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Involvement of Src and epidermal growth factor receptor in the signal-transducing function of Na+/K+-ATPase
1Department of Pharmacology, Medical College of Ohio, Toledo, Ohio 43614, USA.
Abstract:
Nontoxic concentrations of ouabain, causing partial inhibition of the cardiac myocyte Na(+)/K(+)-ATPase, induce hypertrophy and several growth-related genes through signal pathways that include the activation of Ras and p42/44 mitogen-activated protein kinase (MAPK). The aim of this work was to examine the ouabain-induced events upstream of the Ras/MAPK cascade. Treatment of myocytes with genistein antagonized ouabain-induced activation of the MAPK, suggesting that protein tyrosine phosphorylation has a role. Tyrosine phosphorylation of several myocyte proteins was increased rapidly upon cell exposure to ouabain. Lowering of extracellular K(+) had a similar ouabain-like effect. Ouabain also increased protein tyrosine phosphorylation in A7r5, HeLa, and L929 cells. In cardiac myocytes and A7r5 cells, herbimycin A antagonized the ouabain-induced increase in protein tyrosine phosphorylation and MAPK activation. In both cell types, ouabain stimulated Src kinase activity, Src translocation to the Triton-insoluble fraction, Src association with the epidermal growth factor receptor, and the tyrosine phosphorylation of this receptor on site(s) other than its major autophosphorylation site, Tyr(1173). The findings suggest that (a) the ouabain-induced activation of Src and the Src-induced phosphorylation of the growth factor receptor provide the scaffolding for the recruitment of adaptor proteins and Ras and the activation of Ras/MAPK cascade; and (b) the activation of such pathways may be a common feature of the signal-transducing function of Na(+)/K(+)-ATPase in most cells.
Insights
Ouabain activates the cardiac myocyte Na(+)/K(+)-ATPase, initiating a signaling cascade involving Src kinase and growth factor receptors. This pathway leads to Ras and mitogen-activated protein kinase (MAPK) activation, promoting cell growth.
Area of Science:
- Cellular signaling pathways
- Cardiovascular research
- Molecular biology
Background:
- Cardiac myocyte Na(+)/K(+)-ATPase inhibition by ouabain induces hypertrophy and growth gene expression.
- This process involves Ras and p42/44 mitogen-activated protein kinase (MAPK) signaling.
Purpose of the Study:
- To investigate the signaling events upstream of the Ras/MAPK cascade induced by ouabain.
- To elucidate the role of protein tyrosine phosphorylation in ouabain-mediated signaling.
Main Methods:
- Treatment of cardiac myocytes and other cell lines (A7r5, HeLa, L929) with ouabain.
- Use of inhibitors genistein and herbimycin A to block specific signaling pathways.
- Assays for protein tyrosine phosphorylation, MAPK activation, Src kinase activity, and epidermal growth factor receptor (EGFR) phosphorylation.
Main Results:
- Ouabain treatment rapidly increased protein tyrosine phosphorylation in various cell types.
- Genistein and herbimycin A inhibited ouabain-induced MAPK activation and tyrosine phosphorylation.
- Ouabain stimulated Src kinase activity, promoted Src translocation, and induced EGFR tyrosine phosphorylation.
Conclusions:
- Ouabain-induced Src activation and subsequent EGFR phosphorylation serve as a scaffold for Ras recruitment and MAPK activation.
- The Na(+)/K(+)-ATPase signaling pathway, involving Src and EGFR, is crucial for ouabain-induced cardiac cell growth.
- This signaling mechanism may be a conserved function of Na(+)/K(+)-ATPase across different cell types.