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.

We have shown that the caveolar Na/K-ATPase transmits ouabain signals via multiple signalplexes. To obtain the information on the composition of such complexes, we separated the Na/K-ATPase from the outer medulla of rat kidney into two different fractions by detergent treatment and density gradient centrifugation. Analysis of the light fraction indicated that both PLC-␥1 and IP3 receptors (isoforms 2 and 3, IP3R2 and IP3R3) were coenriched with the Na/K-ATPase, caveolin-1 and Src. GST pulldown assays revealed that the central loop of the Na/K-ATPase ␣1 subunit interacts with PLC-␥1, whereas the N-terminus binds IP3R2 and IP3R3, suggesting that the signaling Na/K-ATPase may tether PLC-␥1 and IP3 receptors together to form a Ca 2؉ -regulatory complex. This notion is supported by the following findings.

Short exposure to a low concentration of ouabain protects the heart against ischemia/reperfusion injury. This effect of ouabain on the heart is most likely due to the activation of the Na+,K+-ATPase/c-Src receptor complex and subsequent stimulation of key mediators of preconditioning, namely PLC-gamma1 and PKCepsilon.

a b s t r a c tFlavonoids exhibit a variety of beneficial effects in cardiovascular diseases. Although their therapeutic properties have been attributed mainly to their antioxidant action, they have additional protective mechanisms such as inhibition of signal transducer and activator of transcription 1 (STAT1) activation. Here, we have investigated the cardioprotective mechanisms of strong antioxidant flavonoids such as quercetin, myricetin and delphinidin. Although all of them protect the heart from ischemia/reperfusion-injury, myricetin and delphinidin exert a more pronounced protective action than quercetin by their capacity to inhibit STAT1 activation. Biochemical and computer modeling analysis indicated the direct interaction between STAT1 and flavonoids with anti-STAT1 activity.