Multiple Signal Transduction Pathways Link Na+/K+-ATPase to Growth-related Genes in Cardiac Myocytes

Journal of Biological Chemistry

et al. 2006

151

228

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...

Section: Discussionsupporting

confidence: 59%

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

Liang

Cai

Journal of Biological Chemistry

et al. 2006

151

228

“…Interaction of Ouabain with Na ϩ /K ϩ -ATPase Initiates the Signal Transducing Function of the Enzyme-The earlier work in our laboratory focusing on MAPK and Src activation in response to ouabain was done in both neonatal cardiac myocytes and A7r5 cells (7)(8)(9). These cells as well as mouse SYF ϩ c-Src cells are known to express a relatively ouabain-insensitive Na ϩ /K ϩ -ATPase ( Fig.…”

Section: And Egfrmentioning

confidence: 99%

Src-mediated Inter-receptor Cross-talk between the Na+/K+-ATPase and the Epidermal Growth Factor Receptor Relays the Signal from Ouabain to Mitogen-activated Protein Kinases

Haas

Wang

Journal of Biological Chemistry

et al. 2002

Self Cite

264

260

“…Recent work from several laboratories indicates that the enzyme also functions as a signaltransducing receptor for both endogenous and exogenous cardiotonic steroids such as ouabain (Kometiani et al, 1998;Xie et al, 1999;Haas et al, 2000Haas et al, , 2002Liu et al, 2000;Aizman et al, 2001;Aydemir-Koksoy et al, 2001;Miyakawa-Naito et al, 2003). First, binding of ouabain to the signaling Na/KATPase activates multiple signaling pathways and regulates transcription and translation of many genes in cardiac myocytes and other cell types.…”

Section: Introductionmentioning

confidence: 99%

Na/K-ATPase Tethers Phospholipase C and IP3 Receptor into a Calcium-regulatory Complex

Yuan

Cai

et al. 2005

MBoC

Self Cite

202

209

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.