Altered phosphorylation of RRXS*/T* motif in ouabain-treated renal epithelial cells is not mediated by inversion of the [Na](i)/[K+](i) ratio.
Recently, we reported that the death of ouabain-treated C7-MDCK cells resembling principal cells from collecting ducts of the Madin-Darby canine kidney (MDCK) is caused by ouabain interaction with Na+,K+-ATPase but is not mediated by inversion of the [Na+](i)/[K+](i) ratio. The mechanism of this intriguing phenomenon remains unknown. We therefore examined the action of ouabain on serine/threonine phosphoproteins as possible intermediates of cell death signaling. The death of ouabain-treated C7-MDCK cells proceeded by altered phosphorylation of the RRXS*/T*-motif in 4 proteins with Mr from 80 to 25 kDa. Similarly to cell death, inversion of the [Na+](i)/[K+](i) ratio evoked by Na+,K+-ATPase inhibition in K+-free medium did not affect the phosphorylation of RRXS*/T*-proteins but increased their sensitivity to ouabain. The action of ouabain was preserved in the presence of activators of protein kinases A (forskolin), G (sodium nitroprusside) and C (PMA) as well as inhibitors of protein kinase C (Go 6983, Go 6976) and serine-threonine phosphatases (okadaic acid). Phosphorylation of RRXS*/T*-proteins was also noted in ouabain-sensitive C11-MDCK cells resembling intercalated cells from collecting ducts, but was absent in ouabain-resistant smooth muscle cells from the rat aorta. Our results show that altered phosphorylation of RRXS*/T*-proteins in ouabain-treated C7-MDCK cells is mediated by its interaction with Na+,K+-ATPase but is not caused by inversion of the [Na+](i)/[K+](i) ratio. The molecular origin of serine-threonine kinases and/or phosphatases involved in phosphorylation of ouabain-sensitive proteins and their role in cell death signaling should be examined further.
Akimova
Olga A
OA
Research Centre, Centre hospitalier de l'Université de Montréal (CHUM) - Technopôle - Angus, Montreal, Quebec, Canada.
Lopina
Olga D
OD
Tremblay
Johanne
J
Hamet
Pavel
P
Orlov
Sergei N
SN
D020816:Amino Acid Motifs; D000818:Animals; D002478:Cells, Cultured; D017077:Culture Media, Conditioned; D004285:Dogs; D004847:Epithelial Cells / Q000187:drug effects* / Q000378:metabolism*; D007668:Kidney / Q000166:cytology*; D008297:Male; D010042:Ouabain / Q000494:pharmacology*; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D011188:Potassium / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D051381:Rats; D011921:Rats, Inbred WKY; D012964:Sodium / Q000378:metabolism*; D013379:Substrate Specificity
D017077:Culture Media, Conditioned; D010042:Ouabain; D012964:Sodium; D017346:Protein Serine-Threonine Kinases; D010749:Phosphoprotein Phosphatases; D011188:Potassium