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Functional characterization of Src-interacting Na/K-ATPase using RNA interference assay
Man Liang1, Ting Cai, Jiang Tian
1Department of Physiology, Pharmacology, Metabolism, and Cardiovascular Sciences, Medical University of Ohio, Toledo, Ohio 43614, USA.
Abstract:
We have shown that the Na/K-ATPase and Src form a signaling receptor complex. Here we determined how alterations in the amount and properties of the Na/K-ATPase affect basal Src activity and ouabain-induced signal transduction. Several alpha1 subunit knockdown cell lines were generated by transfecting LLC-PK1 cells with a vector expressing alpha1-specific small interference RNA. Although the alpha1 knockdown resulted in significant decreases in Na/K-ATPase activity, it increased the basal Src activity and tyrosine phosphorylation of focal adhesion kinase, a Src effector. Concomitantly it also abolished ouabain-induced activation of Src and ERK1/2. When the knockdown cells were rescued by a rat alpha1, both Na/K-ATPase activity and the basal Src activity were restored. In addition, ouabain was able to stimulate Src and ERK1/2 in the rescued cells at a much higher concentration, consistent with the established differences in ouabain sensitivity between pig and rat alpha1. Finally both fluorescence resonance energy transfer analysis and co-immunoprecipitation assay indicated that the pumping-null rat alpha1 (D371E) mutant could also bind Src. Expression of this mutant restored the basal Src activity and focal adhesion kinase tyrosine phosphorylation. Taken together, the new findings suggest that LLC-PK1 cells contain a pool of Src-interacting Na/K-ATPase that not only regulates Src activity but also serves as a receptor for ouabain to activate protein kinases.
Insights
The sodium-potassium pump (Na/K-ATPase) forms a complex with Src kinase, regulating its activity and acting as a receptor for ouabain signaling. Altering Na/K-ATPase levels impacts Src activity and downstream signaling pathways.
Area of Science:
- Cellular signaling
- Molecular cell biology
- Biochemistry
Background:
- The sodium-potassium pump (Na/K-ATPase) is crucial for maintaining cellular ion balance.
- Src kinase is a non-receptor tyrosine kinase involved in various cellular processes.
- The interaction between Na/K-ATPase and Src suggests a role in signal transduction.
Purpose of the Study:
- To investigate how Na/K-ATPase quantity and properties influence basal Src activity.
- To determine the effect on ouabain-induced signal transduction pathways.
- To elucidate the role of Na/K-ATPase as a signaling receptor complex.
Main Methods:
- Generation of alpha1 subunit knockdown LLC-PK1 cell lines using small interference RNA.
- Assessment of Na/K-ATPase activity and Src activity.
- Analysis of tyrosine phosphorylation of focal adhesion kinase and ERK1/2 activation.
- Rescue experiments with rat alpha1 subunit and mutant alpha1 (D371E).
- Fluorescence resonance energy transfer (FRET) and co-immunoprecipitation assays.
Main Results:
- Alpha1 knockdown decreased Na/K-ATPase activity but increased basal Src activity and focal adhesion kinase phosphorylation.
- Ouabain-induced activation of Src and ERK1/2 was abolished in knockdown cells.
- Rescue with rat alpha1 restored Na/K-ATPase and basal Src activity, with altered ouabain sensitivity.
- A pumping-null alpha1 mutant (D371E) bound Src and restored basal Src activity and focal adhesion kinase phosphorylation.
Conclusions:
- A pool of Src-interacting Na/K-ATPase regulates basal Src activity in LLC-PK1 cells.
- Na/K-ATPase functions as a receptor for ouabain, mediating protein kinase activation.
- The Na/K-ATPase-Src complex is a key regulator of cellular signaling pathways.
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