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Vol. 17, Issue 1, 317-326, January 2006
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* Departments of Pharmacology and Medicine, Medical University of Ohio, Toledo, OH 43614;
Department of Physiology, Cornell University, Weill Medical College, New York, NY 10021
Submitted August 9, 2005;
Revised September 27, 2005;
Accepted October 21, 2005
Monitoring Editor: Guido Guidotti
We have shown that ouabain activates Src, resulting in subsequent tyrosine phosphorylation of multiple effectors. Here, we tested if the Na+/K+-ATPase and Src can form a functional signaling complex. In LLC-PK1 cells the Na+/K+-ATPase and Src colocalized in the plasma membrane. Fluorescence resonance energy transfer analysis indicated that both proteins were in close proximity, suggesting a direct interaction. GST pulldown assay showed a direct, ouabain-regulated, and multifocal interaction between the 
1 subunit of Na+/K+-ATPase and Src. Although the interaction between the Src kinase domain and the third cytosolic domain (CD3) of 1 is regulated by ouabain, the Src SH3SH2 domain binds to the second cytosolic domain constitutively. Functionally, binding of Src to either the Na+/K+-ATPase or GST-CD3 inhibited Src activity. Addition of ouabain, but not vanadate, to the purified Na+/K+-ATPase/Src complex freed the kinase domain and restored the Src activity. Consistently, exposure of intact cells to ouabain apparently increased the distance between the Na+/K+-ATPase and Src. Concomitantly, it also stimulated tyrosine phosphorylation of the proteins that are associated with the Na+/K+-ATPase. These new findings illustrate a novel molecular mechanism of signal transduction involving the interaction of a P-type ATPase and a nonreceptor tyrosine kinase.
Abbreviations used: BRET, bioluminescence resonance energy transfer; ECFP, enhanced cyan fluorescent protein; EYFP, enhanced yellow fluorescent protein; FRET, fluorescence resonance energy transfer; GST, glutathione-S-transferase; IB, immunoblotting; IP, immunoprecipitation; PKE, purified pig kidney enzyme (Na+/K+-ATPase); PP2, 4-amino-5-[4-chlorophenyl]-7-[t-butyl]pyrazolo[3,4-d]pyrimidine; PP3, 4-amino-7-phenylpyrazol[3,4]pyromidine; RIPA, radioimmunoprecipitation.
These authors contributed equally to this work.
Address correspondence to: Zi-Jian Xie (zxie{at}meduohio.edu).
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