title: 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.
authors.affiliation: From the Department of Biomedicine, Aarhus University, DK-8000 Aarhus C, Denmark ape@biomed.au.dk.
authors.forename: Anja P
authors.initials: AP
authors.lastname: Einholm
authors.affiliation: From the Department of Biomedicine, Aarhus University, DK-8000 Aarhus C, Denmark.
authors.forename: Hang N
authors.initials: HN
authors.lastname: Nielsen
authors.affiliation: From the Department of Biomedicine, Aarhus University, DK-8000 Aarhus C, Denmark.
authors.forename: Rikke
authors.initials: R
authors.lastname: Holm
authors.affiliation: From the Department of Biomedicine, Aarhus University, DK-8000 Aarhus C, Denmark.
authors.forename: Mads S
authors.initials: MS
authors.lastname: Toustrup-Jensen
authors.affiliation: From the Department of Biomedicine, Aarhus University, DK-8000 Aarhus C, Denmark.
authors.forename: Bente
authors.initials: B
authors.lastname: Vilsen
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D016296:Mutagenesis; D020125:Mutation, Missense; D010766:Phosphorylation / Q000235:genetics; D051381:Rats; D012964:Sodium / Q000737:chemistry / Q000378:metabolism*; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D012964:Sodium; D017868:Cyclic AMP-Dependent Protein Kinases; D000254:Sodium-Potassium-Exchanging ATPase
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