Down-regulation of SPAK prevents activation of NKCC1 in Calu-3 cells. Calu-3 cells were electroporated without or with 2 pmol of siSPAK/10⁶ cells, seeded on filter inserts, and incubated in a humidified, 5% CO₂ atmosphere for 48 h. Uptake of ⁸⁶Rb was measured for a 4-min time period, as described under “Experimental Procedures.” In cells incubated with HPSS (Veh), baseline ⁸⁶Rb uptake (white bars, Veh; strippled bars, sucrose) and bumetanide-sensitive ⁸⁶Rb uptake (black bars) in cells electroporated without (left panel) or with siSPAK (right panel) were not significantly different. Stimulation of cells by imposing hyperosmotic stress significantly increased total and bumetanide-sensitive ⁸⁶Rb uptake in cells electroporated without siSPAK (left panel) but had no effect on cells electroporated with siSPAK (right panel). Values are mean ± S.E. for four separate experiments.

In vitro and in vivo SPAK kinase activity. A, in vitro SPAK kinase activity was measured using 3.45 μg of NT-NKCC1 as substrate, as described under “Experimental Procedures.” GST-SPAK was pretreated with activated PKCδ for 15 min at 30 °C. Rottlerin was then added to the mixture to block PKCδ activity. An aliquot of kinase reaction mixture, containing NT-NKCC1, ATP, and [γ-³²P]ATP, was added to SPAK, incubated for 20 min at 37 °C, and the reaction terminated by addition of 9 μl of glacial acetic acid. Phosphorylated NT-NKCC1 was recovered on phosphocellulose discs, washed with phosphoric acid and deionized water, and subjected to Cerenkov counting. Pretreatment of SPAK with activated PKCδ significantly increased phosphorylation of NT-NKCC1. The results also indicate that NKCC1 is a substrate for SPAK. B, in vivo SPAK kinase activity. Calu-3 cells were grown to confluence, serum-deprived overnight, then stimulated with or without vehicle (Veh), 10 μm methoxamine (MOX), or sufficient sucrose (SUC) to increase medium osmolarity to 500 mOsm. Cells were lysed in 1 ml of lysis buffer and SPAK immunoprecipitated, as described under “Experimental Procedures.” SPAK kinase activity was measured in immune complexes using 2 μg of CATCHtide peptide as substrate. Stimulation of Calu-3 cells with MOX or SUC significantly increased SPAK kinase activity. Preincubation of cells with the PKCδ inhibitor rottlerin (Rott) blocked the stimulatory effects of MOX and SUC, indicating that PKCδ activity is necessary for SPAK kinase activity.

NKCC1 phosphorylation detected using a R5 antibody. Calu-3 cells were electroporated with 2 pmol of siSPAK/10⁶ cells, seeded onto 100-mm dishes (6 × 10⁶ cells per dish), and incubated in a humidified, 5% CO₂ atmosphere for 48 h. As a control, an aliquot of Calu-3 was directly seeded onto 100-mm dishes without electroporation. On the day of sucrose stimulation, cell culture medium was discarded and replaced with HPSS, preheated to 35 °C. Cells were treated with HPSS (Veh) or stimulated by imposing hyperosmotic stress for 4 min (Suc). Stimulation was stopped by transferring dishes to an ice bath and washing the cell monolayer rapidly three times with ice-cold PBS. NKCC1 was immunoprecipitated, as described under “Experimental Procedures,” and immune complexes probed for phosphorylated NKCC1 using the R5 antibody then reprobed for NKCC1. Exposed bands were quantified by densitometry and densitometry values used to calculate a ratio of R5 (phosphorylated NKCC1) to total NKCC1. The panels illustrate typical results from three experiments each in which each experimental condition was done in replicates of 2–4. Sucrose increased phosphorylation of NKCC1 by 1.81 ± 0.07 (n = 3)-fold in cells not treated with siSPAK (right panel) and by 1.38 ± 0.09 (n = 3)-fold in cells electroporated with siSPAK (left panel). Treatment with silencing RNA significantly decreased hyperosmotic induced phosphorylation of NKCC1 (p < 0.02).

Down-regulation of SPAK using silencing RNA. A, down-regulation of endogenous SPAK. Double-stranded silencing RNA directed against SPAK (siSPAK) was delivered into Calu-3 cells by electroporation, as described under “Experimental Procedures.” Cells were incubated for 48 h after electroporation with 0, 1, 2, or 5 pmol of siSPAK per 1 × 10⁶ cells and harvested as total cell lysate (TCL). As controls, equal aliquots of cells were not electroporated (0-) or electroporated without siSPAK (0+). Immunoblot analysis for SPAK was performed on a 30-μg aliquot of TCL and exposed bands were quantitated by laser densitometry (LD). Immunoblots were sequentially reprobed for NKCC1, PKCδ, and PP2A, which served as a loading control. Ratios are calculated as LD values divided by PP2A LD value for the specific experimental condition. Maximal loss of SPAK protein was 65.8% after a 48-h incubation immediately following electroporation. siSPAK did not affect the expression of NKCC1, PKCδ, and PP2A. B, non-targeting siCTRL. To test for nonspecific effects of silencing RNA, cells were electroporated with 2 pmol of non-targeting siCONTROL RNA (siCTRL)/10⁶ cells. TCL were collected after 48 h incubation and analyzed for proteins of interest by immunoblot analysis. Exposed bands were quantitated by laser densitometry and normalized to values for actin that served as a loading control. Treatment with siCTRL did not alter expression of SPAK, NKCC1, PKCδ, PP2A, and actin. Typical results from three experiments are illustrated. C, expression of GFP 48 h after delivery of pmaxGFP into Calu-3 cells by electroporation. GFP is detected in multiple cells in this micrograph as a green fluorescent signal.

Interaction between SPAK and PKCδ. A, solid phase slot blot binding assay. Recombinant His₆-full-length (FL)-SPAK or GST-CT-SPAK (aa 316–548), immobilized on paper, was overlaid with solutions containing 1, 25, or 50 ng of inactive or preactivated recombinant PKCδ (Pan Vera). To activate the enzyme, PKCδ was preincubated with 30 μg/ml phosphatidylserine and 2 μg/ml DiC₈. We observed direct binding of PKCδ to FL-SPAK and CT-SPAK. The figure is representative of four experiments. B, solution binding assay. Fifty μg of recombinant His₆-tagged CT-SPAK was mixed without (0, lane 1) or with 25 ng of inactive (I, lanes 2 and 3) or activated (A, lanes 4 and 5) PKCδ. The mixture was incubated for 40 min at 30 °C and bound proteins recovered by pulldown using Talon beads followed by immunoblot analysis for PKCδ. A 20-μg aliquot of Calu-3 total cell lysate (TCL) served as a positive control for the PKCδ immunoblot. Blots were reprobed with antibody to SPAK; His₆-tagged CT-SPAK was detected as a 30-kDa band in lanes 1–5 and endogenous SPAK as a 70-kDa protein band in TCL (data not shown). Densitometry revealed binding of inactive and activated PKCδ to CT-SPAK. The figure is representative of four experiments. C, coimmunoprecipitation of endogenous PKCδ and SPAK. Calu-3 cells were serum-deprived overnight then treated with sufficient sucrose to increase medium osmolarity to 500 mOsm or with vehicle. SPAK or PKCδ was immunoprecipitated from TCL and immunoprecipitates were subjected to immunoblot analysis for pPKCδ, using phospho-PKCδ antibody (pPKCδ, Santa Cruz, sc-1177), then sequentially reprobed for PKCδ, which served as a loading control, and SPAK. Typical results for three experiments are shown. The ratio of SPAK to PKCδ and PKCδ to SPAK was calculated from densitometry values. Hyperosmotic stress increased the amount of pPKCδ detected in immunoprecipitates of PKCδ and SPAK and increased the coimmunoprecipitation of SPAK and PKCδ.

Interaction between SPAK and NKCC1. A, Calu-3 cells were grown to confluence and serum-deprived overnight. TCL was prepared and used for pull-down assays. Pulldowns were performed by adding 50 μg of GST-CT-SPAK (aa 316–548) or 30 μg of GST-NT-NKCC1 (aa 1–286) to 1 ml of TCL followed by a 40-min incubation at 30 °C. GST-tagged protein was recovered using anti-GST-agarose beads to the reaction mixture. Samples were incubated for 60 minat 4 °C. Beads were recovered by centrifugation and washed five times with PBS. Interacting proteins were detected by immunoblot analysis. Typical results for three experiments each using six 1-ml aliquots of TCL are shown. NKCC1 was detected in pulldowns using GST-CT-SPAK and SPAK was detected in pulldowns using GST-NT-NKCC1. B, solid phase slot blot binding assay. Varying amounts (0–60 μg) of the peptide spNT was immobilized on membrane paper and overlaid with 1 μg of GST-SPAK. Samples were incubated for 25 min at room temperature. Unbound protein was removed by repeated washes with PBS. Bound GST-SPAK was detected by immunoblot analysis using anti-GST antibody. Protein bands were detected by chemiluminescence and quantitated by densitometry. Densitometry values were used to calculate an EC₅₀ using the GraphPad Prism software program. C, competitive inhibition of binding of SPAK and NKCC1. GST-SPAK (10 μg, 0.12 nmol) and NT-NKCC1 cleaved from a GST tag (7 μg, 0.32 nmol) were incubated without or with peptide spNT at various molar ratios to GST-SPAK for 20 min at 30 °C. GST-SPAK was pulled down using glutathione-Sepharose beads and proteins bound to SPAK were immunoblotted with antibody to the amino terminus of NKCC1. Immunoblots were reprobed for the GST tag using an anti-GST antibody. Protein bands were detected by chemiluminescence and quantitated by densitometry. In the absence of spNT, the mean binding ratio of NT-NKCC1 to SPAK was 2.7 (lane 1, no spNT); incubation with 100:1 molar excess spNT reduced the binding ratio to 1.7, a 37.0% decrease in binding. Controls consisted of loading spNT alone (lane 2), which was not detected with the antibodies used in these immunoblots, and TCL. A protein band immunoreactive with antibody to NKCC1 was detected at the expected molecular mass of 150 kDa (TCL, lane 9).