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.
Highlights
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]
Na/K-ATPase1⁄7Src complex serves as a sole receptor for ouabain to activate Src and subsequently ERK1/2 in live cells
Manipulation of the Cellular Na/K-ATPase Content by RNA Interference Assays—RNA interference is a cellular mechanism that was first discovered in 1998 in Caenorhabditis elegans and refers to the post-transcriptional gene silencing by doublestranded RNA-triggered degradation of a homologous mRNA [21]
Summary
Materials—Chemicals of the highest purity were purchased from Sigma. The GeneSuppressor vector was purchased from BioCarta (San Diego, CA). Generation of Stable ␣1 Subunit Knockdown and Knock-in Cell Lines—Human embryonic kidney 293T cells were transiently transfected with different siRNA expression vectors along with pEYFP using Lipofectamine 2000 as we described previously [11]. To generate stable cell lines, one batch of LLC-PK1 cells was transfected with the A4 siRNA expression vector (pSuppressor-A4 siRNA) (Table 1) and a puromycin selection marker (pBade-puro). Another batch of cells was co-transfected with pEYFP together with the pSuppressor-A4 siRNA and pBade-puro so that the co-expressed EYFP could be used as a marker to pick clones. FRET analysis was performed in cells co-transfected with Src-ECFP and EYFP-rat ␣1 expression vectors using the acceptor photobleaching protocol as we described previously [10]. Cells transfected with either Src-ECFP and EYFP or EYFP-␣1 and ECFP expression vectors
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