PTH-mediated regulation of Na⁺-K⁺-ATPase requires Src kinase-dependent ERK phosphorylation

Parathyroid hormone (PTH) inhibits Na⁺-K⁺-ATPase activity by serine phosphorylation of the α₁-subunit through ERK-dependent phosphorylation and translocation of protein kinase Cα (PKCα). On the basis of previous studies, we postulated that PTH regulates sodium pump activity through Src kinase, PLC, and calcium-dependent ERK phosphorylation. In the present work utilizing opossum kidney cells, a model of renal proximal tubule, PTH-stimulated ERK phosphorylation and membrane translocation of PKCα were prevented by inhibition of Src kinase, PLC, and calcium entry. Pharmacological inhibition of PLA₂ did not prevent PTH-stimulated ERK phosphorylation but completely prevented PKCα translocation. Silencing the expression of cytosolic or calcium-independent PLA₂ also prevented PTH-mediated phosphorylation of Na ⁺-K⁺-ATPase α₁-subunit and PKCα without blocking ERK phosphorylation. Inhibition of Na⁺-K ⁺-ATPase activity by the PLA₂ metabolites arachidonic acid and 20-hydroxyeicosatetraenoic acid was prevented by specific inhibition of PKCα but not by U0126, a MEK-1 inhibitor. Transient transfection of constitutively active MEK-1 cDNA induced phosphorylation of Na ⁺-K⁺-ATPase α₁-subunit and PKCα, which was prevented by PLA₂ inhibition. We conclude that PTH stimulates Na⁺-K⁺-ATPase phosphorylation and decreases the activity of Na⁺-K⁺-ATPase by a sequential activation of a signaling pathway involving Src kinase, PLC, ERK, PLA₂, and PKCα.