Aurora kinase A activates the vacuolar H+-ATPase (V-ATPase) in kidney carcinoma cells

Abstract

Extracellular proton-secreting transport systems that contribute to extracellular pH include the vacuolar H(+)-ATPase (V-ATPase). This pump, which mediates ATP-driven transport of H(+) across membranes, is involved in metastasis. We previously showed (Alzamora R, Thali RF, Gong F, Smolak C, Li H, Baty CJ, Bertrand CA, Auchli Y, Brunisholz RA, Neumann D, Hallows KR, Pastor-Soler NM. J Biol Chem 285: 24676-24685, 2010) that V-ATPase A subunit phosphorylation at Ser-175 is important for PKA-induced V-ATPase activity at the membrane of kidney intercalated cells. However, Ser-175 is also located within a larger phosphorylation consensus sequence for Aurora kinases, which are known to phosphorylate proteins that contribute to the pathogenesis of metastatic carcinomas. We thus hypothesized that Aurora kinase A (AURKA), overexpressed in aggressive carcinomas, regulates the V-ATPase in human kidney carcinoma cells (Caki-2) via Ser-175 phosphorylation. We found that AURKA is abnormally expressed in Caki-2 cells, where it binds the V-ATPase A subunit in an AURKA phosphorylation-dependent manner. Treatment with the AURKA activator anacardic acid increased V-ATPase expression and activity at the plasma membrane of Caki-2 cells. In addition, AURKA phosphorylates the V-ATPase A subunit at Ser-175 in vitro and in Caki-2 cells. Immunolabeling revealed that anacardic acid induced marked membrane accumulation of the V-ATPase A subunit in transfected Caki-2 cells. However, anacardic acid failed to induce membrane accumulation of a phosphorylation-deficient Ser-175-to-Ala (S175A) A subunit mutant. Finally, S175A-expressing cells had decreased migration in a wound-healing assay compared with cells expressing wild-type or a phospho-mimetic Ser-175-to-Asp (S175D) mutant A subunit. We conclude that AURKA activates the V-ATPase in kidney carcinoma cells via phosphorylation of Ser-175 in the V-ATPase A subunit. This regulation contributes to kidney carcinoma V-ATPase-mediated extracellular acidification and cell migration.

Keywords: cancer; epithelial cell; pH regulation; plasma membrane; proton pump.

Fig. 1.

Informatic analysis of ATP6V1A phosphorylation. Top: consensus phosphorylation target motifs for PKA and aurora kinase A (AURKA) are shown, where X represents any residue, S or T is the phosphorylated residue, and Φ represents a hydrophobic residue at the P+1 position. Bottom: sequence alignment of residues near ATP6V1A at position 175 across various species (frog, chicken, mouse, pig, and human).

Fig. 2.

AURKA and the vacuolar (V)-ATPase in the Caki-2 kidney cancer cell line are coexpressed in cytoplasm and form a complex. A: confocal stacks and X-Z and Y-Z reconstructions of Caki-2 cells immunolabeled for AURKA (red) and the V-ATPase E subunit (green). B: epifluorescence images of immunolabeling for AURKA (green) and the V-ATPase A subunit (red) show extensive colocalization of V-ATPase subunits with mislocalized cytosolic AURKA with V₁ sector subunits of the V-ATPase. Scale bar = 10 μm. C: immunoblot of HEK-293 and Caki-2 cell lysate using the pThr288-Aurora A antibody (top) and β-actin (bottom). The bands in the top blot (arrows) likely represent the 3 phosphorylated Aurora kinase isoforms, as determined by their known molecular weights. D: representative immunoprecipitation (IP) experiments from HEK-293 cells and Caki-2 cells, using mouse anti-AURKA antibody. The samples were then immunoblotted for the V-ATPase V₀ sector a4 subunit (ATP6V0A4; top) and AURKA (bottom) using antibodies raised in rabbits. These results are consistent with the existence of protein complexes containing AURKA and V-ATPase V₀ and V₁ sector subunits in cell lines of kidney origin.

Fig. 3.

AURKA regulates V-ATPase expression at the leading edge of Caki-2 cells. A: representative images of immunolabeling of Caki-2 cells after a wound assay in the absence and presence of the AURKA activator anacardic acid (25 μM) for 4 h. Incubation with CY3-tagged concanavalin A (red) was performed for 3 min before fixation, a condition which lightly labels the cell surface. After fixation, the cells were labeled with an antibody against the V-ATPase E subunit (green) followed by TOPRO-3 to stain the nuclei (blue). The fluorescence intensity profiles along the yellow lines marked in the images were determined for the V-ATPase E subunit as the mean gray value. B: the ratio of the means ± SE gray values corresponding to the leading edge and the cytoplasm areas was calculated for >45 cells/condition and reported as the ratio of the fluorescence intensity measured in the leading edge vs. that in the cytoplasm for the V-ATPase E subunit. V-ATPase concentrates at the wound edge in Caki-2 cells treated with AURKA activator, while it is more diffuse in the cytoplasm in cells incubated in vehicle (DMSO). *P < 0.05 relative to vehicle control. C: immunolabeling against the V-ATPase E subunit (green) in cells treated with the Aurora kinase inhibitor III (50 nM) vs. vehicle control revealed that the inhibitor qualitatively decreased the number of cellular projections (arrow) 4 h after a scratch assay.

Fig. 4.

The AURKA activator anacardic acid increases AURKA phosphorylation in Caki-2 cells. A: AURKA activity as measured by immunoblotting of pThr288-aurora A for IP AURKA (top) relative to AURKA as a loading control (bottom) in Caki-2 cells incubated in CCD media in the absence or presence of AURKA activator anacardic acid (25 μM, 4 h). B: anacardic acid treatment induced a significant increase in phosphorylated pThr288-Aurora A compared with untreated cells. *P < 0.05 relative to vehicle control; n = 3.

Fig. 5.

AURKA activation increases cell surface expression of the V-ATPase in Caki-2 cells. A: representative immunoblots of V-ATPase a4 subunit (ATP6V0A4 in the V-ATPase V₀ integral membrane domain) in whole cell lysates and cell surface biotinylation protein samples from Caki-2 cell monolayers grown under either control conditions or treated with anacardic acid for 60 min. B: relative V-ATPase band intensity measured in the biotinylated fraction and normalized to cell lysates revealed that the presence of the AURKA activator more than doubled V-ATPase surface expression relative to vehicle control. *P < 0.05; n = 3.

Fig. 6.

AURKA regulates V-ATPase activity at the plasma membrane in cultured Caki-2 cells. The rate of extracellular acidification in each set of treatments was measured in a low-buffering capacity solution in the extracellular buffer of cells preincubated in the absence or presence of anacardic acid. The mean ± SE rate of extracellular acidification [(final buffer pH − initial buffer pH)/Δt] was obtained in the absence and presence of bafilomycin, a specific V-ATPase inhibitor, for each treatment condition. There was negligible cell death after these experiments as determined by a trypan blue exclusion assay across conditions (not shown). *P = 0.05; n = 3.

Fig. 7.

AURKA phosphorylates the V-ATPase A subunit in vitro. A: typical phosphoscreen image (top) revealing the signal of AURKA in the in vitro phosphorylated wild-type (WT) A subunit compared with S175A (phosphorylation-deficient) or S175D (phosphomimetic) mutants. Immunoblotting of the FLAG-tagged A subunits (bottom) confirm similar protein expression and loading of the gel for the different conditions. B: quantification of V-ATPase A subunit phosphorylation signal normalized for protein loading as assessed by densitometry of the Western blot. Compared with the WT Flag-A subunit, both mutations (S175A or S175D) showed a significant reduction in phosphorylation level by ∼70%. **P < 0.05; n = 3.

Fig. 8.

AURKA-dependent phosphorylation of the V-ATPase A subunit occurs at Ser-175 in Caki-2 cells. A: typical phosphoscreen image (top) revealing the signal of AURKA in the in vivo phosphorylated A subunit compared with the phosphorylation-deficient (S175A) mutant. The immunoblots (lower) confirm similar protein expression and loading of the gel for the different conditions. B: quantification of the V-ATPase A subunit phosphorylation signal normalized for protein loading as assessed by densitometry of the immunoblot. Compared with the WT-A vehicle control, the WT-A subunit showed a significant increase in phosphorylation by the AURKA activator (anacardic acid, 25 μM, 4 h). Values are means ± SE of 5 independent experiments. *P < 0.05 relative to WT.

Fig. 9.

AURKA regulates V-ATPase A subunit expression at the leading edge of Caki-2 cells via Ser-175. A: representative confocal images of wound assays performed on Caki-2 cells transiently transfected with either the WT or S175A mutant A subunit in the absence or presence of anacardic acid. After the different treatments, the cell monolayers were incubated with concanavalin A (red), fixed, and then labeled with an anti-FLAG antibody (green) followed by TOPRO-3 to stain the nuclei (blue). Fluorescence intensity profiles (see Fig. 3) were determined for the V-ATPase A subunit as the mean gray value. All confocal images shown were acquired using identical laser setting in cells immunolabeled on the same day and under the same conditions. B: ratio of the mean gray value corresponding to the leading edge and the cytoplasm areas were calculated for >45 cells/condition and expressed as the ratio of the fluorescence intensity measured in the leading edge vs. that in the cytoplasm for the V-ATPase A subunit. V-ATPase concentrates at the wound edge in Caki-2 cells treated with the AURKA activator, while it is more diffuse in the cytoplasm in cells incubated in vehicle control, *P < 0.05 relative to vehicle control.

Fig. 10.

The phosphorylation-deficient A subunit S175A mutant decreases V-ATPase-dependent extracellular acidification with AURKA activation in Caki-2 cells. A: the mean ± SE rate of extracellular acidification [(final buffer pH − initial buffer pH)/Δt] was obtained in the absence and presence of bafilomycin for each treatment condition with expression of WT vs. S175A mutant FLAG-tagged A subunit. There was negligible cell death after these experiments, as determined by a trypan blue exclusion assay across conditions (data not shown). Data were obtained from 4 separate experiments/condition. Immunoblots of cell lysates from these experiments showed no significant differences in FLAG-A subunit expression across the 4 conditions (not shown). *P < 0.05. **P = 0.06; n = 4.

Fig. 11.

AURKA activation increases coimmunoprecipitation of AURKA to WT-A subunit in Caki-2 cells. A: representative immunoblots of immunoprecipitations from Caki-2 cells expressing FLAG-tagged A subunit mutants. Cells expressing the WT A subunit and incubated with anacardic acid exhibited a significantly larger AURKA signal (top) in samples immunoprecipitated using an anti-FLAG antibody compared with untreated cells or those transfected with the S175A A subunit mutant independently of treatment. The AURKA signal was normalized for the immunoprecipitated FLAG-tagged subunit in each sample (bottom). Immunoprecipitations in the absence of the anti-FLAG antibody (No antibody) or no lysate (no protein) were also performed as controls and revealed no significant, nonspecific immunoprecipitation of either the A subunit or AURKA. B: quantification of the means ± SE of AURKA coimmunoprecipitated to the V-ATPase A subunit normalized for protein loading as assessed by densitometry of the immunoblot. *P < 0.05 relative to vehicle control; n = 3.

Fig. 12.

AURKA activation and V-ATPase Ser-175 phosphorylation increase Caki-2 cell motility. Brightfield images of untransfected Caki-2 cell confluent monolayers at 0 and 4 h postexposure to AURKA activator (anacardic acid, 25 μM) after wounding of the monolayer are shown. A: wound closure was measured in each image at 3 different locations (white arrows); approximate wound edges (yellow lines). The wound repair was then tracked by obtaining 3 random microscopic images of each wound at each time point with a total of 9 images/condition and time point. B: quantification of wound closure. The data represent the mean distance of cell migration of the area at 4 h after wounding normalized to the control at each time point. Values are means ± SE of 3 independent experiments. *P < 0.05 relative to vehicle control. C: representative images at 3 and 5 h after wounding monolayers of Caki-2 cells transfected with pTracer-S175D (top), -S175A (middle), or -WT (bottom). The top white line in each image demarcates the approximate upper edge of the original scratch at time 0. For illustrative purposes, different colored asterisks (*) were placed in each of the images to demonstrate movement of individual cells between 3 and 5 h. For each condition, 3 typical GFP-expressing cells were identified at the 3-h time point and labeled with a large asterisk (*) of different colors (red, white, and blue). In the 5-h time point panels, the prior 3-h position of each of the cells was marked for reference as well as the current 5-h position of each of the cells, indicated by the small asterisk (*) of the same color. D: means ± SE cell migration scores from 4 blinded observers who scored the overall relative movements of green cells under the 3 conditions as described in materials and methods. *P ≤ 0.01 relative to WT and S175D mutant; n = 3–4 wound assays/condition.