Ouabain-regulated phosphoproteome reveals molecular mechanisms for Na+, K+-ATPase control of cell adhesion, proliferation, and survival

Abstract

The ion pump Na⁺, K⁺-ATPase (NKA) is a receptor for the cardiotonic steroid ouabain. Subsaturating concentration of ouabain triggers intracellular calcium oscillations, stimulates cell proliferation and adhesion, and protects from apoptosis. However, it is controversial whether ouabain-bound NKA is considered a signal transducer. To address this question, we performed a global analysis of protein phosphorylation in COS-7 cells, identifying 2580 regulated phosphorylation events on 1242 proteins upon 10- and 20-min treatment with ouabain. Regulated phosphorylated proteins include the inositol triphosphate receptor and stromal interaction molecule, which are essential for initiating calcium oscillations. Hierarchical clustering revealed that ouabain triggers a structured phosphorylation response that occurs in a well-defined, time-dependent manner and affects specific cellular processes, including cell proliferation and cell-cell junctions. We additionally identify regulation of the phosphorylation of several calcium and calmodulin-dependent protein kinases (CAMKs), including 2 sites of CAMK type II-γ (CAMK2G), a protein known to regulate apoptosis. To verify the significance of this result, CAMK2G was knocked down in primary kidney cells. CAMK2G knockdown impaired ouabain-dependent protection from apoptosis upon treatment with high glucose or serum deprivation. In conclusion, we establish NKA as the coordinator of a broad, tightly regulated phosphorylation response in cells and define CAMK2G as a downstream effector of NKA.-Panizza, E., Zhang, L., Fontana, J. M., Hamada, K., Svensson, D., Akkuratov, E. E., Scott, L., Mikoshiba, K., Brismar, H., Lehtiö, J., Aperia, A. Ouabain-regulated phosphoproteome reveals molecular mechanisms for Na⁺, K⁺-ATPase control of cell adhesion, proliferation, and survival.

Keywords: apoptosis; calcium and calmodulin–dependent protein kinase; inositol triphosphate receptor; kidney; phosphoproteomics.

Figure 1

Treatment with ouabain for 10 and 20 min mediates extensive changes of the phosphoproteome of COS-7 cells. A) Schematic representation of the interaction between NKA and InsP₃ receptor (IP3R) upon ouabain binding to NKA. The interaction leads to the release of calcium ions from the ER. B) Representative measurement of the intensity of the fluorescence emitted by the calcium-sensitive dye GCaMP6 in a single cell over time. F/F₀, fluorescence signal:fluorescence at t = 0; OB, ouabain. The arrow indicates the time point of ouabain addition. C) Representative measurement of the intensity of the fluorescence emitted by the sodium-sensitive dye Asante NaTrium Green-2 AM in single cells over time. Arrows indicate the time points of addition of the indicated drugs. D) Experimental workflow employed for phosphoproteomic and proteomic analyses [Phospho HiRIEF LC-MS and Standard HiRIEF LC-MS (24), respectively]. The number of phospho-sites and proteins identified and quantified is displayed. TiO₂, titanium dioxide. E) Volcano plots representing significantly regulated events in the proteomic analysis (red dots). Left and right panels display the 10-min and the 20-min time points, respectively. The average fold-change of the 3 replicates for each experimental condition is represented on the x axis, and the significance of the regulation based on Student’s t test is represented on the y axis. F) Correlation of phospho-site log₂-transformed ratio values for each replicate pair; Pearson correlation coefficient is displayed. Ratio values are calculated using the average of the 4 untreated samples as a denominator. R#, replicate no. G) Volcano plots representing significantly regulated events in the phosphoproteomic analysis (red dots). The average fold-change of the 3 replicates for each experimental condition is represented on the x axis, and the significance of the regulation based on Student’s t test is represented on the y axis.

Figure 2

STIM and the InsP₃ receptor play central roles in mediating ouabain-dependent [Ca²⁺]_i oscillations. A, B) Recordings of the intensity of the fluorescence emitted by the calcium-sensitive dye GCaMP6 in single cells over time. Time periods when drugs were applied are indicated by solid horizontal lines. The measurements are representative of those obtained from 6 to 8 individual cells identified within each field of view and repeated in 4 different preparations performed on different days. F/F₀, fluorescence signal:fluorescence at t = 0. C) Simulation of the structural rearrangement induced by dephosphorylation of S1832 of InsP₃ type 1 receptor (right panel) compared with the phosphorylated form of the protein (left panel), based on the crystal model with PDB ID 5X9Z (39). Purple coloring represents the receptor loop region. 5Ga, region of 5 amino acids mutated in the orginal study (39, red-colored); HD2, α-helical domain 2; HD3, α-helical domain 3.

Figure 3

NKA triggers temporally regulated phosphorylation events impacting cell adhesion and proliferation. A) Heatmap representing Ward’s method hierarchical clustering based on Euclidian distance of the significantly regulated phospho-site ratios. Line plots (bottom left panel) display the average phospho-site log₂-transformed ratio across experimental conditions for each of the identified clusters. Color shading represents the 25th and 75th percentile of the distribution of all the phospho-site ratios in the cluster. For each enriched GO term (right panel), E indicates the fold enrichment, and S indicates the q-value representing the significance of the enrichment. O, ouabain; U, untreated; R#, replicate no. B) Representative images of Western blot analyses of phosphorylated ERK1 (p-ERK1) Y204 and tubulin levels in COS-7 cells protein extracts. C) Line plot representing the average level of p-ERK1 Y204 normalized to tubulin in ouabain-treated vs. untreated cells. Error bars represent se of the 3 replicates’ means. *P < 0.05.

Figure 4

Ouabain modulates the activation status of several protein kinases. A) Relative number of genes identified for each of the indicated classes. Protein class enrichment is evaluated by Fisher’s exact test. UBL, ubiquitin-like. B) Enrichment of phospho-motifs for each of the identified cluster of regulation, evaluated using motif-x. Motif score represents the significance of the enrichment (negative log₂-transformed P value). No. of matches indicates the number of phospho-sites containing the motif in the subset of significantly regulated phospho-sites. Fold increase indicates the fold enrichment of the motif in the subset of significantly regulated phospho-sites, as compared with the background (all identified phospho-sites). Putative kinases are indicated based on known kinase recognition motifs (–37). CDK5, cyclin dependent kinase 5; GSK-3, glycogen synthase kinase-3.

Figure 5

CAMK2G acts downstream of NKA, mediating protection from apoptosis. A, B) Top: Time plan of transfection of primary rat proximal tubular cells with CAMK2G or control (ctrl) siRNA followed by the indicated treatment. A) Top: 48 h after CAMK2G or ctrl siRNA transfection, cells were cultured with 10% (v/v) or 0.2% (v/v) FBS in the presence or absence of 10 nM ouabain for 24 h. B) Top: 48 h after CAMK2G or ctrl siRNA transfection, cells were exposed to normal (5.5 mM) or high (20 mM) glucose concentration in the presence or absence of 10 nM ouabain for 6 h. Nanomolar concentration of ouabain yields physiologic effects after long exposure time because ouabain binding to NKA is characterized by a slower off-rate than on-rate (68). A, B) Bottom: Apoptotic index of rat proximal tubular cells (y axis) as measured by TUNEL assay. At least 8 (A) or 4 (B) replicates were employed for analysis. Individual data points are represented as gray dots. OB, ouabain. *P < 0.05.