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

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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.

Original languageEnglish
JournalJournal of Biological Chemistry
Volume291
Issue20
Pages (from-to)10934-47
Number of pages14
ISSN0021-9258
Publication statusPublished - 13 May 2016

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