Ral-GTPase interacts with the beta1 subunit of Na+/K+-ATPase and is activated upon inhibition of the Na+/K+ pump.
Na+/K+-ATPase functions as both an ion pump and a signal transducer. Cardiac glycosides partially inhibit Na+/K+-ATPase, causing activation of multiple interrelated growth pathways via the Na+/K+-ATPase/c-Src/epidermal growth factor receptor complex. Such pathways include Ras/MEK/ERK and Ral/RalGDS cascades, which can lead to cardiac hypertrophy. In search of novel Ral-GTPase binding proteins, we used RalB as the bait to screen a human testes cDNA expression library using the yeast 2-hybrid system. The results demonstrated that 1 of the RalB interacting clones represented the C-terminal region of the beta1 subunit of Na+/K+-ATPase. Further analysis using the yeast 2-hybrid system and full-length beta1 subunit of Na+/K+-ATPase confirmed the interaction with RalA and RalB. In vitro binding and pull-down assays demonstrated that the beta1 subunit of Na+/K+-ATPase interacts directly with RalA and RalB. Ral-GTP pull-down assays demonstrated that short-term ouabain treatment of A7r5 cells, a rat aorta smooth muscle cell line, caused activation of Ral GTPase. Maximal activation was observed 10 min after ouabain treatment. Ouabain-mediated Ral activation was inhibited upon the stimulation of Na+/K+-ATPase activity by Ang II. We propose that Ral GTPase is involved in the signal transducing function of Na+/K+-ATPase and provides a possible molecular mechanism connecting Ral to cardiac hypertrophy during diseased conditions.
Bhullar
Rajinder P
RP
Department of Oral Biology, University of Manitoba, 744 Bannatyne Avenue, Winnipeg, MB R3E 0W2, Canada. bhullar@cc.umanitoba.ca
Clough
Richard R
RR
Kanungo
Juddy
J
Elsaraj
Sherif M
SM
Grujic
Ognjen
O
D000804:Angiotensin II / Q000494:pharmacology; D000818:Animals; D002460:Cell Line; D018076:DNA, Complementary / Q000235:genetics; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D008297:Male; D010042:Ouabain / Q000494:pharmacology*; D010957:Plasmids; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D013737:Testis / Q000378:metabolism; D020798:Two-Hybrid System Techniques; D015003:Yeasts / Q000235:genetics; D020725:ral GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*
C503554:Atp1b1 protein, rat; D018076:DNA, Complementary; C082190:Ralb protein, rat; D011994:Recombinant Proteins; D000804:Angiotensin II; D010042:Ouabain; D005982:Glutathione Transferase; C492197:Rala protein, rat; D020725:ral GTP-Binding Proteins; D000254:Sodium-Potassium-Exchanging ATPase