Here we show that ouabain-induced cell growth regulation is intrinsically coupled to changes in the cellular amount of Na/KATPase via the phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway. Ouabain increases the endocytosis and degradation of Na/K-ATPase in LLC-PK1, human breast (BT20), and prostate (DU145) cancer cells. However, ouabain stimulates the PI3K/Akt/mTOR pathway and consequently up-regulates the expression of Na/K-ATPase in LLC-PK1 but not BT20 and DU145 cells. This up-regulation is sufficient to replete the plasma membrane pool of Na/K-ATPase and to stimulate cell proliferation in LLC-PK1 cells. On the other hand, ouabain causes a gradual depletion of Na/K-ATPase and an increased expression of cell cycle inhibitor p21 cip , which consequently inhibits cell proliferation in BT20 and DU145 cells. Consistently, we observe that small interfering RNA-mediated knockdown of Na/K-ATPase is sufficient to induce the expression of p21 cip and slow the proliferation of LLC-PK1 cells. Moreover, this knockdown converts the growth stimulatory effect of ouabain to growth inhibition in LLC-PK1 cells. Mechanistically, both Src and caveolin-1 are required for ouabaininduced activation of Akt and up-regulation of Na/K-ATPase. Furthermore, inhibition of the PI3K/Akt/mTOR pathway by rapamycin completely blocks ouabain-induced expression of Na/K-ATPase and converts ouabain-induced growth stimulation to growth inhibition in LLC-PK1 cells. Taken together, we conclude that changes in the expression of Na/K-ATPase dictate the growth regulatory effects of ouabain on cells.The Na/K-ATPase, a member of P-type ATPase family, was discovered as an energy transducing ion pump. It transports Na ϩ and K ϩ across the cell membrane and maintains ion homeostasis in animal cells (1, 2). Recent studies indicate that the Na/K-ATPase is also an important receptor that can transduce ligand binding into the activation of protein kinase cascades (3). Specifically, the Na/K-ATPase interacts with Src, which provides at least two important cellular regulations (4, 5). First, association with Na/K-ATPase keeps Src in an inactive state. Thus, the Na/K-ATPase serves as a native negative Src regulator (4). Second, this interaction forms a functional receptor complex for cardiotonic steroids (CTS) 3 (3), a group of well characterized ligands of the Na/K-ATPase. Cardiotonic steroids include cardenolides (e.g. ouabain) and bufadienolides (e.g. marinobufagenin) (6). Although CTS are known cardiac drugs, some of them have now been identified as endogenous steroid hormones (6 -8). Binding of CTS to the receptor complex activates the Na/K-ATPase-associated Src. Subsequently, the activated Src transactivates other tyrosine kinases, and together they recruit and further phosphorylate multiple membrane and soluble proteins, which results in the activation of protein kinase cascades and the generation of second messengers (3, 4, 6). Ultimately, this chain of signaling events would alter cellular functions and cell growth in a cell-...

Background: It has not been possible to study the pumping and signaling functions of Na/K-ATPase independently in live cells. Results: Both cell-free and cell-based assays indicate that the A420P mutation abolishes the Src regulatory function of Na/K-ATPase. Conclusion: A420P mutant has normal pumping but not signaling function. Significance: Identification of Src regulation-null mutants is crucial for addressing physiological role of Na/K-ATPase.

Pase is a fundamental component of ion transport. Four ␣ isoforms of the Na-K-ATPase catalytic ␣ subunit are expressed in human cells. The ubiquitous Na-K-ATPase ␣1 was recently discovered to also mediate signal transduction through Src kinase. In contrast, ␣2 expression is limited to a few cell types including myocytes, where it is coupled to the Na ϩ /Ca 2ϩ exchanger. To test whether rat Na-KATPase ␣2 is capable of cellular signaling like its ␣1 counterpart in a recipient mammalian system, we used an ␣1 knockdown pig renal epithelial cell (PY-17) to create an ␣2-expressing cell line with no detectable level of ␣1 expression. These cells exhibited normal ouabain-sensitive ATPase, but failed to effectively regulate Src. In contrast to ␣1-expressing cells, ouabain did not stimulate Src kinase or downstream effectors such as ERK and Akt in ␣2 cells, although their signaling apparatus was intact as evidenced by EGF-mediated signal transduction. Additionally, ␣2 cells were unable to rescue caveolin-1. Unlike the NaKtide sequence derived from Na-K-ATPase ␣1, which downregulates basal Src activity, the corresponding ␣2 NaKtide was unable to inhibit Src in vitro. Finally, coimmunoprecipitation of cellular Src was diminished in ␣2 cells. These findings indicate that Na-K-ATPase ␣2 does not regulate Src and, therefore, may not serve the same role in signal transduction as ␣1. This further implies that the signaling mechanism of Na-K-ATPase is isoform specific, thereby supporting a model where ␣1 and ␣2 isoforms play distinct roles in mediating contraction and signaling in myocytes.rat Na-K-ATPase isoforms; Src tyrosine kinase; membrane transporters; ouabain; signal transduction THE NA-K-ATPASE, discovered in 1957 by Jens Skou, is a member of the P-type ATPase family and an integral membrane protein maintaining cellular ion homeostasis by pumping Na ϩ and K ϩ across the cell membrane (32). The protein consists of two noncovalently linked subunits, ␣ and ␤. The ␣-subunit contains the binding sites for substrates (e.g., ions and ATP) and ligands (e.g., ouabain) as it undergoes E1 and E2 conformational changes during a transport cycle. Four isoforms of the Na-K-ATPase ␣-subunit are expressed in humans. While ␣1 is expressed ubiquitously, ␣2 and ␣3 are primarily found in myocytes and neurons, respectively, and ␣4 is detected in sperm (3,4,31,45,50). Recent studies have revealed major differences in the physiological functions of each isoform. For example, the ␣2 isoform appears to possess the unique ability to regulate intracellular Ca 2ϩ levels in myocytes and coresides with the Na ϩ /Ca 2ϩ exchanger (5, 24). In addition, recent experiments using SWAP mice (ouabainsensitive ␣1 Na-K-ATPase mutant and ouabain-resistant ␣2 Na-K-ATPase mutant) suggest that the ␣2 isoform plays a more prominent role in calcium release in cardiac and smooth muscle myocytes than ␣1 (12).Over the last decade, we have also come to realize that the Na-K-ATPase may have many regulatory functions other than pumping ions across cell membranes. Studies fr...

Background:We propose that Na/K-ATPase regulates Src in an E1/E2 conformation-dependent manner. Results: Expression of ␣1 mutants defective in E1/E2 transition altered both basal and stimuli-induced Src regulation. Conclusion: Na/K-ATPase is necessary for dynamic regulation of Src and Src-mediated pathways. Significance: This is the first demonstration that E1/E2 transition-defective mutants can affect both pumping and signaling functions of Na/K-ATPase.

We have shown that Na/K-ATPase interacts with Src. Here, we test the role of this interaction in H2O2-induced activation of Src and ERK. We found that exposure of LLC-PK1 cells to H2O2 generated by the addition of glucose oxidase into the culture medium activated Src and ERK1/2. It also caused a modest reduction in the number of surface Na/K-ATPases and in ouabain-sensitive Rb(+) uptake. These effects of H2O2 seem similar to those induced by ouabain, a specific ligand of Na/K-ATPase, in LLC-PK1 cells. In accordance, we found that the effects of H2O2 on Src and ERK1/2 were inhibited in α1 Na/K-ATPase-knockdown PY-17 cells. Whereas expression of wild-type α1 or the A420P mutant α1 defective in Src regulation rescued the pumping activity in PY-17 cells, only α1, and not the A420P mutant, was able to restore the H2O2-induced activation of protein kinases. Consistent with this, disrupting the formation of the Na/K-ATPase/Src complex with pNaKtide attenuated the effects of H2O2 on the kinases. Moreover, a direct effect of H2O2 on Na/K-ATPase-mediated regulation of Src was demonstrated. Finally, H2O2 reduced the expression of E-cadherin through the Na/K-ATPase/Src-mediated signaling pathway. Taken together, the data suggest that the Na/K-ATPase/Src complex may serve as one of the receptor mechanisms for H2O2 to regulate Src/ERK protein kinases and consequently the phenotype of renal epithelial cells.