Simultaneous Phosphorylation of Ser11 and Ser18 in the α-Subunit Promotes the Recruitment of Na⁺,K⁺-ATPase Molecules to the Plasma Membrane

Abstract

Renal sodium homeostasis is a major determinant of blood pressure and is regulated by several natriuretic and antinatriuretic hormones. These hormones, acting through intracellular second messengers, either activate or inhibit proximal tubule Na(+),K(+)-ATPase. We have shown previously that phorbol ester (PMA) stimulation of endogenous PKC leads to activation of Na(+),K(+)-ATPase in cultured proximal tubule cells (OK cells) expressing the rodent Na(+), K(+)-ATPase alpha-subunit. We have now demonstrated that the treatment with PMA leads to an increased amount of Na(+),K(+)-ATPase molecules in the plasmalemma, which is proportional to the increased enzyme activity. Colchicine, dinitrophenol, and potassium cyanide prevented the PMA-dependent stimulation of activity without affecting the increased level of phosphorylation of the Na(+), K(+)-ATPase alpha-subunit. This suggests that phosphorylation does not directly stimulate Na(+),K(+)-ATPase activity; instead, phosphorylation may be the triggering mechanism for recruitment of Na(+),K(+)-ATPase molecules to the plasma membrane. Transfected cells expressing either an S11A or S18A mutant had the same basal Na(+),K(+)-ATPase activity as cells expressing the wild-type rodent alpha-subunit, but PMA stimulation of Na(+),K(+)-ATPase activity was completely abolished in either mutant. PMA treatment led to phosphorylation of the alpha-subunit by stimulation of PKC-beta, and the extent of this phosphorylation was greatly reduced in the S11A and S18A mutants. These results indicate that both Ser11 and Ser18 of the alpha-subunit are essential for PMA stimulation of Na(+), K(+)-ATPase activity, and that these amino acids are phosphorylated during this process. The results presented here support the hypothesis that PMA regulation of Na(+),K(+)-ATPase is the result of an increased number of Na(+),K(+)-ATPase molecules in the plasma membrane.

Structural Basis for α1 Versus α2 Isoform-distinct Behavior of the Na,K-ATPase

“…Thus, targets for protein kinase C phosphorylation present in the N terminus of ␣1 are absent in ␣2. The significance of this difference is underscored by the observation that in transfected opossum kidney cells (24) protein kinase C␤-mediated phosphorylation of ␣1 at these residues promotes its translocation to the plasma membrane. Similarly, in ␣1 but not ␣2, the presence of Tyr-5 within a consensus sequence for phosphorylation by tyrosine kinases of the Src family has been implicated in the insulin stimulation of pump activity in the proximal convoluted tubules of the rat kidney (25).…”

Agonist-dependent Regulation of Renal Na+, K+-ATPase Activity Is Modulated by Intracellular Sodium Concentration

“…The expression vector pCMV containing the rodent Na ϩ -pump ␣1-subunit cDNA was obtained from PharMingen. Mutants of ␣1 were prepared, as previously described (12)(13)(14)(15), from a plasmid containing the wild type ␣-subunit sequence and complementary oligonucleotides containing the desired change. Briefly, annealed plasmid and oligonucleotides were subjected to PCR amplification with Pfu polymerase, followed by restriction of the original wild type template with DpnI.…”

“…After transformation of bacteria, the recovered mutant plasmids were evaluated by restriction analysis and direct sequencing of the altered region. Plasmids containing the wild type and mutated ␣-subunit cDNAs were transfected into OK cells using liposomes, as described previously (12)(13)(14)(15). Selection for cells expressing the highest level of rodent ␣-subunit was achieved by exposing them to a medium containing 3 M ouabain.…”

“…These amino acids are phosphorylated by different PKC isoforms during these processes (10 -15). We also described that although dopamine inhibition of Na ϩ ,K ϩ -ATPase is mediated by endocytosis of plasma membrane Na ϩ ,K ϩ -ATPase molecules, PMA stimulation is due to recruitment of Na ϩ ,K ϩ -ATPase molecules from intracellular compartments to the plasma membrane (10,15).…”

Analysis of Na+,K+-ATPase Motion and Incorporation into the Plasma Membrane in Response to G Protein–coupled Receptor Signals in Living Cells

“…The action of these networks ultimately results in activation of protein kinases and phosphorylation of the Na ϩ ,K ϩ -ATPase catalytic ␣-subunit (Therien and Blostein, 2000). Depending on the type and isoform of the kinase their action could lead to a decrease or increase in cell Na ϩ ,K ϩ -ATPase activity (Carranza et al, 1998;Efendiev et al, 1999Efendiev et al, , 2000Ridge et al, 2002). However, phosphorylation may not affect the intrinsic properties of the Na ϩ ,K ϩ -ATPase (i.e., catalytic activity) but their subcellular distribution.…”