Importance of a potential protein kinase A phosphorylation site of Na+,K+-ATPase and its interaction network for Na+ binding.

TY - JOUR

T1 - Importance of a potential protein kinase A phosphorylation site of Na+,K+-ATPase and its interaction network for Na+ binding.

AU - Einholm, Anja P.

AU - Nielsen, Hang Nguyen

AU - Holm, Rikke

AU - Toustrup-Jensen, Mads Schak

AU - Vilsen, Bente

PY - 2016/5/13

Y1 - 2016/5/13

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=84969134829&partnerID=8YFLogxK

M3 - Journal article

SN - 0021-9258

VL - 291

SP - 10934

EP - 10947

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

IS - 20

ER -