Clathrin-mediated Endocytosis of Na⁺,K⁺-ATPase in Response to Parathyroid Hormone Requires ERK-dependent Phosphorylation of Ser-11 within the α₁-Subunit

Parathyroid hormone (PTH) inhibits Na⁺,K⁺-ATPase activity through protein kinase C- (PKC) and extracellular signal-regulated kinase- (ERK) dependent pathways and increases serine phosphorylation of the α₁-subunit. To determine whether specific serine phosphorylation sites within the Na⁺,K⁺-ATPase α₁-subunit are involved in the Na⁺,K ⁺-ATPase responses to PTH, we examined the effect of PTH in opossum kidney cells stably transfected with wild type rat Na⁺,K ⁺-ATPase α₁-subunit (WT), serine 11 to alanine mutant α₁-subunit (S11A), or serine 18 to alanine mutant α₁-subunit (S18A). PTH increased phosphorylation and endocytosis of the Na⁺,K⁺-ATPase α ₁-subunit into clathrin-coated vesicles in cells transfected with WT and S18A rat Na⁺,K⁺-ATPase α₁-subunits. PTH did not increase the level of phosphorylation or stimulate translocation of Na⁺,K⁺-ATPase α₁-subunits into clathrin-coated vesicles in cells transfected with the S11A mutant. PTH inhibited ouabain-sensitive ⁸⁶Rb uptake and Na⁺,K ⁺-ATPase activity (ouabain-sensitive ATP hydrolysis) in WT- and S18A-transfected opossum kidney cells but not in S11A-transfected cells. Pretreatment of the cells with the PKC inhibitors and ERK inhibitor blocked PTH inhibition of ⁸⁶Rb uptake, Na⁺,K⁺-ATPase activity, α₁-subunit phosphorylation, and endocytosis in WT and S18A cells. Consistent with the notion that ERK phosphorylates Na ⁺,K⁺-ATPase α₁-subunit, ERK was shown to be capable of causing phosphorylation of Na⁺,K⁺-ATPase α₁-subunit immunoprecipitated from WT and S18A but not from S11A-transfected cells. These results suggest that PTH regulates Na ⁺,K⁺-ATPase by PKC and ERK-dependent α ₁-subunit phosphorylation and that the phosphorylation requires the expression of a serine at the 11 position of the Na⁺,K ⁺-ATPase α₁-subunit.