Na/K-ATPase tethers phospholipase C and IP3 receptor into a calcium-regulatory complex.
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-gamma1 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 alpha1 subunit interacts with PLC-gamma1, whereas the N-terminus binds IP3R2 and IP3R3, suggesting that the signaling Na/K-ATPase may tether PLC-gamma1 and IP3 receptors together to form a Ca(2+)-regulatory complex. This notion is supported by the following findings. First, both PLC-gamma1 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-gamma1 at Tyr(783) and activated PLC-gamma1 in a Src-dependent manner, resulting in increased hydrolysis of PIP2. It also stimulated Src-dependent tyrosine phosphorylation of the IP3R2. Finally, ouabain induced Ca(2+) release from the intracellular stores via the activation of IP3 receptors in LLC-PK1 cells. This effect required the ouabain-induced activation of PLC-gamma1. Inhibition of Src or depletion of cholesterol also abolished the effect of ouabain on intracellular Ca(2+).
Yuan
Zhaokan
Z
Department of Pharmacology, Medical College of Ohio, Toledo, OH 43614, USA.
Cai
Ting
T
Tian
Jiang
J
Ivanov
Alexander V
AV
Giovannucci
David R
DR
Xie
Zijian
Z
D000818:Animals; D002118:Calcium / Q000502:physiology*; D015220:Calcium Channels / Q000378:metabolism*; D002460:Cell Line; D053496:Inositol 1,4,5-Trisphosphate Receptors; D051379:Mice; D010042:Ouabain / Q000378:metabolism; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D051381:Rats; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology; D000254:Sodium-Potassium-Exchanging ATPase / Q000502:physiology*; D013552:Swine; D010738:Type C Phospholipases / Q000378:metabolism*
D015220:Calcium Channels; D053496:Inositol 1,4,5-Trisphosphate Receptors; D018160:Receptors, Cytoplasmic and Nuclear; D010042:Ouabain; D010738:Type C Phospholipases; D000254:Sodium-Potassium-Exchanging ATPase; D002118:Calcium