Interaction between Na⁺/K⁺-ATPase and Src in LLC-PK1 cells. (A) Colocalization of the Na⁺/K⁺-ATPase (red) and Src (green) in LLC-PK1 cells at a resolution of 1024 × 1024 pixels. Left and center images showed the membrane localization of the Na⁺/K⁺-ATPase α1 and Src, respectively, and the merged image (right) showed the colocalization of these two proteins. Scale bar, 20 μm. (B) FRET analysis of the interaction between EYFP-rat α1 (yellow) and Src-ECFP (cyan) in LLC-PK1 cells. The boxed area (ROI 1) was photobleached and analyzed for FRET. We also measured FRET at the circled area (ROI 2) that was not photobleached. The same studies were performed in 16 cells from 6 independent experiments. Scale bar, 8 μm.

Identification of the Src domains involved in the interaction with the Na⁺/K⁺-ATPase. (A) Schematic presentation of structures of Src. (B) Coomassie blue staining of GST-Src, GST-SH2, GST-SH3, GST-SH3SH2, and GST-kinase. (C) Binding of GST-Src, GST-SH3SH2, GST-kinase, GST-SH2, but not GST-SH3, domains to the Na⁺/K⁺-ATPase. An aliquot (2 μg) of the purified Na⁺/K⁺-ATPase was used for each binding assay. The same experiments were repeated three times.

Regulation of Src by the Na⁺/K⁺-ATPase and GST-CD3. (A) Indicated amount of purified Na⁺/K⁺-ATPase (PKE) were incubated with recombinant Src (4.5 U) for 30 min in PBS, then 2 mM ATP/Mg²⁺ was added and incubated for another 5 min. After the samples were resolved on SDS-PAGE, the membranes were probed with antibodies as indicated. (B) GST (100 ng) or different amount of GST-CD3 was incubated with recombinant Src (4.5 U) for 30 min in PBS. The phosphorylation of Src was analyzed as in A. Values are mean ± SE of at least four independent experiments. ^* p < 0.05; ^** p < 0.01 compared with control.

Activation of Src by freeing the kinase domain from the Na⁺/K⁺-ATPase. (A) A control experiment showing that Src could be cosedimented with Na⁺/K⁺-ATPase. Src (4.5 U) incubated with or without 5 μg Na⁺/K⁺-ATPase in 0.5 ml PBS was centrifuged at 100,000 × g for 30 min. The pellets were resuspended in PBS and subjected to phosphorylation assay as described in Materials and Methods. As an input control, 4.5 U of Src were directly suspended in PBS and assayed for pY418 phosphorylation. (B) Src (4.5 U) was preincubated with 5 μg of the purified Na⁺/K⁺-ATPase in PBS and then exposed to 10 μM ouabain for 15 min. Both control and ouabain-treated Na⁺/K⁺-ATPase/Src complexes were then collected by centrifugation, resuspended in PBS, and subjected to phosphorylation assay as in A. Two representative Western blots are shown in A and B, and the values are mean ± SE of at least three independent experiments. (C) A representative Western blot of four separate experiments showing that ouabain induced the release of the kinase domain from the Na⁺/K⁺-ATPase. GST-Src, GST-SH3SH2, or GST-kinase was incubated with 1 μg purified Na⁺/K⁺-ATPase for 30 min at room temperature in 500 μl PBS. Complexes were then pulled down on glutathione beads, washed three times, resuspended in 500 μl PBS, and exposed to 10 μM ouabain for 15 min. The beads were then washed for three more times using PBS, and the pulled down Na⁺/K⁺-ATPase was analyzed by Western blot using anti-α1 antibody. (D) A representative Western blot of three independent experiments showing the activation of Src by GST-kinase domain fusion protein. GST, GST-SH3SH2, or GST-kinase (5 μg each) was preincubated with 2 μg of the purified Na⁺/K⁺-ATPase for 15 min at room temperature. Recombinant Src (4.5 U) was then added to the mixture for additional 30 min. Phosphorylation reaction was started by addition of 2 mM ATP/Mg²⁺ and Src pY418 was measured as in A.

Ouabain-activated Na⁺/K⁺-ATPase/Src phosphorylates and recruits downstream effectors. (A) LLC-PK1 cells were treated with 1 μM ouabain for 5 min, and cell lysates were immunoprecipitated with anti-α1 antibody and analyzed for tyrosine phosphorylated proteins. (B) Both SYF and SYF + Src cells were treated with 100 μM ouabain for 5 min and analyzed as in A. Representative Western blots of three experiments are shown in both A and B. (C) Inhibition of Src blocks ouabain-induced recruitment of Src to the Na⁺/K⁺-ATPase signaling complex. LLC-PK1 cells were pretreated with 1 μM PP2 or PP3 for 15 min and then exposed to 1 μM ouabain for 5 min. Cell lysates were immunoprecipitated and analyzed. Values are mean ± SE of at least four independent experiments. (D) Caveolae were isolated and treated with 100 nM ouabain for 5 min in the presence or absence of 2 mM ATP as we previously described (Wang et al., 2004). Afterward, caveolae were lysed in RIPA buffer, and the lysates were cleared by centrifugation and immunoprecipitated with anti-caveolin-1 antibody. Immunoprecipitates were probed for the α1, Src, and caveolin-1 by Western blot. A representative Western blot of three independent experiments is shown.

Binding of the purified pig kidney Na⁺/K⁺-ATPase (PKE) to GST-Src. Purified Na⁺/K⁺-ATPase was solubilized in 1% Triton X-100. After centrifugation at 100,000 × g, indicated amounts of the cleared supernatants were incubated with 5 μg GST-Src in the presence of 0.5% Triton X-100 for 30 min and followed by four washes with the same buffer. (A and B) The Coomassie blue-stained GST-Src and purified Na⁺/K⁺-ATPase (PKE). (C) A representive Western blot from three independent experiments showing the pulldown products probed with anti-Na⁺/K⁺-ATPase α1 antibody. (D) The same pulldown assay as in C was performed, and 650 ng (one-third of the total input) of the purified Na⁺/K⁺-ATPase (PKE) was directly loaded as an input control.

Identification of the Na⁺/K⁺-ATPase domains involved in the interaction with Src. (A) Schematic presentation of α1 subunit of Na⁺/K⁺-ATPase. NT, N-terminus; CD2, cytosolic domain 2; CD3, cytosolic domain 3; PD, phosphorylation domain; ND, nucleotide-binding domain; CT, C-terminus. (B) A representative Western blot of four independent experiments shows the binding of purified Src (lacking of first 84 amino acids) to the CD3, but not the NT of the α1 subunit when 200 ng of Src was used. (C) A Western blot showing that Src was pulled down by GST-CD3 of Na⁺/K⁺-ATPase (Na/K) and H⁺/K⁺-ATPase (H/K), but not SERCA from 1 mg LLC-PK1 cell lysates. (D) A Western blot showing the domain interaction between the Na⁺/K⁺-ATPase and Src. Different GST-fused Na⁺/K⁺-ATPase domain constructs were incubated with either His-tagged SH3SH2 domain or kinase domain of Src, and the pulldown products were analyzed by Western blot.

Stimulation of the Na⁺/K⁺-ATPase/Src complex by ouabain. (A) The preformed Na⁺/K⁺-ATPase/Src complex was treated with different concentrations of ouabain in the presence of 2 mM ATP/Mg²⁺ for 5 min, and the phosphorylated Src was analyzed using site-specific antibodies as indicated. Values are mean ± SE of at least four independent experiments. (B) Src or Src/Na⁺/K⁺-ATPase complex were treated with 10 μM ouabain, and the Src activity was measured. (C) A representative Western blot of four experiments showing the effects of ouabain and vanadate on the Na⁺/K⁺-ATPase/Src complex. A similar experiment as in A was repeated to assess the effects of either vanadate (Van) or vanadate plus ouabain (Oua) on Src phosphorylation. ^** p < 0.01 compared with control.

Ouabain dissociates Src kinase domain from the Na⁺/K⁺-ATPase in live cells. (A) A representative trace of ouabain-induced changes in FRET signal in an LLC-PK1 cell. (B) 293T cells were cotransfected with Src-Rluc and GFP-α1. 293T cells transfected with Rluc-GFP fusion protein were used as a positive control, and cells that cotransfected with Rluc and GFP-Na⁺/K⁺-ATPase were used as a negative control. (C) Ouabain treatment reduced BRET signal between GFP-Na⁺/K⁺-ATPase and Src-Rluc in a dose-dependent manner. Values are mean ± SE of at least four experiments. ^*p < 0.05; ^** p < 0.01.

Schematic presentation shows how ouabain regulates the Na⁺/K⁺-ATPase/Src receptor complex.