Multiple pathways for protein phosphatase 1 (PP1) regulation of Na-K-2Cl cotransporter (NKCC1) function: the N-terminal tail of the Na-K-2Cl cotransporter serves as a regulatory scaffold for Ste20-related proline/alanine-rich kinase (SPAK) AND PP1

Abstract

The Na-K-2Cl cotransporter (NKCC1) participates in epithelial transport and in cell volume maintenance by mediating the movement of ions and water across plasma membranes. Functional studies have previously demonstrated that NKCC1 activity is stimulated by protein phosphatase 1 (PP1) inhibitors. In this study, we utilized both in vivo (heterologous cRNA expression in Xenopus laevis oocytes) and in vitro ((32)P-phosphorylation assays with glutathione S-transferase fusion proteins) experiments to determine whether PP1 exerts its inhibitory effect directly on the cotransporter, or indirectly by affecting the activating kinase. We found that PP1 reduced NKCC1 activity in oocytes under both isotonic and hypertonic conditions to the same level as in water-injected controls. Interestingly, mutation of key residues in the PP1 binding motif located in the N-terminal tail of NKCC1 significantly reduced the inhibitory effect of PP1. In vitro experiments performed with recombinant PP1, SPAK (Ste20-related proline/alanine-rich kinase, which activates NKCC1), and the N terminus of NKCC1 fused to glutathione S-transferase demonstrated that PP1 dephosphorylated both the kinase and the cotransporter in a time-dependent manner. More importantly, PP1 dephosphorylation of SPAK was significantly greater when protein-protein interaction between the kinase and the N-terminal tail of NKCC1 was present in the reaction, indicating the necessity of scaffolding the phosphatase and kinase in proximity to one another. Taken together, our data are consistent with PP1 inhibiting NKCC1 activity directly by dephosphorylating the cotransporter and indirectly by dephosphorylating SPAK.

FIGURE 1.

PP1 inhibits the activity of Na-K-2Cl cotransporters located at the plasma membrane. A, K⁺ uptake of X. laevis oocytes expressing 15 ng of fluorescently tagged cotransporter (EGFP-NKCC1) in the presence or absence of 10 ng of wild-type PP1 measured under isosmotic (195 mosmol, white bars) and hyperosmotic (260 mosmol, black bars) conditions. Bars represent mean ± S.E. (n = 20 oocytes). Uptake experiments were repeated twice with similar results. Asterisk denotes statistical significance (p < 0.001). B, confocal laser microscopy of membrane fluorescence in oocytes 3 days after injection with 15 ng of EGFP-NKCC1 cRNA and 10 ng of PP1 cRNA. C, representative surface biotinylation of oocytes injected with 18.5 ng of wild-type NKCC1 cRNA in the presence or absence of 10 ng of PP1 cRNA. Samples were immunoblotted with a polyclonal antibody raised against a portion of the C-terminal domain of NKCC1. Open arrow indicates NKCC1-specific signal. D, densitometry analysis of cotransporter surface expression from three separate biotinylation experiments. Paired t tests found no statistical difference (p = 0.38, n = 3).

FIGURE 2.

Catalytic activity and binding of PP1 are necessary for NKCC1 inhibition. K⁺ uptake of X. laevis oocytes expressing 15 ng of wild-type cotransporter (NKCC1 (wt)) or PP1 binding-deficient cotransporter (NKCC1 (bd)) in the presence or absence of 10 ng of wild-type phosphatase (PP1 (wt)) or catalytically inactive phosphatase (PP1 (ci)) under isosmotic (195 mosmol, white bars) and hyperosmotic (260 mosmol, black bars) conditions. All conditions were repeated twice with similar results. Bars represent mean ± S.E. (n = 20 oocytes). Asterisk denotes statistical significance (p < 0.001).

FIGURE 3.

PP1 mediates direct inhibition of NKCC1. K⁺ uptake of X. laevis oocytes expressing 15 ng of mutant NKCC1 (F79A, white bars) or wild-type NKCC1 (gray bar) in the presence or absence of 10 ng of wild-type SPAK (SPAK (wt)), wild-type WNK4 (WNK4 (wt)), wild-type PP1 (PP1 (wt)), and constitutively active SPAK (SPAK (ca)) under isosmotic (195 mosmol) conditions. All conditions were repeated twice with similar results. Bars represent mean ± S.E. (n = 20 oocytes). Asterisk denotes statistical significance (p < 0.001). Inset, amino acid sequence of mouse NKCC1 (residues 132–141) with overlapping binding sites for SPAK and PP1 highlighted by horizontal bars.

FIGURE 4.

AATYK inhibition of NKCC1 activity is prevented by a constitutively active SPAK. K⁺ uptake of X. laevis oocytes expressing 15 ng of wild-type NKCC1 in the presence or absence of 10 ng of wild-type SPAK (SPAK (wt)), wild-type WNK4 (WNK4 (wt)), wild-type AATYK (AATYK (wt)), and constitutively active SPAK (SPAK (ca)) under isosmotic (195 mosmol) conditions. All conditions were repeated twice with similar results. Bars represent mean ± S.E. (n = 20 oocytes). Asterisk denotes statistical significance (p < 0.001). Letters a, b, and c indicate specific comparisons discussed in the text. Inset, amino acid sequence of mouse AATYK (residues 1170–1178 and 1277–1294) with the one PP1 and two SPAK binding sites highlighted by horizontal bars.

FIGURE 5.

Scaffolding of SPAK and PP1 increases the rate of kinase dephosphorylation. A, autoradiograph of a 45-min kinase reaction involving wild-type SPAK and wild-type NKCC1, followed by the addition of 2.5 units of recombinant PP1 for 5–30 min. One unit of PP1 hydrolyzes 1 nmol of substrate in 1 min. Upper panel, SPAK signal from reaction containing wild-type SPAK only; middle panel, SPAK signal from reaction containing both wild-type SPAK and wild-type NKCC1; bottom panel, NKCC1 signal from reaction containing both wild-type SPAK and wild-type NKCC1. B, densitometry analysis of the upper panel A (open triangles), middle panel A (open squares), and bottom panel A (filled circles). C, autoradiograph of a 45-min kinase reaction involving wild-type SPAK, wild-type NKCC1, and SPAK binding-deficient NKCC1 (F79A and F135A), followed by the addition of specified units of recombinant PP1 for 10 min. Upper panel, SPAK signal from the reaction containing both wild-type SPAK and SPAK binding-deficient NKCC1; middle panel, SPAK signal from reaction containing both wild-type SPAK and wild-type NKCC1; bottom panel, NKCC1 signal from reaction containing both wild-type SPAK and wild-type NKCC1. D, densitometry analysis of upper panel C (open diamonds), middle panel C (open squares), and bottom panel C (filled circles). Western blot analysis demonstrates that both wild-type NKCC1 (wt) and SPAK binding-deficient NKCC1 (bd) were present in sufficient amounts in their respective experiments (D, inset). Assays were repeated twice with similar results.