Parathyroid hormone-mediated regulation of Na⁺-K ⁺-ATPase requires ERK-dependent translocation of protein kinase Cα

Parathyroid hormone (PTH) inhibits Na⁺-K⁺-ATPase activity by serine phosphorylation of the α₁ subunit through protein kinase C (PKC)- and extracellular signal-regulated kinase (ERK)-dependent pathways. Based on previous studies we postulated that PTH regulates sodium pump activity through isoform-specific PKC-dependent activation of ERK. In the present work utilizing opossum kidney cells, a model of renal proximal tubule, PTH stimulated membrane translocation of PKCα by 102 ± 16% and PKCβI by 41 ± 7% but had no effect on PKCβII and PKCζ. Both PKCα and PKCβI phosphorylated the Na ⁺-K⁺-ATPase α₁ subunit in vitro. PTH increased the activity of PKCα but not PKCβI. Coimmunoprecipitation assays demonstrated that treatment with PTH enhanced the association between Na⁺-K⁺-ATPase α₁ subunit and PKCα, whereas the association between Na⁺-K⁺-ATPase α₁ subunit and PKCβI remained unchanged. A PKCα inhibitory peptide blocked PTH-stimulated serine phosphorylation of the Na ⁺-K⁺-ATPase α₁ subunit and inhibition of Na⁺-K⁺-ATPase activity. Pharmacologic inhibition of MEK-1 blocked PTH-stimulated translocation of PKCα, whereas transfection of constitutively active MEK-1 cDNA induced translocation of PKCα and increased phosphorylation of the Na⁺-K⁺-ATPase α₁ subunit. In contrast, PTH-stimulated ERK activation was not inhibited by pretreatment with the PKCa inhibitory peptide. Inhibition of PKCα expression by siRNA did not inhibit PTH-mediated ERK activation but significantly reduced PTH-mediated phosphorylation of the Na⁺-K ⁺-ATPase α₁ subunit. Pharmacologic inhibition of phosphoinositide 3-kinase blocked PTH-stimulated ERK activation, translocation of PKCα, and phosphorylation of the Na⁺-K⁺-ATPase α₁ subunit. We conclude that PTH stimulates Na ⁺-K⁺-ATPase phosphorylation and decreases the activity of Na⁺-K⁺-ATPase by ERK-dependent activation of PKCα.