Importance of a Potential Protein Kinase A Phosphorylation Site of Na+,K+-ATPase and Its Interaction Network for Na+ Binding

Abstract

The molecular mechanism underlying PKA-mediated regulation of Na(+),K(+)-ATPase was explored in mutagenesis studies of the potential PKA site at Ser-938 and surrounding charged residues. The phosphomimetic mutations S938D/E interfered with Na(+) binding from the intracellular side of the membrane, whereas Na(+) binding from the extracellular side was unaffected. The reduction of Na(+) affinity is within the range expected for physiological regulation of the intracellular Na(+) concentration, thus supporting the hypothesis that PKA-mediated phosphorylation of Ser-938 regulates Na(+),K(+)-ATPase activity in vivo Ser-938 is located in the intracellular loop between transmembrane segments M8 and M9. An extended bonding network connects this loop with M10, the C terminus, and the Na(+) binding region. Charged residues Asp-997, Glu-998, Arg-1000, and Lys-1001 in M10, participating in this bonding network, are crucial to Na(+) interaction. Replacement of Arg-1005, also located in the vicinity of Ser-938, with alanine, lysine, methionine, or serine resulted in wild type-like Na(+) and K(+) affinities and catalytic turnover rate. However, when combined with the phosphomimetic mutation S938E only lysine substitution of Arg-1005 was compatible with Na(+),K(+)-ATPase function, and the Na(+) affinity of this double mutant was reduced even more than in single mutant S938E. This result indicates that the positive side chain of Arg-1005 or the lysine substituent plays a mechanistic role as interaction partner of phosphorylated Ser-938, transducing the phosphorylation signal into a reduced affinity of Na(+) site III. Electrostatic interaction of Glu-998 is of minor importance for the reduction of Na(+) affinity by phosphomimetic S938E as revealed by combining S938E with E998A.

Keywords: Na+/K+-ATPase; P-type ATPase; PKA site; membrane transport; post-translational regulation; protein kinase A (PKA); site-directed mutagenesis; sodium binding; sodium transport.

FIGURE 1.

Reaction cycle and structure of Na⁺,K⁺-ATPase. A, simplified scheme of the Na⁺,K⁺-ATPase reaction cycle (reactions 1–6). E₁ and E₂ denote the two major conformational states of the enzyme. P represents phosphorylation of the conserved aspartate in the catalytic site of the P-domain. Occluded ions are shown in parentheses, and boxed ATP indicates low affinity binding of ATP. B, overall structure of the Na⁺-bound E₁ form of Na⁺,K⁺-ATPase with the L8-9 loop containing the potential PKA site highlighted in yellow. Details of this region (framed by a broken line) are shown in C. The cytoplasmic A-, P-, and N-domains of the α-subunit as well as the β- and γ-subunits are indicated. C, close-up showing the relations of the potential PKA site at Ser-938 in L8-9 (yellow), the C terminus (light blue), and the ion-binding transmembrane segments M4, M5, M6, and M8. The view is from the cytoplasmic side of the membrane. The residues studied by mutagenesis are depicted as sticks (Ser-938, Asp-997, Glu-998, Arg-1000, Lys-1001, and Arg-1005). In addition, Thr-1016 and the C-terminal tyrosine (Tyr-1018) are shown as sticks as are the interaction partners of the C terminus, Arg-935 in L8-9 and Lys-768 in M5. The three bound Na⁺ ions are depicted as purple spheres labeled I, II, and III according to site nomenclature. B and C were prepared from the Protein Data Bank structure with code 3WGV (8) using PyMOL.

FIGURE 2.

The phosphomimetic S938D/E mutations interfere with the binding of Na⁺ at the intracellularly facing E₁ sites. A, Na⁺ dependence of phosphorylation from [γ-³²P]ATP. Each line shows the best fit of the Hill equation, and the extracted K_0.5 values are listed in Table 1. B, ATP dependence of Na⁺,K⁺-ATPase activity. Each line shows the best fit of the Hill equation, and the extracted K_0.5 values are listed in Table 1. C, Na⁺ dependence of Na⁺-ATPase activity. Inhibition of Na⁺-ATPase activity by high Na⁺ concentrations is indicated semiquantitatively in Table 1. D, K⁺ dependence of Na⁺,K⁺-ATPase activity. Each line shows the best fit of the Hill equation, and the extracted K_0.5 values are listed in Table 1. E, distribution of the phosphoenzyme between E₁P and E₂P at 150 mm NaCl. Each line shows the best fit of a biexponential decay function. The initial amounts of E₂P, which correspond to the amplitude of the slow phase, are listed in Table 1. A–E, experimental conditions and equations used for data fitting are described under “Experimental Procedures.” Statistical information is given in Table 1. Symbol and error bars (seen only when larger than the size of the symbols) represent mean ± S.E. Dotted lines reproduce the wild type for direct comparison in the same panel.

FIGURE 3.

Several charged residues in M10 near Ser-938, but not Arg-1005, are crucial to binding of Na⁺ at the intracellularly facing E₁ sites. A, Na⁺ dependence of phosphorylation from [γ-³²P]ATP. Each line shows the best fit of the Hill equation, and the extracted K_0.5 values are listed in Table 1. B, ATP dependence of Na⁺,K⁺-ATPase activity. Each line shows the best fit of the Hill equation, and the extracted K_0.5 values are listed in Table 1. C, K⁺ dependence of Na⁺,K⁺-ATPase activity. Each line shows the best fit of the Hill equation or, for the mutants showing inhibition at high K⁺ concentrations, a two-component Hill equation with the inhibition represented by a negative term. K_0.5 values for the rising parts of the curves are listed in Table 1. A–C, experimental conditions and equations used for data fitting are described under “Experimental Procedures.” Statistical information is given in Table 1. Symbol and error bars (seen only when larger than the size of the symbols) represent mean ± S.E. Dotted lines reproduce the wild type for direct comparison in the same panel.

FIGURE 4.

Several charged residues in M10 near Ser-938, but not Arg-1005, are important for binding of Na⁺ from the extracellular side of the membrane. A, Na⁺ dependence of Na⁺-ATPase activity. Inhibition of Na⁺-ATPase activity by high Na⁺ concentrations is indicated semiquantitatively in Table 1. B, distribution of the phosphoenzyme between E₁P and E₂P at 150 mm NaCl. Each line shows the best fit of a biexponential decay function. The initial amounts of E₂P, which correspond to the amplitude of the slow phase, are listed in Table 1. A and B, experimental conditions and equations used for data fitting are described under “Experimental Procedures.” Statistical information is given in Table 1. Symbol and error bars (seen only when larger than the size of the symbols) represent mean ± S.E. Dotted lines reproduce the wild type for direct comparison in the same panel.

FIGURE 5.

The phosphomimetic mutation S938E mediates its effect through interaction with Arg-1005. A, representative phosphorimaging autoradiograph of transiently expressed double mutants. The double mutants S938E/R1005A, S938E/R1005M, and S938E/R1005S, which could not sustain cell viability in the presence of ouabain, were transiently expressed in the presence of siRNA-targeted knockdown of the endogenous COS-1 Na⁺,K⁺-ATPase. The autoradiograph shows ³²P incorporation from [γ-³²P]ATP at the catalytic site aspartate in the P-domain following separation by SDS-PAGE of enzyme phosphorylated in the presence of the Na⁺ concentrations indicated in mm. It can be seen that all the double mutants tested were phosphorylation-inactive. B, Na⁺ dependence of phosphorylation from [γ-³²P]ATP. Each line shows the best fit of the Hill equation, and the extracted K_0.5 values are listed in Table 1. C, ATP dependence of Na⁺,K⁺-ATPase activity. Each line shows the best fit of the Hill equation, and the extracted K_0.5 values are listed in Table 1. D, K⁺ dependence of Na⁺,K⁺-ATPase activity. Each line shows the best fit of a two-component Hill equation with the inhibition represented by a negative term. K_0.5 values for the rising parts of the curves are listed in Table 1. E, Na⁺ dependence of Na⁺-ATPase activity in the absence of K⁺. Inhibition of Na⁺-ATPase activity by high Na⁺ concentrations is indicated semiquantitatively in Table 1. B–E, experimental conditions and equations used for data fitting are described under “Experimental Procedures.” Statistical information is given in Table 1. Symbol and error bars (seen only when larger than the size of the symbols) represent mean ± S.E. Dotted lines reproduce the wild type for direct comparison in the same panel.

FIGURE 6.

PKA-mediated phosphorylation of Na⁺,K⁺-ATPase mutants. Representative phosphorimaging autoradiographs showing ³²P incorporation following incubation with [γ-³²P]ATP with PKA or without PKA catalytic subunit and separation by SDS-PAGE of COS-1 cell plasma membrane proteins containing expressed Na⁺,K⁺-ATPase mutants or wild type (WT) or purified Na⁺,K⁺-ATPase from pig kidney. See “Experimental Procedures” for further details. Two SDS-PAGE gels, A and B, are shown. A, lane 1, WT − PKA; lane 2, WT + PKA; lane 3, S938A + PKA; lane 4, S938A − PKA. B, lane 1, S938A − PKA; lane 2, S938A +PKA; lane 3, R1005A − PKA; lane 4, R1005A + PKA; lane 5, R1005K − PKA; lane 6, R1005K + PKA; lane 7, R1005M + PKA; lane 8, purified Na⁺,K⁺-ATPase (15 μg) +PKA; lane 9, purified Na⁺,K⁺-ATPase (20 μg) + PKA. The arrow indicates the migration position corresponding to the purified Na⁺,K⁺-ATPase. For both gel A and gel B, 35 μg of total plasma membrane protein from COS-1 cells expressing mutant or wild type were loaded in each lane. Plasma membrane preparations containing approximately equal amounts (±5%) of expressed exogenous mutant or wild type rat Na⁺,K⁺-ATPase per mg of total protein were used based on determination of the active site concentration.

FIGURE 7.

Structural relations of L8-9 containing the potential PKA site Ser-938. Shown is the relevant part of the structure of the Na⁺-bound E₁ form of Na⁺,K⁺-ATPase (Protein Data Bank code 3WGV) (8) viewed from the side along the membrane surface. Depicted is the extensive bonding network that connects L8-9 (yellow) harboring the potential PKA site at Ser-938 with M10 (gray), the C terminus (light blue), and the Na⁺ binding segment M8 (gray). The residues studied by mutagenesis are depicted as sticks (Ser-938, Asp-997, Glu-998, Arg-1000, Lys-1001, and Arg-1005). Stick representation of backbone atoms (blue nitrogen and red oxygen atoms) is shown for the entire L8-9. In addition, Gln-925 and Asp-928 known to contribute to the binding of the Na⁺ ion at site III are shown as sticks as are Ile-931 and Thr-934 that interact with Lys-1001 in M10. The Na⁺ ion bound at site III is depicted as a large purple sphere. The figure was prepared using PyMOL.