期刊
MOLECULAR BIOLOGY OF THE CELL
卷 16, 期 9, 页码 4034-4045出版社
AMER SOC CELL BIOLOGY
DOI: 10.1091/mbc.E05-04-0295
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- NHLBI NIH HHS [HL-67963, P01 HL036573, R01 HL067963, HL-36573] Funding Source: Medline
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-gamma 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 alpha 1 subunit interacts with PLC-gamma 1, whereas the N-terminus binds IP3R2 and IP3R3, suggesting that the signaling Na/K-ATPase may tether PLC-gamma 1 and IP3 receptors together to form a Ca2+-regulatory complex. This notion is supported by the following findings. First, both PLC-gamma 1 and IP3R2 coimmunoprecipitated 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-gamma 1 at Tyr(783) and activated PLC-gamma 1 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-gamma 1. Inhibition of Src or depletion of cholesterol also abolished the effect of ouabain on intracellular Ca2+.
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