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Vol. 16, Issue 9, 4034-4045, September 2005
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Department of Neurosciences, Medical College of Ohio, Toledo, OH 43614;
* Department of Pharmacology, Medical College of Ohio, Toledo, OH 43614
Submitted April 14, 2005;
Revised June 3, 2005;
Accepted June 13, 2005
Monitoring Editor: Guido Guidotti
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 Ca2+-regulatory complex. This notion is supported by the following findings. First, both PLC-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-1 at Tyr783 and activated PLC-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-1. Inhibition of Src or depletion of cholesterol also abolished the effect of ouabain on intracellular Ca2+.
Abbreviations used: EGFR, EGF receptor; GST, glutathione S-transferase; RTK, receptor tyrosine kinases; ERK, extracellular signal-regulated kinase; RKE, rat kidney Na/K-ATPase; MALDI-TOF MS, matrix-assisted laser desorption/ionization-time of flight mass spectrometry; RIPA, radioimmunoprecipitation buffer; [Ca2+]i, intracellular calcium; IP, immunoprecipitation; IB, immunoblotting; PIP2, phosphatidylinositol-4,5-bisphosphate; IP3, inositol 1,4,5-trisphosphate; IP3R2, IP3 receptor isoform 2; DAG, 1,2-diacylglycerol; PLC, phospholipase C; GFP, green fluorescence protein; PH domain, pleckstrin homology domain; ER, endoplasmic reticulum; PMSF, phenylmethanesulfonyl fluoride; PP2, 4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo [3,4-d] pyrimidine; M
-CD, methyl-- cyclodextrin.
Address correspondence to: Zijian Xie (zxie{at}mco.edu).
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