| Signaling Function of Na/K-ATPase in Ouabain-induced Regulation of Intracellular Calcium (2005) | |||||||||||||
Abstract | |||||||||||||
| 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-g1 and IP3 receptors (isoforms 2 and 3, IP3R2 and IP3R3) were co-enriched with the Na/K-ATPase, caveolin-1 and Src. GST pull-down assays revealed that the central loop of the Na/K-ATPase a1 subunit interacts with PLC-g1 while the N-terminus binds IP3R2 and IP3R3, suggesting that the signaling Na/K-ATPase may tether PLC-g1 and IP3 receptors together to form a Ca2+-regulatory complex. This notion is supported by the following findings. First, both PLC-g1 and IP3R2 co-immunoprecipitated with the Na/K-ATPase and ouabain increased this interaction in a dose- and time-dependent manner in LLC-PK1 cells. Depletion of cholesterol abolished the effects of ouabain on this interaction. Second, ouabain induced phosphorylation of PLC-g1 at Tyr783 and activated PLC-g1 in a Src-dependent manner, resulting in increased hydrolysis of PIP2. It also stimulated Src-dependent tyrosine phosphorylation of the IP3R2. Finally, ouabain induced Ca2+ release from the intracellular stores via the activation of IP3 receptors in LLC-PK1 cells. This effect required the ouabain-induced activation of PLC-g1. Inhibition of Src or depletion of cholesterol also abolished the effect of ouabain on intracellular Ca2+. Following the activation of PLC, PKC isozymes were also activated by ouabain signaling in a Src-dependent manner and recruited to the Na/K-ATPase signaling complex together with RACK-1, which is the receptor of activated PKCs and believed to facilitate PKCs signal transduction. | |||||||||||||
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