Functional Characterization of Src-interacting Na/K-ATPase Using RNA Interference Assay

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 ␣1 subunit knockdown cell lines were generated by transfecting LLC-PK1 cells with a vector expressing ␣1-specific small interference RNA. Although the ␣1 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 ouabaininduced activation of Src and ERK1/2. When the knockdown cells were rescued by a rat ␣1, 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 ␣1. Finally both fluorescence resonance energy transfer analysis and co-immunoprecipitation assay indicated that the pumping-null rat ␣1 (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. Na/K-ATPase was discovered by Skou (1) as the molecular machinery of the cellular sodium pump. It belongs to a family of evolutionarily ancient ATPases that couple the hydrolysis of ATP to membrane ion translocation (2, 3). A major difference between the Na/K-ATPase and other ATPases is its ability to bind cardiotonic steroids such as ouabain. Studies from many laboratories have now established that the binding of ouabain to this enzyme not only inhibits the ATPase activity but also stimulates protein tyrosine kinases such as Src (4, 5). The activated Src in turn transactivates epidermal growth factor receptor, resulting in the assembly and activation of multiple signaling cascades such as the ERK1/2 2 and phospholipase C-␥/ protein kinase C pathways (5, 6).Because several laboratories have demonstrated that the activation of Src is essential for ouabain-induced changes in many cellular activities including the regulation of intracellular calcium, gene expression, and cell growth (6 -9), we have recently examined whether the Na/K-ATPase interacts directly with Src to form a functional signaling receptor (10). Using in vitro glutathione S-transferase pulldown assays we have identified that the second and the third intracellular domains of the Na/K-ATPase ␣1 subunit interact with the Src SH2 and the kinase domains, respectively. Functionally these interactions keep Src in an inactive state, and binding of ouabain to this inactive Na/K-ATPase⅐Src complex frees and then activates the associated Src (10). These new findings suggest that the cellular Src-interacting Na/K-ATPase may play an...

Involvement of Reactive Oxygen Species in a Feed-forward Mechanism of Na/K-ATPase-mediated Signaling Transduction

“…Both PY-17 and C2-9 cells have disrupted Na/K-ATPase⅐c-Src signaling and do not respond to ouabain stimulation in terms of c-Src activation (11,31,32). As shown in Fig.…”

Expression of Mutant α1 Na/K-ATPase Defective in Conformational Transition Attenuates Src-mediated Signal Transduction*

“…The mutants were verified by DNA sequencing. To generate stable cell lines, the ␣1 knockdown PY-17 cells were transfected with different vectors expressing either I279A or F286A ␣1 mutant using Lipofectamine 2000 as previously described (5). The cells were then selected with ouabain (3 M) 24 h after transfection.…”

“…The generation of ␣1 knockdown PY-17 cells and the rat ␣1-rescued PY-17 cells (called AAC-19) were done as previously described (5). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% FBS (14).…”

“…The ouabain-resistant colonies were isolated and expanded into stable cell lines. Cells were then cultured in the absence of ouabain for three generations before being used for the experiments (5).…”

“…Lines-Site-directed mutagenesis of rat ␣1 subunit (I279A or F286A, numbered according to the GenBank TM accession number NM_01254) was performed by the QuikChange mutagenesis kit using pRc/CMV-␣1 AACm1 vector as a template (5). The pRc/CMV-␣1 AACm1 carries silent mutations that prevent the binding of ␣1-specific siRNA to the transcript of rat ␣1 mRNA (5).…”