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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 - J. Biol. Chem. (2013)

Bottom Line: Two phosphorylation sites (Thr-991 and Thr-1048) in KCC3 have been found to be critical for its regulation.Mutation of Ser-96 alone (KCC3-S96A) had no effect on the activity of the cotransporter when compared with wild type KCC3.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.

View Article: PubMed Central - PubMed

Affiliation: From the Molecular Physiology Unit, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México and Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Tlalpan, 14000 Mexico City, Mexico.

ABSTRACT
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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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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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.

Mentions: Because Ser-96 residue in the double mutant KCC3-T991A/T1048A was phosphorylated and dephosphorylated similarly as in the wild type KCC3a, but elimination of this site alone had no effect on the cotransporter behavior (Fig. 4), to find out the role of serine 96 on the regulation of KCC3, we substituted the Ser-96 for alanine in the double mutant KCC3-T991A/T1048A to create the triple mutant KCC3-S96A/T991A/T1048A. In parallel experiments, functional expression of the double and triple mutant revealed that both were active in isotonic conditions (Fig. 6A). However, the level of activity was significantly higher in the triple mutant than in the double mutant (4.97 ± 0.57 versus 1.86 ± 0.15 nmol/oocyte/h; p < 0.0001), suggesting that the absence of phosphorylation in serine 96 contributed to higher activity of the triple mutant KCC3 in isotonic medium. This is supported by the findings observed during incubation of oocytes in hypotonic conditions. Further activation of the double mutant was noted (Figs. 1, A and C, and 6B), whereas no additional effect of hypotonicity was detected in the triple mutant (Fig. 6B). The Cl−-dependent, 86Rb+ uptake of the triple mutant in isotonicity and hypotonicity was 4.97 ± 0.57 and 5.23 ± 0.57 nmol/oocyte/h, respectively (p = not significant). In addition, co-injection of oocytes with WNK3 cRNA was able to significantly reduce the activity of wild type and double mutant KCC3 in hypotonic conditions, but no inhibitory effect was observed upon the triple mutant (Fig. 6C).


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 - J. Biol. Chem. (2013)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3814743&req=5

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.
Mentions: Because Ser-96 residue in the double mutant KCC3-T991A/T1048A was phosphorylated and dephosphorylated similarly as in the wild type KCC3a, but elimination of this site alone had no effect on the cotransporter behavior (Fig. 4), to find out the role of serine 96 on the regulation of KCC3, we substituted the Ser-96 for alanine in the double mutant KCC3-T991A/T1048A to create the triple mutant KCC3-S96A/T991A/T1048A. In parallel experiments, functional expression of the double and triple mutant revealed that both were active in isotonic conditions (Fig. 6A). However, the level of activity was significantly higher in the triple mutant than in the double mutant (4.97 ± 0.57 versus 1.86 ± 0.15 nmol/oocyte/h; p < 0.0001), suggesting that the absence of phosphorylation in serine 96 contributed to higher activity of the triple mutant KCC3 in isotonic medium. This is supported by the findings observed during incubation of oocytes in hypotonic conditions. Further activation of the double mutant was noted (Figs. 1, A and C, and 6B), whereas no additional effect of hypotonicity was detected in the triple mutant (Fig. 6B). The Cl−-dependent, 86Rb+ uptake of the triple mutant in isotonicity and hypotonicity was 4.97 ± 0.57 and 5.23 ± 0.57 nmol/oocyte/h, respectively (p = not significant). In addition, co-injection of oocytes with WNK3 cRNA was able to significantly reduce the activity of wild type and double mutant KCC3 in hypotonic conditions, but no inhibitory effect was observed upon the triple mutant (Fig. 6C).

Bottom Line: Two phosphorylation sites (Thr-991 and Thr-1048) in KCC3 have been found to be critical for its regulation.Mutation of Ser-96 alone (KCC3-S96A) had no effect on the activity of the cotransporter when compared with wild type KCC3.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.

View Article: PubMed Central - PubMed

Affiliation: From the Molecular Physiology Unit, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México and Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Tlalpan, 14000 Mexico City, Mexico.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus