Angiotensin II-dependent phosphorylation at Ser11/Ser18 and Ser938 shifts the E2 conformations of rat kidney Na+/K+-ATPase

Abstract

Kidney plasma membranes, which contain a single α-1 isoform of Na+/K+ -ATPase, simultaneously contain two sub-conformations of E2P, differing in their rate of digoxin release in response to Na+ and ATP. Treating cells with Ang II (angiotensin II) somehow changes the conformation of both, because it differentially inhibits the rate of digoxin release. In the present study we tested whether Ang II regulates release by increasing phosphorylation at Ser11/Ser18 and Ser938. Opossum kidney cells co-expressing the AT1a receptor and either α-1.wild-type, α-1.S11A/S18A or α-1.S938A were treated with or without 10 nM Ang II for 5 min, increasing phosphorylation at the three sites. Na+/K+ -ATPase was bound to digoxin-affinity columns in the presence of Na+, ATP and Mg2+. A solution containing 30 mM NaCl and 3 mM ATP eluted ~ 20 % of bound untreated Na+/K+ -ATPase (Population #1). Pre-treating cells with Ang II slowed the elution of Population #1 in α-1.wild-type and α-1.S938A, but not α-1.S11A/S18A cells. Another 50 % of bound Na+/K+ -ATPase (Population #2) was subsequently eluted in two phases by a solution containing 150 mM NaCl and 3 mM ATP. Ang II increased the initial rate and slowed the second phase in α-1.wild-type, but not α-1.S938A, cells. Thus Ang II changes the conformation of two forms of EP2 via differential phosphorylation.

Ang II acutely stimulates Na,K‐pump in cells from proximal tubules by increasing its phosphorylation at S938 via a PI3K / AKT pathway

“…On the other hand, the idea that PKA-dependent phosphorylation of S938 was required was a surprise, because the binding of Ang II to OK cells stably expressing the AT 1 receptor markedly decreases intracellular concentrations of cAMP (Thekkumkara et al, 1998), which is the intracellular second messenger that directly activates PKA. Also, in our earlier studies we found that Ang II dependent phosphorylation of the Na,K-pump at S938 is not blocked by H-89 (Massey et al, 2012), which selectively inhibits PKA at the concentration used in our experiments. Thus, we investigated whether S938 could be phosphorylated by another kinase.…”

“…As our study only involved cultured cells, it was exempt from institutional approval at Wayne State University. Cell cultures were maintained in DMEM-F12 (HyClone) supplemented with 10% fetal bovine serum (Corning), 1% penicillin-streptomycin (Gibco) and 1 μM ouabain (Sigma) (Massey et al, 2012(Massey et al, , 2016, which was included to kill any cells that were not primarily expressing the ouabain-resistant rat α-1 subunit of the Na,K-pump. For immunoblotting experiments, cultures were switched to DMEM-F12 without serum or ouabain supplementation on the day prior to stimulation and preparation of cell lysates (Massey et al, 2016;Yingst et al, 2008).…”

Stimulation of Na+/K+-ATPase with an Antibody against Its 4thExtracellular Region Attenuates Angiotensin II-Induced H9c2 Cardiomyocyte Hypertrophy via an AMPK/SIRT3/PPARγSignaling Pathway

“…An important function of NKA is to regulate cell volume [38, 39]. Recently, NKA expression and activity were also found to be closely regulated by Ang II [14, 40–42]. For instance, Ang II can inhibit NKA activity via induction of NADPH oxidase-derived O 2 ∗ −15 .…”

“…Rasmussen's group reported that Ang II induced NKA inhibition in cardiac myocytes via PKC-dependent activation of NADPH oxidase [13]. Massey et al also reported that Ang II-dependent phosphorylation of the rat kidney NKA at specific sites can regulate how the NKA releases bound cardiac glycoside [14]. Moreover, Ang II inhibits the NKA activity accompanied with the involvement of an increase in NADPH oxidase-derived O 2 ∗ - [15].…”

Kinetic Comparisons of Heart and Kidney Na+,K+-ATPases

“…Both phosphorylation and glutathionylation have been suggested as possible acute regulatory mechanisms. Recently, Massey et al (33) reported that in the kidney, a differential regulatory phosphorylation of the Na þ ,K þ -ATPase occurs depending on the enzyme's conformational state. Liu et al (34) found that the b-subunit of Na þ ,K þ -ATPase in the heart undergoes glutathionylation, also depending on the pump's conformational state.…”