the endogenous Na ϩ -K ϩ -2Cl Ϫ (NKCC1) was assessed using the heterologous expression system of Xenopus laevis oocytes following our standard procedures (10,36,37,39). Briefly, mature oocytes were injected with water alone or containing 0.2 g/l of KCCs cRNA alone or together with 0.1 g/l of the various isoforms and mutants of L-WNK1 kinase cRNA.…”

“…Two days after injection, the activity of the KCCs was determined by measuring the Cl Ϫ -dependent 86 Rb ϩ uptake under hypotonic conditions (110 mosmol/kgH2O). The activity of endogenous NKCC1 was determined by bumetanide-sensitive 86 Rb ϩ uptake under hypotonic (155 mosmol/ kgH2O) conditions, which is known to promote inhibition of the cotransporter (16,27 (10,36,37,39). Tracer activity was determined for each oocyte by ␤-scintillation counting.…”

“…The total and phospho-specific KCC3a antibodies for KCC3a and SPAK/OSR1 used in this study were previously described and validated (11,36). Polyclonal anti-c-Myc antibody was used to identify L-WNK1 (Sigma).…”

“…The full-length human KCC3a and L-WNK1-⌬11 cDNAs were previously subcloned into the mammalian expression vector pCMV5 and pcDNA3.1, respectively. KCC3a was tagged with Flag and L-WNK1-⌬11 with c-Myc (7,36). To generate the transient cell lines, HEK-293 cells underwent passages every 2-3 days to maintain the cells in their exponential growth phase.…”