Src-independent ERK signaling through the rat α3 isoform of Na/K-ATPase

Abstract

The Na/K-ATPase α1 polypeptide supports both ion-pumping and signaling functions. The Na/K-ATPase α3 polypeptide differs from α1 in both its primary structure and its tissue distribution. The expression of α3 seems particularly important in neurons, and recent clinical evidence supports a unique role of this isoform in normal brain function. The nature of this specific role of α3 has remained elusive, because the ubiquitous presence of α1 has hindered efforts to characterize α3-specific functions in mammalian cell systems. Using Na/K-ATPase α1 knockdown pig kidney cells (PY-17), we generated the first stable mammalian cell line expressing a ouabain-resistant form of rat Na/K-ATPase α3 in the absence of endogenous pig α1 detectable by Western blotting. In these cells, Na/K-ATPase α3 formed a functional ion-pumping enzyme and rescued the expression of Na/K-ATPase β1 and caveolin-1 to levels comparable with those observed in PY-17 cells rescued with a rat Na/K-ATPase α1 (AAC-19). The α3-containing enzymes had lower Na⁺ affinity and lower ouabain-sensitive transport activity than their α1-containing counterparts under basal conditions, but showed a greater capacity to be activated when intracellular Na⁺ was increased. In contrast to Na/K-ATPase α1, α3 could not regulate Src. Upon exposure to ouabain, Src activation did not occur, yet ERK was activated through Src-independent pathways involving PI3K and PKC. Hence, α3 expression confers signaling and pumping properties that are clearly distinct from that of cells expressing Na/K-ATPase α1.

Keywords: Na+/K+-ATPase; Src; caveolin; extracellular‐signal‐regulated kinase (ERK); phosphatidylinositide 3‐kinase (PI 3‐kinase); signal transduction.

Fig. 1.

Generation of stable cell lines expressing Na/K-ATPase α3. Whole cell lysates from AAC-19, LM-α3-1, and PY-17 were separated by SDS-PAGE and analyzed by Western blot for the expression of Na/K-ATPase α1 (A), Na/K-ATPase α3 (B), and Na/K-ATPase β1 (C). A representative Western blot is shown. Cells were immunostained with anti-Na/K-ATPase α3 primary and Alexa Fluor-conjugated secondary antibody (D). The scale bar represents 25 µM (n = 3–6). *P < 0.05, ** P < 0.01 compared with AAC-19. E: ³H ouabain binding. Cells were treated for 2 µM ouabain for 30 min and then assayed for ouabain binding as described in materials and methods. Quantification of data is shown as means ± SE from at least three separate experiments.

Fig. 2.

Na/K-ATPase and dose-effect of ouabain in rescued cells. Ouabain-sensitive ATPase activity was obtained as described in materials and methods. Data are shown as means ± SE from at least three separate experiments.

Fig. 3.

Substrate-dependent ATPase activity. Crude membranes were prepared from AAC-19 and LM-α3-1 cells, and ATPase activity was measured under varying concentrations of potassium chloride (KCl) (A) and sodium chloride (NaCl) (B). Data are shown as means ± SE from at least three separate experiments.

Fig. 4.

Na/K-ATPase-mediated ⁸⁶Rb⁺ influx. Basal level (A) and basal and monensin-treated (B) ouabain-sensitive ⁸⁶Rb⁺ influx was measured as described in materials and methods. Values were normalized per protein amount. Data are shown as means ± SE and from at least three separate experiments. *P < 0.05.

Fig. 5.

Caveolin expression in LM-α3-1. Whole cell lysates from AAC-19, LM-α3-1, and PY-17 cells were separated by SDS-PAGE and analyzed by Western blotting for the expression of caveolin-1 (Cav-1). A representative Western blot is shown. Quantification of data is shown as means ± SE from at least three separate experiments. **P < 0.01 compared with AAC-19.

Fig. 6.

Basal phosphorylation levels of protein kinases. Total cell lysates collected from AAC-19 and LM-a3-1 cells were separated by SDS-PAGE and analyzed by Western blotting for pScr (Src pY418) (A), pERK (B), and pAkt (C). A representative Western blot is shown, and the quantitative data are means ± SE from at least three independent experiments. *P < 0.05 compared with AAC-19.

Fig. 7.

Effect of Na/K-ATPase peptides on Src by in vitro kinase assay. A: amino acid sequence of α1 NaKtide and α3 NaKtide among different species. The amino acids of α3 NaKtide that are different from those of α1 NaKtide are in bold and underlined. B: Src (4.5 units) was incubated with different concentrations of NaKtide and α3 NaKtide for 15 min and then assayed for pY418 Src. Quantitative data from at least three independent experiments are shown as means ± SE. *P < 0.05 compared with control.

Fig. 8.

Effect of ouabain on cell signaling. Cells were serum starved, treated with 10 and 100 µM ouabain for 10 min, and harvested. Total cell lysates collected from AAC-19 and LM-α3-1 cells were separated by SDS-PAGE and analyzed by Western blotting for Src pY418 and c-Src (A), pERK and ERK (B), and pAkt and Akt (C). A representative Western blot is shown, and the quantitative data are means ± SE from at least three independent experiments. *P < 0.05 and **P < 0.01 compared with control.

Fig. 9.

Effect of inhibitors on ouabain-stimulated ERK activation. LM-α3-1 cells were treated with 10 µM ouabain for 10 min without pretreatment (A) or following preincubation with selected inhibitors as follows: 5 µM Src inhibitor PP2 for 30 min (B), 2 µM EGFR inhibitor PD153035 (PD) for 60 min (C), 50 µM PI3K inhibitor LY 29400 (LY) for 60 min (D), or 1 µM PKC inhibitor GO6983 (GO) for 60 min (E). A representative Western blot is shown, and the quantitative data are means ± SE from at least three independent experiments. *P < 0.05 compared with control.

Fig. 10.

Effect of extracellular calcium (Ca²⁺). LM-α3-1 cells were preincubated in Krebs-Henseleit (KH) buffer with (1.8 mM) or without Ca²⁺ for 30 min and then treated with 10 µM ouabain for 10 min and harvested. Total cell lysates were collected and separated by SDS-PAGE and analyzed by Western blotting for pERK and ERK. A representative Western blot is shown, and the quantitative data are means ± SE from at least three independent experiments. *P < 0.05 compared with control (no ouabain).