Activation of protein kinase A acutely inhibits and phosphorylates Na/H exchanger NHE-3

O. W. Moe, M. Amemiya, Y. Yamaji

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Abstract

In the mammalian renal proximal tubule, protein kinase A (PKA) plays an important role in mediating hormonal regulation of apical membrane Na/H exchanger activity. This exchanger is likely encoded by NHE-3. The present studies examined regulation of NHE-3 by PKA. NHE-3 was stably expressed in Na/H exchanger-deficient fibroblasts (AP-1/NHE-3 cells). PKA activation (0.1 mM 8-BrcAMP x 20 min) inhibited NHE-3 activity by 39% (P < 0.01) with no change in NHE-3 protein abundance in the plasma membrane. To define the structural requirements for PKA-mediated inhibition, full-length NHE-3 and a cytoplasmic domain.truncated mutant (NHE-3(Δcyto)) were expressed in Xenopus laevis oocytes. 8-BrcAMP inhibited NHE-3 activity by 27% (P < 0.05), an effect that was blocked by 10-7 M PKA inhibitor peptide. NHE-3(Δcyto) bad baseline activity similar to that of full-length NHE-3 but its activity was not regulated by 8-BrcAMP. The purified recombinant cytoplasmic domain of NHE-3 was phosphorylated in vitro by the catalytic subunit of PKA on serine residues. In AP-1/NHE-3 cells, NHE-3 was immunoprecipitated as a ~87-kD phosphoprotein. Addition of 0.1 mM 8-BrcAMP increased the phosphocontent of NHE-3 by threefold. In summary, acute activation of PKA inhibits NHE-3 activity, an effect that is likely mediated by phosphorylation of its cytoplasmic domain.

Original languageEnglish (US)
Pages (from-to)2187-2194
Number of pages8
JournalJournal of Clinical Investigation
Volume96
Issue number5
StatePublished - 1995

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Sodium-Hydrogen Antiporter
Cyclic AMP-Dependent Protein Kinases
Transcription Factor AP-1
Cyclic AMP-Dependent Protein Kinase Catalytic Subunits
Proximal Kidney Tubule
Phosphoproteins
Xenopus laevis
Serine
Oocytes
Fibroblasts
Phosphorylation
Cell Membrane
Membranes

Keywords

  • hormonal regulation
  • hydrogen ion transport
  • Na transport

ASJC Scopus subject areas

  • Medicine(all)

Cite this

Activation of protein kinase A acutely inhibits and phosphorylates Na/H exchanger NHE-3. / Moe, O. W.; Amemiya, M.; Yamaji, Y.

In: Journal of Clinical Investigation, Vol. 96, No. 5, 1995, p. 2187-2194.

Research output: Contribution to journalArticle

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abstract = "In the mammalian renal proximal tubule, protein kinase A (PKA) plays an important role in mediating hormonal regulation of apical membrane Na/H exchanger activity. This exchanger is likely encoded by NHE-3. The present studies examined regulation of NHE-3 by PKA. NHE-3 was stably expressed in Na/H exchanger-deficient fibroblasts (AP-1/NHE-3 cells). PKA activation (0.1 mM 8-BrcAMP x 20 min) inhibited NHE-3 activity by 39{\%} (P < 0.01) with no change in NHE-3 protein abundance in the plasma membrane. To define the structural requirements for PKA-mediated inhibition, full-length NHE-3 and a cytoplasmic domain.truncated mutant (NHE-3(Δcyto)) were expressed in Xenopus laevis oocytes. 8-BrcAMP inhibited NHE-3 activity by 27{\%} (P < 0.05), an effect that was blocked by 10-7 M PKA inhibitor peptide. NHE-3(Δcyto) bad baseline activity similar to that of full-length NHE-3 but its activity was not regulated by 8-BrcAMP. The purified recombinant cytoplasmic domain of NHE-3 was phosphorylated in vitro by the catalytic subunit of PKA on serine residues. In AP-1/NHE-3 cells, NHE-3 was immunoprecipitated as a ~87-kD phosphoprotein. Addition of 0.1 mM 8-BrcAMP increased the phosphocontent of NHE-3 by threefold. In summary, acute activation of PKA inhibits NHE-3 activity, an effect that is likely mediated by phosphorylation of its cytoplasmic domain.",
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