Melo Z et al. (2013), N-terminal serine dephosphorylation is required...

XB-ART-47897

J Biol Chem 2013 Nov 01;28844:31468-76. doi: 10.1074/jbc.M113.475574.

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N-terminal serine dephosphorylation is required for KCC3 cotransporter full activation by cell swelling.

Melo Z , de los Heros P , Cruz-Rangel S , Vázquez N , Bobadilla NA , Pasantes-Morales H , Alessi DR , Mercado A , Gamba G .

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The K(+):Cl(-) cotransporter (KCC) activity is modulated by phosphorylation/dephosphorylation processes. In isotonic conditions, KCCs are inactive and phosphorylated, whereas hypotonicity promotes their dephosphorylation and activation. Two phosphorylation sites (Thr-991 and Thr-1048) in KCC3 have been found to be critical for its regulation. However, here we show that the double mutant KCC3-T991A/T1048A could be further activated by hypotonicity, suggesting that additional phosphorylation site(s) are involved. We observed that in vitro activated STE20/SPS1-related proline/alanine-rich kinase (SPAK) complexed to its regulatory MO25 subunit phosphorylated KCC3 at Ser-96 and that in Xenopus laevis oocytes Ser-96 of human KCC3 is phosphorylated in isotonic conditions and becomes dephosphorylated during incubation in hypotonicity, leading to a dramatic increase in KCC3 function. Additionally, WNK3, which inhibits the activity of KCC3, promoted phosphorylation of Ser-96 as well as Thr-991 and Thr-1048. These observations were corroborated in HEK293 cells stably transfected with WNK3. Mutation of Ser-96 alone (KCC3-S96A) had no effect on the activity of the cotransporter when compared with wild type KCC3. However, when compared with the double mutant KCC3-T991A/T1048A, the triple mutant KCC3-S96A/T991A/T1048A activity in isotonic conditions was significantly higher, and it was not further increased by hypotonicity or inhibited by WNK3. We conclude that serine residue 96 of human KCC3 is a third site that has to be dephosphorylated for full activation of the cotransporter during hypotonicity.

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Species referenced: Xenopus laevis
Genes referenced: actl6a prss1 slc12a1 slc12a6 stk39 wnk3

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	FIGURE 1. Evidence for additional phosphorylation sites in KCC3. A, functional expression assay shows the activity of wild type (WT) KCC3 and KCC3-T991A/T1048A under isotonic (gray bars) and hypotonic (open bars) conditions. , p < 0.01 versus human KCC3a (hKCC3a) in isotonic condition. B and C, -fold activity of WT KCC3a (B) or KCC3a-T991A/T1048A (C) taking isotonic conditions as 100% (gray bars) and normalizing accordingly the effect of hypotonic conditions alone (open bars) or hypotonic conditions plus co-injection with WNK3 cRNA (hatched bars). , p < 0.01 versus isotonic control. *, p < 0.05 versus hypotonicity without WNK3. D, time dependence of WT KCC3a activity (open bars) when compared with KCC3a-T991A/T1048A (black bars) in hypotonic conditions was measured after 5 or 30 min of pre-uptake incubation. , p < 0.01 versus wild type.
	FIGURE 2. In vitro identification of KCC3 phosphorylation sites by SPAK. A, GST-KCC3(1–175) and GST-NKCC2(1–174) were expressed in E. coli and phosphorylated with the active and kinase inactive (DA) forms of SPAK in the presence of 10 m MO25α. Dotted lines between autoradiographs and gels indicate that these were undertaken on separate gels. p, phosphorylated. B, phosphorylated GST-KCC3(1–175) was digested with trypsin and chromatographed on a C18 column. The peak fraction containing the major 32P-labeled peptides is labeled P1. C, summary of the mass spectrometry and solid-phase Edman sequencing data obtained after phospho-peptide analysis.
	FIGURE 3. Serine 96 is unique to KCC3. A, KCCs sequence alignment of the N-terminal domain fragment revealed that Ser-96 of KCC3a (Ser-45 in KCC3b as shown the gray shaded box) is not present in other KCCs and thus is unique to KCC3. h, human; m, mouse. B, KCC4 Rb+ uptake of WT KCC4 and KCC4-T926A/T980A under isotonic (gray bars) and hypotonic (open bars) conditions. n = 8–9 experiments, *, p < 0.0001 versus KCC4 in isotonicity.
	FIGURE 4. Elimination of serine 96 by itself had no effect on the KCC3 behavior. A, functional expression assay of WT KCC3a and mutant KCC3a-S96A shows no difference in hypotonic activation (open bars), with no basal expression under isotonic conditions (gray bars). n = 4, p < 0.0001 versus hypotonicity. B, similarly to WT KCC3a, the activity of mutant KCC3a S96A in hypotonic conditions is inhibited by WNK3 (hatched bars). n = 4, *, p < 0.0001 versus control.
	FIGURE 5. Serine 96 becomes phosphorylated under isotonic conditions and in co-expression with WNK3. A, characterization of KCC3a phosphorylation sites using phospho-specific antibodies. HEK 293 cells were transfected with WT human KCC3a or the indicated mutant forms (pT or pS). At 36 h after transfection, cells were lysed, and total cell extracts were immunoblotted with KCC3a total and phospho-specific antibodies. Similar results were obtained in 2 separate experiments. B, representative immunoblotting of total protein extracted from oocytes injected with WT KCC3a or KCC3a-T991A/T1048A cRNA with or without WNK3 cRNA and under hypotonicity or isotonicity maneuvers, as stated. C, representative immunoblotting of total proteins extracted from HEK293 cells transfected as stated. Blots were performed using specific antibody against total KCC3 and phospho-antibodies directed to Ser-96, Thr-991, and/or Thr-1048 of KCC3a and β-actin as loading control.
	FIGURE 6. Serine 96 is a third site involved in regulation of KCC3a activity. A and B, functional expression assay was assessed in isotonic (A) or hypotonic (B) conditions in oocytes injected with cRNA for WT KCC3, KCC3 double mutant (T991A/T1048A), or KCC3 triple mutant (S96A/T991A/T1048A) as stated. , p < 0.001 versus WT. C, the triple mutant KCC3a S96A/T991A/T1048A is no longer sensitive to WNK3 (hatched bars) inhibition. The open bar shows the control uptake for each clone taken as 100%. The hatched bars show the effect of WNK3 upon wild type or mutants KCC3, as stated. n = 3, , p < 0.001 versus white bar.

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